Scientific Program - International BioIron Society

Transcription

Welcome to Bethesda and BioIron 2003!
Dear Colleagues,
It is with great pleasure that we welcome you to BioIron 2003, the 2nd World Congress on Iron
Metabolism. This marks the first time that the BioIron meeting has been held on the grounds of
the National Institutes of Health in Bethesda, Md.
Meeting highlights will cover advances in our understanding of iron transport and regulation
in organisms ranging from bacteria to man, discussion of important new therapeutic agents for
treatment of iron overload, evaluation of the role of genetic screening for hemochromatosis,
mitochondrial iron metabolism, the role of iron in neurodegenerative diseases, and many other
interesting topics.
The meeting also offers many social opportunities. Afternoon poster sessions can be followed by
dinner with new friends in any of the various and wonderful restaurants of downtown
Bethesda. Our dinner cruise to Mount Vernon, home of George Washington, will offer views
of Washington from the Potomac and a private viewing of the home of the first president of
the United States. The Thursday evening banquet will give everyone a chance to unwind after
a week of intense scientific interactions.
We hope for a wonderful meeting!
Tracey Rouault
Victor Gordeuk
Caroline Philpott
Esther Meyron-Holtz
Table of Contents
Local Organizing Committee & International Scientific Committee ......................... 1
Student Bursary Awardees...................................................................................... 2
Acknowledgements ................................................................................................. 3
General Meeting Information ................................................................................... 4
Social Activities ....................................................................................................... 5
Scientific Program ................................................................................................... 6
Poster and Podium Session Presenter Index .......................................................... 20
Podium Presentations – Abstracts .......................................................................... 27
Poster Session 1 – Abstracts ..................................................................................111
Poster Session 2 – Abstracts ..................................................................................195
Poster Session 3 – Abstracts .................................................................................271
BioIron 2003 Local Organizing Committee
Tracey Rouault, M.D. (Co-Chair)
Head, Section on Human Iron Metabolism
Cell Biology and Metabolism Branch
NICHD
Bldg. 18, Room 101
Bethesda, MD 20892
Phone: 301-496-6368 or 301-402-3428
Fax: 301-402-0078
Victor R. Gordeuk, M.D. (Co-Chair)
Professor of Medicine, Center for Sickle Cell
Disease and Department of Medicine
Howard University
2121 Georgia Ave. NW
Washington, DC 20059
Phone: 202-806-7930 or 202-865-1941
Fax: 202-865-7985
E-Mail: vgordeuk@howard.edu
Caroline C. Philpott, M.D.
Senior Clinical Investigator
Liver Diseases Section, NIDDK, NIH
Building 10, Room 9B16
10 Center Drive
Bethesda, MD 20892-1800
Phone: 301-435-4018
Fax: 301-402-0491
E-mail: carolinep@intra.niddk.nih.gov
Esther Meyron-Holtz, Ph.D.
NIH, NICHD, CBMB
Building 18T, room 101
Bethesda, MD 20892
Phone: 301.496-4743
Fax: 301.402-0078
E-mail: meyron@mail.nih.gov
BioIron 2003 International Scientific Committee
Nancy Andrews
Children's Hospital, Boston, Mass., USA
Bruce Bacon
Saint Louis University, Saint Louis, Mo., USA
Carole Beaumont
Faculte Xavier Bichat, Paris, France
Matthias Hentze
EMBL, Heidelberg, Germany
Antonello Pietrangelo
University of Modena, Modena, Italy
Prem Ponka
McGill University, Montreal, Quebec, Canada
Lawrie Powell
QIMR, Herston, Australia
Elizabeth Theil
CHORI, Oakland, Calif., USA
Günther Winkelmann
University of Tuebingen, Tuebingen, Germany
BioIron 2003 Student Bursary Awardees
A limited number of student bursaries were offered for the 2003 meeting to assist bona fide
scholars who submited an abstract to BioIron 2003 as the presenting author. The following are
the awardees, listed alphabetically by last name, that were selected by the program committee:
Brice Courselaud
Rennes, France
Alessandro Campanella
Milan, Italy
Elizabeth Skovran
Madison, Wis.
Maite Courel
Paris, France
Suh Young Jeong
McGill University, Montreal, Quebec, Canada
Deborah Johnson
University of Surrey, England
Jeung Hyoun Kim
Berkeley, Calif.
Stefania Recalcati
Milan, Italy
Aeisha Robb
Harvard School of Public Health
BioIron 2003 Acknowledgements
The International BioIron Society gratefully acknowledges the contributions
of the following companies:
Platinum – $40,000+
Novartis
Gold – $20,000+
Centers for Disease Control (CDC)
National Institute of Child Health and Human Development (NICHD)
National Institute of Neurologic Disease and Stroke (NINDS)
Silver – $10,000+
National Institute of Digestive Disease and Kidney (NIDDK)
National Heart Lung and Blood Institute (NHLBI)
Cooley’s Anemia Foundation
Blackfan-Diamond Disease Association
Bronze – up to $10,000
Nestle Corporation
Bio-rad
Amgen
Office of Dietary Supplements (ODS), National Institutes of Health
Iron Disorders Institute
BioIron 2003 General Meeting Information
Speaker Ready Room
The speaker ready room is Room H of the lower level. For your convenience, it will open at 8:00 a.m.
daily.
Poster Sessions
Poster sessions will be in the Atrium, located on the upper level, and the Foyer, located on the lower
level. Please remove all posters immediately following each session.
Host Hotel
The Hyatt Regency Bethesda in Bethesda, Md., will play host to our 2003 Conference. Completely
renovated and located at Bethesda Metro Center in the heart of Bethesda, Md., the hotel is six miles from
downtown Washington, D.C., two miles inside the Capital Beltway, and located on the Metro Red Line.
The Hyatt Regency Bethesda is conveniently within walking distance of restaurants, theaters and worldclass shopping. Located nearby are the National Institutes of Health, the Food and Drug Administration,
the Naval Medical Center, the American University, the Department of Homeland Security and the
National Zoo. From the hotel, it is just 18 miles to Reagan National Airport, 24 miles to Washington-Dulles
International Airport and 32 miles to Baltimore Washington International Airport.
Hyatt Regency Bethesda
One Bethesda Metro Center
Bethesda, Maryland
20814 USA
Phone: (301) 657 1234, (800) 233-1234
Fax: (301) 657 6453
Tourist Information
Tourist information is available from the hotel concierge.
Registration Fee
Registration fee includes: program book, scientific sessions, breakfasts, lunches, coffee breaks and (1)
ticket to Welcome Reception. Tickets to additional events can be purchased with Visa, Mastercard or
check at the registration booth during the meeting.
Name Badges
Attendees are requested to wear their name badges while at the meeting.
Lunches
Boxed lunches will be served daily at the Natcher Center in the lower level foyer area.
Cancellations and Refunds
Cancellations must be made in writing to the BioIron 2003 Meeting Office. Cancellations received prior to
April 4, 2003, will receive a refund of registration fees less an administration charge of $100. This will be
provided at the conclusion of the meeting. Cancellations after April 4, 2003, will be considered after the
meeting at the discretion of the Organizing Committee.
Dress
Casual attire is suggested for the meeting and social events.
Special Needs
If you have specific needs to facilitate your comfort during the conference (i.e. wheelchair access, dietary,
auditory or other assistance), all concerns should be addressed at the registration desk.
BioIron 2003 Social Activities
Welcome Reception
Gather with old friends and new acquaintances at the BioIron Welcome Reception. Heavy hors d'oeuvres
will be served. This event will be held at the Hyatt Regency Bethesda Hotel in the Crystal Ballroom from
6:00 p.m. – 8:00 p.m. on Sunday, May 4, 2003, and is included in the registration fee for meeting
attendees. Additional tickets can be purchased for $25.00 per person at the registration desk, located
outside of the Crystal Ballroom.
Poster Sessions
Wine, beer and cheese will be served at Poster Session 1 on Monday, May 5, from 4:00 p.m. – 6:00 p.m;
at Poster Session 2 on Wednesday, May 7, from 4:00 p.m. – 6:00 p.m.; and at Poster Session 3 on
Thursday, May 8, from 4:00 p.m. – 6:00 p.m.
Banquet
The Banquet will be held in the Hyatt Regency’s Crystal Ballroom on Thursday, May 8, from 7:00 p.m. –
11:00 p.m. Enjoy an elegant evening of dining and dancing with colleagues and friends. Smart, casual
attire is appropriate. Additional tickets may be purchased for $55.00 per person at the registration desk
until Wednesday, May 7.
Spirit Dinner Cruise on the Potomac River
Tuesday, May 6, 2003
5:00 p.m. – 10:30 p.m. (Departure from the Hyatt: 4:00 p.m.)
On Tuesday, May 6, join us as we dine on the Spirit of Washington and sail from our nation’s capitol
down the Potomac River. While dining on delicious entrees, fresh vegetables and rolls, tasty salads and
dessert, take advantage of the ever-changing scenery through the large panoramic view window. The
ship will pass Old Town Alexandria, a restored colonial village from the 1600’s; Hains Point/The
Awakening, a unique sculpture left behind after an artists’ convention and now on sale for $150,000;
Washington Monument, the tallest free-standing stone sculpture in the world; and General’s Row, the
historic homes at Fort McNair, where generals and their families live while on duty in D.C.
After dinner, come with us on a private evening tour of Mount Vernon, George Washington’s home for
more than 45 years. This peaceful, exclusive visit to the estate happens after the gates have closed to the
public. You can enjoy a leisurely stroll around the gardens, explore the outbuildings and have the
opportunity to visit the rarely seen third floor.
After your Mount Vernon tour, treat yourself to some delicious desserts and dancing on the dock, as we
sail into D.C.!
We have a limited number of tickets for this enjoyable evening. While available, tickets will remain on sale
at the registration desk for $50.00 for scientific program attendees and $90 for additional guests.
BioIron 2003 Scientific Program
SUNDAY, MAY 4, 2003
3:00 p.m. - 6:00 p.m.
Registration
Hyatt Hotel on the Ballroom Level
6:00 p.m. - 8:00 p.m.
Welcome Reception
Hyatt Hotel in the Crystal Ballroom
MONDAY, MAY 5, 2003
7:30 a.m. - 5:00 p.m.
Registration
Natcher Entrance
8:00 a.m. - 8:30 a.m.
Light Breakfast
Natcher Auditorium Foyer
8:30 a.m. - 8:40 a.m.
Welcome to Washington
Tracey Rouault, Victor Gordeuk
8:40 a.m. - 9:30 a.m.
PODIUM PRESENTATION 1
Location: Main Auditorium
Keynote Address: "Heme, Gas and Redox Sensing at the Heart of
Circadian Rhythm"
Steven McKnight
9:30 a.m. - 12:00 p.m.
Plenary Session 1:
Regulation of Cellular and Systemic Iron Metabolism
Chairpersons: Matthias Hentze, David Haile
9:30 a.m. - 9:50 a.m.
New Insights in Iron Metabolism from the Study of Juvenile
Hemochromatosis
Camaschella C, Papanikolaou G, De Gobbi M, Roetto A
9:50 a.m. - 10:10 a.m.
Regulation of Human Hepcidin by Inflammation and Iron
T. Ganz, E. Nemeth
10:10 a.m. - 10:40 a.m.
Coffee Break in the Foyer (lower level)
10:40 a.m. - 11:00 a.m.
Iron Regulation in Primary Cell Cultures From Mice With
Targeted Deletions of IRP1 and IRP2
Esther Meyron-Holtz, Manik Ghosh, Tracey A. Rouault
11:00 a.m. - 11:20 a.m.
Ceruloplasmin Maintains Iron Homeostasis in Astrocytes
Via Interactions With Ireg1
S.Y. Jeong, S. David
11:20 a.m. - 11:40 a.m.
Heme-Iron Transport Across the Cell Wall of Gram Positive
Bacteria
E. Skaar, S. Mazmanian, A. Gaspar, A. Joachmiak, D.
Missiakas, O. Schneewind
11:40 a.m. - 12:00 p.m.
Characterisation of a Novel Gene Highly Expressed in
Duodenum
Andrew McKie, Majid Shayeghi, Neil Halliday, Jonathan Oakhill,
Yemisi Latunde-Dada, David M. Frazer, Christopher D. Vulpe,
Gregory J. Anderson, Robert J. Simpson
12:00 p.m. - 1:30 p.m.
Lunch in the Foyer (lower level)
1:30 p.m. - 3:30 p.m.
Simultaneous Session 1:
Iron, Inflammation and Oxidative Stress
Location: Room E1/E2
Chairpersons: Gunther Weiss, Maria de Sousa
1:30 p.m. - 1:45 p.m.
Cellular and Molecular Approaches to Study Heme Iron
Recycling in Macrophages
F. Canonne-Hergaux, C. Delaby, G. Hetet, B. Grandchamp, C.
Beaumont
1:45 p.m. - 2:00 p.m.
The Processing of Polymeric Iron by Macrophages Follows
the Major Stages of Erythrophagocytosis: A Cellular Model
for On-line Monitoring of Iron Recycling by Fluorescence
Virginie Leib, Abraham M. Konijn, Z. Ioav Cabantchik
2:00 p.m. - 2:15 p.m.
MDL1 is a High Copy Suppressor of ATM1, and Regulates
Resistance to Oxidative Stress in Wild Type Cells
M. Chloupková, L.M. LeBard, D.M. Koeller
2:15 p.m. - 2:30 p.m.
Increased Ferroportin 1 (FPN1) Expression After
Erythrophagocytosis by J774 Macrophages
M. Knutson, D. Haile, M. Wessling-Resnick
2:30 p.m. - 2:45 p.m.
Down-Regulation of Hydroxyacid Oxidase 1 Expression by
Iron: An Oxidative Stress Mediated Response?
S. Recalcati, L. Tacchini, A. Alberghini, D. Conte, G. Cairo
2:45 p.m. - 3:00 p.m.
The Nature of Extracellular Iron Chelates, Macrophage HFE
Status, and Interferon-g Influence Iron Acquisition by
Intraphagosomal Mycobacterium Tuberculosis
O. Olakanmi, L.S. Schlesinger, A. Ahmed, B.E. Britigan
3:00 p.m. - 3:15 p.m.
Iron, Infection and the Inflammatory Response
Roberta J Ward, Stephanie Wilmet, Jacques Piette, Robert R
Crichton
3:15 p.m. - 3:30 p.m.
Cytokine Mediated Regulation of Iron Transport in Human
Monocytic Cells
S Ludwiczek, E Aigner, I Theurl, G Weiss
1:30 p.m. - 3:30 p.m.
Molecular Regulation of Iron Metabolism
Location: Balcony B
Chairpersons: Carole Beaumont, Robert Fleming
1:30 p.m. - 1:45 p.m.
Regulation of DMT1 and Dcytb But Not IREG1 or Hephaestin
by Intracellular Iron Levels in Duodenal Enterocytes
G J Anderson, S J Wilkins, E M Becker, T L Murphy, C D Vulpe,
A T McKie, D M Frazer
1:45 p.m. - 2:00 p.m.
Novel Divalent Metal Transporter 1 Isoforms: Functional and
Regulatory Implications
N. Hubert, M.W. Hentze
2:00 p.m. - 2:15 p.m.
Differential Regulation of Iron-Related Proteins in Polarized
and Non-Polarized HT-29 Cells
P. S. Davies, E. L. Anderson, C. A. Enns
2:15 p.m. - 2:30 p.m.
Sorting of HFE to the Apical Membrane Correlates With
Inhibition of Intestinal Iron Absorption
M. Arredondo, C. Mura, V. Tapia, P. Muñoz, DI. Mazariegos, MT.
Núñez
2:30 p.m. - 2:45 p.m.
Structural Studies of Human Hepcidin: a Peptide-Hormone
and Antimicrobial Peptide
H.N. Hunter, D.B.Fulton, A.J. Waring, T. Ganz, H.J. Vogel
2:45 p.m. - 3:00 p.m.
Iron Efflux as a Novel Mechanism for Control of Intracellular
Iron Concentrations in Salmonella
Marie-Laure Crouch, Ferric Fang
3:00 p.m. - 3:15 p.m.
The Response To Iron Deprivation in Saccharomyces
Cerevisiae: Regulation of Iron-Dependent Metabolic
Pathways
C. C. Philpott, M. Shakoury-Elizeh, J. Tiedeman, J. Rashford, T.
Ferea, D. Botstein, P.O. Brown
3:15 p.m. - 3:30 p.m.
Non-Transferrin Bound Iron Uptake by Hepatocytes is
Increased in a Hfe Knockout Mouse Model of Hereditary
Hemochromatosis
Anita CG Chua, John K Olynyk, Deborah Trinder
3:30 p.m. - 4:00 p.m.
4:00 p.m. - 6:30 p.m.
POSTER SESSION 1
Posters 1 - 46 upstairs in Atrium
Posters 47 - 92 downstairs in Foyer
4:00 p.m. - 6:00 p.m.
Poster topics: HFE and non-HFE Hemochromatosis; Iron
Deficiency; Iron Overload in Sickle Cell Disease, Thalassemia;
Iron Chelation and Chelators; Screening for HFE and non-HFE
Hemochromatosis
6:00 p.m. - 6:30 p.m.
Poster Discussion
TUESDAY, MAY 6, 2003
8:00 a.m. - 12:00 p.m.
Registration
8:00 a.m. - 8:30 a.m.
Light Breakfast
8:30 a.m. - 10:00 a.m.
Plenary Session 2:
HFE and Non-HFE Hemochromatosis
Chairpersons: Clara Camaschella, James Kushner
8:30 a.m. - 9:00 a.m.
The Disease Due to Mutations of the SLC11A3 Gene
Antonello Pietrangelo
9:00 a.m. - 9:15 a.m.
Failure of Hepcidin Upregulation in HFE-Associated
Haemochromatosis Implicates the Liver in the Regulation of
Body Iron Homeostasis
D M Frazer, K R Bridle, S J Wilkins, J L Dixon, D M Purdie, D H
G Crawford, V N Subramaniam, L W Powell, G A Ramm, G J
Anderson
9:15 a.m. - 9:30 a.m.
Hereditary Hemochromatosis Mutations Abrogate the
Hepcidin Response to Iron Overload Without Affecting
mRNA Expression of Intestinal Iron Transporters
Martina Muckenthaler, Cindy N. Roy, Ángel O. Custodio, Belén
Minãna, Jos deGraaf, Lynne K. Montross, Nancy C. Andrews,
Matthias W. Hentze
9:30 a.m. - 9:45 a.m.
Role for the HFE H63D Mutation in Murine Hereditary
Hemochromatosis
S. Tomatsu, R.E. Fleming, R.S. Britton, B.R. Bacon, A. Waheed,
W.S. Sly, Edward A. Doisy
9:45 a.m. - 10:00 a.m.
Functional Consequences of N144H and Dval 162
Ferroportin Mutations
D.J. Haile, X.B. Liu
10:00 a.m. - 10:30 a.m.
10:30 a.m. - 12:00 p.m.
Plenary Session 3: Iron Chelation and Iron Overload in Sickle Cell
Disease, Thalassemia, Diamond Blackfan Anemia and Other IronLoading Anemias
Chairpersons: Gary Brittenham, John Porter
10:30 a.m. - 10:45 a.m.
Pattern and Dynamics of Iron Deposition in Transfusion
Dependent Thalassemic Patients on Regular Chelation
Treatment
M.D. Cappellini, D. Prati, M. Maggioni, R. Gramignoli, M.
Maiocchi, D. DiCataldo, M. Cerino, G. Fiorelli
10:45 a.m. - 11:00 a.m.
Relationship of Deferoxamine (DFO) Dose to Plasma NTBI
Removal, Transferrin Saturation and Plasma and Urine
Ferrioxamine (FO)
P. Evans, F. Shah, B. Davis, F. Kotynia, C. Kim, L. Merson, N.
Olivieri, J. Porter
11:00 a.m. - 11:15 a.m.
A Phase Ib Study of the Safety Pharmacokinetics, Acute
Tolerability, and Efficacy of Ascending Single Doses of
40SD02 (CHF1540) in Iron-Loaded Patients
P. Harmatz, J. Madden, E. Vichinsky, M. Jeng, P.J. Giardina,
K.A. Nugent, R.W. Grady, P. Dragsten, B. Hedlund
11:15 a.m. - 11:30 a.m.
ICL670: Mobilization of Iron in Animals
HP. Nick, S. Hauffe, R. Lattmann, F. Waldmeier
11:30 a.m. - 11:45 a.m.
Clinical Overview of ICL670A
M.D. Cappellini
11:45 a.m. - 12:00 p.m.
Intralysosomal Iron and Oxidant-Mediated Cell Death
J.W. Eaton, H.L. Persson, Z.Q. Yu, U.T. Brunk
12:00 p.m. - 1:00 p.m.
Annual Business Meeting in the Main Auditorium
1:00 p.m. - 2:30 p.m.
4:00 p.m.
Depart from the Hyatt for the Potomac River Cruise (registration
required)
5:00 p.m. - 10:30 p.m.
Spirit Dinner Cruise on the Potomac River
WEDNESDAY, MAY 7, 2003
8:00 a.m. - 5:00 p.m.
Registration
8:00 a.m. - 8:30 a.m.
Light Breakfast
8:30 a.m. - 10:00 a.m.
Plenary Session 4:
Iron and Neurodegenerative Diseases
Chairpersons: Tracey Rouault, George Perry
8:30 a.m. - 8:45 a.m.
Introduction
George Perry
8:45 a.m. - 9:05 a.m.
Iron Misregulation and Neurodegeneration in IRP2-/- Mice
T. Rouault, S. Smith, S. Cooperman, E. Meyron-Holtz, M.
Ghosh, W. Land, H.Olivierre Wilson, T. LaVaute, C. Grabill, L.
Jui-chen
9:05 a.m. - 9:25 a.m.
Multicopper Oxidase Deficient Mice Reveal an Essential
Role for Ceruloplasmin and Hephaestin in Central Nervous
System Iron Homeostasis
Z. Leah Harris, Xueying Xu, Hoon Shim
9:25 a.m. - 9:40 a.m.
Ceruloplasmin and Hephaestin Serve Overlapping
Regulatory Functions in Retinal Iron Homeostasis
Paul Hahn, Robert W. Wong, Lin Chen, Tzvete Dentchev, Z.
Leah Harris, Joshua L. Dunaief
9:40 a.m. - 10:00 a.m.
Development of Mouse and Cellular Models to Decipher the
Function of Frataxin in Iron and Iron-sulfur Metabolisms
H. Puccio, H. Seznec, N. Carelle, A. Hertzog, C. Bouton, D.
Simon, L. Reutenauer, M. Koenig
10:00 a.m. - 10:30 a.m.
10:30 a.m. - 12:15 p.m.
Plenary Session 5:
Penetrance of HFE Hemochromatosis
Chairpersons: Pierre Brissot, Lawrie Powell
10:30 a.m. - 10:50 a.m.
The Penetrance of Hereditary Hemochromatosis
E. Beutler, V. Felitti, T. Gelbart, J. Waalen, P. Lee
10:50 a.m. - 11:10 a.m.
Longitudinal and Penetrance Studies of HFE-associated
Hemochromatosis in Probands and Relatives in an
Australian Population
L. Powell, J. Dixon, D. Hewett, G. Ramm, G. Anderson, N.
Subramaniam, L. Fletcher, D. Crawford, J. Cavanaugh, M.
Bassett
11:10 a.m. - 11:30 a.m.
Prevalence of Self-reported Symptoms in the Heirs Study:
Differences by HFE Genotype
Adams P, Reboussin D, Acton R, Barton J, Gordeuk V, Dawkins
F, McLaren C, McLaren G, Harris E, Press N, Speechley M,
Mellen B, Thomson E, Eckfeldt J, Sholinsky P
11:30 a.m. - 12:15 p.m.
Panel discussion with Paul Adams, Bruce Bacon, Ernest
Beutler, Pierre Brissot, Kris Kowdley, Antonello Pietrangelo,
Lawrie Powell
Moderator: Tim Cox
12:15 p.m. - 1:30 p.m.
1:30 p.m. - 3:30 p.m.
Epithelial Iron Transport
Location: Balcony A
Chairpersons: Greg Anderson, Matthias Hediger
1:30 p.m. - 1:45 p.m.
Introduction to Epithelial Iron Transport
Matthias Hediger
1:45 a.m. - 2:00 p.m.
Iron Transporters and the Kidney
Craig Smith
2:00 p.m. - 2:15 p.m.
New Insights Into the Biophysical Properties of DMT1
Bryan Mackenzie, M L Ujwal, Min-Hwang Chang, Michael F
Romero, Matthias A Hediger
2:15 p.m. - 2:30 p.m.
Patch Clamp Characterization of the Mouse Iron Transporter
DMT1 Transiently Expressed in Mammalian Cell Lines
Haoxing Xu, Jie Jin, David E. Clapham, Nancy C. Andrews
2:30 p.m. - 2:45 p.m.
DMT1 Protein Expression in the Apical Membrane of Human
Intestinal Caco-2 Cells is Rapidly Decreased Following
Exposure to Iron
Johnson, Tennan, Sharp
2:45 p.m. - 3:00 p.m.
The Endocytic Pathway of DMT1 in Caco-2 Cells
Y. Ma, K-Y Yeh, J. Rodriquez-Paris, Y. Chen, M. Yeh, J. Glass
3:00 p.m. - 3:15 p.m.
Regulation of Lenramp1 is Dependent on the bHLH Gene fer
in Plants
P. Bauer, Z. Bereczky, H.-Y. Wang, T. Brumbarova, V. Schubert
3:15 p.m. - 3:30 p.m.
Discussion
1:30 p.m. - 3:30 p.m.
Cellular Iron Metabolism and Transport
Location: Balcony B
Chairpersons: Nancy Andrews, Caroline Philpott
1:30 p.m. - 1:45 p.m.
Identification of the Ubiquitin-Protein Ligase That
Recognizes Heme-Oxidized IRP2
K. Iwai, K. Yamanaka, H. Ishikawa, T.A. Rouault, K. Ishimor
1:45 p.m. - 2:00 p.m.
Redox Properties of Human Transferrin Bound to its
Receptor
A. L. Crumbliss, S. Dhungana, C. H. Taboya, M. Larvie, O. Zak,
P. Aisen
2:00 p.m. - 2:15 p.m.
The Mechanism for Iron-Dependent Degradation of IRP2 is
Saturable, Stimulated by Antioxidants and Independent of
Site-specific Oxidation of C168, C174 and C178
J. Wang, G. Chen, M. Muckenthaler, M. W. Hentze, K.
Pantopoulos
2:15 p.m. - 2:30 p.m.
Enhanced Nitric Oxide Sensitivity of the Fe-S Cluster in
Phosphomimetic Mutants of Iron Regulatory Protein 1
K.M. Deck, S.A. Anderson, M.C. Kennedy, R.S. Eisenstein
2:30 p.m. - 2:45 p.m.
Molecular Control of Mammalian Iron Metabolism:
Molecules, Mice and Microarrays
M.W. Hentze
2:45 p.m. - 3:00 p.m.
Iron Homeostasis Abnormalities in HFE and Transferrin
Receptor 2 Compound Mutant Mice
R.E. Fleming, H.D. Hall, J.R. Ahmann, M.C. Migas, R.S. Britton,
B.R. Bacon, A. Waheed, W.S. Sly
3:00 p.m. - 3:15 p.m.
Nuclear Import and Export of AFT1 Protein, The Major IronResponsive Transcriptional Activator in Yeast
Y. Yamaguchi-Iwai, A. Fukunaka, R. Ueta
3:15 p.m. - 3:30 p.m.
POSTER 200 (PRESENTATION)
Hemin Uptake and Use as an Iron Source By Candida
albicans: Role of CaHMX1 Encoded hemE Oxygenase
R. Santos, N. Buisson, S. Knight, A. Dancis, J.-M. Camadro,
E. Lesuisse
1:30 p.m. - 3:30 p.m.
Scientific Symposium: Dietary Iron Fortification and Iron Deficiency
Location: Balcony C
Chairpersons: James Cook, Leif Hallberg
1:30 p.m. - 1:55 p.m.
Increased Prevalence of Iron Deficiency Due to Withdrawal
of Iron Fortification of Flour in Sweden
Leif Hallberg, Lena Hultheń
1:55 p.m. - 2:20 p.m.
The Assessment of Iron Fortification Programs Using
Quantitative Measurements of Body Iron
James Cook
2:20 p.m. - 2:30 p.m.
Evaluation of Elemental Iron Powder Bioavailability and
Strategies for Enhancing Iron
Elizabeth Turner
2:30 p.m. - 2:50 p.m.
Importance of Bioavailability in Iron Fortification
Sean Lynch
2:50 p.m. - 3:10 p.m.
Program Experience with Iron Fortification
Frances Davidson
3:10 p.m. - 3:30 p.m.
Changing Spectrum of Treatment-resistant Iron Deficiency
Anemia (IDA)
C Hershko, B Roth, J Ashkenazi, J Heyd, D Kereth
3:30 p.m. - 4:00 p.m.
Coffee Break
4:00 p.m. - 6:30 p.m.
POSTER SESSION 2
4:00 p.m. - 6:00 p.m.
Poster topics: Aging, Oxidative Stress, and Neurological
Disease; Animal Models of Iron Metabolism; Hepatic Iron
Metabolism and Treatment of Hepatitis; Iron and Inflammation;
Iron and Neurodegenerative Disease; Iron as a Co-factor of
Disease; Iron Toxicity; Iron, Infection, and Lactoferin; Iron,
Inflammation, and Oxidative Stress; Iron, Oxygen Sensing, and
the Response to Hypoxia
6:00 p.m. - 6:30 p.m.
Poster Discussion
THURSDAY, MAY 8, 2003
8:00 a.m. - 5:00 p.m.
Registration
8:00 a.m. - 8:30 a.m.
Light Breakfast
8:00 a.m. - 10:00 a.m.
Plenary Session 6:
Microbial Iron Metabolism
Chairpersons: Günther Winkelmann, Volkmar Braun
8:30 a.m. - 9:00 a.m.
A Systematic View of the FecA, FhuA and FepA Structures
Dick van der Helm
9:00 a.m. - 9:30 a.m.
Structural Studies of the Escherichia Coli Periplasmic
Binding Protein FhuD and its Complexes With Several
Hydroxamate Siderophores
H.J.Vogel
9:30 a.m. - 9:45 a.m.
Mechanism of Action of a Small RNA Regulator of
Intracellular Iron Metabolism in Bacteria
Susan Gottesman Eric Massé, Freddie Escorcia, Paula
Wilderman, Urs Ochsner, Michael Vasil, David Fitzgerald, Peter
Fitzgerald
9:45 a.m. - 10:00 a.m.
Hemophore Dependent Heme Acquisition Systems in Gram
Negative Bacteria
Cécile Wandersman
10:00 a.m. - 10:30 a.m.
10:30 a.m. - 12:00 p.m.
Plenary Session 7:
Mitochondrial Iron Metabolism and Iron Sulfur Cluster Biogenesis
Chairpersons: Andrew Dancis, Jerry Kaplan
10:30 a.m. - 10:50 a.m.
Mechanism and Regulation of Iron-Sulfur Cluster
Biosynthesis in Bacteria
Dennis Dean
10:50 a.m. - 11:10 a.m.
A Role for Molecular Chaperones in Fe/S Center Biogenesis
Elizabeth Craig
11:10 a.m. - 11:30 a.m.
Factors Affecting Fe-S Cluster Assembly/Disassembly in
Iron Regulatory Protein 1
W. E. Walden, A. Roy, N. Solodovnikova, W. E. Antholine
11:30 a.m. - 11:45 a.m.
Mitochondrial Ferritin: Functional and Expression Studies
S. Levi, B. Corsi, A. Cozzi, P. Santambrogio, A. Campanella, F.
Sanvito, S. Olivieri, G. Biasiotto, P. Arosio
11:45 a.m. - 12:00 p.m.
Targeted Disruption of the Murine X-lined Sideroblastic
Anemia with Ataxia Gene
C. Pondarré, D. Campagna, B. Antiochos, S. Clarke, E. Greer,
R. Eisenstein, M.D. Fleming
12:00 p.m. - 1:30 p.m.
1:30 p.m. - 3:30 p.m.
Ferritin and Ferroxidases
Location: Balcony A
Chairpersons: Paolo Arosio, Elizabeth Theil
1:30 p.m. - 1:45 p.m.
Gated Pores in Ferritin: Structure and Chelator Targets
Xiaofeng Liu, Elizabeth C. Theil
1:45 p.m. - 2:00 p.m.
Chemopreventive Agents and Xenobiotics Activate the
Murine Ferritin H Gene Via a NRF2 Dependent Mechanism
EC Pietsch, JY Chan, FM Torti, SV Torti
2:00 p.m. - 2:15 p.m.
A New Pathway for Mineral Core Formation in Mammalian
Apoferritin: The Role of Hydrogen Peroxide
N. D. Chasteen, G. Zhao, P. Arosio, S. Levi, C. Janus-Chandler,
F. Bou-Abdallah
2:15 p.m. - 2:30 p.m.
Nuclear Distribution and Function of Ferritin
N. Surguladze, K.J. Thompson, M.G. Fried, J.R. Connor
2:30 p.m. - 2:45 p.m.
Exploring the Biological Function of Ferritins and Proteins
of Iron Metabolism in HeLa Cells by the Use of Small
Interfering RNAS
P. Arosio, A. Cozzi, S. Levi, B. Corsi, G. Biasiotto, G.M. Gerardi,
P. Santambrogio, A. Campanella, I. Zanella
2:45 p.m. - 3:00 p.m.
Recent Developments With Non Transferrin Bound Iron
Measurement
R C Hider, Z D Liu, D Devanur, D Y Liu
3:00 p.m. - 3:15 p.m.
Biochemical and Biological Characterization of Mutated LFerritin Involved in Neuroferritinopathy, a Rare
Neurodegenerative Disorder
P. Santambrogio, A. Cozzi, B. Corsi, A. Campanella, P. Arosio,
S. Levi
3:15 p.m. - 3:30 p.m.
Discussion
1:30 p.m. - 3:30 p.m.
Iron Metabolism of Microorganisms
Location: Balcony B
Chairpersons: Günther Winkelmann, Volkmar Braun
1:30 p.m. - 1:45 p.m.
Functional Analysis of the Fhua and Feca Transport
Proteins
V. Braun, M. Braun, F. Endriß, H. Killmann, S. Mahren, M.
Ogierman, A. Sauter
1:45 p.m. - 2:00 p.m.
Global Iron-Dependent Gene Regulation in Escherichia Coli:
A New Mechanism for Iron Homeostasis
J. P. McHugh, H. Abdul-Tehrani, D. A. Svistunenko, R. K. Poole,
C. E. Cooper, F. Rodríguez-Quiñones
2:00 p.m. - 2:15 p.m.
FHUF- The First Example Of A Siderophore-Reductase
Berthold F. Matzanke, Klaus Hantke, Stefan Anemüller
2:15 p.m. - 2:30 p.m.
Salmochelins are the First Sugar Containing Siderophores
and Major Iron Chelators of Salmonella Enterica
G. Winkelmann, K. Hantke, G. Nicholson, W. Rabsch
2:30 p.m. - 2:45 p.m.
Novel Features of Gene Regulation for Iron Nutrition and
Uptake in the Symbiotic Bacteria Rhizobium
Andrew W. B. Johnston, Jonathan D. Todd, Margaret Wexler,
Kay H. Yeoman
2:45 p.m. - 3:00 p.m.
A Novel Gene Required for Vacuolar Acidification Identified
Through a Saccharomyces Cerevisiae Genome Wide
Analysis of Iron-Dependent Growth
J Kaplan, DM Ward, SL Shiflett, SR Davis-Kaplan
3:00 p.m. - 3:15 p.m.
The Unique Process Heme Crystallization is a Method of
Iron Sequestration in Plasmodium that is Vulnerable to
Chemotherapy by the Quinolines
D. Sullivan
3:15 p.m. - 3:30 p.m.
Discussion
1:30 p.m. - 3:30 p.m.
Genotype-phenotype Correlations in HFE Hemochromatosis and
Screening
Location: Balcony C
Chairpersons: Ernest Beutler, Lawrie Powell
1:30 p.m. - 1:45 p.m.
Mutational Analysis of Ferritin and Ferroportin Genes in
Patients With Unexplained Hyperferritinemia:
An Interim Report
Laura Cremonesi, Barbara Foglieni, Francesca Ferrari, Nadia
Soriani, Gaetano Bergamaschi, Daniela Caprino, José Antonio
García Erce, Silvia Fargion, Renzo Galanello, Maurizio
Longinotti, Sonia Levi, Maurizio Ferrari, Paolo Arosio, Mario
Cazzola
1:45 p.m. - 2:00 p.m.
A Study of 492 French Centenarians Suggests That
Longevity is Not Affected in Carriers of the
Haemochromatosis Gene C282Y Mutation
H. Coppin, M. Bensaid, S. Fruchon, N. Borot, H. Blanché, M.P.
Roth
2:00 p.m. - 2:15 p.m.
Digenic Alteration (HFE/HAMP) Associated With Adult
Hemochromatosis Phenotype
S.Jacolot, G.Le Gac, I.Quéré, C.Mura, C.Férec
2:15 p.m. - 2:30 p.m.
Comparison of Screening Serum Ferritin Concentrations
and Transferrin Saturations in HFE Wild-type AfricanAmerican and Caucasian HEIRS Participants
F.W. Dawkins, B.G. Mellen, D.M. Reboussin, C.E. McLaren, P.
Sholinsky, R.T. Acton, P.C. Adams, J.C. Barton, G.D. McLaren,
E.L. Harris, N. Press, M. Speechley, E. Thomson, J.H. Eckfeldt,
V.R. Gordeuk
2:30 p.m. - 2:45 p.m.
Determinants of Iron Accumulation in the HFE Knockout
Mouse Model of Hereditary Hemochromatosis
R.E. Fleming, K.A. Ahmad, J.R. Ahmann, B. Roshan, M.C.
Migas, R.S. Britton, B.R. Bacon, A. Waheed, W.S. Sly
2:45 p.m. - 3:00 p.m.
Risk of Hepatocellullar Carcinoma in Patients With
Hereditary Hemochromatosis and Their First-Degree
Relatives
Hultcrantz R, Elmberg M, Ekbom A, Brandt L, Olsson S, Olsson
R, Lindgren S, Lööf L, Stål P, Wallerstedt S, Almer S, SandbergGertzén H, Askling J
3:00 p.m. - 3:15 p.m.
Hemochromatosis Subjects as Allogeneic Blood Donors:
A Prospective Study
Susan F. Leitman, Janet N. Browning, Yu Ying Yau, Glorice
Mason, Harvey G. Klein, Cathy Conry-Cantilena, Charles D.
Bolan
3:15 p.m. - 3:30 p.m.
Hepatic Iron Concentration and Total Body Iron Stores in
Genetic Hemochromatosis and Thalassemia Major
S. Jacquelinet, G.M. Brittenham, E. Angelucci, C.E. McLaren, P.
Brissot
3:30 p.m. - 4:00 p.m.
4:00 p.m. - 6:00 p.m.
POSTER SESSION 3
4:00 p.m. - 6:00 p.m.
Poster topics: Cellular Iron Metabolism -Transport, Storage,
Regulation; Iron Absorption; Iron and Kidney Cells; Iron
Metabolism of Microorganisms; Iron Sulfur Cluster Biogenesis;
Mitochondrial Iron Metabolism; Regulation of Iron Metabolism
6:00 p.m. - 6:30 p.m.
Poster Discussion
7:00 p.m. - 11:00 p.m.
Banquet
Hyatt Ballroom
FRIDAY, MAY 9, 2003
8:00 a.m. - 5:00 p.m.
Registration
8:00 a.m. - 8:30 a.m.
Light Breakfast
8:30 a.m. - 10:00 a.m.
Plenary Session 8:
Iron and the Response to Hypoxia
Chairpersons: Patrick Maxwell, Prem Ponka
8:30 a.m. - 8:50 a.m.
Iron and the Response to Hypoxia
Patrick Maxwell
8:50 a.m. - 9:15 a.m.
HIF and VHL
William Kaelin
9:15 a.m. - 9:40 a.m.
Fe(II)-dependent Dioxygenases in the Mammalian Hypoxic
Response Pathway
Rick Bruick
9:40 a.m. - 10:00 a.m.
Structural and Mechanistic Studies on the Dioxygen
Dependant Modification of Hypoxia Inducible Factor
Chris Schofield
10:00 a.m. - 10:30 a.m.
10:30 a.m. - 12:00 p.m.
Plenary Session 9:
Iron as a Cofactor in Disease
Chairpersons: Bruce Bacon, Antonello Pietrangelo
10:30 a.m. - 10:50 a.m.
Congenital Disorder of Oxygen-Sensing: Clinical
Manifestations of the Homozygous Chuvash Polycythemia
VHL Mutation
Victor R. Gordeuk,M.D., Adelina I. Sergueeva, M.D., Galina Y.
Miasnikova, M.D., Daniel Okhotin, B.S., Yaroslav Voloshin, B.S.,
Katerina Jedlickova, M.S., Josef T. Prchal, M.D., Lydia A.
Polyakova, M.D.
10:50 a.m. - 11:10 a.m.
Iron, HFE, HCV Genotype and Hepatic MHC Class I
Expression in Chronic Hepatitis C
EMP Cardoso, MA Duarte,M de Sousa,E Ribeiro, P Rodrigues,
G Porto, P Carvalho, J Fraga
11:10 a.m. - 11:30 a.m.
Induction of Transferrin Receptor by Ethanol in Rat Primary
Hepatocyte Culture
Y. Suzuki, K. Ikuta, M. Suzuki, T. Otake, H. Saito, Y. Fujimoto, Y.
Torimoto, Y. Kohgo
11:30 a.m. - 11:50 a.m.
Iron Chelators Inhibit HIV-1 TAT-Dependent Transcription
from HIV-1 Promoter in Cultured Cells
S. Nekhai, J. Kurantsin-Mills, T. Ammosova, V. Gordeuk
11:50 a.m. - 12:00 p.m.
Farewell Remarks
12:00 p.m. - 1:30 p.m.
Presenter Index
Following are the names, listed alphabetically by last name, of those listed as the
PRESENTER of the abstract (upon submission of the abstract) along with their podium
and/or poster number(s).
A
Adams, Paul
Aguilar-Martinez, P.
Ajioka, Richard
Andersen, Rolf
Anderson, Greg
Andrews, Simon
Arosio, Paolo
Arredondo, Miguel
Podium 41; Posters 12, 21
Poster 54
Poster 172
Poster 52
Podium 16
Podium 79
Podium 75; Posters 9, 111, 205, 208
Podium 19; Poster 184
B
Babady, Ngolela
Baker, Erica
Bauer, Petra
Beard, John
Beaumont, Carole
Bensaid, Mounia
Beutler, Ernest
Bilello, John
Blanck, Heidi
Bolan, Charles
Bonnah, Robert
Boretskyy, Yuriy
Bou-Abdallah, Fadi
Bouton, Cecile
Bozzini, Claudia
Brandhagen, David
Braun, Volkmar
Brazzolotto, Xavier
Briggs, Sara
Brooks, David
Broyles, Robert
Bruick, Rick
Poster 143
Poster 6
Podium 48
Poster 116
Posters 42, 226
Poster 132
Podium 39
Poster 98
Poster 22
Poster 170
Poster 233
Posters 243, 244, 245
Poster 202
Poster 144
Posters 48, 49
Podium 78
Poster 219
Poster 253
Posters 79, 171
Posters 74
Podium 95
C
Cabantchik, Ioav
Camaschella, Clara
Campanella, Alessandro
Canonne-Hergaux Francois
Cappellini, Marie Domenica
Cazzola, Mario
Cellier, Mathieu
Chasteen, N. Dennis
Chen, Huijun
Chen, Opal
Cherukuri, Srujana
Chiueh, Chuang
Chloupkova, Maja
Chong, Curtis
Chorney, Michael
Chua-Anusorn, Wanida
Poster 26
Podium 2
Poster 178
Podium 8
Podiums 29, 33
Podium 85
Posters 250
Posters 80, 81
Poster 120
Poster 163
Poster 167
Podium 10
Poster 156
Poster 61
Poster 68
Chua, Anita
Clarke, Stephen
Connor, James
Cook, James
Coppin, Helene
Corna, Gianfranca
Cortopassi, Gino
Courel, Maite
Courselaud, Brice
Craig, Elizabeth
Crichton, Robert
Crouch, Marie-Laure
Crowe, William
Crumbliss, Alvin
Cruz, Eugenia
Podium 23
Poster 262
Posters 33, 194
Podium 57
Podium 86
Poster 164
Poster 175
Poster 201
Poster 227
Podium 67
Poster 270
Podium 21
Poster 147
Podium 50
Posters 45, 47
D
Dailey, Harry
Davidson, Frances
Davies, Paige
Dawkins, Fitzroy W.
De Sousa, Maria
Dean, Dennis
Deck, Kathryn
Delatycki, Martin
Desai, Tusar
Deugnier, Yves
Djaman, Ouliana
Dolezal, Pavel
Dongiovanni, Paola
Drakesmith, Hal
Dunaief, Joshua
Poster 145
Podium 60
Podium 18
Podium 88
Podium 98
Podium 66
Podium 52
Poster 4
Poster 77
Poster 65
Poster 206
Poster 139
Posters 105, 108
Poster 40
E
Eaton, John
Eckard, Jonathan
Eisenstein, Richard
Epsztejn, Silvina
Evans, Patricia
Evans, Robert
Podium 34
Poster 127
Poster 264
Poster 159
Posters 82, 266
F
Fernaeus, Sandra
Fillebeen, Carine
Fiorelli, Gemino
Fox, Paul
Fleming, Mark
Fleming, Robert
Frazer, David
Poster 137
Poster 247
Poster 60
Poster 149
Podiums 27, 54, 89
G
Gaboriau, Francois
Gakh, Oleksandr
Gallardo, Viviana
Galy, Bruno
Ganz, Tomas
Poster 13
Poster 141
Poster 96
Poster 133
Podium 3
Garrick, Michael
Gerhard, Glenn
Ghosh, Manik
Glass, Jonathan
Gleit, Merav
Gottesman, Susan
Gordeuk, Victor
Gunshin, Hiromi
Poster 23
Poster 155
Poster 234
Posters 57
Poster 91
Podium 64
Podium 97
Poster 150
H
Hadley, Kevin
Hager, Ward
Haile, David
Halbrooks, Peter
Hall, Heather
Hallberg, Leif
Han, Okhee
Hansen, Lotte Fynbo
Hanson, Eric
Hanspeter, Nick
Harmatz, Paul
Harris, Wesley
Harris, Z. Leah
Hassen, Mohamed
Hayashi, Hisao
Hediger, Matthias
Henderson, Rebecca
Hentz, Matthias
Hershko, Chaim
Hider, Robert
Hodges, Marcus
Hubert, Nadia
Hultcrantz, Rolf
Hunt, Janet
Hunter, Howard
Poster 18
Poster 17
Podium 28
Poster 251
Poster 83
Podium 56
Poster 181
Poster 5
Poster 100
Podium 32
Podium 31
Poster 44
Podium 36
Poster 75
Poster 110
Podium 42
Poster 195
Podium 53
Podium 61
Podium 76
Poster 166
Podium 17
Podium 90
Poster 62
Podium 20
I
Ishikawa, Haruto
Iwai, Kazuhiro
Poster 186
Podium 49;
J
Jacobson, Eric
Jacolot, Sandrine
Jacquelinet, Sylvie
Jeong, Suh Young
Jeong, Jinsook
Jin, Jie
Johnson, Deborah
Johnston, Andrew
Johnston, Kelly
Jones, Byron
Jouanolle, Anne-Marie
Poster 174
Podium 87
Podium 5
Poster 236
Podium 46
Podium 82
Poster 8
Poster 140
Poster 25
K
Kaelin, William
Kaplan, Jerry
Kayyali, Reem
Khomenko, Tetyana
Kim, Jeung Hyoun
Kim, Sangwon
Kim, Youngwoo
Kimura, Fumiaki
Knutson, Mitchell
Konijin, Abraham
Kple-Faget, Paul
Krewulak, Karla
Kriegshaeuser, Gernot
Kumar, S.
Kuo, Hung-Chieh
Kurantsin-Mills, Joseph
Podium 94
Podium 83
Poster 7
Poster 131
Poster 222
Posters 215, 216
Poster 258
Poster 161
Podium 11
Podium 9
Poster 24
Podium 63
Poster 3
Poster 92
Poster 209
Poster 239
L
Land, William
Latunde-Dada, Oluyemisi
Le Brun, Nick
Le Lan, Caroline
Lee, Sang
Leibold, Elizabeth
Lepape, A.
Lesuisse, Emmanuel
Levi, Sonia
Linder, Maria
Long, Joanna
Loyevsky, Mark
Ludwiczek, Susanne
Lundgren, Hans
Lumppio, Heather
Lynch, Sean
Poster 119
Poster 267
Poster 224
Poster 204
Posters 28, 112
Poster 257
Poster 54
Posters 106, 148
Podium 69
Posters 86, 87, 269
Poster 242
Poster 218
Poster 225
Poster 228
Podium 59
M
Ma, Yuxiang
Macedo, Fatima
Mackenzie, Bryan
Marx, Joanes J.M.
Matzanke, Berthold
Maxwell, Patrick
McClain, Danielle
McLaren, Gordon
McKie, Andrew
Medlock, Amy
Merryweather-Clarke, Alison
Meyron-Holtz Esther
Milman Nils
Minotti, Giorgio
Missirlis, Fanis
McKnight, Steven
Muckenthaler, Martina
Mura, Catherine
Podium 47
Poster 122
Podium 44
Posters 94, 136, 160
Podium 80
Podium 93
Poster 138
Poster 63
Podium 7
Poster 151
Posters 50, 51
Poster 1
Poster 135
Poster 130
Podium 1
Poster 43
N
Nekhai, Sergei
Nielsen, Peter
Nunez, Marco T.
Nunez-Millacura, Claudia
Podium 100
Posters 66, 67
Poster 95
Poster 101
O
Oates, Phillip
Oh, Eric
Okada, Shigeru
Olakanmi, Oyebode
Olsson, K. Sigvard
Olynyk, John
O’Neill, Heather
Outten, Wayne
Posters 37, 38
Poster 260
Poster 241
Podium 13
Poster 85
Poster 115
Poster 142
Poster 271
P
Pantopoulos, Kostas
Patch, Christine
Patton, Stephanie
Peacock, Sean
Pedersen, Palle
Perkins-Balding, D.
Phatak, Pradyumna
Phillips, John
Philpott, Caroline
Pietrangelo, Antonello
Piperno, Alberto
Pietsch, E. Christine
Pitula, Joseph
Planalp, Roy
Ponka, Prem
Pontre, Beau
Porto, Graca
Powell, Lawrie
Protchenko, Olga
Puccio, Helene
Podium 51
Poster 55
Poster 203
Poster 259
Poster 14
Poster 273
Poster 2
Poster 107
Podium 22
Podium 24
Poster 41
Podium 72
Poster 263
Poster 78
Poster 126
Poster 72
Poster 29
Podium 40
Poster 255
Podium 38
R
Rabsch, Wolfgang
Raha-Chowdhury, Ruma
Rafique, Roozina
Ramm, Grant
Rasmusser, A.R.
Reaves, Scott
Recalcati, Stefania
Reindel, Sabine
Rivarola, Maria
Robb, Aeisha
Robinson, Andrea
Rochette, Jacques
Rodrigues, Pedro
Rodriguez-Paris, Juan
Roetto, Antonella
Romao, Celia
Rossi, Enrico
Rouault, Tracey
Poster 207
Poster 97
Poster 199
Poster 192
Posters 182, 274
Poster 114
Poster 188
Poster 237
Poster 254
Poster 183
Poster 70
Poster 117
Poster 30
Poster 64
Poster 198
Poster 19
Podium 35
S
Sanchez, Mayka
Sanchez, Yasmin
Santambrogio, Paolo
Santos, Renata
Schalk, Isabelle
Schofield, Chris
Schranzhofer, Matthias
Seshadri, Vasudevan
Severance, Scott
Sharma, Naveen
Shayeghi, Majid
Sheftel, Alex
Shindo, Motohiro
Sinclair, Peter
Skaar, E.
Skovran, Elizabeth
Smith, Craig
Smith, James
Smith, Sophia
Soares, Ricardo
Solanky, Nita
Solodovnikova, Natalia
St. Pierre Timothy
Stoehr, Stephanie
Sturm, Brigitte
Subramaniam, V. Nathan
Sullivan, David
Surguladze, Nodar
Suzuki, Yasuaki
Szuber, Natasha
Poster 196
Poster 179
Podium 77
Poster 200
Poster 187
Podium 96
Poster 230
Poster 238
Poster 231
Poster 197
Poster 268
Posters 113, 121, 217
Poster 185
Poster 129
Podium 6
Poster 190
Podium 43
Poster 123
Poster 173, 275
Posters 153, 154
Poster 27
Poster 256
Posters 69, 162
Poster 99
Poster 189
Posters 220, 221
Podium 74
Podium 99
Poster 53
T
Tachezy, Jan
Tennant, Jason
Theil, Elizabeth
Theurl, Igor
Tolosano, Emanuela
Tong, Wing-Hang
Torti, Suzy
Trinder, Deborah
Turner, Elizabeth
Turner, JoLyn
Poster 229
Poster 10
Podium 71; Posters 17, 18, 34, 210, 242
Poster 58
Poster 134
Poster 240
Poster 73
Posters 36, 223
Podium 58
Poster 76
U
Ulvik, Rune
Poster 168
V
Vahdati-Ben Arieh, Sayeh
Valenti, Luca
van der Helm, Dick
Vogel, Hans
Voloshin, Yaroslav
Poster 90
Poster 16
Podium 62
Poster 261
Poster 118
W
Walcourt, Asikiya
Walden, William
Walker, Ann
Wallace, Daniel
Wandersman, Cécile
Wang, Jian
Wang, Xinsheng
Ward, Diane
Ward, Roberta
Warek, Ujwala
Watkins, Joseph
Wilkinson IV, John
Winkelmann, Guenther
Weiss Guenter
West, Adrian
Poster 32
Podium 68
Poster 165
Poster 35
Podium 65
Poster 59
Poster 93
Poster 214
Poster 272
Posters 84, 265
Poster 125
Podium 81
Poster 177
Poster 15
X
Xiaofeng, Liu
Poster 210
Y
Yamaguchi-Iwai, Yuko
Yamaji, Sachie
Yano, Motoyoshi
Yeh, Kwo-yih
Yiakouvaki, Anthie
Yikilmaz, Emine
Podium 55
Poster 11
Poster 20
Posters 103, 212
Poster 109
Poster 235
Z
Zaahl, Monique
Zanella, I.
Zhang, An-Sheng
Poster 71
Poster 180
Posters 56, 124
PODIUM
PRESENTATIONS
PODIUM 1
HEME, GAS AND REDOX SENSING AT THE HEART OF CIRCADIAN RHYTHM
Steven McKnight
The regulatory system controlling circadian rhythm is composed of a transcription feedback cycle. An
activating transcription factor turns genes on and off with a 24 hour rhythm. Certain of the genes
activated by this positively acting factor encode proteins that function in a direct and dedicated manner to
repress the system. The negatively acting factors decay with time, allowing the system to rebound in an
oscillatory manner. This same strategy is employed in flies, mice, humans and even simple molds such
as Neurospora crassa. In mammals, the activating transcription factor is a heterodimeric protein
composed of the Clock and BMAL polypeptides. In certain tissues, Clock is replaced by a paralogous
protein designated NPAS2. Both the Clock:BMAL and NPAS2:BMAL heterodimers serve to activate
expression of genes encoding the Period (Per) and Cryptochrome (Cry) proteins. The Per and Cry
proteins form a multimeric complex dedicated to the inhibition of Clock:BMAL- and NPAS2:BMALmediated gene activation. Recent studies have indicated the possibility of a reciprocal connection
between circadian rhythm and intermediary metabolism. Many genes encoding enzymes vital to
metabolism appear to be regulated rhythmically as a function of the 24 hour day:night cycle, pointing to
the possibility that metabolic processes may themselves cycle. Moreover, in vitro activity of the
Clock:BMAL and NPAS2:BMAL transcription factors has been shown to be sensitive to metabolites that
may vary as a function of the 24 hour cycle. These observations indicate that the circadian cycle may
actually constitute an integrated metabolic cycle, possibly as fundamental to life as the cell cycle.
PODIUM 2
NEW INSIGHTS IN IRON METABOLISM FROM THE STUDY OF JUVENILE HEMOCHROMATOSIS
Camaschella C’. Papanikolaou G*, De Gobbi M’, Roetto A’.
Juvenile (JH or Type 2) Hemochromatosis is a rare autosomal recessive disorder which leads to severe
iron overload and clinical complications at young age. The natural history of the disease has been
recently reviewed (De Gobbi et al Br J Haematol 117: 973-979, 2002). Comparing the clinical data of JH
patients with HFE-related or TFR2-related Hemochromatosis patients we have suggested that the protein
encoded by the JH gene exerts the strongest inhibitory effect on intestinal iron absorption. The JH locus
maps on chromosome 1q (Roetto et al, Am J Hum Genet 64: 1389-1393, 1999), but the gene remains
elusive. The antimicrobial peptide hepcidin has been recently identified as the putative main regulator of
iron absorption in animal models (Nicolas et al, PNAS 98;8780-8785, 2001; Nicolas et al, PNAS 99;45964601, 2001). Although hepcidin is excluded as a positional candidate for 1q-linked disease, we have
evaluated it as a possible functional candidate in a subset of JH families and recently reported hepcidin
mutations at the homozygous state in two unrelated Mediterranean families (Roetto et al, Nat Genet 33:
21-22, 2003).
Our findings indicate that hepcidin plays the same regulatory role on iron absorption in humans as in
mice. They have implications for the heterogeneity of hemochromatosis and for the molecular diagnosis
of JH. Finally they are consistent with the hypothesis that hepcidin is the final pathway where both the
store and the erythroid regulators converge.
‘Dipartimento di Scienze Cliniche e Biologiche, Università di Torino, Italy
*First Department of Medicine, University of Athens, School of Medicine, Greece
PODIUM 3
REGULATION OF HUMAN HEPCIDIN BY INFLAMMATION AND IRON
T. Ganz and E. Nemeth, University of California, Los Angeles
Hepcidin is a liver-made, circulating cationic peptide excreted in urine. Evidence from various mouse
models makes a strong case that hepcidin is an inhibitory regulator of intestinal absorption of dietary iron
and of the release of recycled iron from macrophages, and that hepcidin mRNA is induced by iron stores
and by inflammation. Hepcidin may also be the long sought mediator of anemia of inflammation (chronic
disease). To explore the role of hepcidin in humans, we analyzed hepcidin production in patients with
different iron disorders, and in human tissues in vitro.
Patients’ urine was processed using cationic matrix-exchange and urinary hepcidin levels analyzed by
Western blotting using rabbit anti-human hepcidin antibody. We found that urinary excretion of hepcidin
was greatly increased in patients with iron overload, infections or inflammatory diseases and was
undetectable in iron-deficiency anemia. Hepcidin excretion correlated well with levels of serum ferritin, a
protein known to be increased by inflammation and body iron stores.
To explore the molecular basis of hepcidin induction, we used fresh human hepatocytes which were
treated with different inflammatory and iron-loading stimuli. Hepcidin mRNA expression was determined
by quantitative Northern analysis. To simulate inflammation, cells were treated with lipopolysaccharide
(LPS) or the conditioned medium of monocytes exposed to LPS. Both stimuli induced hepcidin mRNA but
the latter response was greater. It appears to be a type II acute-phase response, as direct addition of IL6, but not IL-1 or TNF-α, dramatically induced hepcidin mRNA in hepatocytes. For iron loading,
hepatocytes were treated with ferric-ammonium citrate (FAC) or iron-saturated transferrin (Fe2-Tf), which
surprisingly resulted in a decrease in hepcidin expression. We then postulated that other iron-sensing
cells such as macrophages provide signal to hepatocytes to modulate hepcidin expression according to
the body iron load. When conditioned medium of monocytes exposed to FAC and Fe2-Tf was added to
hepatocytes culture, it resulted in hepcidin mRNA induction.
Our data support the role of hepcidin as a mediator of anemia of inflammation in humans and point to an
indirect effect of iron stores on hepcidin production by hepatocytes.
PODIUM 4
IRON REGULATION IN PRIMARY CELL CULTURES FROM MICE WITH TARGETED
DELETIONS OF IRP1 AND IRP2
Esther Meyron-Holtz, Manik Ghosh and Tracey A. Rouault, National Institute of Child Health and
Human Development, Bethesda, MD 20892
In mammalian cells, the two Iron Regulatory Proteins IRP1 and IRP2 mediate the regulation of
several important iron metabolism proteins. IRPs bind to an RNA stem-loop structure called the
Iron Responsive Element (IRE) located in the transcript of target genes such as Transferrin
Receptor (TfR) and Ferritin (Ft). Both IRPs are regulated by the status of a chelatable iron pool in
the cell and respond to levels of O2 and NO 1,2.
IRP1 IRE binding activity is enhanced by oxygen and repressed by iron, whereas IRP2 IRE
binding activity is repressed by both oxygen and iron. The balance between intracellular oxygen,
chelatable iron levels and cell specific ratios between IRP1 and IRP2 expression leads to a
complex iron regulatory mechanism.
IRP2-/- mice suffer from neurodegeneration that colocalizes with misregulated high Ft and low
TfR expression in specific areas of the brain, suggesting that certain cell types are more
susceptible to misregulation in response to the lack of IRP2 than others. In order to understand
the combined effects of iron, redox status and the two IRPs on iron metabolism, we monitor the
activities and protein levels of IRPs and their downstream targets, Ft and TfR in primary cellcultures. Typically oxygen concentrations in tissues range between 3-5%. Therefore we exposed
bone marrow derived macrophages (MPs) or embryonic fibroblasts (EFs) from IRP1-/- or IRP2-/mice to 3% or 21% O2. Gel retardation assays indicate marked decrease in the IRP1/IRP2 ratio
and in total IRP content in MPs cultured at 3% O2 compared to 21% O2. TfR synthesis is notably
repressed, whereas Ft level is significantly increased in MPs from IRP2-/- mice at 3% O2,
consistent with our previous finding that IRP2-/- mice have elevated serum Ft which suggested
misregulation of Ft synthesis in MPs. Quite the opposite, MPs from IRP1-/- mice have Ft levels
similar to wt. In contrast, EFs have a much higher total IRE binding activity (IRP1 and 2)
compared to MPs which lead only to a mildly compromised Ft and TfR synthesis in the IRP2-/EFs at 3% O2. Thus, iron regulation in cells with a low IRP1/IRP2 ratio and low total IRP binding
activity appeared to be more affected by the targeted deletion of IRP2. These results suggest that
primary cultures of single cell types from IRP1-/- or IRP2-/- mice, when cultured at 3% O2, can
serve as a model for the misregulation of iron metabolism in the whole mouse. In addition, these
models give us insight into the complexity and robustness of a regulatory system with two
apparently redundant but subtly different regulators.
Reference:
1. Rouault T. Klausner R. Regulation of iron metabolism in eukaryotes. Curr Top Cell Regul.
1997;35:1-19
2. Hanson ES. Leibold EA. Regulation of the iron regulatory proteins by reactive nitrogen and
oxygen species. Gene Expr. 1999;7(4-6):367-76
PODIUM 5
CERULOPLASMIN MAINTAINS IRON HOMEOSTASIS IN ASTROCYTES VIA INTERACTIONS WITH
IREG1
S.Y. Jeong*, S. David.
Centre for Research in Neuroscience, McGill University Health Centre, Montreal General Hospital
Research Institute, 1650 Cedar Ave., Montreal, Quebec, Canada, H3G 1A4
Iron is an essential metal but because it is highly reactive, it can cause tissue damage when present in
high levels. Therefore the amount of iron at the cellular level must be tightly regulated in organs,
particularly the brain. The brain expresses mainly the GPI-anchored form of ceruloplasmin (GPI-Cp)
which is produced by astrocytes. Mutations in the Cp gene in humans and mice lead to iron accumulation
in the CNS, and neurodegeneration. In this study we examined the role of ceruloplasmin in iron flux in
vitro in astrocytes purified from the brains of neonatal Cp-/- and Cp+/+ mice. Iron influx and efflux was
studied using [59Fe]ferrous ascorbate in serum-free, transferrrin-free culture medium over a 48h period.
Our results show that iron influx is not significantly different between astrocytes from Cp+/+ and Cp-/- mice.
In contrast, iron efflux was markedly impaired in astrocyte cultures from Cp-/- mice. In Cp+/+ cultures, 70%
of the intracellular radiolabeled iron was effluxed by 48 hours, while iron efflux failed to occur in the 48h
period in Cp-/- cultures. As Cp must work with iron transporters to efflux iron, we examined the expression
of such transporters in purified astrocyte cultures. Expression of IREG1 and DMT1 was detected by RTPCR and Western blotting. Double-immunofluorescence studies showed that Cp co-localizes with IREG1
but not DMT1 on the surface of cultured astrocytes. Co-immunoprecipitation studies using an anti-Cp
antibody confirmed that GPI-Cp is associated with IREG1 but not DMT1. These results suggest that GPICp and IREG1 work likely in concert to regulate iron homeostasis in the CNS.
This work was supported by a grant from the Canadian Institute of Health Research.
PODIUM 6
HEME-IRON TRANSPORT ACROSS THE CELL WALL OF GRAM POSITIVE BACTERIA
E. Skaar*, S. Mazmanian*, A. Gaspar*, A. Joachmiak&, D. Missiakas*, O. Schneewind*
*
Committee on Microbiology, University of Chicago, and &Argonne National Laboratory
Many bacterial pathogens require iron for growth and virulence. While several iron acquisition systems
have been characterized in Gram-negative bacteria, the mechanisms for iron transport into Gram-positive
bacteria have hitherto not been revealed in detail. We report that the Gram-positive human pathogen,
Staphylococcus aureus, utilizes heme or hemoglobin as an iron source. An iron-regulated locus, called
iron responsive surface determinants (isd), was identified. isd is composed of eight genes isdA, isdB,
isdC, isdD, isdE, isdF, isdG and srtB. IsdB, a cell wall anchored polypeptide that is tethered to the
bacterial peptidoglycan by a transpeptidation mechanism involving an LPXTG motif sorting signal, binds
to both hemoglobin and heme-iron. In contrast, IsdA, another LPXTG motif anchored protein, as well as
IsdC, carrying an NPQTN type sorting signal, bind only to heme. IsdC is tethered to the staphylococcal
envelope by sortase B (SrtB), whereas IsdA and IsdB are both substrates for sortase A-mediated
anchoring. IsdE, a membrane lipoprotein, and IsdG, a cytoplasmic protein, also bind heme-iron, while
IsdF assembles as a polytopic membrane protein. Deletion of sortases or isd genes reduces the ability of
mutant staphylococci to bind radiolabeled heme-iron or to transport this molecule into staphylococci.
These variants also display a step-wise decrease in staphylococcal growth on heme-iron as the sole iron
source. Together with the observation that the staphylococcal genome encodes multiple genes with
identity to isdB, isdE and isdF, these results suggest that S. aureus employs multiple cell wall-anchored
surface proteins and membrane transporters to retrieve heme-iron from hemoglobin and to transport
heme-iron across the cell wall envelope and plasma membrane into the cytoplasm. It is hypothesized that
the isd locus functions as a cell wall import apparatus for heme-iron during human infection, employing
multiple anchored proteins to sequentially relay heme across the bacterial envelope. This process
appears to involve sortase A and sortase B mediated-cell wall anchored proteins, membrane lipoproteins
and integral membrane proteins as well as cytoplasmic factors. Future work will need to test these
hypotheses by elucidating the biochemical reactions involved in heme-iron removal from hemoglobin or
heme transport, and by measuring the non-redundant role of isd genes in heme-iron uptake and bacterial
pathogenesis.
PODIUM 7
CHARACTERISATION OF A NOVEL GENE HIGHLY EXPRESSED IN DUODENUM
Majid Shayeghi, Neil Halliday, Jonathan Oakhill, Yemisi Latunde-Dada, David M Frazer, Christopher D
Vulpe, Gregory J Anderson, Robert J Simpson, Andrew T McKie. Division of Life Sciences, King's College,
London SE1 9NN, UK, .Joint Clinical Sciences Program, the Queensland Institute of Medical Research and
the University of Queensland, PO Royal Brisbane Hospital, Brisbane, Queensland 4029, Australia
Department of Nutritional Sciences, University of California, Berkeley, Berkeley CA 94720, USA
Using a previously described subtractive cloning strategy(1; 2), we have identified a number of novel
cDNAs that are highly expressed in the duodenal mucosa and may be involved in the iron absorption
pathway. We are currently focusing on several cDNAs encoding novel membrane proteins. By Northern
blotting the mRNA of one cDNA is up-regulated by hypoxia and hypotransferrinaemia in mouse duodenum.
We have investigated the sub-cellular localisation of the protein within duodenal enterocytes using specific
peptide antibodies. Preliminary experiments on duodenal sections show that the novel protein is found
associated in distinct vesicles within the enterocytes as well as the apical membrane. This would indicate
the protein is involved in a vesicular transport process. Due to its localisation in the proximal duodenum
and its regulation by hypoxia we are investigating whether this cDNA plays a role either in iron or haem
transport and/or trafficking. To test these possibilities we are expressing the protein in mammalian cells
and Xenopus oocytes. Data will be presented from these studies.
This work is supported by grants from the NIH, MRC, HFSP and EU
Reference List
1. McKie AT, Barrow D, Latunde-Dada GO, Rolfs A, Sager G, Mudaly E, Mudaly M, Richardson C,
Barlow D, Bomford A, Peters TJ, Raja KB, Shirali S, Hediger MA, Farzaneh F and Simpson RJ. An
iron-regulated ferric reductase associated with the absorption of dietary iron. Science 291: 17551759, 2001.
2. McKie AT, Marciani P, Rolfs A, Brennan K, Wehr K, Barrow D, Miret S, Bomford A, Peters TJ,
Farzaneh F, Hediger MA, Hentze MW and Simpson RJ. A novel duodenal iron-regulated
transporter, IREG1, implicated in the basolateral transfer of iron to the circulation. Mol Cell 5: 299309, 2000.
PODIUM 8
CELLULAR AND MOLECULAR APPROACHES TO STUDY HEME IRON RECYCLING IN
MACROPHAGES
F. Canonne-Hergaux, C. Delaby, G. Hetet, B. Grandchamp and C. Beaumont. Inserm U409, Génétique et
Pathologie Moléculaire de Hematopoièse, Faculté de Médecine Xavier Bichat, Paris.
Introduction: Senescent erythrocytes are phagocytosed by macrophages present in the bone marrow,
spleen and liver. During this process of « erythrophagocytosis », iron is released from heme through the
action of heme oxygenase 1 (HO-1). Iron is then transported into the circulation to respond to the need of
immature red blood cells in the bone marrow. In mammals, this process of iron recycling by macrophages
plays a key role in iron homeostasis. In chronic inflammatory diseases, anemia is frequently observed
due to the retention of iron in cytokines activated macrophages. On the other hand, in hereditary HFEhemochromatosis, macrophages seem to loose their ability to store iron thereby participating in the onset
of the disease. Different studies suggest the possible role of transporters such as Nramp1, DMT1/Nramp2
and ferroportin in macrophage iron recycling. However the molecular mechanisms involved in transport,
distribution and secretion of iron by macrophages are still poorly documented and more physiological
cellular models need to be developed. Here we describe an ex vivo model of erythrophagocytosis by
bone marrow derived macrophages.
Methods: Murine red blood cells (mRBC) were aged artificially at different temperatures in the presence
++
or in the absence of Ca and a calcium ionophore A23187. The effect of these treatments on mRBC was
monitored by flow cytometry analysis and annexin-V labelling. After treatment, erythrocytes were
incubated with resting or activated (with Interferon γ; INFγ and/or Lipopolysaccharide; LPS) bone marrow
derived macrophages (BMDM). In these different conditions of culture, the expression and the subcellular
localisation of Nramp1, Nramp2, and ferroportin were studied by western blot analysis and confocal
microscopy. Results: In vitro treatments that lead to morphological changes of mRBC with
externalisation of phosphatidyl serine (features of natural senescence) were found. Untreated mRBC are
poorly phagocytosed whereas Ca++/A23187 pre-treated mRBC are captured and ingested by resting
BMDM. Moreover, the phagocytic activity (binding and internalisation) of BMDM is strongly increased
after INFγ/LPS treatment. As previously reported at mRNA level, INFγ/LPS increase the expression of
Nramp1 and Nramp2/DMT1 proteins but decrease ferroportin protein expression in macrophages.
Interestingly, Nramp1 is found to be associated to membranes of phagosomes that contain mRBC. This
association is particularly important in activated BMDM. Nramp2/DMT1 presents a distinct vesicular
localisation and does not seem to be recruited to the phagosomal membrane during erythrophagocytosis
even in the absence of Nramp1 (experiment done with BMDM from Nramp1-deficient strain of mice).
Conclusions and perspectives: In order to dissect the molecular mechanisms of iron recycling by
macrophages, we established a cellular model of erythrophagocytosis that tends to mimic the in vivo
clearance of senesent RBC. Preliminary results indicate that Nramp1 is a strong marker of the
“hemophagosome” but its possible role in iron recycling from macrophages needs further investigation.
Activated macrophages have a high ability to ingest mRBC associated with a lower expression of the iron
exporter ferroportin. Such observation may explain in part the retention of iron in macrophages during
inflammation. With this model of erythrophagocytosis, other approaches using cDNA microarray and
proteomic analysis will be developed to find new genes involved in iron recycling by macrophages.
PODIUM 9
THE PROCESSING OF POLYMERIC IRON BY MACROPHAGES FOLLOWS THE MAJOR
STAGES OF ERYTHROPHAGOCYTOSIS. A CELLULAR MODEL FOR ON-LINE
MONITORING OF IRON RECYCLING BY FLUORESCENCE
Virginie Leib1, Abraham M. Konijn2 and Z. Ioav Cabantchik 1
1
Department of Biological Chemistry, Institute of Life Sciences and 2Department of Human
Nutrition, Hadassah Medical School, Hebrew University of Jerusalem, Jerusalem 91904, Israel.
The major route of iron trafficking in the organism is iron recycling by macrophages whereby
aged erythrocytes are phagocytosed, their Hb is degraded and its iron is released into the cytosol
and secreted into the medium. The molecular mechanisms underlying those steps remain to be
elucidated primarily because of lack of experimental devices to assess cell iron trafficking. In an
attempt to dissect the putative steps of iron recycling by macrophages we studied macrophage
processing of polymeric iron preparations used in the clinic to intravenously treat iron-deficient
patients. For that purpose we have used: a. J774 macrophage cells in culture as the
experimental cell model; b. iron saccharate polymer (Venofer, Vifor, Switzerland) as the
macromolecular iron substrate, either as the native polymer (NPI) or the modified polymer (MPI)
that was rendered devoid of free iron by chelator treatment (and in the process the iron particles
grew in size and decreased in solubility) and c. fluorescent metalosensors targeted either to
different cell compartments (calcein to endosomes and calcein-acetomethoxy ester and phengreen to the cytosol) or to the extra-cellular medium as cell-impermeant fluorescent iron probes
(fluorescein-deferrioxamine FL-DFO or fluoresceinated-apotransferrin –FL-ATf). Uptake of MPI
and NPI into cells was revealed by assessing the number and nature of polymeric iron-containing
vesicles, using Perl’s stain, transmission electron microscopy (TEM), phase microscopy and
fluorescence microscopy (standard and confocal). The MPI or NPI particles were cophagocytosed with either fluoresceinated dextran (as pH sensitive indicator) or free calcein (as
iron-sensitive probe). Incubation with MPI gave rise to a multiplicity of relatively large intracellular
vesicles (phagosomes) containing polymeric iron, whereas NPI was detected mainly in smaller
cytosolic vesicles of pinocytotic character. MPI-containing vesicles were detected within 1hr of
incubation, peaked at 3-4 hrs and gradually decreased over the following 16 hrs. This process
was sensitive to inhibitors of the cytoskelon that affect endocytic processes. Release of free iron
into the vesicle interior was detected following 2 hrs of incubation, peaked at 8 hrs and decreased
concomitantly with the appearance of iron in the cytosolic labile iron pool (LIP). These processes
were inhibited by the action of acidotropic agents that alkalinize the vesicle content. On the other
hand, NPI uptake led first to a gradual rise in cytosolic LIP over a 16 hr period, followed by a slow
but steady phase of iron egress into the medium. The time profiles of the different phases of
polymeric iron processing by macrophages were obtained also on-line using attached cells grown
in 96-well plates and monitored for changes in respective probe fluorescence by a fluorescence
plate reader. The intracellular and extracellular factors controlling the various steps of iron
recycling by macrophages are presently under evaluation.
th
This work was supported in part by The European Community 5 Framework and by Apotex,
Ltd., Ont. Canada.
PODIUM 10
MDL1 IS A HIGH COPY SUPRESSOR OF ATM1, AND REGULATES RESISTANCE TO OXIDATIVE
STRESS IN WILD TYPE CELLS
M. Chloupková, L.M. LeBard, and D.M. Koeller*, Oregon Health & Science University, Portland, Oregon
INTRODUCTION: The yeast Atm1p is essential for normal cellular iron homeostasis. The protein is
localized to the inner mitochondrial membrane, and it belongs to the family of ATP-binding cassette
(ABC) transporters. Deletion of ATM1 results in mitochondrial iron accumulation, cytosolic iron starvation,
and deficiencies in cytosolic Fe-S proteins. Mutations in the human ABC7, an ortholog of ATM1, result in
X-linked sideroblastic anemia with ataxia, in which patients accumulate mitochondrial iron in
erythroblasts. The specific functions of Atm1p and ABC7 have not been determined, though roles in both
mitochondrial iron export and cytosolic Fe-S cluster assembly have been proposed. To better understand
the function of Atm1p, and thus of ABC7, we undertook a screen for yeast genes capable of suppressing
the abnormalities of cellular iron metabolism demonstrated by ∆atm1 cells. One of the identified genes
was MDL1, which codes another mitochondrial inner membrane ABC transporter. Mdl1p has previously
been proposed to function in the export of peptides from the mitochondrial matrix. In this work, we
analyze the phenotype of ∆atm1 cells overexpressing MDL1 (∆atm1/MDL1), and describe novel
phenotypes for ∆mdl1 and wild type cells overexpressing MDL1 (WT/MDL1). METHODS: The procedures
employed in this work include bacterial and yeast genetic manipulations, northern blotting, analysis of
growth phenotype, metal and H2O2 toxicities, mitochondrial non-heme iron determination, estimation of
catalase activity, isolation of mitochondria, protein determination, and β-galactosidase assay. RESULTS:
MDL1 overexpression resulted in partial rescue of ∆atm1 cells. The ∆atm1/MDL1 cells grew on galactose,
but did not utilize non-fermentable carbon sources. The extreme sensitivity of the ∆atm1 cells to metals
and H2O2 was partially suppressed by MDL1. In addition, MDL1 overexpression resulted in a 50%
reduction in the mitochondrial iron level of ∆atm1 cells; however, it remained nearly 8-fold increased over
wild type. To address whether the mechanism by which MDL1 rescues ∆atm1 cells is the direct role of
Mdl1p in cellular iron homeostasis, we constructed two additional strains, ∆mdl1 and WT/MDL1. The
FET3 and FET4 mRNA levels, and mitochondrial iron content were not significantly changed in these
strains compared to wild type cells. Thus, we looked at metal toxicities and growth of various strains
under oxidative stress. Neither deletion nor overexpression of MDL1 did specifically affect metal toxicities.
In contrast, ∆mdl1 showed an extreme resistance to H2O2, diamide, and Cd compared to wild type cells.
Catalase activity was also increased in ∆mdl1 strain. We saw the opposite results for the H2O2, diamide,
and Cd toxicities, as well as catalase activity, in WT/MDL1 cells. DISCUSSION: The complex phenotype
of an ATM1 deletion results from a combination of primary (iron accumulation) and secondary (oxidative
stress) effects. Based on the phenotype of ∆atm1/MDL1 cells, it seems that MDL1 suppresses the
secondary effects of ATM1 deletion. The analysis of ∆mdl1 cells supports this hypothesis. MDL1 does not
affect the growth of wild type cells under non-stressed conditions. In contrast, in the presence of an
oxidative stress, we observed marked differences between the ∆mdl1 and WT/MDL1 strains, suggesting
a novel function for Mdl1p in modulation of oxidative stress resistance. CONCLUSIONS: 1. Suppression
of ATM1 deletion by MDL1 is not due to the function of Mdl1p in iron homeostasis. 2. Discrepancies
between the phenotype of ∆atm1/MDL1 and WT/MDL1 cells imply a possible functional interaction
between Atm1p and Mdl1p, the two of three inner membrane mitochondrial ABC transporters. *Contact
person: koellerd@ohsu.edu. This work was sponsored by National Institutes of Health (PO1 HD08315).
PODIUM 11
INCREASED FERROPORTIN 1 (FPN1) EXPRESSION AFTER ERYTHROPHAGOCYTOSIS BY J774
MACROPHAGES
M. Knutson, D. Haile, M. Wessling-Resnick. Harvard School of Public Health, Boston, MA and
University of Texas Health Science Center, San Antonio, Texas.
BACKGROUND: Macrophages play a major role in iron metabolism by recycling iron and by serving as a
large storage reservoir. Iron recycling is achieved mainly by reticuloendothelial (RE) macrophages, which
phagocytose senescent red cells, liberate the iron from heme, and return the metal to the circulation. Iron
export from the macrophage to the plasma represents the largest flux of iron within the body;
approximately 25 mg of iron is recycled through this pathway each day. Perturbations in iron efflux from
RE macrophages appear to contribute to the etiology of hereditary hemochromatosis and the anemia of
chronic disease. However, despite the critical role of the RE macrophage in whole-body iron
homeostasis and its clinical importance, the molecular mechanisms of iron handling in the RE system
remain poorly characterized. Recent studies suggest that the duodenal iron-export protein FPN1 (also
known as MTP1, IREG1, or SLC11A3), which is abundantly expressed in RE macrophages, may also
play a role in erythrocyte iron recycling. The goal of the present study was to determine if macrophage
FPN1 mRNA and protein expression change after phagocytosis of red cells.
METHODS: J774 cells (a murine macrophage cell line) were incubated with opsonized erythrocytes, and
after 1.5 h of erythrophagocytosis, non-ingested erythrocytes were lysed and removed. Cells were then
incubated at 37ºC for various times before harvesting for subsequent extraction of total RNA and protein.
FPN1 mRNA and protein levels were determined by Northern and Western analysis, respectively. To
test whether the increase in FPN1 after erythrophagocytosis was due to iron, the lipophilic iron chelator,
salicylaldehyde isonicotinoyl hydrazone, SIH (0, 5, 20, 50, 100 µM), was added to the cell culture media
after erythrophagocytosis, and cells were harvested 6 h later for analysis of FPN1 mRNA and protein
levels. Changes in FPN1 mRNA levels after erythrophagocytosis were also compared to those of other
iron-regulated genes such as heme oxygenase 1 (HO-1), natural resistance-associated macrophage
protein 1 (Nramp1), and divalent metal transporter 1 (DMT1).
RESULTS: FPN1 mRNA levels increased transiently after erythrophagocytosis, reaching maximal levels
(8-fold higher than basal levels) after 4 h and returning to basal levels by 16 h. FPN1 protein increased to
maximal levels by 10 h and then decreased. SIH added immediately after the erythrophagocytosis period
resulted in a dose-dependent decrease in FPN1 protein induction, with no detectable expression following
the addition of 50 or 100 µM SIH. Although a dose-responsive decrease in FPN1 mRNA induction was
also observed with increasing doses of SIH, FPN1 mRNA levels in cells treated with 100 µM SIH were
still 3-fold higher than basal levels. The marked changes in FPN1 mRNA expression after
erythrophagocytosis were similar to those of HO-1, whereas levels of Nramp1 and DMT1 mRNA did not
change appreciably.
CONCLUSIONS: The rapid, strong, and transient induction of FPN1 (but not Nramp1 or DMT1)
expression after erythrophagocytosis suggests that FPN1 plays a unique role in iron recycling by the
macrophage. The demonstration that FPN1 induction can be suppressed by iron chelation indicates that
the increase in the levels of this protein after erythrophagocytosis most likely results from iron released
from red cell heme degradation.
PODIUM 12
DOWN-REGULATION OF HYDROXYACID OXIDASE 1 EXPRESSION BY IRON. AN OXIDATIVE
STRESS MEDIATED RESPONSE ?
S. Recalcati1,2, L. Tacchini1, A. Alberghini1,2, D. Conte2 and G. Cairo1, 1Inst. General Pathology and 2Dept.
Medical Sciences, University of Milan, Italy
Hydroxy acid oxidase 1 (Hao1) is a liver specific protein targeted to peroxisomes (1) where it converts αhydroxy acids to α-keto acids and concomitantly reduces molecular oxygen to hydrogen peroxide. The
mRNA for Hao1 bears an iron responsive element (IRE) with good homology to the consensus (2).
However, the involvement of this sequence in iron regulatory proteins (IRP) -mediated regulation of Hao1
is still unclear. In fact, the finding that the IRE of the Hao1 mRNA was not able to impart translational
regulation to a reporter gene and the lack of Hao1 mRNA accumulation in hepatoma cells treated with an
iron chelator suggested that Hao1 is not subjected to IRP-dependent post-transcriptional regulation (2).
However, Hao1 expression in a mouse liver cell line was stimulated by exposure to iron (2). In the present
study, to assess whether iron controls Hao1 expression in vivo, we analyzed Hao1 mRNA levels in ironrich and depleted rat livers. Hao1 mRNA content was markedly reduced below control levels in the livers
of animals overloaded with iron by feeding an iron-enriched diet, which also showed decreased IRP
activity and higher ferritin content, as expected. Immunoblot analysis also showed a strong induction of
heme oxygenase (HO-1), a marker of oxidative damage, in iron overloaded livers, thus suggesting that
production of reactive oxygen species (ROS) caused by accumulation of high levels of iron may be
involved in the down-regulation of Hao1. To separate the effects of iron from those of ROS, we treated
rats acutely with repeated doses of parenteral iron given as ferric ammmonium citrate that were found to
be sufficient to significantly induce ferritin expression and to repress IRP activity, but did not cause
oxidative stress (detected as HO-1 induction). This form of iron loading did not significantly alter Hao1
mRNA levels. Similarly, Hao1 expression was not affected in the livers of rats repeatedly treated with
desferrioxamine to induce iron deficiency. To further investigate the role of ROS, we analyzed the
expression of Hao1 in the liver of rats subjected to oxidative stress induced by glutathione (GSH)
depletion (3) and ischemia/reperfusion (I/R) (4,5). We found a strong down-regulation of Hao1 mRNA
after treatment with ROS-generating drugs (nitrofurantoin, phorone, buthionine-sulfoximine), in parallel
with a marked decrease of GSH content. Similarly, Hao1 mRNA was strongly reduced at early times after
restoration of the blood supply to a formerly ischemic liver, concomitantly to HO-1 induction. The
demonstration that rat liver Hao1 mRNA levels are down-regulated in two in vivo models of oxidative
stress in which we have previously shown that IRP activity is repressed (3-5) would argue in favor of a
functional Hao1 IRE. However, we also found a lack of modulation of Hao1 in both acutely iron
overloaded and iron-deficient livers, two conditions characterized by an opposite response of IRP activity.
Moreover, the results obtained with GSH-depleted and reperfused livers suggest that the down-regulation
in chronically iron-loaded livers may be the consequence of the oxidative stress that accompanies dietary
iron overload. The present results are therefore not consistent with a regulation of Hao1 through the
IRE/IRP pathway and suggest a transcriptional effect of ROS. Peroxisomes are responsible for formation
of H2O2 by several oxidases and degradation of H2O2 by catalase and are therefore involved in the
oxidative stress response. We have previously shown that hepatic catalase expression is activated as an
adaptive response to oxidative stress caused by GSH depletion (6). Since the oxidation of glycolate to
glyoxylate by Hao1 is accompanied by production of H2O2, the down-regulation of Hao1 expression
during oxidative stress might represent an additional mechanism to prevent excessive H2O2 formation in
peroxisomes.
1. Recalcati et al. 2001 J. Cell Sci.
2. Kohler et al 1999 J. Biol.Chem.
3. Cairo et al. 1995 J. Biol.Chem.
4. Tacchini et al. 1997 Gastroenterology
5. Tacchini et al. 2002 Hepatology
6. Tacchini et al. 1996 Redox Report
PODIUM 13
THE NATURE OF EXTRACELLULAR IRON CHELATES, MACROPHAGE HFE STATUS, AND
INTERFERON-γ INFLUENCE IRON ACQUISITION BY INTRAPHAGOSOMAL MYCOBACTERIUM
TUBERCULOSIS
O. Olakanmi, L.S. Schlesinger, A. Ahmed, B.E. Britigan. VAMC-Iowa City, The University of Iowa, and
The Ohio State University
Iron (Fe) acquisition is critical to the growth of most microbes. In humans, extracellular Fe is chelated to
transferrin (TF) and lactoferrin (LF), as a strategy of host defense to limit Fe availability to pathogenic
microbes. Mycobacterium tuberculosis (M.tb) is a major human intracellular pathogen that enters and
multiplies within lung macrophages in a unique phagosomal compartment. We have recently
demonstrated that M.tb residing within the phagosomes of human monocyte-derived macrophages
(MDM), termed intraphagosomal M.tb, can acquire Fe from extracellular TF, as well as from sites within
the MDM containing Fe acquired from TF (J.Biol.Chem.277:29727-34, 2002). In the lung, more Fe is
bound to LF than TF and some Fe present is also present in the form of low molecular weight chelates.
Therefore, we compared the ability of intraphagosomal M.tb to acquire Fe from these biologically relevant
extracellular Fe chelates, as well as intracellular MDM sites preloaded with Fe from these same chelates.
To measure Fe uptake from the extracellular chelates, human MDM were infected with virulent M.tb
59
(Erdman strain) and then non-phagocytosed M.tb were removed. Fe chelated to TF, LF or citrate was
added for 24h to the MDM monolayer, after MDM- and M.tb-associated 59Fe was determined. To assess
Fe acquisition from endogenous MDM sources, MDM were incubated with the 59Fe chelates for 24h,
extensively washed, and then infected with M.tb. 59Fe associated with the MDM and M.tb was then
determined after another 24h. We found that the amount of MDM-associated 59Fe from all the exogenous
chelates was similar. For all chelates, the presence of intracellular M.tb reduced MDM Fe uptake from the
exogenous chelates, relative to uninfected MDM: greatest with TF (40%) and least with LF (15%). M.tb
acquired 59Fe from exogenous TF and citrate to a similar degree, whereas acquisition from LF was 2-5
fold greater. M.tb acquisition from endogenous MDM sites preloaded with 59Fe from the various chelates
was similar in magnitude to that from exogenous TF and citrate. Interferon-γ (IFN-γ) plays a major role in
host defense against intracellular pathogens, in part by its ability to decrease macrophage-associated Fe.
IFN-γ (1000 units, 5 d) treatment reduced MDM Fe uptake from each of the 3 chelates by ~50%,
augmenting the M.tb-induced decrease in MDM Fe uptake from TF, but not the other chelates. IFN-γ
decreased Fe acquisition from TF by intraphagosomal M.tb 25%, whereas there was >2 fold increase with
LF and citrate. In contrast to most other cell types, the Fe content of MDM from individuals with
hereditary hemochromatosis (HH) is low. We have reported (JBC above) that MDM from HH subjects
acquire similar amounts of Fe from TF over 24h, but M.tb residing within HH MDM acquire significantly
less Fe (about 60-70%) compared to bacilli within healthy control MDM. We have extended these studies
59
to LF and citrate. HH and control MDM exhibited similar uptake of Fe from TF, LF and citrate. In
59
contrast, to the results with TF, we found no difference in M.tb Fe acquisition from exogenous citrate or
from MDM preloaded with 59Fe from citrate. In the case of LF, M.tb in the HH MDM acquired more 59Fe
from exogenous LF compared to M.tb in control MDM, whereas no difference was observed with MDM
preloaded with 59Fe from LF. IFN-γ treatment enhanced the decrease in 59Fe uptake by M.tb residing
within HH MDM, but had no impact on Fe acquisition from citrate or LF. The nature of the Fe chelate
appears to affect the ability of intraphagosomal M.tb to acquire Fe from both the extracellular environment
and sites within the infected macrophage. The nature of the Fe chelate also alters the influence of IFN-γ
and the macrophage HFE phenotype on M.tb Fe acquisition. M.tb seems to have developed efficient
mechanisms of acquiring Fe from a variety of Fe chelates that it would potentially encounter within the
human lung. More investigation is required to define these mechanisms and the possible role of the HH
phenotype as an evolutionary strategy of host defense against M.tb.
PODIUM 14
IRON, INFECTION AND THE INFLAMMATORY RESPONSE
Roberta J Ward1, Stephanie Wilmet1, Jacques Piette2 and Robert R Crichton1.
1
Unité de Biochimie, University of Louvain, 2 Virology Unit, University of Liege, BELGIUM
NFκappaB orchestrates the inflammatory response in cells. However the stimulus for its cytoplasmic
activation and translocation to the nucleus remains conjectural. The involvement of reactive oxygen
intermediates (ROIs) has been indicated in some studies (Baeuerle and Henkel 1994; Bonizzi et al.,2000)
but not in others (Bowie and O’Neill, 2000) which may, in part, be due to the different cell types in the
experiments as well as the use of agents, such as hydrogen peroxide, to mimick ROIs.
In the present studies primary cell cultures of alveolar macrophages of different iron status have been
investigated to ascertain the role of NADPHoxidase in the activation of this transcription factor.
Iron overload or iron deficiency was induced in rats by injection of low (10mg) or high doses (125mg) of
iron dextran while iron deficiency was induced by dietary means. NADPH oxidase was assayed by
chemiluminescence after stimulation with N-formylated methionylpeptide, FMLP, or phorbol-12 myristate13 acetate, PMA, and NFκB by band-shift assay after activation with lipolysaccharide and TNFα.
Chemiluminescence was present in the macrophages from each of the groups prior to activation which
was caused by mitochondrial activity. Small but insignificant differences were apparent between the
three groups, iron overload, iron deficiency and controls. However NFκB activity was significantly
enhanced in both iron loaded groups and diminished in the iron deficiency group by comparison to
controls. After activation of the macrophages, significant increases in chemiluminescence was evident in
both iron loaded groups-however no further activation of NFκB was apparent. In the control
macrophages an increase in both NADPH oxidase and NF
B wa s e vide nt a fte r s
administration of desferrioxamine for a two week period after iron loading did not significantly reduce
either the iron content of the macrophages or NFκB activation (Legssyer et al., 2003).
These results indicate that in control macrophages there is a correlation between the activation of
NADPH oxidase and NFκB activation. However in iron loaded macrophages no correlation was apparent,
iron inducing NFκB activation without an increase in NADPH oxidase activity. Iron loaded monocytic
cells are able to induce NFκB independently of NADPH oxidase although the mechanism remains to be
identified. DFO did not remove iron from iron loaded macrophages which could be of significance in its
use for the treatment of iron loading syndromes.
Bonizzi G. Piette J., Merville M-P and Bours V. Biochem Pharmacol, 59 7-11 2000
Baeuerle and Henkel Annu Rev Immunol 12 141-179 1994
Bowie A and O’Neill L. Biochem Pharmacol 59 13-23 2000
Legssyer R., Josse C., Piette J., Crichton R., Ward R.J. J Inorg Biochem, in press 2003
PODIUM 15
CYTOKINE MEDIATED REGULATION OF IRON TRANSPORT IN HUMAN MONOCYTIC CELLS
S Ludwiczek, E Aigner, I Theurl, G Weiss
Department of Internal Medicine, University Hospital, A-6020 Innsbruck, AUSTRIA
Under chronic inflammatory conditions cytokines induce a diversion of iron traffic leading to hypoferremia
and retention of the metal within the reticuloendothelial system. However, the regulatory pathways
underlying these disturbances of iron homeostasis are poorly understood.
We investigated transferrin receptor (TfR) dependent and independent iron transport mechanisms in
cytokine stimulated human monocytic cell lines, THP-1 and U937.
Combined treatment of cells with IFN-γ and LPS reduced TfR mRNA levels, surface expression and iron
uptake, and these effects were reversed by IL-10 thus stimulating TfR mediated iron acquisition.
IFN-γ and LPS dose-dependently increased the cellular expression of divalent metal transporter-1, a
transmembrane transporter of ferrous iron, and stimulated the uptake of non-transferrin bound iron (NTBI)
into cells. At the same time, IFN-γ and LPS down-regulated the expression of ferroportin mRNA, a
putative iron exporter, and decreased iron release from monocytes. Pre-incubation with IL-10 partly
counteracted these effects.
Our results demonstrate that the pro-inflammatory stimuli, IFN-γ and LPS, increase the uptake of NTBI via
stimulation of divalent metal transporter-1 expression and cause retention of the metal within monocytes
by down-regulating ferroportin synthesis. Opposite, the anti-inflammatory cytokine IL-10 stimulates TfR
mediated iron uptake into activated monocytes. The regulation of iron transport by cytokines is a key
mechanism in the pathogenesis of anemia of chronic disease and a promising target for therapeutic
intervention.
PODIUM 16
REGULATION OF DMT1 AND DCYTB BUT NOT IREG1 OR HEPHAESTIN BY INTRACELLULAR
IRON LEVELS IN DUODENAL ENTEROCYTES.
G J Anderson1, S J Wilkins1, E M Becker1, T L Murphy1, C D Vulpe2, A T McKie3 and D M Frazer1.
1
Joint Clinical Sciences Program, The Queensland Institute of Medical Research and The University of
Queensland, PO Royal Brisbane Hospital, Brisbane, Queensland 4029 Australia, 2Department of
Nutritional Sciences, University of California, Berkeley, Berkeley CA 94720 USA and 3Division of Life
Sciences, King’s College, London SE19NN United Kingdom.
Background: A large oral dose of iron will reduce the absorption of a subsequent smaller dose of iron in a
phenomenon known as mucosal block. An examination of the molecular basis of this process may
provide insights into the regulation of intestinal iron absorption.
Aims: To determine the effect of an oral bolus of iron on the duodenal expression of molecules involved in
iron metabolism. These include the brush border uptake system consisting of the ferrireductase Dcytb
and the iron transporter divalent metal transporter 1 (DMT1), the intracellular ferroxidase hephaestin and
the basolateral transporter Ireg1. Changes in the expression of these molecules were compared with
changes in iron absorption.
Methods: Rats were given a 10mg oral dose of iron as ferrous sulfate and duodenal tissue was collected
at various intervals thereafter. The expression of DMT1 (both the IRE containing and the non-IRE
containing splice variants), Dcytb, hephaestin and Ireg1 mRNAs was determined using ribonuclease
protection assays. Changes in DMT1 and Dcytb protein levels were determined by immunofluorescence
using frozen sections of duodenum. The levels of Ireg1, hephaestin and ferritin protein were determined
by western blotting. The RNA-binding activity of the iron regulatory proteins IRP1 and IRP2 was
determined using an RNA mobility shift assay. Iron absorption was measured in duodenal loops from
59
duplicate animals using radioactive Fe.
Results: A decrease in intestinal iron absorption occurred within 3 hours following an oral dose of iron and
this was associated with an increase in enterocyte iron levels as assessed by IRP RNA-binding activity
and immunoblotting for ferritin. The reduced absorption was also accompanied by a rapid decrease in
the expression of the mRNAs encoding the brush border iron transport molecules Dcytb and the IREcontaining splice variant of DMT1. No such change was seen in the expression of the non-IRE splice
variant of DMT1, hephaestin or the basolateral iron transporter Ireg1. Similar changes were observed at
the protein level. Both iron absorption and gene expression had returned to control or near-control levels
72 hours after iron administration. In contrast, Ireg1 levels were influenced by variations in systemic iron
stores with expression of the transporter increasing in iron deficiency and decreasing in iron-loaded
animals.
Conclusions: These data suggest that the IRP binding is involved in the regulation of duodenal DMT1
expression as the decrease in mRNA level was due solely to a reduction in the IRE containing splice
variant of this gene. However, such a regulatory mechanism cannot be responsible for the change in
Dcytb expression that occurred as this mRNA sequence does not contain an IRE motif. The results of
this study suggest that brush border, but not basolateral, iron transport components are regulated locally
by enterocyte iron levels. This supports the hypothesis that systemic signals to change iron absorption
primarily affect basolateral transfer and that this in turn alters enterocyte iron levels. Changes in
intracellular iron levels then adjust the expression of the brush border components such that an adequate
supply of iron is provided to the basolateral transport machinery.
PODIUM 17
NOVEL DIVALENT METAL TRANSPORTER 1 ISOFORMS : FUNCTIONAL AND REGULATORY
IMPLICATIONS
N. Hubert, M.W. Hentze, European Molecular Biology Laboratory, Gene Expression Programme,
Meyerhofstrasse 1, Heidelberg, Germany.
The Divalent Metal Transporter 1(DMT1) is involved in two critical steps of iron homeostasis in mammals:
systemic iron uptake from the apical surface of the duodenum and iron release from endosomes following
cellular iron uptake via the transferrin cycle. Not surprisingly, the expression of DMT1 is tightly regulated
by iron status. In iron deficiency, the level of DMT1 protein and mRNA is strongly increased in the
proximal duodenum.
Alternative splicing and 3’ end processing generates two forms of mRNA that differ in the encoded Cterminal region of the protein and the 3' untranslated region. Interestingly, the processing variant of DMT1
mRNA that is increased in iron deficiency contains an Iron Responsive Element (IRE) in its 3'UTR,
suggesting post-transcriptional regulation by the IRE/IRP system.
However, the IRE-containing 3’ UTR cannot account for iron regulation of DMT1 expression (Hubert and
Hentze, 2002): We identified a novel first exon (called exon 1A) at the 5’end of the human DMT1 gene,
which is used alternatively to the previously determined first exon (exon 1B). Analysis of DMT1
expression in human Caco2 cells and in mouse tissues revealed that the exon1A-containing DMT1
mRNA (i) is expressed in a tissue specific way whereas exon1B-containing DMT1 mRNA is expressed
ubiquitously, (ii) is strongly iron regulated in Caco2 cells treated with desferrioxamine and in the
duodenum of iron-deficient mice, irrespective of whether or not it carries the 3'UTR IRE variant.
An important feature of the exon 1A-containing mRNA is the presence of an upstream, in-frame AUG
codon that specifies an N-terminal extension of the DMT1 protein. This extension is highly conserved
between the human, mouse and rat. To determine whether this additional domain was expressed, we
generated specific antibodies against a specific murine N-terminal peptide. Analysis of duodenal protein
extracts from control and iron-deficient mice shows that the extended version of the protein encoded by
the exon 1A containing mRNA is expressed and highly iron regulated. To study the specific function of
this novel isoform of DMT1 protein, we are currently investigating its subcellular localization using tagged
versions of the protein transfected into polarized cells (MDCK). Additional experiments are being
performed to confirm this observation in mouse tissues.
The identification of novel isoforms of DMT1 mRNA sheds new light on the complexity of DMT1
expression and its regulation. It will be important to consider the heterogeneity of DMT1protein in the
context of its different physiological functions and the regulation of its activity.
Hubert N & Hentze MW (2002). PNAS, 99, 12345.
PODIUM 18
DIFFERENTIAL REGULATION OF IRON-RELATED PROTEINS IN POLARIZED AND NONPOLARIZED HT-29 CELLS
P. S. Davies, E. L. Anderson, and C. A. Enns; Department of Cell & Developmental Biology, Oregon
Health & Science University, Portland, OR, USA
HT-29 cells, a human colonic carcinoma cell line, have many duodenal characteristics and become
polarized when seeded on microporous filters. These cells were used as a model system to study the
regulation and expression of iron-related genes in an environment similar to the polarized enterocyte of
the duodenum. Using real-time quantitative RT-PCR and western analysis, we tested changes in the
mRNA and protein levels of a number of iron-related genes as a function of polarization, iron treatment,
and HFE expression. We show that the mRNAs had distinct expression patterns depending on the stage
of polarization. When the cells were fully polarized, the mRNA levels of the iron transporters, DMT1 and
ferroportin, increased at least 25-fold compared to the non-polarized state, and Hephaestin expression
increased approximately 10-fold. This expression pattern for DMT1 and ferroportin followed what has
been reported in mature differentiated villus cells compared to undifferentiated crypt cells. Cytoplasmic
iron regulatory proteins (IRPs) regulate the levels of several key proteins involved in iron uptake, storage
and utilization by binding to iron responsive elements (IREs) in their respective mRNAs. Under low iron
conditions, IRPs bind to the 3’ IREs of TfR and protect a cleavage site causing an increase in TfR
message, while in ferritin, they bind to the 5’ IRE blocking translational machinery causing a decrease in
ferritin protein levels. While these responses were seen in non-polarized cells, there was a lack of such
regulation of TfR mRNA in response to iron stimulus when the cells were polarized. No significant
changes in DMT1 or ferroportin mRNA levels were detected either. However, polarized cells were still
sensitive to the iron treatment as reflected in increased ferritin levels. Furthermore, when HT-29 cells
were stably transfected with HFE, an increase in ferritin was observed in conjunction with a drastic
decrease in Hephaestin mRNA levels. The result of an iron-overload phenotype in the presence of HFE
is opposite of what is seen in other cell lines, such as HeLa and HEK 293 cells. Such findings indicate
that changes in iron homeostasis due to HFE expression in HT-29 cells is similar to the macrophage-like
cell line THP1, which also shows increased ferritin levels when transfected with HFE.
PODIUM 19
SORTING OF HFE TO THE APICAL MEMBRANE CORRELATES WITH INHIBITION OF INTESTINAL
IRON ABSORPTION.
M. Arredondo*,#, C. Mura*, V. Tapia*, P. Muñoz*, DI. Mazariegos*, and MT. Núñez*.
# Laboratorio de Micronutrientes, Instituto de Nutrición y Tecnología de los Alimentos. * Departmento de
Biología, Facultad de Ciencias, and Millennium Institute for Advanced Studies in Cell Biology and
Biotechnology, Universidad de Chile.
Introduction: Mutations in the hfe gene results in hereditary hemochromatosis, a disorder of iron
metabolism characterized by increased intestinal iron absorption. Based on the observation that ectopic
expression of HFE strongly inhibits apical iron uptake in Caco-2 cells (Arredondo et al. 2001. FASEB J.
15:1276-1278), we tested the hypothesis that HFE regulates iron absorption by interacting in the apical
membrane with the Fe2+ transporter DMT1. To that end, we investigated in polarized intestinal cells the
effects of overexpressing wild type HFE or HFE containing the H63D mutation on iron homeostasis, and
correlated these effects with HFE, β-2 microglobulin and DMT1 membrane distribution.
Methods: Caco-2 cells transfected with either wtHFE, HFE carrying the H63D mutation, HFE plus β2M or
control (pcDNA3) were obtained. 55Fe2+ uptake and transport was performed in these cells grown in
bicameral inserts. HFE apical/basolateral distribution: Control, HFE and H63D cells were grown in
bicameral chamber, and then incubated from either the apical or the basal side with NHS imino biotin.
Cellular extracts were precipitated with streptavidin and resolved by 8% SDS-PAGE. For HFE transcytosis,
the cells were labeled from the basolateral side at 4 °C with the reversible biotinylation reagent sulfoNHS-S-S-biotin. After a chase at 37 °C, cells were cooled, incubated from the apical side with GSH (50
mM), and a cell extracts were prepared. The extracts were precipitated with streptavidin and HFE, DMT1,
TfR and β2-M were recognized by Western blot.
Results: HFE but not H63D cells had a markedly diminished apical 55Fe uptake activity, despite increased
synthesis of DMT1. Co-expression of HFE and β2M did not change the inhibition pattern. Selective
biotinylation indicated preferential localization of DMT1 and HFE in the apical and basolateral
membranes, respectively. Basolateral HFE underwent transcytosis to the apical membrane with a half
time of 2.4 hours. HFE, but not HFE-H63D overexpression, resulted in the re-distribution of HFE and β2M to the apical membrane.
Discussion and Conclusions. HFE is a negative regulator of DMT1 activity, since strong inhibition of apical
iron uptake was observed in HFE-overexpressing cells. The property of HFE to act as a negative
regulator of DMT1 was lost when the protein has the H63D mutation. The findings that basolateral HFE
transcytosed to the apical membrane, and that inhibition of apical iron uptake by HFE was concurrent with
increased levels of HFE and β2M in this membrane, are in line with the proposal that the HFE-β2M
complex moves between the basolateral and the apical membrane, and that apical HFE inhibits DMT1
activity.
Supported by project P99-031 of the Millennium Institute for Advanced Studies in Cell Biology and
Biotechnology, by grant 1010657 and 2990116 from Fondo de Ciencia y Tecnología, Chile, and by a
grant of Departamento de Investigación y Desarrollo (DID), Universidad de Chile to MA.
PODIUM 20
STRUCTURAL STUDIES OF HUMAN HEPCIDIN; A PEPTIDE-HORMONE AND ANTIMICROBIAL
PEPTIDE
H.N. Hunter1, D.B.Fulton2, A.J. Waring3, T. Ganz3 and H.J. Vogel1
1
University of Calgary, Calgary, Canada; 2Iowa State University, Ames, IA, USA; 3UCLA, Los Angeles,
CA, USA
The liver expressed peptide hepcidin (LEAP-1) was originally discovered as an antimicrobial and
antifungal peptide. It has recently been found to act as a signaling molecule in iron metabolism. Because
of its peptide hormone-like action, hepcidin has been suggested to play a major role in hereditary
hemochromatosis and consequently the peptide may have clinical applications. Here we present the
solution structures of the hepcidin-20 and -25 amino acid peptides determined by standard two
dimensional 1H NMR spectroscopy. The peptides were assigned through TOCSY and NOESY data
recorded at 700 MHz. Structures were calculated using ARIA and CNS. The two small cysteine-rich
peptides exhibit an overall amphipathic beta sheet-like structure with 6 of the 8 cysteines involved in
maintaining inter-strand connectivity. Hepcidin-25 assumes major and minor conformations centred about
the proline residue near the N-terminal end. Further NMR diffusion studies indicate that hepcidin-20 exists
as a monomer while hepcidin-25 readily aggregates in solution. In addition we will discuss the possible
significance of the two cyteines which form a rare vicinal disulphide. (This work was supported by CIHR
and AHFMR.)
PODIUM 21
IRON EFFLUX AS A NOVEL MECHANISM FOR CONTROL OF INTRACELLULAR IRON
CONCENTRATIONS IN SALMONELLA
Marie-Laure Crouch and Ferric Fang
Salmonella enterica serovar Typhimurium can be killed by reactive oxygen species (ROS) produced by
the NADPH oxidase of phagocytic cells. Intracellular iron is an important determinant of susceptibility to
ROS because Fe(II) can catalyze the formation of highly toxic oxyradicals. S. Typhimurium is known to
control intracellular iron levels by using the iron-binding protein Fur to repress expression of genes
involved in iron uptake. Although measurement of desferrioxamine-chelatable iron by eletroparamagnetic
resonance (EPR) spectroscopy shows that fur mutant S. Typhimurium grown in minimal medium contains
3-fold more iron than wild type cells, fur mutants grown in rich medium maintain levels of chelatable iron
similar to wild type cells. These results suggest that Salmonella possesses additional means of controlling
intracellular iron concentrations. An S. Typhimurium mudJ transposon library was screened for mutants
with increased sensitivity to streptonigrin, an antibiotic that generates ROS and kills bacteria in an irondependent manner. Three mudJ mutants with heightened streptonigrin-susceptibility were found to be
located in the mdtABCD baeSR operon encoding a Resistance-Nodulation-Division type efflux pump, a
Major Facilitator Superfamily transporter, and a two-component regulatory system. The transposon
insertions were also found to confer enhanced susceptibility to hydrogen peroxide, elevated total bacterial
iron content, and reduced virulence in mice. Targeted mutations of the operon and measurement of total
iron suggest that mdtD (renamed fieA) encodes an iron efflux pump. Preliminary studies suggest that
mdtABC fieA baeSR expression is enhanced when intracellular iron concentrations are elevated. The
mdtABC fieA baeSR locus may promote Salmonella virulence by reducing intracellular iron
concentrations and limiting susceptibility to ROS.
PODIUM 22
THE RESPONSE TO IRON DEPRIVATION IN SACCHAROMYCES CEREVISIAE: REGULATION OF
IRON-DEPENDENT METABOLIC PATHWAYS
C. C. Philpott, M. Shakoury-Elizeh, J. Tiedeman, J. Rashford, T. Ferea, D. Botstein, and P.O. Brown.
Liver Diseases Section, NIDDK, NIH, Bethesda, MD
Iron is an essential nutrient, yet it is also potentially toxic; therefore, the uptake and utilization of iron is a
carefully controlled process in cells. Organisms respond to a scarcity of iron in the environment by upregulating systems involved in the acquisition of iron. Other metabolic alterations that might be made in
response to iron deprivation are not known. We have used cDNA microarrays to study the response to
iron deprivation in the budding yeast Saccharomyces cerevisiae and have identified the target genes for
Aft1p, the major iron-dependent transcription factor in yeast, as well as genes that encode the
components of metabolic pathways that are regulated by iron. The Aft1p regulon consists of 23 genes, 17
of which are involved in the uptake of iron at the plasma membrane. These include cell wall proteins that
bind siderophore-iron, the plasma membrane metallo-reductases, the high-affinity ferrous iron transport
complex, the siderophore-iron transporters, and the copper loading system that activates the ferrous
transport complex. In addition to these genes involved in iron uptake, the Aft1p regulon contains genes
involved in the intracellular transport and utilization of iron. FTH1, SMF3, and COT1 encode permeases
involved in the transport of metals in and out of the vacuole. HMX1 encodes a protein with homology to
heme oxygenases that can degrade heme and facilitate the utilization of heme iron. Surprisingly, the gene
encoding the plasma membrane biotin permease, VHT1, is also a direct target of Aft1p. Although yeast
are auxotrophic for biotin, they can synthesize biotin from intermediates in the biosynthetic pathway. The
ultimate, rate-limiting step in biotin synthesis is catalyzed by Bio2p, which is predicted to contain a 4Fe-4S
sulfur cluster. We determined that each of the genes in the biotin biosynthetic pathway (BIO2, BIO3, and
BIO4) is down-regulated under conditions of iron deprivation, and yeast strains bearing a deletion of
VHT1 are unable to synthesize biotin from precursors under conditions of iron deprivation. Genes
involved in nitrogen and amino acid metabolism were also found, by cDNA microarrays, to be upregulated in response to iron deprivation. GLT1, which encodes glutamate synthtase and is involved in
nitrogen assimilation, is an iron-sulfur cluster enzyme that was also found to be transcriptionally upregulated when iron availability was high. The promoter region of GLT1 was mapped and found to contain
sequences that confer iron-regulated expression to a lacZ reporter construct. These sequences suggest
that a transcription factor of the binuclear zinc cluster family may be involved in mediating the
transcriptional response to iron. We propose that under conditions of iron deprivation, yeast undergo
transcriptional remodeling, which results in a shift from iron-dependent metabolic pathways to ironindependent pathways.
PODIUM 23
NON-TRANSFERRIN BOUND IRON UPTAKE BY HEPATOCYTES IS INCREASED IN A Hfe
KNOCKOUT MOUSE MODEL OF HEREDITARY HEMOCHROMATOSIS
Anita CG Chua, John K Olynyk, Deborah Trinder, University of Western Australia School of Medicine and
Pharmacology, Fremantle Hospital Unit, Fremantle 6160, Western Australia, Australia
Hereditary hemochromatosis (HH) is an autosomal recessive disorder of iron metabolism that causes
iron overload. In most HH patients, the genetic defect is due to a C282Y mutation in the HFE gene. Hfe
knockout mice have many characteristics of the human disease, accumulating excess iron in the liver, as
a result of increased iron absorption by the duodenum. In HH, plasma non-transferrin bound iron (NTBI)
levels are increased and NTBI is bound mainly by citrate. The aim of this study was to examine the
importance of iron citrate in the pathogenesis of hepatic iron loading in the Hfe knockout mouse.
Hepatocytes were isolated from Hfe knockout and control C57BL/6J mice and cultured for 24h prior to
59
incubation with Fe-citrate for 0-2h at 37°C. The rate of ferric citrate uptake (10µM Fe: 1000µM citrate)
was increased by 2-fold by cells from Hfe knockout mice (34.1±2.8nmol Fe/g protein/min) compared with
control mice (17.8±2.7nmol Fe/g protein/min; p<0.001; Mean±SEM; n=7). The maximal rate of iron uptake
(Vmax) was 70% greater by cells from Hfe knockout mice while Km was similar. In the presence of
ferrous ion chelators, bathophenanthroline disulfonate and 2’,2-bipyridine, ferric citrate uptake by the
hepatocytes from both types of mice was inhibited. Divalent metal ions such as Fe, Mn, Co and Zn
inhibited ferric citrate uptake by the hepatocytes, as did diferric transferrin. Expression of DMT1 mRNA by
hepatocytes from Hfe knockout mice was increased 2-fold compared with control hepatocytes.
We conclude that the uptake of NTBI as ferric citrate by hepatocytes from Hfe knockout mice
contributed to hepatic iron loading. Ferric ion was reduced to ferrous ion and taken up by the hepatocytes
by a pathway shared by diferric transferrin. Inhibition of uptake by divalent metals and upregulation of
DMT1 mRNA suggested that NTBI uptake was mediated by DMT1.
PODIUM 24
THE DISEASE DUE TO MUTATIONS OF THE SLC11A3 GENE
A. Pietrangelo, Centre for Hemochromatosis, Department of Internal Medicine, University of Modena and
Reggio Emilia, Modena , Italy.
In 1999, a hereditary disease associated to systemic iron overload in adults and not linked to 6p was
described 1. This clinical entity showed features distinct from classical HFE-hemochromatosis including
autosomal-dominant inheritance, early increase in serum ferritin despite normal transferrin saturation and
predominant iron accumulation in reticuloendothelial (RE) macrophagic cells, suggesting defective ironrecycling in RE cells1. In 2001, a genome-wide screen was performed in the original pedigree and
evidence of linkage for 2q32 was found2. One compelling candidate gene, SLC11A3 (encoding
ferroportin1/IREG1/ MTP1; GenBank accession # NM_014585)3-5 was identified and all affected patients
were heterozygous for a C to A substitution in exon 3 that results in replacement of alanine 77, a small,
hydrophobic, amino acid, with aspartate, a large, negatively charged amino acid, within the first predicted
transmembrane domain 2. A N144H change in the SLC11A3 gene product has been also reported in a
dutch pedigree with an hereditary iron overload disease 6, and, more recently, a common 3–base pair
deletion in exon 5 of SLC11A3 leading to a Val162 deletion has been found in patients in different ethnic
groups 7, 8. Additional families with the disease are being described worldwide.
The SLC11A3 gene encodes for a main iron export protein in mammals, ferroportin1/IREG1/MTP-1.
This multiple transmembrane domain protein is likely to function in placental materno-fetal iron transfer, in
intestinal iron absorption and in release of iron from hepatocytes and reticuloendothelial macrophages.
Although the physiological role of this protein and its regulation by iron and non-iron stimuli have not been
fully understood, we can speculate that the disease is due to a selective disturbance of iron egress from
cells, particularly from RE macrophages. In this context, a loss-of-function mutation might cause a mild
but significant impairment of iron recycling by RE macrophages, which normally must process and
release a large quantity of iron derived from the lysis of senescent erythrocytes. A defective iron exit from
RE cells may explain the early rise in serum ferritin levels and low transferrin saturation, the latter due to
inability of RE cells to load circulating transferrin with iron. As a consequence, in subjects with increased
erythron demands, iron retention by macrophages could lead to decreased availability of iron for the
hematopoietic system and anemia, as may occur in young female patients at the time of menstruation or
in some patients after aggressive phlebotomy regimen. The clinical manifestations are milder as
compared to expressed HFE hemochromatosis since macrophagic iron overload is safer than
parenchymal iron overload. Mutations in the SLC11A3 gene should be searched in all subjects with
unexplained hyperferritinemia, particularly in the presence of a normal transferrin saturation.
The increasing number of families with this hereditary iron overload disorder indicates that the disease
is spread worldwide and it may represent the most common hereditary iron overload disorder beyond
classical HFE-associated hemochromatosis.
1.
2.
3.
4.
5.
6.
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Pietrangelo A, et al. N Engl J Med 1999;341:725-32.
Montosi G, et al. J Clin Invest 2001;108:619-23.
Abboud S, Haile DJ. J Biol Chem 2000;275:19906-12.
Donovan A, et al. Nature 2000;403:776-81.
McKie AT, et al. Mol Cell 2000;5:299-309.
Njajou OT, et al. Nat Genet 2001;28:213-4.
Devalia V, et al. Blood 2002;100:695-7.
Wallace DF, et al. Blood 2002;100:692-4.
PODIUM 25
FAILURE OF HEPCIDIN UPREGULATION IN HFE-ASSOCIATED HAEMOCHROMATOSIS
IMPLICATES THE LIVER IN THE REGULATION OF BODY IRON HOMEOSTASIS
D M Frazer1, K R Bridle2, S J Wilkins1, J L Dixon1, D M Purdie3, D H G Crawford2,5, V N Subramaniam4, L
W Powell1, G A Ramm2, G J Anderson1.
1
The Iron Metabolism, 2Hepatic Fibrosis, 3Biostatistics Research and 4Membrane Transport Groups, The
Queensland Institute of Medical Research and 5Department of Gastroenterology and Hepatology,
Princess Alexandra Hospital, Brisbane, QLD, Australia, 4029.
Background: The mechanisms responsible for disturbed iron homeostasis in hereditary
haemochromatosis, and in particular how intestinal iron absorption is misregulated in this disorder, are
poorly understood. However, recent evidence indicating a link between hepcidin, a liver-derived peptide,
and intestinal iron transport suggests that this molecule may play a central role in the maintenance of
body iron levels. In normal individuals, body iron loading is associated with an increase in hepcidin
expression and a decrease in iron absorption. In contrast, iron absorption remains relatively high in
haemochromatosis patients despite elevated body iron stores.
Aims: In this study we sought to examine the relationship between the hepatic expression of hepcidin
and iron stores in patients with HFE-associated haemochromatosis, normal controls and HFE-knockout
mice to determine whether mutation of the HFE gene can influence hepcidin expression.
Methods: Total RNA was extracted from the liver tissue of 27 HFE-associated haemochromatosis
patients and 7 transplant donors (controls). Of the patients, 23 were C282Y homozygotes (8 of whom had
been treated by venesection therapy) and 4 were C282Y/H63D compound heterozygotes. Hepcidin
mRNA levels were examined by ribonuclease protection assay and expressed relative to the
housekeeping gene GAPDH. Hepcidin expression was also determined in liver tissue from HFE knockout
mice and from wild-type mice maintained on control and on iron loaded diets.
Results: The untreated haemochromatosis patients had a mean hepatic iron concentration (HIC) of
164±32 micromoles per gram dry weight and a hepatic iron index of 4.2±0.9. The corresponding HIC
concentrations for the treated patients and controls were significantly lower at 25±9 and 8.6±1.4,
respectively. There was a significant decrease in hepcidin expression in both untreated (5.4-fold;
p<0.0001) and treated (7.7-fold; p<0.0003) haemochromatosis patients compared to controls despite
significantly increased iron loading in the untreated group. A decrease in hepcidin mRNA was also seen
in the small group of expressing compound heterozygote patients. There was a significant correlation
between hepatic iron concentration and hepcidin (r=0.59, p=0.02) expression in untreated, but not treated
haemochromatosis patients. Similar results were obtained in mice, with a decrease in hepcidin
expression being observed in HFE knockout mice when compared to with wild-type control mice with an
equal iron load.
Conclusions: The failure of hepcidin upregulation in HFE-associated haemochromatosis despite
significant hepatic iron loading suggests that HFE plays an important role in regulating hepcidin
expression in response to iron overload. As hepcidin is expressed primarily in the liver, these data imply
that the liver plays a major role in the pathophysiology of HFE-associated haemochromatosis. The results
presented in this study have significant implications for understanding the pathogenesis of
haemochromatosis and the regulation of intestinal iron absorption.
PODIUM 26
GENES ENCODING HEPCIDIN AND DUODENAL IRON TRANSPORT PROTEINS ARE REGULATED
DIFFERENTLY IN HFE HEMOCHROMATOSIS AND SECONDARY IRON OVERLOAD
Martina Muckenthaler1,3, Cindy N. Roy2,3, Ángel O. Custodio2,3, Belén Minãna1, Jos deGraaf1, Lynne K.
Montross2, Nancy C. Andrews2, 3 and Matthias W. Hentze1, 3
1
European Molecular Biology Laboratory, Germany, 2Children’s Hospital, Harvard Medical School and
Howard Hughes Medical Institute, Boston, MA, 3The first three authors contributed equally to this work;
the last two authors are co-senior authors.
Patients with hereditary hemochromatosis due to mutations in the HFE gene develop systemic iron
overload as a result of duodenal hyperabsorption of iron. It was previously hypothesized that this
regulatory defect was associated with depletion of iron in maturing enterocytes, and consequent induction
of duodenal iron transporter gene expression. That model did not incorporate a role for hepcidin, a
putative peptide hormone that appears to play an important part in regulating iron homeostatis. In our
study we asked two questions: (1) is the pattern of liver hepcidin expression similar in genetic (primary)
and induced (secondary) iron overload and (2) does the hemochromatosis enterocyte have a pattern of
transporter expression similar to that seen in iron deficiency? Our experiments took advantage of a
mouse IronChip cDNA microarray representing approximately 300 genes involved directly or indirectly in
iron metabolism.
We studied mice homozygous for either null (Hfe-/-) or missense (Hfe C282Y) mutations in the murine
Hfe gene. In contrast to mice loaded parenterally with iron, neither mutant strain induced hepcidin
expression in response to increased iron stores. Furthermore, both homozygous mutants failed to
increase hepcidin expression when additional iron was administered parenterally. This suggests that
regulation of hepcidin expression is defective when Hfe is not active.
We went on to show that the pattern of duodenal iron transporter expression was distinct from that seen
in iron deficiency. Previous studies have shown that duodenal expression of Dmt1 (Nramp2, DCT-1), Fpn
(ferroportin, Ireg1, MTP1) and Dcytb mRNAs is substantially increased in iron deficient mice. In contrast,
we found that levels of Dmt1 mRNA were not increased and levels of Fpn mRNA were decreased in Hfe
mutant mice, similar to the pattern we detected in animals with secondary iron overload. Only Dcytb
mRNA was differentially expressed – it was increased in Hfe mutant mice but unaffected in mice with
secondary iron overload.
We propose that these findings should inform new models for the pathogenesis of HFE
hemochromatosis.
PODIUM 27
ROLE FOR THE HFE H63D MUTATION IN MURINE HEREDITARY HEMOCHROMATOSIS
S. Tomatsu3, R.E. Fleming1,3, R.S. Britton2, B.R. Bacon2, A. Waheed3, W.S. Sly3. 1Pediatrics; 2Internal
Medicine and 3Edward A. Doisy Department of Biochemistry & Molecular Biology, Saint Louis University
School of Medicine, St. Louis, MO.
Introduction: The C282Y mutation in HFE accounts for ~90% of cases of hereditary hemochromatosis
(HH) in populations of northern European ancestry. While the C282Y mutation is common in these
populations (2-5%), another HFE mutation, H63D, has a much greater overall allele frequency (15-20%).
Population studies suggest that the H63D mutation is associated with an increased risk for iron loading,
but has a much lower penetrance than C282Y. However, the relationship between the H63D allele and
HH is controversial.
Objective: We generated mice carrying the murine orthologue of the human H63D mutation to determine
the contribution of this allele to the iron homeostasis abnormalities observed in HH.
Methods: We introduced murine orthologues to the human H63D (D) and C282Y (Y) alleles in separate
mouse lines by targeted mutagenesis. Mice carrying each mutation were bred to homozygosity and
crossed to yield compound mutant mice. The offspring were genotyped for the Hfe alleles, and liver nonheme iron concentrations and serum transferrin saturations were measured. Tissue iron distribution was
determined by modified Perls’ staining.
Results: Mice heterozyogous for either the D mutation or Y mutation did not differ significantly in hepatic
iron concentrations or transferrin saturations from wild-type mice. However, mice with genotypes D/D,
Y/D, or Y/Y showed evidence of iron loading. Even on a standard diet, by 10 weeks of age, hepatic iron
concentrations in each of these groups were higher than that of wild-type mice. Hepatic iron
concentrations from each genotype and each sex were as follows: female Y/Y (n=7), 1670; male Y/Y
(n=8), 765; female Y/D (n=6), 875, male Y/D (n=8), 696; female D/D, (n=10), 523; male D/D, (n=9), 363;
female wild-type (n=14), 306; male wild-type (n=8), 286 µg/g dry weight. Transferrin saturations were as
follows: female Y/Y (n=7), 90; male Y/Y (n=8), 77; female Y/D (n=6), 57, male Y/D (n=8), 70; female D/D,
(n=10), 50; male D/D, (n=9), 46; female wild-type (n=14), 46; male wild-type (n=8), 58%. The hepatic iron
staining in homozygous mutant and compound mutant mice was in periportal hepatocytes.
Conclusions: The H63D mutation is associated with iron loading in the mouse. The consequences of this
mutation are significantly less than C282Y and are most apparent in C282Y/H63D compound
heterozygous mice. These observations suggest that the H63D allele leads to partial loss of HFE
function.
PODIUM 28
FUNCTIONAL CONSEQUENCES OF N144H AND ∆VAL 162 FERROPORTIN MUTATIONS
D.J. Haile and X.B. Liu, Audie Murphy Veterans Administration Hospital and University of Texas Health
Science Center at San Antonio
Ferroportin 1 is a cellular iron exporter and is believed to the primary iron exporter in the
duodenal epithelial cell and cells of the monocyte/phagocyte system (MPS). Iron export by FPN has been
directly demonstrated in frog oocytes and additionally, zebra fish embryos with a mutation in FPN are
deficient in export of iron from the yolk sac. Several mutations in FPN also appear to cause an autosomal
dominant form of hereditary hemochromatosis. The phenotypes of patient with these mutations have not
yet been fully characterized but it appears as if the N144H mutation results in both parenchymal and MPS
iron overload, whereas the ∆Val 162 mutation results in a primary MPS iron overload. For the purpose of
this study, mutations corresponding to those found in the Dutch pedigree (N144H) and the more widely
distributed valine deletion (∆Val 162) have been made in the mouse FPN 1 gene and a variety of assays
have been used to measure the functional consequences of these mutations.
HEK293 and COS cells were transiently transfected with wild type and mutant (N144H and ∆Val
162) FPN encoding plasmids and cellular iron content and iron metabolism assessed by measurements
of cellular ferritin (Western blotting), iron-responsive protein activation (gel-retardation assay with labeled
ferritin IRE RNA), and cellular iron uptake and export (Fe-55 labeled iron). In addition, the subcellular
distribution of wt and mutant FPN molecules were assessed by cell surface biotinylation and
immunoflourescence. Controls consisted of cells transfected with an empty vector and a mutant FPN 1
encoding plasmid with a deletion of a conserved five amino acid region (RRKGC, ∆364-8).
Transient transfection of cells with the wt or N144H molecules resulted in equivalent cellular
ferritin depletion and IRP1 activation compared to control transfected cells. Additionally, the wt or N144H
transfected cells had diminished cellular iron uptake and enhanced iron efflux compared to control
transfected cells. Pulse/chase labeling of wt or N144H transfected cells with S-35 labeled amino acids
failed to demonstrate a difference between wt and N144H FPN protein half-life. Cells transfected with the
∆Val 162 FPN had a lesser degree of cellular ferritin depletion and IRP activation compared to wt or ∆Val
162 transfected cells. In addition, the ∆Val 162 transfected cells showed almost normal cellular iron
uptake and a small enhancement in iron efflux. Western blot analysis of lysates from transfected cells
demonstrated a greater tendency of ∆Val 162 FPN protein to aggregate and migrate as a higher
molecular weight form compared to the wt and N144H FPN molecules. Pulse/chase experiments showed
an increased protein half-life of the ∆Val 162 compared to the wt and N144H molecules. Cell surface
biotinylation experiments of transfected cells demonstrated a smaller fraction of ∆Val 162 on the cell
surface compared to the wt and N144H FPN 1 molecules. Immunofluorescence experiments confirmed
that wt and N144H molecules were expressed strongly at the cell surface whereas the ∆Val 162 molecule
was primarily expressed in the endoplasmic reticulum.
There was no apparent difference in cellular iron metabolism in N144H or wt FPN transiently
transfected cells. These data suggest that N144H is likely a gain of function mutation, although the assay
may not be sensitive to small changes in function. In contrast, the ∆Val 162 mutation appears to result in
a FPN molecule that is capable of iron transport, but probably at a lower efficiency. Importantly, the ∆Val
162 FPN molecules is retained in the ER rather than exported to the cell surface. The ER retention of the
protein may be a consequence of the tendency of this protein to aggregate. The MPS iron excess
phenotype of the ∆Val 162 mutant patients may result from diminished MPS cell surface expression and
overall diminished FPN MPS iron export activity. These differences in the behavior of the N144H and
∆Val 162 mutants may explain the differences observed in the phenotypes of the affected patients.
PODIUM 29
PATTERN AND DYNAMICS OF IRON DEPOSITION IN TRANSFUSION DEPENDENT THALASSEMIC
PATIENTS ON REGULAR CHELATION TREATMENT.
MD Cappellini, D.Prati, M.Maggioni, R.Gramignoli, M.Maiocchi, D.DiCataldo, M.Cerino, G.Fiorelli for the
Cooleycare Cooperative Group. IRCCS Ospedale Maggiore and University of Milan, Italy
Background and Aim: The main causes contributing liver injury in thalassemic patients are hepatitis C
virus (HCV) infection and hepatic siderosis, both secondary to the regular transfusion regimen. We
conducted a multicenter study within Cooleycare Cooperative Group, to describe the pattern and
dynamics of iron deposition among patients on regular chelation therapy.
Patients and Method: Two-hundred-three β-thalassemic patients with laboratory signs of liver disease,
attending 5 centers, were eligible for the study. Anti-HCV antibodies were found in 118 patients (91%), 85
of whom were HCV RNA positive. Ninety-one patients (70%) had abnormal aminotrasferase levels. Liver
biopsy was performed in the 129 who accepted (63.5%; males/females 65/64, age 26±7 years). Biological
samples were sent to the central laboratory in Milano. 117 liver biopsy samples met the criteria for
histological evaluation. Iron accumulation was assessed according the total iron score (TIS) , developed
by Deugnier et al.
Results. The TIS was not significantly different in males (22.0±8.9) as compared to females (19.6±8.5)
(p=0.120). In two of 117 patients (1.7%; age 19 and 37 years; mean serum ferritin, 514 and 450 ng/ml
respectively), there was no stainable iron in the liver (i.e., TIS was 0). Iron was absent from hepatocytes
and limited to the Kupffer cells in other 3 patients (2.5%), all with mean ferritin levels ≤900 ng/ml and TIS
≤ 5. Among the remaining 112 patients, parenchymal iron predominated in zone 1, with a decreasing
gradient throughout the lobule from zone 1 to zone 3. The lobular gradient was appreciable in all the 112
patients but 2, both with massive iron overload (i.e. TIS of 34 and 35 respectively). The dynamics of iron
deposition in patients with different degrees of hepatic siderosis are summarized in the Table. The figures
died not significantly differ in anti-HCV positive as compared to anti-HCV negative patients.
Low TIS
(0-15)
(n=36)
Intermediate TIS
(16-25)
(n=44)
High TIS
(17-60)
(n=37)
Parenchymal Iron Score [0-12]
• Zone 1
• Zone 2
• Zone 3
4.2±2.1
0.9±1.4
0.2±0.7
7.3±1.5
3.3±1.6
1.2±1.6
8.4±1.5
6.2±1.3
4.5±2.0
Sinusoidal Iron Score [0-12]
3.3±2.3
6.4±2.2
8.4±2.2
Portal Iron Score [0-4]
• Connective tissue
• Biliary ducts
• Vascular walls
0.4±0.7
0.0±0.0
0.3±0.6
1.1±0.9
0.1±0.5
0.8±0.9
2.9±1.3
1.0±1.1
1.5±1.1
Total Iron Score [0-60]
9.2±4.2
20.3±3.0
32.8±5.1
Mean Serum Ferritin (ng/mL)
796±474
1398±790
3168±2114
Comments: Our data challenge previous results on the liver pathology of transfusion-associated
hemosiderosis. Transfusional iron was previously thought to be absorbed by hepatocytes only in the more
advanced stage of accumulation, as a consequence of a redistribution from the saturated
reticuloendothelial system. Furthermore, the presence of a lobular gradient was considered a distinctive
feature of hereditary hemochromatosis and nontransfused ineffective erythropoiesis, being typically
absent from transfusional iron overload. A critical interpretation of these divergences should take into
account that currently treated thalassemics receive regular chelation therapy from childhood, while
previous studies focused on poorly or not chelated patients, with massive siderosis and cirrhosis. The
latter conditions can attenuate or even abolish the evidence for a lobular gradient. In addition, it is
possible that the reduction of the pre-transfusion hemoglobin thresholds (from 11 to 9.5 g/dL), adopted in
the early 90s to limit the transfusional iron burden in thalassemia, had the simultaneous effect of
stimulating ineffective erythropoiesis, leading to a relative increase of the quote of iron absorbed from the
portal circulation. In conclusion, the pattern and dynamics of liver iron deposition in currently treated
thalassemics underwent profound modifications as compared to the “classical” form of post-transfusion
siderosis, described before deferoxamine therapy became a standard practice.
PODIUM 30
RELATIONSHIP OF DEFEROXAMINE (DFO) DOSE TO PLASMA NTBI REMOVAL, TRANSFERRIN
SATURATION AND PLASMA AND URINE FERRIOXAMINE (FO)
P. Evans¹, F. Shah¹, B. Davis², F. Kotynia³, C. Kim³, L. Merson³, N. Olivieri ³, J. Porter ¹
1
Dept. of Haematology University College London, WC1E 6HX, UK 2 Dept. of Haematology, Whittington
Hospital, London N14 UK, 3 University Health Network Toronto, ON M5G 2C4, CA
Non transferrin bound iron species (NTBI) in the plasma of transfusionally iron loaded patients cause
excess iron uptake into the liver, heart and other tissues. The influence of chelation dose and regimen on
plasma NTBI level is largely unexplored. We have shown previously that plasma NTBI is highly labile,
increasing within minutes of stopping DFO chelation therapy. However NTBI values obtained by the
method used might be underestimated due to ‘shuttling’ of iron onto DFO during the assay procedure. A
method was therefore developed to ‘block’ this shuttling during the assay procedure. This method also
stabilized free DFO, which is unstable in the iron free form in plasma, as aluminoxamine, allowing the
measurement of both DFO and FO. Thus it has been possible to investigate for the first time the
relationship of NTBI removal by DFO to plasma levels of FO, DFO and their metabolites. In this study we
have sought to explore how escalating doses of DFO affect the kinetics of NTBI, DFO and FO in order to
determine how dose and regimen may be optimized to remove NTBI. Adult thalassaemia major patients
(TM, n=9) were rested from DFO for 48 hours. IV DFO infusions at 10, 40 and 60 mg/kg were then given
at intervals but always at the same stage of the transfusion cycle. Blood samples for serum preparation
were taken pre-infusion and at timed intervals up to 24 hours of infusion.
Surprisingly, at all doses of infused DFO, the plasma concentrations of FO exceeded those of DFO.
Steady state plasma DFO concentrations, which equilibrated by 30 minutes of commencing DFO infusion,
increased with DFO doses of 10, 40 and 60mg/kg to 0.5µM, 3µM, and 5 µM respectively. By contrast FO
values increased biphasically with the first phase complete by 2h and the second not at steady state by
24h. Furthermore, no increment in the 24h FO concentration was seen above 40mg/kg. Trends on the 24
hour urine collections of these patients showed a similar pattern to plasma: firstly iron bound species (FO
plus FO metabolite B) exceeded concentrations of iron free forms (DFO plus DFO metabolite B) at all
doses except 60mg/kg. Secondly, while iron free forms increased between 10, 40 and 60 mg/kg, the ironbound forms did not increase above 40 mg/kg. NTBI removal was significantly less using the AlCl3
blocking method, than without blocking. Using the blocking method, falls in NTBI during DFO infusion
were biphasic, the first phase complete by 1h at all doses, with nadir values generally obtained by 12h.
NTBI was not completely cleared from plasma at any dose but the fall in NTBI was the most rapid at 60
mg/kg. After 12h infusion, NTBI subsequently increased until the infusion ceased at 24h. There was then
a sharp rebound in NTBI above baseline, which stabilized 2 hours post-infusion. Changes in NTBI level
were generally paralleled by changes in transferrin saturation.
A surprising finding is that plasma concentrations of FO exceed those of DFO in both plasma and urine at
40mg/kg and less. This is also true for metabolites. This implies that the low proportion of the applied
dose of DFO resulting in iron excretion is not due to excess plasma or urinary DFO species but to hepatic
elimination or metabolism of DFO. The low concentration of iron free DFO in plasma may partly explain
the inefficient removal of NTBI. Also access of DFO to some citrate-iron species may be slow. We
suggest that the rapid initial decrement of NTBI represents chelation of monomeric or dimeric forms of
iron-citrate species and the slower subsequent removal represents interaction with oligomeric/polymeric
forms. In these TM patients, increasing DFO dose above 40mg/kg did not remove NTBI more rapidly or
increase FO concentrations in plasma or urine but was instead associated with higher levels of free DFO
in both plasma and urine. It may not be coincidental that 40mg/kg is the dose generally associated with
the optimal therapeutic safety margin in these patients. Measurement of iron free and iron bound forms of
chelators may help to optimize the dosing schedule and therapeutic safety margin particularly for novel
iron chelators.
This work is supported by grant number 1R01DK55462-02
PODIUM 31
A PHASE Ib STUDY OF THE SAFETY, PHARMACOKINETICS, ACUTE TOLERABILITY, AND
EFFICACY OF ASCENDING SINGLE DOSES OF 40SD02 (CHF1540) IN IRON-LOADED PATIENTS
P. Harmatz, MD, J. Madden, PNP, E. Vichinsky MD, M. Jeng, MD, P.J. Giardina, MD, K.A. Nugent, R.W.
Grady, PhD, P. Dragsten, PhD and B. Hedlund, PhD.
Children's Hospital & Research Center at Oakland, CA, Stanford Medical Center, Palo Alto, CA, Weill
Medical College of Cornell University, New York, NY, and Biomedical Frontiers, Minneapolis, MN.
Introduction: Currently, the only drug approved in the U.S. for clinical use as an iron chelator is
deferoxamine mesylate (Desferal®, Novartis). However, it has several undesirable properties. The drug
is excreted very rapidly after intravenous injection. In addition, rapid intravenous administration is
associated with hypotension. Therefore, Desferal® is customarily given by subcutaneous continuous
infusion daily over an 8 to 12 hr period using a portable pump. While the drug is effective in promoting
iron excretion in β-thalassemia patients, compliance with this regimen can be poor, particularly in
adolescent patients. 40SD02 (CHF1540) is a new chemical entity synthesized by chemically attaching
deferoxamine to a modified starch polymer. The resulting high molecular weight chelator retains
deferoxamine’s affinity and specificity for iron, but has a prolonged vascular retention, and exhibits none
of the acute toxicity of Desferal® such as hypotension. The purpose of this study is to investigate the
safety, pharmacokinetics and efficacy of a single intravenous administration of three ascending doses of
40SD02 in transfusion-dependent patients with β-thalassemia.
Methods: Ascending doses of 40SD02 (150, 300 or 600 mg/kg) were given by intravenous infusion over 1
hour. Patients were hospitalized for 7 days beginning with the infusion day, and followed for a total of 21
days. Safety assessments included complete blood count (CBC), chemistry panel, coagulation studies
and urinalysis. Pharmacokinetics and efficacy were assessed by serum and urine iron and chelator
levels.
Results: Fourteen iron-overloaded patients with β-thalassemia who receive regular blood transfusions
have been screened thus far and 10 were eligible for study. Two patients were excluded due to a positive
skin test to 40SD02; and two because of elevated transaminase. Four subjects (2F, 2M) have received
the study drug at 150 mg/kg, four (1F, 3M) have received 300 mg/kg and two have been dosed with the
highest dose (600 mg/kg). The mean age of the subjects was 29 years (range = 17-51). The drug was
well tolerated. Drug related adverse events were limited to three urticarial reactions: two localized and
one generalized. The latter was treated with diphenhydramine with resolution and none required
termination of the infusion. There were no consistent abnormalities in safety laboratory parameters.
Approximately 50% and 90% of the infused drug was cleared from the blood within 8 and 96 hours,
respectively. The average urinary iron excretion over 7 days was 0.46 mg/kg (range 0.35 - 0.62) in the
lowest dose group and 0.72 mg/kg (range 0.41 - 1.04) in the middle dose group.
Conclusions: Single doses of up to 600 mg/kg of 40SD02 are safe and well tolerated in β-thalassemia
patients with transfusion-dependent iron overload. Clinically significant iron excretion was stimulated.
Excretion data from the high dose group (600 mg/kg) will be reported at the meeting. In summary,
40SD02 offers significant potential to improve compliance and chelation therapy in β-thalassemia
patients.
Sponsored by Biomedical Frontiers, Minneapolis, MN. Supported in part by Chiesi Farmaceutici, S.p.A.,
Parma, Italy, and GensiaSicor Pharmaceuticals, Irvine, CA.
PODIUM 32
ICL670: MOBILIZATION OF IRON IN ANIMALS
HP. Nick, S. Hauffe, R. Lattmann, F. Waldmeier
Novartis Institute for Biomedical Research, Basel, Switzerland
In 1963 the introduction of Desferal (DFO), a hexadentate chelator, marked a breakthrough in the
treatment of ß-thalassemia. DFO significantly reduces body iron burden in ß-thalassemia patients who
accumulated iron through repeated blood transfusions. Desferal is still the only drug for general use in the
treatment of transfusion dependent iron overload. However, its very short plasma half-life and poor oral
activity necessitate special modes of application which are impractical, and difficult to accept by many
patients. ICL670, a bis-hydroxyphenyl-triazole, marked another breakthrough by proving that oral activity,
high affinity for iron, high efficiency and efficacy can be achieved in combination with acceptable
tolerability in animals and, as we are showing in on-going clinical studies, in patients.
Three differing properties are outstanding between DFO and ICL670 in animals:
ICL670 is orally bioavailable, it has a long duration of action and the bulk of the iron is excreted in feces.
The differential behavior of DFO and ICL670 with respect to their route of iron excretion can most
dramatically be shown by preformation of the respective [59]iron complexes ex vivo, followed by i.v.
administration of the mixtures to rats. With DFO, virtually all radiolabelled iron is found in the urine.
Conversely, with ICL670, the bulk of radiolabelled iron is found in feces. From this it is concluded that
whenever ICL670-Fe complexes reach the blood stream, these will be taken up by the liver and
1
transported into bile. A recent publication by Hershko et al. is supporting this view by showing that
ICL670 promotes excretion of iron, both from rat hepatocytes and from the reticuloendothelial system into
feces.
The efficacy of ICL670 has been assessed in marmoset monkeys after single dose administration and by
measurement of liver iron after 39 weeks of daily administration of ICL670. From single dose
administration of ICL670 the efficiency of the chelator was calculated to be close to 30%. In the 39-week
study ICL670 was given at daily doses of 0 (control) 20, 40 and 80 mg/kg. While liver iron was
dramatically decreased, levels of copper and zinc remained virtually unchanged, thus confirming in a long
term in vivo study the selectivity of ICL670 for iron.
An important question with respect to efficacy is the metabolism of ICL670 which has been studied in rat
and marmoset monkey. Five major metabolites have been detected. Two important ones were
synthesized and tested in the bile duct cannulated rat. The potential of these metabolites to promote iron
excretion was dramatically reduced as compared to ICL670 despite predicted high affinity of the
metabolites for iron.
PODIUM 33
CLINICAL OVERVIEW OF ICL670A
M.D. Cappellini
No abstract available at time of printing.
PODIUM 34
INTRALYSOSOMAL IRON AND OXIDANT-MEDIATED CELL DEATH
J. W. Eaton1,2, H. L. Persson2, Z. Q. Yu2 and U. T. Brunk2. 1James Graham Brown Cancer Center,
University of Louisville, Louisville, KY and 2Department of Pathology II, University of Linköping, Linköping,
Sweden
Intracellular iron powerfully synergizes oxidant-induced cell damage and death (apoptotic or necrotic
depending on the level of oxidant exposure). The hexadentate Fe chelator, desferrioxamine (DFO),
protects cultured cells against oxidant-induced killing but pharmacologically effective concentrations of
this drug cannot readily be achieved in vivo. DFO is thought to passively localize almost exclusively within
the lysosomal compartment following endocytic uptake, suggesting that truly lysosomotropic chelators
might be even more effective. We hypothesized that an amine derivative of α-lipoamide (LM), 5[1,2]dithiolan-3-yl-pentanoic acid (2-dimethylamino-ethyl)-amide (‘α-lipoic acid-plus’ or LAP; pKa = 8.0)
would concentrate via proton trapping within lysosomes, and that the vicinal thiols of the reduced form of
this agent would interact with intralysosomal Fe, preventing oxidant-mediated cell damage. Using a thiolreactive fluorochrome and fluorescence microscopy, we find that reduced LAP does accumulate within
the lysosomes of cultured J774 cells. Furthermore, in protection against H2O2-induced cell death, LAP
(ED100 = 0.2 µM) is approximately 5,000 times more effective than DFO (ED100 ~1 mM) and 1,000 times
more effective than the cell permeable but charge neutral LM (ED100 ~200 µM) in protecting lysosomes
against oxidant-induced rupture and in preventing ensuing cell death. (None of these protective agents
affects the rate of H2O2 clearance.) Suppression of lysosomal accumulation of LAP (by ammonium
chloride-mediated lysosomal alkalinization) blocks these protective effects. Electron paramagnetic
resonance reveals that the intracellular generation of hydroxyl radical following addition of H2O2 to J774
cells is totally eliminated by pre-treatment with DFO (1 mM) or LAP (0.2 µM) whereas LM (200 µM) is
much less effective. By contrast, LM and LAP are equally effective in preventing hydroxyl radical
formation by a chemical system comprised of Fe2+ and H2O2, once again supporting the importance of
intracellular concentration via proton trapping. These results raise the concept of developing antioxidants
which restrain the reactivity of intracellular Fe and which are targeted to the lysosomal compartment.
Protection of this very sensitive organelle from Fe-mediated oxidant attack may, in itself, suffice to prevent
cell death. We believe that LAP, or other agents similarly targeted to the lysosomal apparatus, have great
therapeutic potential for the treatment of a variety of disease states wherein reactive oxygen species
conspire with iron to initiate lysosomal rupture and cell death.
PODIUM 35
IRON MISREGULATION AND NEURODEGENERATION IN IRP2-/- MICE
T. Rouault, S. Smith, S. Cooperman, E. Meyron-Holtz, M. Ghosh, W. Land, H.Olivierre Wilson, T.
LaVaute, C. Grabill, L. Jui-chen
We have previously observed that IRP2-/- mice develop adult-onset neurodegeneration characterized by
tremor and abnormal gait. In IRP2-/- mice that also lack one copy of IRP1, disease is much worse.
Ferritin overexpression occurs in specific subsets of neurons. Axonal degeneration is a prominent early
feature of disease. As the disease progresses, we observe necrosis of neuronal cell bodies and
vacuolization. Abnormal accumulations of ferritin iron can be visualized by MRI scanning and electron
tomography. Our results imply that neurodegeneration can be caused by primary abnormalities in
neuronal iron metabolism.
PODIUM 36
MULTICOPPER OXIDASE DEFICIENT MICE REVEAL AN ESSENTIAL ROLE FOR CERULOPLASMIN
AND HEPHAESTIN IN CENTRAL NERVOUS SYSTEM IRON HOMEOSTASIS
Xueying Xu, Hoon Shim and Z. Leah Harris
To further delineate and characterize the roles of the multicopper oxidases in iron metabolism, a mouse
lacking ceruloplasmin and hephaestin was generated. Total tissue iron, hematologic profiles, serum iron
measurements, northern blot analysis, western blot analysis, in-situ hybridization, histologic staining,
electron microscopy, neurobehavioral testing and rodent magnetic resonance (MR) imaging were
performed. Multicopper oxidase (MCO) deficient mice manifest a neurodegenerative phenotype at
approximately 6 months of age (5-9 months). Presenting symptoms included gait abnormalities,
weakness and poor grooming. Tissue iron content studies (µg Fe/dry weight tissue) reveal a significant
contribution by hephaestin in regulating iron homeostasis in all tissues except the liver (Figure 1).
Presumably reflecting the contribution of ceruloplasmin in regulating hepatic iron metabolism.
Pancreas
Heart
500.00
1500.00
Cp +/+
1000.00
1038.87
500.00
300.00
Sla -/-
200.00
Cp -/- Sla -/27.59
54.03
400.00
Cp -/-
35.99
0.00
100.00
Cp +/+
Cp -/Sla -/314.55
66.73
69.96
Brain
600.00
120.00
500.00
Cp +/+
400.00
Cp -/-
300.00
200.00
0.00
46.84
0.00
Liver
100.00
Cp -/- Sla -/-
393.96
368.84
36.26
Sla -/Cp -/- Sla -/-
39.90
100.00
Cp +/+
80.00
Cp -/-
60.00
81.43
40.00
20.00
Sla -/Cp -/- Sla -/-
17.96
20.78
16.16
0.00
Figure 1. Tissue iron content determination following overnight acid digestion (µg Fe/dry weight tissue) in
6 month-old mice. Results are expressed as means +/- standard deviations, n=6 minimum.
The neurodegenerative phenotype coupled with the increased whole brain tissue iron content suggested
an essential role for multicopper oxidases in central nervous system iron homeostasis. Histologic
analysis of brain tissue by Perls and modified DAB-Perls staining demonstrate enhanced iron
accumulation within the specific brain regions. MR imaging on a 9.4T scanner non-invasively confirm
pathologic iron deposition. Neurobehavior testing identifies neurologic deficits consistent with the
regional iron deposition. Electron microscopy is suggestive of a mitochondrial defect associated with the
increased iron deposition. The multicopper oxidases appear to regulate the redox state of the central
nervous system and protect tissues from iron mediated oxidative damage.
PODIUM 37
CERULOPLASMIN AND HEPHAESTIN HAVE OVERLAPPING REGULATORY ROLES IN RETINAL
IRON HOMEOSTASIS
J.L. Dunaief1, P. Hahn1, R.W. Wong1, L. Chen1, T. Dentchev1, and Z. L. Harris2
1: F.M. Kirby Center for Molecular Ophthalmology,Scheie Eye Institute,University of Pennsylvania
2: Department of Pediatric Anesthesiology, Johns Hopkins University
Corresponding author: jdunaief@mail.med.upenn.edu
Purpose: Ceruloplasmin and hephaestin are ferroxidases important for the export of iron from cells to
plasma. The purpose of this study was to investigate the roles of ceruloplasmin and hephaestin in
regulating retinal iron transport.
Methods: Mice deficient in ceruloplasmin (cp) were generated and crossed with hephaestin-deficient (sla)
mice to generate cp-/-sla-/Y mice. Iron levels in these retinas (cp-/-, sla-/Y, and cp-/-sla-/Y) were
compared to wild-type by the enhanced Perl’s stain and indirectly by immunolabeling retinas for ferritins.
The effects of ferroxidase deficiency on iron regulated proteins, ferroportin and transferrin receptor, were
also studied by immunohistochemistry. Ultrastructural changes were examined by electron microscopy.
Ceruloplasmin and hephaestin in normal retinas were localized by immunohistochemistry, Western
analysis, and RT-PCR.
Results: Increases in iron were observed in the retinal pigment epithelium (RPE) of cp-/-sla-/Y retinas
only (5-9 months old, n=5), with corresponding increases in ferritins. Both H- and L-ferritin were also
increased in retinas of cp-/-sla-/Y mice. These increases were accompanied by increased ferroportin and
decreased transferrin receptor. These changes appear to be age-dependent, as 4 week cp-/-sla-/Y
retinas were Perl’s stain negative. At the ultrastructural level, the RPE of 5 month cp-/-sla- eyes only was
highly vacuolated and contained electron-dense siderosomes. Both ceruloplasmin and hephaestin are
expressed in the retina.
Conclusions: Ceruloplasmin and hephaestin serve overlapping functions in the regulation of ocular iron,
such that combined deficiency results in age-dependent accumulation of iron in the RPE and retina with
alterations in iron regulated proteins. Increased iron in the RPE results in vacuolization, possibly as a
result of iron induced oxidative damage. Iron accumulates in the RPE in some age-related macular
degeneration eyes (P. Hahn et al., submitted), and the cp-/-sla- mouse may provide a model of agerelated macular degeneration and/or other retinal diseases.
PODIUM 38
DEVELOPMENT OF MOUSE AND CELLULAR MODELS TO DECIPHER THE FUNCTION OF
FRATAXIN IN IRON AND IRON-SULFUR METABOLISMS
H. Puccio, H. Seznec, N. Carelle, A. Hertzog, C. Bouton*, D. Simon, L. Reutenauer, M. Koenig
Institut de Génétique et Biologie Moléculaire et Cellulaire, 1 rue Laurent Fries, BP 10142, 67404 IllkirchStrasbourg, France * Institut de Chimie des Substances Naturelles, Centre National de la Recherche
Scientifique, avenue de la Terrasse, 91190 Gif-sur-Yvette, France
Friedreich ataxia (FRDA), a progressive neuro- and cardio-degenerative disease, is due to the
partial loss of function of frataxin, an essential mitochondrial protein. The exact function of frataxin is still
unclear. However, loss of frataxin causes mitochondrial iron accumulation, deficiency in the activities of
iron-sulfur (Fe-S) proteins, and increase oxidative stress in FRDA patients. Recently, both phylogenic and
experimental data suggested that frataxin is more particularly involved in the biogenesis of Fe-S cluster.
We have recently generated several conditional mouse models that reproduce the
pathophysiology of the disease. These models demonstrate that the Fe-S deficit precedes iron
accumulation, indicating that unlike previous suggestions, iron accumulation is a secondary event. To
investigate the effect of frataxin deletion on iron metabolism and Fe-S cluster biogenesis, we performed
expression analysis of several genes known to be involved in the respective pathways, in parallel to
investigating both the aconitase and trans-regulator activities of IRP-1. Our preliminary results further
suggest that frataxin is involved in Fe-S metabolism rather than cellular iron import. In parallel, to further
characterise the molecular and biochemical events in these mouse models in order to define the
functional pathway in which frataxin is involved DNA microarray analysis has been performed. Preliminary
results using RNA from cardiac muscle at 5 weeks of age reveal 35 genes that show differential
expression some of which can be grouped into various categories : oxidative stress, iron metabolism,
cardiac failure, apoptosis…etc… Further functional studies using classical biochemical and molecular
biology techniques are being perform to refine the genetic interaction.
Deciphering the molecular mechanisms involved in FRDA and finding therapeutics for this
disease would be facilitated by the availability of cellular models. To date, only two cellular models are
available. The absence of spontaneous phenotype and the genetic heterogeneity in cell lines derived from
FRDA patients make them unsuitable for large-scale drug screening. We have therefore decided to
develop mouse cellular models deleted for frataxin, using the Cre-lox homologous recombination system,
derived from mouse models that have been generated in the lab.
Using our different mouse models, our first strategy was to generate inducible immortalized
fibroblasts cell lines in which the deletion of frataxin was obtained by adding tamoxifen to the media. Even
under “anaerobic” conditions (supplemented with uridine and sodium pyruvate), no clone with
homozygous deletion for frataxin was isolated. Indeed, after tamoxifen treatment, a certain percentage of
floating (dead) cells were observed. Enrichment of these floating cells by pre-plating enabled us to verify
by PCR that these cells were homozygous for the frataxin deletion, confirming the lethality of frataxin
deficiency at the cellular level. These results seem at first in contradiction with the 10 weeks survival of
cardiomyocytes and the complete absence of skeletal muscle phenotype in our conditional cardiac
knockout where we showed complete frataxin deletion in both skeletal and cardiac muscle. In fact, our
results indicate that the absence of frataxin is more deleterious in dividing cells than in post-mitotic cells,
even rich in mitochondria, a surprising result. However, this is in concordance with the very early
embryonic lethality that we demonstrated in complete frataxin knockout mice.
We then tried to establish primary cultures of frataxin-deficient myoblasts (derived from our
conditional cardiac models) and fibroblasts derived from new inducible mouse models in order to study
the lethality phenotype. Our results show that primary myoblasts can be established and maintained for at
least 3 weeks, and can be differentiated into contracting myotubes. Preliminary results show that the
recombinase responsible for the deletion is expressed at the myoblast stage, and that the primary
myoblast derived are deficient for frataxin. These frataxin deficient cells are currently being analyzed for
morphological and biochemical phenotype.
PODIUM 39
THE PENETRANCE OF HEREDITARY HEMOCHROMATOSIS
E. Beutler, V. Felitti, T. Gelbart, J. Waalen, P. Lee, The Scripps Research Institute.
Hereditary hemochromatosis, once considered to be a rare disease, has been more recently regarded as
the most common genetic disorder of Northern Europeans. It has been suggested that many patients
with this disease die unnecessarily because of the failure to recognize that the cirrhosis, diabetes, and
cardiomyopathies from which they suffer are due to easily-treated iron overload. Indeed, some five
persons per thousand have the hereditary hemochromatosis genotype and many of them also manifest
the characteristic blood findings, viz., elevated transferrin saturation and increased serum ferritin.
To determine the actual clinical penetrance of the HFE mutation we genotyped more than 41,000
subjects attending the Health Appraisal Clinic at Kaiser Permanente in San Diego, California for the
C282Y and H63D mutation. We found 156 homozygotes for the common 845G→A (C282Y) mutation of
the HFE gene. There were 630 compound heterozygotes for the C282Y and 187C→G (H63D) mutation.
Laboratory studies including serum iron, transferrin saturation, ferritin, and SGOT were compared with
ethnically and age-matched wt/wt controls. Participants recorded responses to 400 questions concerning
symptoms and medical history. Although there was a strong relationship between HFE genotype and
transferrin saturation and serum ferritin, we found no statistically significant increases in the symptoms
commonly associated with hemochromatosis: diabetes, cardiac arrythmias, impotence, darkening of the
skin, or arthropathy in homozygotes or compound heterozygotes. The only significant differences in the
responses of homozygotes was that more of them had been told by physicians that they had "liver
trouble". There was also a small but statistically significant increase in prevalence of elevated SGOT and
serum collagen IV (a surrogate for hepatic fibrosis) in the homozygous group. Importantly, the number of
homozygotes among the white patients exceeded slightly the Hardy-Weinberg expectation and there was
no significant effect of homozygosity or compound heterozygosity on age distribution.
It is clear from these investigations that the clinical penetrance, in contrast to the biochemical penetrance,
of the HFE mutations is extremely low. We have therefore tried to identify genes that may modify the
expression of HFE, sequencing genes encoding the following proteins in 5 expressing homozygotes, 5
non-expressing homozygotes, 5 wt/wt subjects with iron overload, and 5 wt/wt subjects without iron
overload: transferrin, transferrin receptor-1, transferrin receptor-2, ferroportin, NRamp1, DMT-1, β2microglobulin, ferroportin, USF-2, hepcidin, ceruloplasmin, TNF promoter, haptoglobin, ferritin heavy
chain, and ferritin light chain. We have not found a polymorphism in any of these genes that incline
patients toward iron overload.
PODIUM 40
LONGITUDINAL AND PENETRANCE STUDIES OF HFE-ASSOCIATED HEMOCHROMATOSIS IN
PROBANDS AND RELATIVES IN AN AUSTRALIAN POPULATION
L. Powell1, J. Dixon1, D. Hewett2, G. Ramm1, G. Anderson1, N. Subramaniam1, L. Fletcher3, D. Crawford3,
J. Cavanaugh4 and M. Bassett4. 1The Queensland Institute of Medical Research, The Departments of
Gastroenterology and Hepatology 2Royal Brisbane Hospital, and 3Princess Alexandra Hospital, Brisbane,
and 4Canberra Hospital, Canberra, Australia.
Introduction: Hereditary hemochromatosis (HH) is due to homozygosity for the C282Y mutation in HFE in
over 90% of Caucasian patients with the disorder, with a prevalence of approximately 1:200 for
homozygotes and 1:10 for heterozygotes. However, the penetrance of the disease is highly variable
between different populations with frequencies for significant clinical disease varying from 1% to 50% of
homozygotes. In addition, the natural history of non-expressing C282Y homozygotes and compound
heterozygotes (C282Y/H63D), is unclear.
Aims: To 1. Determine the mode of presentation and disease severity in C282Y homozygous probands.
2. Assess the degree of biochemical and clinical expression in homozygous relatives. 3. Follow nonexpressing C282Y homozygotes by serial iron studies. 4. Ascertain the degree of biochemical and
clinical expression in compound heterozygous subjects.
Methods: We analysed (a) the mode of presentation and disease severity in 376 probands [242 males
(M) and 134 females (F)]; (b) the expression of HH in 366 C282Y homozygous first or second degree
relatives (189M,177F); and (c) the biochemical and clinical expression in 157 compound heterozygous
subjects. All probands and available relatives underwent clinical evaluation, genotyping, assessment of
serum ferritin (SF) and transferrin saturation (TS), liver function tests (LFT) and liver biopsy where
clinically indicated. Biochemical expression was defined as SF >150µg/L if aged <30 years, or >250 if 30
years or older for females; SF >250 and >350 respectively for males (Leggett B.A. et al., Clin. Chem.
1990, 36/7. 1350-1355).
Results: All subjects were of European descent. 1. Of the probands, 119 (32%) presented with clinical
disease (88M,31F), 82 (22%) presented specifically because of lethargy (42M,40F) and 154 (41%) were
detected coincidentally (94M,60F). The remaining 21 (5%) of subjects are still being assessed. Although
significant clinical disease (including cardiac arrhythmia, diabetes, cirrhosis and hepatocellular cancer)
was observed more frequently in the first group, it was present in all three groups.
2. In the C282Y homozygous relatives, biochemical expression was seen in 280 (76%), (169M,111F).
Symptoms other than lethargy (arthralgia, hypogonadism, abdominal pain) were present in 64 subjects
(17%), the most common being arthralgia in 54 (15%). Diabetes mellitus was present in 6 (1.6%).
Hepatomegaly was present in 27 (7.4%) (24M,3F), arthritis in 32 (8.7%) (22M,10F), gonadal atrophy in 6
(3%) of males, and cardiomyopathy in 2 (0.5%). Abnormal LFTs were present in 66 (18%) (49M,17F). Of
these, daily alcohol intake of 60 grams or more was present in 11 (17%), all males. Of the 183 subjects
who underwent liver biopsy (106M,77F), grade 3 or 4 iron stores were found in 84 (44% of all males) and
44 (25% of all females); 52 (14%) had hepatic fibrosis and 11 (3%) (10M,1F) had cirrhosis. Four of the
11 cirrhotic subjects drank 60g or more of alcohol per day (all males). 3. In 37 of 91 (41%) nonexpressing relatives, the serum ferritin rose after the initial observation into the abnormal range and 25 of
these were subjected to phlebotomy therapy. 4. Amongst the compound heterozygous subjects, although
the SF and TS were often elevated, there was no significant clinical disease without cofactors such as
heavy alcohol intake.
Conclusions: 1. Case detection of C282Y homozygous subjects is important because significant clinical
disease can be present in those detected coincidentally or who present because of lethargy. 2. The
majority of homozygous relatives demonstrated biochemical expression and 17% had either hepatic
fibrosis or cirrhosis. 3. Compound heterozygosity does not lead to progressive clinical disease. Thus,
early case detection and screening of relatives of patients with HFE-associated HH remains a very
important strategy to detect early disease and prevent complications.
PODIUM 41
PREVALENCE OF SELF-REPORTED SYMPTOMS IN THE HEIRS STUDY: DIFFERENCES BY HFE
GENOTYPE.
Adams P1, Reboussin D2, Acton R3, Barton J4, Gordeuk V5, Dawkins F6, McLaren C7, McLaren G8, Harris
E9, Press N10, Speechley M11, Mellen B12, Thomson E13, Eckfeldt J14, Sholinsky P15.
1
Department of Medicine, London Health Sciences Center, London, Ontario, Canada. 2,12Wake Forest
University School of Medicine, Department of Public Health Sciences, Winston-Salem, NC 3University of
Alabama at Birmingham, AL, 4Southern Iron Disorders Center, Birmingham, AL, 5,6Division of
Hematology/Oncology, Department of Medicine, Howard University, Washington, DC,7Epidemiology
Division, College of Medicine, University of California, Irvine, CA,8Division of Hematology/Oncology,
College of Medicine, University of California, Irvine,. 9Kaiser Permanente Center for Health Research,
Portland, OR,10Departments of Microbiology, Medicine, and Epidemiology and International Health,
Department of Public Health and Preventative Medicine, Oregon Health Sciences University, Portland,
OR, 11Department of Epidemiology and Biostatistics, University of Western Ontario, London, Ontario,
Canada, 13National Human Genome Research Institute, Bethesda, MD. 14Department of Laboratory
Medicine and Pathology, University of Minnesota, Minneapolis, MN, 15Epidemiology and Biometry
Program, National Heart Lung and Blood Institute, Bethesda, MD.
Background: Persons with HFE genotypes typically associated with hemochromatosis appear to be
much more common than persons with the clinical manifestations of hemochromatosis. Attribution of any
symptoms in hemochromatosis to genotype is problematic because of the high prevalence of these
symptoms in the general population and the inconsistent response of some symptoms to iron depletion
therapy. Objectives: To analyze the prevalence of self-reported symptoms by HFE genotype in a large
primary care population. Methods: The HEIRS study has screened primary care participants in 5 Field
Centers in the United States and Canada with genetic testing for the C282Y and H63D mutations of the
HFE gene, serum ferritin, transferrin saturation and a questionnaire which included questions on specific
complications attributed to hemochromatosis and iron overload in previous studies (liver disease,
diabetes mellitus, arthritis, heart failure, impotence and infertility). Questions were answered prior to the
participants being informed of their test results.
Results: Interim data were available on 45,619 participants (17,149 men and 28,740 women. mean age
= 51 years). The sample is multi-ethnic and includes 53% Caucasians, 22% African Americans, 12%
Asians and Pacific Islanders and 10% Hispanics. The sample included 176 C282Y homozygotes (74
men, 102 women), 532 C282Y/H63D compound heterozygotes (205 men, 327 women), 628 H63D
homozygotes (247 men, 381 women), 2,941 C282Y heterozygotes (1134 men, 1807 women), 7,884
H63D heterozygotes (2963 men, 4921 women) and 33,458 wild-type participants (12,526 men, 20,932
women). In an analysis restricted to Caucasians who were C282Y homozygotes or wild-type, odds ratios
for presence of symptoms in C282Y homozygotes versus wild-type participants, adjusted for age and
Field Center were computed using logistic regression separately for men and women. Men who were
C282Y homozygotes were more than twice as likely to report liver disease (OR 2.1, 95% CI 1.4 to 3.2)
and 1.5 times more likely to report impotency (OR 1.5, 95% CI 1.1 to 2.1) compared men who were wildtype. Women who were C282Y homozygotes were 1.2 times more likely to report arthritis (OR 1.2, 95%
CI 1.0 to 1.6) than women who were wild-type. No other symptoms showed significant differences. A
similar analysis including all participants examined the odds ratios for presence of symptoms among
participants with serum ferritin (SF) greater than 1000 µg/L versus others, adjusted for age, Field Center,
race and genotype. Men SF > 1000 µg/L were 5.6 times more likely (OR 5.6, 95% CI 2.2 to 14) and
women with SF > 1000 µg/L were 2.9 times more likely (OR 2.9, 95% CI 1.5 to 5.3) to report liver disease
compared to men and women with SF less than or equal to 1000 µg/L. No other odds ratios were
significant. Conclusions: C282Y male homozygotes report more liver disease and impotence and
female C282Y homozygotes report more arthritis compared to wild-type control Caucasian participants.
Liver disease is more prevalent amongst all participants with serum ferritin greater than 1000 µg/L.
Clinical assessment of these iron-loaded patients and controls is in progress and may add further
information on the role of genotype, iron overload, and other environmental factors to their clinical
symptoms.
PODIUM 42
INTRODUCTION TO EPITHELIAL IRON TRANSPORT
Matthias A. Hediger, Membrane Biology Program, Brigham & Women’s Hospital and Harvard Medical
School, Boston, MA 02115
Dietary iron is taken up into the plasma via specific transport molecules located in the enterocytes.
There are two pathways for intestinal iron absorption: Heme-iron and inorganic iron uptake. While the
absorption of heme iron is still poorly understood, the transport molecules involved in inorganic iron
absorption across the apical (DMT1) and basolateral (ferroportin/IREG1 and hephaestin) membranes
have been identified and characterized in great detail.
Maintenance of body iron homeostasis occurs predominantly at the level of intestinal iron absorption,
which depends on the regulation of DMT1 and IREG1. In the blood, iron is bound and stabilized by
transferrin. Little iron exists in free form under normal circumstances and hence iron is maintained in a
non-toxic form that is available to the tissues. Small amounts of iron, either bound to transferrin or in the
form of free iron, however, may be filtered in the kidney glomerulus. Recent studies by Smith and
colleagues (J Physiol. 2000, 524:581-6) indicated that filtered iron may be reabsorbed by the kidney
tubular system via DMT1 and/or the transferrin receptor.
The recent studies of DMT1, ferroportin and hephaestin have revealed an intricate net of interlinking
pathways involved in regulated acquisition, transport and storage of iron. Understanding these pathways
enables us to decipher the mechanisms of iron transport-related diseases.
PODIUM 43
IRON TRANSPORTERS AND THE KIDNEY
Craig P. Smith
School of Biological Sciences, University of Manchester, Manchester, UK
Using renal micropuncture we have shown that iron is present in glomerular ultrafiltrate and that iron is
reabsorbed in proximal and distal nephron segments (Wareing et al. 2000, Wareing et al. unpublished).
The divalent metal transporter DMT1 (also known as DCT1 or NRAMP2) transports iron, copper, and
manganese and to a lesser extent other divelent metals, but not calcium or magnesium. Using light
microscopy and immuno-gold/electron microscopy we have shown that DMT1 is expressed intracellularly
in the proximal tubule, on the apical membrane of early distal convoluted tubule, and is distributed both
apically and basolaterally in cortical and outer medullary collecting ducts (Ferguson et al. 2001, Gburek et
al. unpublished). This distribution suggests that DMT1 may be involved in iron reabsorption in these
segments. In support of this suggestion, DMT1 expression in the kidney, like in the duodenum, is
sensitive to dietary iron intake and the expression level of DMT1 is inversely related to the dietary iron
content. Feeding iron restricted or iron enriched diets causes changes in DMT1 expression intracellularly
in the proximal tubule and in the apical and sub-apical regions of the distal convoluted tubule. Increased
DMT1 expression was accompanied by a decrease in urinary iron excretion rate. The opposite was true
when DMT1 expression was reduced by feeding an iron enriched diet. Together these findings suggest
that modulation of renal DMT1 expression may influence renal iron handling.
Wareing, M., Ferguson, C.J.,Green, R, Riccardi, D. Smith, C.P. (2000) In vivo characterization of renal
Iron (Fe) transport in the anaesthetized rat. J Physiol (London)., 524, 581-6
Ferguson, C.J., Wareing, M., Ward, D.T., Green, R, Smith, C.P. & Riccardi, D. (2001) Cellular localization
of the divalent metal transporter DMT1 in rat kidney. Am. J Physiol., 280(5):F803-14
PODIUM 44
COMBINING KINETIC AND MUTAGENESIS APPROACHES TO BETTER UNDERSTAND THE
MOLECULAR MECHANISMS OF THE THE IRON TRANSPORTER DMT1
Bryan Mackenzie1, M L Ujwal1, Min-Hwang Chang2, Michael F Romero2, Matthias A Hediger1 1Membrane
Biology Program, Brigham & Women's Hospital, 77 Ave Louis Pasteur, Boston, MA 02115, and
2
Physiology & Biophysics, Case Western Reserve University, Cleveland, OH 44106
DMT1 is a widely-expressed H+-coupled metal-ion transporter [Nature 388, 482 (1997)]. We are
exploring the driving forces, kinetics and the involvement of other ions by using voltage clamp,
intracellular pH microelectrodes and radiotracer assays in cRNA-injected Xenopus oocytes. Evidence for
the coupling of Fe2+ transport to H+ comes from the observation of (1) pH-dependent presteady-state
charge movements (in the absence of metal ion), which indicate that H+ is a ligand, and (2) intracellular
acidification associated with Fe2+ transport. DMT1 does not mediate a Na+ flux, but analysis of the
Belgrade / mk mutation G185R (which abolished 55Fe transport) reveals a non-obligatory Fe2+-gated Cl–
conductance which, in wild-type DMT1, may counter the depolarization that results from H+/Fe2+
cotransport. Mathematical fits of steady-state kinetic data at low pHo (pH 5.2-6.1) indicate that H+/ binding
precedes the binding of Fe2+ and its simultaneous translocation. However, data at higher pHo diverge
from the ‘textbook’ model and reveal uncoupled Fe2+ transport at neutral pHo. Meanwhile, we have
identified two critical histidyl residues in TM6. Mutations at His-267 and His-272 each reduce Fe2+
transport, although H267A activity resembled that of wild-type DCT1. H272A, in the absence of metal ion,
behaves as a H+ uniporter, increasing ten-fold the ‘leak’ current observed for wild-type DMT1. The H272A
leak (1) was associated with a significant intracellular acidification, and (2) was carried solely by H+ (ΔVrev
of −53 ± 2 mV per pHo unit), but — despite Fe2+ inhibiting the H+ leak — H272A mediated pHindependent 55Fe transport. Coupling of Fe2+ transport to the H+ gradient, such as in the acidic
microclimate of the intestinal brush-border or acidified late endosomes, will allow DMT1 to be highly
concentrative. However, our data reveal for the first time that DMT1 will also be operational at the plasma
membrane in cells facing a neutral-pH environment, where facilitative Fe2+ transport will be driven by the
electrochemical gradient for Fe2+ alone.
PODIUM 45
PATCH CLAMP CHARACTERIZATION OF THE MOUSE IRON TRANSPORTER DMT1 TRANSIENTLY
EXPRESSED IN MAMMALIAN CELL LINES
Haoxing Xu, Jie Jin, David E. Clapham and Nancy C. Andrews
Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA
Divalent metal transporter 1 (DMT1, also known as Nramp2 and DCT1) plays important roles in iron
metabolism. Two-electrode voltage clamp studies in Xenopus oocytes suggest that DMT1 is a pHdependent divalent cation transporter that mediates a “leak” current in the absence of iron and an ironevoked current [Gunshin et al. (1997) Nature 388: 482-488]. Here we established a patch clamp assay in
mammalian cell lines to study an isoform of mouse DMT1. Our results showed that the “leak” was
approximately two to three times larger than the iron-induced current. Ion substitution experiments and
reversal potential analyses suggested that the “leak” was predominantly proton current and the ironinduced current was mediated by both protons and iron. Lowering the pH decreased the percentage of
the iron component despite an increase in the total current, indicating a dynamic stoichiometry between
protons and iron. Our data suggest that the proton leak is an important component of DMT1 function and
might be physiologically relevant, since the proton leak might provide a more acidic microenvironment
that helps solubilize iron and the apparent inefficient “coupling” might prevent iron overload. This assay, in
combination with a mutagenesis approach, provides a fast and efficient tool for structure-function studies
of DMT1.
PODIUM 46
DMT1 PROTEIN EXPRESSION IN THE APICAL MEMBRANE OF HUMAN INTESTINAL CACO-2
CELLS IS RAPIDLY DECREASED FOLLOWING EXPOSURE TO IRON
Deborah Johnson, Jason Tennant and Paul Sharp
Centre for Nutrition and Food Safety, School of Biomedical and Life Sciences, University of Surrey,
Guildford, GU2 7XH.
The transporter DMT1 mediates uptake of iron from the diet by intestinal enterocytes. Recently, we have
shown that DMT1 protein expression in the plasma membrane of human intestinal Caco-2 cells is
decreased by exposure to iron in a dose-dependent fashion (Sharp et al., 2002). Intriguingly, whole cell
levels of DMT1 do not change suggesting that the transporter may be internalised into an intracellular
compartment. In this study we have investigated the time-course of the iron-dependent decease in DMT1
in Caco-2 cells to determine whether changes in expression might occur within a physiologically relevant
period coincident with the digestion and processing of a meal in the normal gastrointestinal tract.
2
Caco-2 cells were grown in 25cm flasks for 21 days. Cells were incubated with 100µM FeCl3 for up to 24
hours. At the end of the experimental period, whole cell and plasma membrane proteins were isolated
and utilised for western blotting and total RNA extracted and subjected to RT-PCR for DMT1. In some
experiments, membrane proteins were biotinylated prior to exposure to iron. In these studies, at the end
of the incubation period, cells were lysed and the amount of biotinylated DMT1 in the cytosol determined
following immunoprecipitation with DMT1 antibody, protein separation by western blotting and
visualisation with streptavidin-HRP and enhanced chemiluminescence.
Plasma membrane DMT1 was significantly reduced by 4 hours exposure to iron (0 h, 91.4 ± 6.7 a.u.; 4 h,
52.9 ± 11.1 a.u., means ± S.E.M., n = 4, p = 0.025 Student’s unpaired t-test). However, whole cell levels
of DMT1 were unaltered by iron exposure. DMT1 mRNA levels isolated from control and iron-treated
cells was not significantly different at this time. Interestingly, there was an increase in biotinylated DMT1
in the cytosol following exposure to iron (control 8.3 a.u., +Fe 23.7 a.u.). Taken together, these data
suggest that the initial cellular response to elevated iron involves a decrease in apical membrane
expression of DMT1, perhaps due to internalisation of the transporter into an intracellular compartment.
These changes are rapid (between 1-4 hours following exposure to iron) and could occur within the time
scale for the digestion and processing of a meal.
This work was funded by BBSRC (project grant 90/D13400).
Reference
Sharp P et al., (2002) FEBS Lett. 510, 71-76.
PODIUM 47
THE ENDOCYTIC PATHWAY OF DMT1 IN CACO-2 CELLS
Y. Ma, K-Y Yeh, J. Rodriquez-Paris, Y. Chen, M. Yeh, J. Glass
Feist-Weiller Cancer Center, LSU Health Sciences Center, Shreveport, LA, USA
The molecular mechanisms ensuring directionality of iron transport across the intestinal epithelium are
still poorly understood. Iron is transported across the brush-border membrane (BBM) by the divalent
metal transporter 1 (DMT1) and then must be targeted to organelles and other transporters such as
Ferroportin 1 and accessory molecules such as Hephaestin for transport across the basolateral
membrane (BLM). Caco-2 cells grown in bicameral chambers exhibit constitutive transport of iron from
the apical (luminal) chamber to the basal (serosal) chamber, and are a model system to study intestinal
iron absorption. We have previously shown that with the addition of iron to the apical surface, DMT1 on
the BBM undergoes endocytosis and fuses with endocytic vesicles derived from the basal surface (1).
These findings suggest that in Caco-2 cells DMT1 undergoes a process of transcytosis and that this
process may direct iron to the transport complex on the BLM. However, what cytosolic proteins mediate
the endocytosis of DMT1 and the interaction of apical-derived vesicles with basal-derived vesicles are still
unknown. Vesicular transport between membrane compartments requires high specificity and tight
regulation and coordination of the participant structures and also involves other proteins common to and
required for vesicular transport. PAP7, a DMT1 interacting protein identified in the yeast 2-hybrid system
(cf. accompanying abstract), may be involved in specific clustering of DMT1 in the BBM for endosome
formation and both early endosome antigen 1 (EEA1) and the small GTPase, Rab5, that are required for
general endosome transport and fusion in mammalian cells, are potential tethering molecules that might
provide directionality to vesicular transport from the membrane to the early endosomes. We investigated
whether EEA1 and/or Rab5 are involved in the transcytosis of DMT1 containing microsomes. We first
investigated by immunofluorescence confocal microscopy, the distribution of EEA1 in Caco2 cells prior to
and after feeding with iron. In polarized Caco-2 cells, EEA1 was localized in a perinuclear region and in
the apical cytoplasm. The co-localization coefficient of EEA1 with DMT1 derived from BBM was 0.2 ± 0.13
prior to iron feeding and increased significantly to 0.45 ± 0.26 at 20 min after exposure of Caco-2 cells to
apical iron (p=0.045). As expected, because of the constitutive endocytosis of BLM vesicles, there was
little change in the co-localization coefficient for EEA1 and BLM vesicles as marked by Texas Red apotransferrin with a coefficient of 0.37 ± 0.16 prior to and 0.54 ± 0.17 after iron feeding (p=0.24). In another
series of experiments, Caco-2 cells were transiently transfected with recombinant green fluorescent
protein (GFP)-DMT1. After iron feeding, the co-localization co-efficiency of GFP-DMT1 with both EEA1
and Rab5 vesicles increased from 0.12 ± 0.1 to 0.46 ± 0.24 (p=0.02) and 0.08 ± 0.1 to 0.3 ± 0.11(p=0.01),
respectively. To further show that DMT1, EEA1, and Rab5 were present in the same subcellular
structures and that the co-localization was increased after iron feeding, we performed subcellular
fractionation of Caco2 cells before and after iron feeding using sucrose–gradient centrifugation. The cosedimentation of EEA1 with DMT1 increased from 17.1% of the total EEA1 prior to feeding to 47.9% at
twenty min after iron feeding (mean of 2 experiments). Similar results were obtained with Rab5. These
results give further support to the observations that DMT1 undergoes transcytosis and the EEA1 and
Rab5, the common molecules required for endosome transport and fusion are involved. We are
currently studying the signals that allow EEA1 and Rab5 to interact with PAP7 as well as BBM derived
endosomes containing DMT1 and the role of transcytosis in iron transport.
1.
Ma, Y., Specian, R. D., Yeh, K. Y., Yeh, M., Rodriguez-Paris, J., and Glass, J. The transcytosis of
divalent metal transporter 1 and apo-transferrin during iron uptake in intestinal epithelium. Am J
Physiol Gastrointest Liver Physiol, 283: G965-974., 2002.
PODIUM 48
REGULATION OF Lenramp1 IS DEPENDENT ON THE bHLH GENE fer IN PLANTS
P. Bauer, Z. Bereczky, H.-Y. Wang, T. Brumbarova, V. Schubert
Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, D-06466 Gatersleben,
Germany, E-mail bauer@ipk-gatersleben.de
Plants, like all other higher eukaryotes have to transport iron short and long distance to meet their
requirements and at the same time cope with potential low solubility and toxicity of this metal. Since
plants play a major role for human nutrition and iron deficiency is a world-wide nutritional problem it is one
of the challenges to produce iron-rich crops. Lack of available iron such as on alkaline or calcareous soils
results in leaf chlorosis and reduced plant growth and yield. Therefore, it is of great interest to study the
mechanisms for iron uptake and transport in plants.
Recent research has primarily focussed on characterizing the structural iron acquisition components for
uptake of iron from the soil into the root epidermis upon iron deficiency (for review Planta, 2003, vol. 216,
p. 541-555). Grasses produce and release phytosiderophores and take up the Fe III complexes via the
transporter YELLOWSTRIPE 1 (YS1) (strategy II). Dicot plants reduce iron due to the activity of ferric
chelate reductase (FRO2) and take up Fe II via the iron-regulated transporter IRT1 (strategy I). Plants
have evolved their own mechanisms for internal transport of iron. In the xylem iron is essentially bound as
iron citrate. Iron-transport protein has strong affinity for iron in the phloem. The plant-specific nonproteinaceous amino acid nicotianamine is essential for metal binding in the cytoplasm. The presence of
evolutionarily conserved iron transport and storage genes in the plant genome suggests that plants also
share iron storage and mobilisation components with other organisms (for example nramp, ABC
transporters, ferritin). The integrative regulation of iron transport, storage and remobilisation inside the
plant is currently not known.
Recently, we have identified the bHLH gene fer which regulates iron acquisition in tomato roots (PNAS,
2002, vol. 99, p. 13938-13943). fer loss of function mutant plants are chlorotic and do not display
physiological, morphological and molecular adaptations to iron deficiency, such as iron reduction,
acidification of the rhizosphere, root hair proliferation and iron transporter gene induction. Interestingly,
FER transcripts are localized to parenchyma cells of the vascular cylinder in the root hair zone rather than
the root epidermis. Since FER mRNA levels and expression pattern are not affected by iron supply we
predict post-transcriptional or post-translational control of the FER protein. To deduce a possible role of
fer we have studied potential downstream target genes of FER. In this context we identified Lenramp1
which encodes a functional iron transporter. Lenramp1 expression is to a great extent dependent on a
functional fer gene in the root as it is down-regulated in fer mutant roots. LENRAMP1 transcripts colocalize with those of FER in the vascular parenchyma and are absent in fer mutant roots. Lack of the
cytoplasmic metal chelator nicotianamine in the chloronerva nicotianamine synthase mutant causes leaf
chlorosis presumably due to the failure of iron to reach its final cellular targets. As a result, chloronerva
mutants mobilise iron upon sufficient iron supply. Lenramp1 is over-expressed in the roots of chloronerva
plants. When expressed in yeast, LENRAMP is stabilized when the cells are grown under iron deficiency.
From these results, we propose that LeNRAMP1 may serve mobilisation of iron upon iron deficiency.
Moreover, our results suggest that the vascular root parenchyma may be involved in storage, mobilisation
and regulation of iron in plants. Our future studies will concentrate on the model plant species Arabidopsis
thaliana.
PODIUM 49
IDENTIFICATION OF THE UBIQUITIN-PROTEIN LIGASE THAT RECOGNIZES HEME-OXIDIZED IRP2
K. Iwai, K. Yamanaka, H. Ishikawa, T.A. Rouault, K. Ishimori. Osaka City University, Osaka, Kyoto
University, Kyoto, CREST, Saitama Japan and CBMB, NICHD, NIH
The expression of proteins involved in mammalian iron metabolism is regulated post-transcriptionally
through interactions between RNA stem-loop structures known as iron responsive elements (IREs) found
on the transcripts encoding those molecules and iron regulatory protein 1 and 2 (IRP1 and 2). The
binding of IRPs to IREs in the 5’ untranslated region (UTR) prevents initiation of the translation of ferritin
mRNA, and the binding of IRPs to IREs in the 3’UTR of TfR mRNA protects the mRNA from degradation
in iron-depleted cells. Both IRP1 and IRP2 bind IREs with high affinity only in iron-depleted cells, but the
mode of regulation by iron differs between the two proteins. IRP1 has been shown to be a stable bifunctional protein and its activity is regulated by the assembly/disassembly of its iron-sulfur cluster. In
contrast, IRP2 is regulated by iron-induced ubiquitination and degradation. A specific 73 amino acid
domain - iron-dependent degradation (IDD) domain - has been shown to be essential for targeting the
protein for degradation following iron-mediated oxidative modification.
Ubiquitination is carried out by a cascade of three reactions catalyzed by enzymes, E1, E2 and E3.
Among them, the ubiquitin-protein ligases (E3s) have shown to play a pivotal role in determining the
specificity of the system by recognizing the target substrates via defined targeting motifs. Recently, RING
finger proteins have been shown to constitute an important family of E3s. However, only a handful of
post-transcriptional modifications and E3s recognizing those modifications have been reported to date.
The E3 recognizing oxidized IRP2 has not been clarified yet.
By using a differential yeast two-hybrid screening in which yeast cells were cultured in either aerobic or
anaerobic conditions using the IDD domain as a bait, we identified a RING finger protein, HOIL-1 as an
E3 for oxidized IRP2. Moreover, the oxidation of IRP2 is generated by heme, which binds to IRP2 in ironrich cells, and oxygen.
These results indicate that oxidation serves as a specific recognition signal for ubiquitination and the iron
sensing of IRP2 depends on synthesis and availability of heme.
PODIUM 50
REDOX PROPERTIES OF HUMAN TRANSFERRIN BOUND TO ITS RECEPTOR
A. L. Crumbliss,a S. Dhungana, a C. H. Taboya M. Larvie, b , O. Zakc and P. Aisenc
a
Department of Chemistry, Duke University, Durham, NC 27708-0346 ; b Harvard-MIT Division of Health
Sciences and Technology, Cambridge, MA 02139; cDepartment of Physiology & Biophysics, Albert
Einstein College of Medicine, Bronx, NY 10461
Transferrin, the iron carrier of the circulation, is a bilobal protein with a single iron-binding site in each
lobe. Iron borne by transferrin is in the ferric state, with a reduction potential too low (< -500 mV) for it to
be reduced to the ferrous state by physiological means either at extracellular pH, 7.4, or at endosomal
pH, near 5.6 [1,2]. Iron exits the endosome, to which it is internalized, and enters the cytoplasm via the
divalent metal ion1ransporter, DMT1, which accepts only ferrous iron. A fundamental question is how
then does iron carried by transferrin gain access to the cytoplasm?
A salient feature of the transferrin-to-cell cycle in iron metabolism is the persistence of the transferrintransferrin receptor complex throughout the cycle: free transferrin is not known to exist within the cell
once iron has been complexed to the receptor at the cell surface. The possibility that the receptor
modulates the reduction potential of transferrin-bound iron as it does the release of iron from transferrin to
chelators was therefore investigated.
The reduction potential of transferrin C-lobe at endosomal pH, free and bound to its receptor, was
measured as described previously [1]. The soluble ectodomain of the transferrin receptor (sTfR)
expressed in a baculovirus system, was provided by Dr. Peter Snow of the California Institute of
Technology, and human transferrin C-lobe in native conformation was obtained as previously described
[3]. The C-lobe/transferrin receptor complex was
prepared by incubating iron loaded transferrin C-lobe
with sTfR and separating the complex of the two proteins
from its uncomplexed components by size-exclusion
chromatography.
We chose to study the complex of transferrin receptor
with the C-lobe of transferrin rather than the full-length
protein because at endosomal pH lability and release of
iron carried by the N-lobe could confound interpretation
of iron reduction. Our results clearly demonstrate that the
energetics of Fe(III)Tf reduction to Fe(II)Tf is facilitated
by transferrin binding to its receptor (see Figure). The
redox potential shifts ca 200 mV positive on binding to
transferrin receptor at pH 5.8. These results indicate that
reduction of iron bound to transferrin in the
transferrin-transferrin receptor complex is physiologically
and thermodynamically feasible, since the reduction
potential of pyridine nucleotides at endosomal pH, -284
mV, is close to that of iron in the Tf/TfR complex, -289
mV. Ferrous iron is bound to transferrin at least 12
orders of magnitude more weakly than ferric iron, so that reductive release of iron from transferrin in the
endosome is at least possible, and perhaps likely. The transferrin receptor is more than a simple
conveyor of transferrin and its iron.
[1] Kraiter, D. C.; Zak, 0.; Aisen, P.; Crumbliss, A. L. Inorg. Chem. 1998, 37, 974.
[2] Kretchmar, S. A.; Reyes, Z. E.; Raymond, K. N. Biochim. Biophys. Acta 1988, 226,187.
[3] Zak, 0., Aisen, P.; Biochemistty2002, 41, 1647.
PODIUM 51
THE MECHANISM FOR IRON-DEPENDENT DEGRADATION OF IRP2 IS SATURABLE, STIMULATED
BY ANTIOXIDANTS AND INDEPENDENT OF SITE-SPECIFIC OXIDATION OF C168, C174 AND C178
J. Wang1, G. Chen1, M. Muckenthaler2, M. W. Hentze2 and K. Pantopoulos1
1
Lady Davis Institute for Medical Research and Department of Medicine, McGill University, Montreal,
Quebec, Canada, and 2European Molecular Biology Laboratory (EMBL), Heidelberg, Germany
Iron regulatory protein (IRP2) is a central regulator of cellular and body iron metabolism and IRP2-/- mice
develop a progressive neurodegenerative disorder. In iron-starved or hypoxic cells, IRP2 binds to “iron
responsive elements” (IREs) within the untranslated regions of several mRNAs and thereby controls their
expression. In iron-replete cells, IRP2 undergoes proteasomal degradation, by a mechanism which is part
of a well-orchestrated homeostatic response to iron supply. The prevailing model postulates that the ironmediated degradation of IRP2 involves site-specific oxidation of 3 cysteine residues (C168, C174 and
C178) within a 73 amino acid “degradation domain”. We have expressed wild type and mutated versions
of IRP2 in H1299 (human lung cancer) cells under the control of a tetracycline-inducible promoter.
Surprisingly, we find that a C168S, C174S and C178S triple mutant is as sensitive to iron-mediated
degradation as wild type IRP2. Antioxidants such as ascorbate, vitamin E and N-acetyl-cysteine not only
fail to stabilize IRP2 in iron-replete cells but, moreover, promote its degradation in the absence of iron.
Transient transfection experiments showed that the degradation pathway of IRP2 is saturable and the
protein is stable when expressed at high concentrations. This observation may explain earlier data
supporting the “cysteine-oxidation” model. Deletion analysis to map the elements involved in IRP2
degradation shows that a mutated version of IRP2 lacking the entire 73 amino acid domain exhibits the
same response to iron as the wild type. These results challenge the current model for the iron-dependent
degradation of IRP2 and allow the examination of alternative hypotheses. Considering that the
degradation of the “hypoxia inducible factor 1α“ (HIF-1α) depends on oxygen, iron and ascorbate and
exhibits a saturable pattern, we examined whether the pathways for HIF-1α and IRP2 degradation may
share additional similarities. However, while HIF-1α degradation can be effectively competed upon
expression of its own “oxygen-degradation domain” or treatment with the specific 4-prolyl-hydroxylase
inhibitor dimethyl-oxalylglycine, the response of IRP2 to iron remains unaffected under these conditions.
PODIUM 52
ENHANCED NITRIC OXIDE SENSITIVITY OF THE FE-S CLUSTER IN PHOSPHOMIMETIC MUTANTS
OF IRON REGULATORY PROTEIN 1
K.M. Deck, S.A. Anderson, M.C. Kennedy, and R.S. Eisenstein, Dept of Nutritional Sciences, University of
Wisconsin, Madison, WI 53706
Iron regulatory proteins (IRPs) are cytosolic RNA-binding proteins that regulate the synthesis of proteins
involved in iron uptake, storage and utilization by binding to the iron responsive elements (IREs) of
specific mRNAs. When iron levels are high, a [4Fe-4S] cluster is assembled in IRP1, allowing it to
function as a cytosolic aconitase (c-acon) and eliminating high-affinity RNA binding. We previously
demonstrated that phorbol 12-myristate-13-acetate (PMA), an activator of protein kinase C (PKC),
increased IRP1 phosphorylation and RNA binding activity in HL-60 cells. Ser138 and Ser711 were
identified as sites where PKC could act. We hypothesized that phosphorylation at S138 might modulate
the interconversion of the RNA-binding and aconitase forms of IRP1 by small reactive species such as
..
O2, O2 and NO that are able to perturb the [4Fe-4S] cluster in c-acon. Using aspartate (D) and
glutamate (E) to mimic the charge and size of a phosphate group, we previously demonstrated increased
oxygen sensitivity of the aconitase form of the protein in the phosphomimetic mutants S138D and S138E
both in vitro and in yeast (Brown et al, 1998). Nitric oxide, an important cellular signaling agent, has been
shown to inactivate both c- and m-acon (Kennedy et al., 1997). Significantly, cellular studies have
demonstrated that NO increases IRP1 RNA binding activity as well (Drapier et al., 1993; Weiss et al.,
1993). We are interested in the mechanism of this interconversion between c-acon and IRP1, and
specifically in the role that phosphorylation plays. Our current study focuses on the effect of NO on
aconitase and RNA binding activities of the phosphomimetic mutants of IRP1 in vitro. The Fe-S cluster
was reconstituted in wildtype (WT), S138Alanine (S138A) (control), S138D, and S138E recombinant IRP1
proteins using NifS with Fe2+, L-cysteine and DTT under anaerobic conditions. The WT and all S138
mutants displayed aconitase activity. Under anaerobic conditions, loss of aconitase activity upon
treatment with the NO donor diethylamine NONOate [DEA(NO)] occurred most readily for S138E,
followed by S138D, and occurred more slowly for S138A and WT. In the presence of 0.35 mM DEA(NO)
the half-lives were 1 min for S138E, 6 min for S138D, and at least 4 h for S138A and WT. Doseresponse studies were consistent with the Fe-S cluster in the phosphomimetic mutants being much less
stable than the Fe-S cluster in WT and S138A. The IC50 of the aconitase activity after 30 min exposure to
DEA(NO) was 0.02 ± 0.01 mM for S138E, 0.07 ± 0.02 mM for S138D, 0.87 ± 0.03 mM for S138A and 1.0
± 0.4 mM for WT. In the presence of 2 mM citrate at pH 7.5, the [4Fe-4S] cluster appeared to be
protected from attack by DEA(NO) in all four proteins WT, S138A, S138D and S138E. To assess the
effect of NO on RNA binding activity, reconstituted WT and S138D were treated with DEA(NO) under
anaerobic conditions. Preliminary data suggest that RNA binding activity can be restored more rapidly in
S138D than in WT, consistent with a less stable cluster in the phosphomimetic mutant. After 60 min of
DEA(NO) exposure, WT had measurable RNA binding activity in the presence of 2-ME, but the 2-ME
induction was blocked by citrate, indicating the presence of substantial amounts of cluster. Within 20 min
of DEA(NO) exposure, S138D had measurable RNA binding activity in the presence of 2-ME, and the 2ME induction of RNA binding was not blocked by citrate, indicating significant loss of cluster. Taken
together, our results indicate that phosphorylation of IRP1 at S138 induces a cluster instability phenotype
in response to multiple cluster denaturants. We suggest that phosphorylation alters the set-point for
regulation of IRP1 RNA binding activity in response to iron, oxidative stress, and NO. (Support: NIH
DK47219)
Brown,N.,Anderson,S.,Steffen,D.,Carpenter,T.,Kennedy,M.,Walden,W.,Eisenstein,R.(1998) Proc. Natl.
Acad. Sci. 95, 15235-15240.
Drapier,J-C., Hirling,H., Wietzerbin,J., Kaldy,P., Kuhn,L.C. (1993) EMBO J. 12, 3643-3649.
Kennedy, M. C., Antholine, W. E., Beinert, H. (1997) J. Biol. Chem. 272, 20340-20347.
Weiss, G., Goossen, B., Doppler, W., Fuchs, D., Pantopoulos, K., Werner-Felmayer, G., Wachter, H.,
Hentze, M. W. (1993) EMBO J. 12, 3651-3657.
PODIUM 53
MOLECULAR CONTROL OF MAMMALIAN IRON METABOLISM: MOLECULES, MICE AND
MICROARRAYS
M.W. Hentze, European Molecular Biology Laboratory, Heidelberg, Germany.
Iron metabolism is regulated at the cellular and the systemic level. In both instances, iron deficiency and
iron overload must be prevented. Experimental approaches using cultured cell lines and focussing on
single genes/molecules have helped to define the regulatory frameworks, including the IRE/IRP
regulatory system, at the cellular level. Human genetics and animal models have been instrumental in the
identification of critical genes involved in systemic iron homeostasis.
We strive to integrate the understanding of cellular and systemic iron homeostasis, to better understand
the regulation of iron metabolism of and by specific tissues and organs, and to explore the regulatory
interactions between the hundreds of genes involved in mammalian iron metabolism. To this end, k.o.
strains of mice were generated (IRP1, IRP2) and different tissues (duodenum, liver, spleen, brain, heart)
were subjected to DNA microarray (“IronChip”) analysis. Together with our collaborating partners, many
additional, well-characterized mouse strains are being analyzed in the same way. Emerging principles will
be discussed, emphasizing pathways under the primary control or independent of the IRE/IRP system.
This abstract is based on work by the members of my lab, past and present, and ongoing, fruitful
collaborations with the Andrews, Connor, deSousa, Hediger, Pietrangelo and Stremmel labs.
PODIUM 54
IRON HOMEOSTASIS ABNORMALITIES IN HFE AND TRANSFERRIN RECEPTOR 2 COMPOUND
MUTANT MICE
R.E. Fleming1,3, H.D. Hall1, J.R. Ahmann1, M.C. Migas1, R.S. Britton2, B.R. Bacon2, A. Waheed3, W.S.
Sly3. 1Pediatrics; 2Internal Medicine and 3Edward A. Doisy Department of Biochemistry & Molecular
Biology, Saint Louis University School of Medicine, St. Louis, MO.
Introduction: Transferrin receptor 2 (TfR2) is a recently discovered iron transport protein with homology to
the classic transferrin receptor. Several pedigrees have been identified with a phenotype similar to HFErelated HH, but caused by mutations in the TfR2 gene. We have generated transgenic mice carrying the
murine orthologue (Y245X) of the first described human TfR2 mutation (Y250X) and demonstrated that
homozygous mutant mice have markedly elevated periportal liver iron and decreased splenic iron
concentrations. Similar findings are observed in Hfe knockout mice.
Objective: We tested whether combined disruption of both TfR2 and Hfe leads to additive effects on
parameters of iron homeostasis.
Methods: We bred mice homozygous for the TfR2 mutation with Hfe knockout mice to generate
compound mutant mice. Mice were sacrificed at 4 weeks of age and genotyped for presence of the
disrupted Hfe and TfR2 alleles by PCR analysis of tail DNA. Non-heme liver and splenic iron
concentrations were measured, and issue sections stained for iron by Perls’ method. Values obtained
from mice homozygous for each single gene disruption were compared by ANOVA with mice
homozygous for both disrupted genes, and with mice carrying at least one wild-type allele for both TfR2
and Hfe.
Results: Hepatic and splenic iron concentrations from each of the four groups are summarized in the
table below. Values represent mean (µg/g dry weight) ± SEM, N = 4 to 6 per group.
Genotype
Hfe
+/±
-/+/±
-/-
TfR2
+/±
+/±
-/-/-
Iron Concentration
Liver
297 ± 68
1118 ± 590
2172 ± 547
1815 ± 466
Spleen
618 ± 90
414 ± 09
369 ± 18
385 ± 21
Mice homozygous for either disrupted gene demonstrated greater hepatic iron concentrations and lower
splenic iron concentrations than did mice carrying at least one wild-type allele for both genes (P<0.05).
While mice homozygous for the TfR2 gene disruption demonstrated somewhat higher hepatic iron
concentrations than mice homozygous for the Hfe disruption, there was no statistical difference with this
sample size. Homozygosity for disruption of both alleles did not lead to greater hepatic iron loading or
splenic iron sparing than did disruption of the TfR2 allele alone. Iron deposition was predominantly in
periportal hepatocytes in mice homozygous for disruption of either or both genes.
Conclusion: The phenotypic consequences of loss of Hfe and TfR2 on iron homeostasis are qualitatively
similar, suggesting that they each modulate a common effector. The consequences on iron homeostasis
by the combined functional loss of Hfe and TfR2 are no greater than that observed with loss of TfR2
alone.
PODIUM 55
NUCLEAR IMPORT AND EXPORT OF AFT1 PROTEIN, THE MAJOR IRON-RESPONSIVE
TRANSCRIPTIONAL ACTIVATOR IN YEAST
Y. Yamaguchi-Iwai, A. Fukunaka, R. Ueta, Graduate School of Biostudies, Kyoto University
The major iron-responsive transcriptional activator, Aft1p, plays critical roles in maintaining iron
homeostasis in budding yeast; its activity is induced in response to iron starvation, and as a consequence
the expression of iron-regulon is activated. We have shown previously that the Aft1p is localized in the
cytoplasm under iron-replete condition, but that upon the exposure to iron-starvation it localized to the
nucleus. We show here the mechanisms involved in the import and export of Aft1p. We found that Aft1p
is imported into the nucleus by the importin-β-family member, Pse1p/Kap121p. Aft1p was found in the
cytoplasm in pse1-1 cells carrying a temperature-sensitive mutation of PSE1/KAP121 at the restrictive
temperature. In an in vitro assay, we showed that Aft1p could directly bind to Pse1p and that this
interaction was dissociated by Ran-GTP. We also identified the two nuclear localization signals of Aft1p.
Furthermore, We found that the importin-β-family member, Msn5p is required for the nuclear export of
Aft1p. Aft1p was found in the nucleus in ∆msn5 cells and interacted with Msn5p in the two-hybrid assay.
We are now investigating how the import and export of Aft1p is regulated in response to changes in iron
availability.
PODIUM 56
INCREASED PREVALENCE OF IRON DEFICIENCY DUE TO WITHDRAWAL OF IRON
FORTIFICATION OF FLOUR IN SWEDEN.
Leif Hallberg and Lena Hultheń.
From the Department of Clinical Nutrition, Sahlgrenska Academy of Medicine, University of Göteborg,
Sweden.
Swedish millers decided to withdraw the voluntary fortification of floor in Sweden, at January 1 1955. The
Swedish National Food Administration accepeted to temporarily accept the decision provided an effect
on iron status was made. Our group was recommended to accept a study on the effect on menstruating
teenage girls. Menstrual iron losses were determined in a random sample of 15-16 year old girls. In a
sample of more than 500 girls iron status were examined ( s-Hb and s- ferritin). About 39.3% were iron
deficient based on s-ferritin. Six years later after discarding those who were on iron tablets, who have
changed to a vegetarian lifestyle and who have changed to contraceptive tablets the iron deficient girls
had increased to 50.4%. The iron deficient girls had now increased with 28.3%.
PODIUM 57
THE ASSESSMENT OF IRON FORTIFICATION PROGRAMS USING QUANTITATIVE
MEASUREMENTS OF BODY IRON.
James D. Cook and Barry S. Skikne, Kansas University Medical Center, Kansas City, Kansas 66160.
The continuing uncertainty about the efficacy of iron fortification programs stems is due largely to the
limitations of the laboratory methods used to assess iron status. In this presentation, a new method for
measuring body iron quantitatively is described based on the ratio of the serum transferrin receptor to
serum ferritin. The method was developed and calibrated using quantitative phlebotomy. When a
convenience sample of 2057 specimens collected in US National Health and Examination Survey III was
examined, the observed changes in body iron were consistent with our knowledge of the effect of gender,
growth, and menstruation on iron status. Using age ranges in which iron status was relatively constant,
body iron stores averaged 9.82±2.82 mg/kg in 649 adult men aged 20-65 years and 5.5±3.35 mg/kg in
409 women aged 20-45 years. The utility of the method for assessing the impact of iron fortification was
examined by analyzing a small subset of anemic Vietnamese women participating in a randomized trial of
iron fortification with fish sauce 10 mg iron as NaFeEDTA daily. Absorption of the added iron as
determined by the changes in body iron agreed closely with radioisotopic studies of iron absorption. A
major advantage of body measurements is the ability to determine iron status in each individual rather
then relying on cut-off points of laboratory indices. Quantitative estimates of body iron will facilitate the
assessment of iron fortification programs by reducing the size and duration of intervention trials
conducted under field conditions.
PODIUM 58
EVALUATION OF ELEMENTAL IRON POWDER BIOAVAILABILITY AND STRATEGIES FOR
ENHANCING IRON ABSORPTION
Elizabeth Turner
Elemental iron powders are the most common iron fortificants used worldwide because they cause the
fewest problems with color, flavor, and rancidity in food products, and are relatively inexpensive. However
research conducted over the past forty-five years has produced highly variable results with respect to the
bioavailability of the powders (5%-145% RBV). To address these concerns, SUSTAIN has launched a
comprehensive program for evaluating the five types of elemental iron powders currently used in
fortification. Preliminary results from metallurgical, solubility, caco-2, rat hemoglobin repletion and human
iron tolerance tests indicate that the relative bioavailability of these iron powders with respect to ferrous
sulfate varies from 24% to 64%. Human efficacy studies are in progress. In addition, SUSTAIN
convened a workshop in March 2003 to assess ingredient technologies that could be used to improve the
bioavailability of iron fortificants. The workshop evaluated cost, product stability, and commercial
feasibility of each strategy to improve iron absorption including the addition of ascorbic acid, sodium iron
EDTA, amino acid chelates, phytate removal, and iron encapsulation. These appeared promising and
commercially feasible in specific food vehicles. No technology appeared feasible in all widely consumed
food vehicles due to cost, stability and organoleptic factors.
PODIUM 59
THE IMPORTANCE OF BIOAVAILABILITY FOR IRON FORTIFICATION
Sean Lynch, Eastern Virginia Medical School
Nutritional iron deficiency remains a major global health problem affecting an estimated 2 billion people.
Most of them live in developing countries. Cereal flour staple foods are fortified with iron in many
countries. However there is little evidence to indicate that iron fortification of food has had a significant
impact on the prevalence of nutritional iron deficiency in most developing countries. There are several
possible reasons for the apparent failure of these programs. One of the most important may be the
widespread use of inadequate quantities of poorly bioavailable iron compounds. Elemental iron powders
are favored for the fortification of cereal flour staples because they do not promote fat oxidation and
rancidity during storage, and they do not affect the taste and color of meals prepared from the fortified
product. However absorption of the iron may be inadequate. Preliminary screening studies carried out on
elemental iron powders that are currently used in most fortification programs indicate that only about half
of the iron in the more bioavailable iron powders (carbonyl, electrolytic and some types of reduced iron)
enters the nonheme common pool. Programs that employ these powders will probably not have a
significant impact unless the fortified meals are rich in enhancers of iron absorption. The less bioavailable
iron powders are unlikely to be effective under any circumstances. There are several well documented
examples of successful fortification. They include the use of ferrous sulfate and ascorbic acid in powdered
milk for infants and young children in Chile, encapsulated ferrous sulfate in school children in Morocco
and NaFeEDTA in fish sauce and soy sauce for mass (universal) fortification in Vietnam and China
respectively. In each case a sufficient quantity of iron was employed and adequate bioavailability was
ensured by the use of a water-soluble iron compound and, in three of the examples, an enhancer of
absorption. These observations demonstrate that careful attention must be paid to the delivery of
adequate quantities of iron in a bioavailable form for iron fortification of food to be successful.
PODIUM 60
PROGRAM EXPERIENCE WITH IRON FORTIFICATION
Frances Davidson, USAID.
The addition of iron to foods through fortification is widely practiced in developed countries, and
has been used for decades in cereal flours and cereal-based complementary foods for infants
and young children. Iron fortification of foods is increasingly being implemented in developing
countries. For example, more than 20 countries in Latin America have universal iron fortification
programs-for wheat or maize flours. Chile fortifies its dried milk powder for infants while Brazil
fortifies its whole milk. Fish sauce fortification is currently underway in Vietnam and China is
scaling up fortification of soy sauce. The evidence-base, challenges, and prospects for the
successful implementation of these programs will be presented as well as promising options such
as the double fortification of salt with iodine and iron and the use of fortified complementary food
supplements for infants and young children
PODIUM 61
CHANGING SPECTRUM OF TREATMENT-RESISTANT IRON DEFICIENCY ANEMIA (IDA): C
Hershko1, B Roth1, J Ashkenazi1, J Heyd1 and D Kereth2. 1Department of Hematology, Shaare Zedek Med. Ctr. and
2
Gastorenterology Clinic, Kupat Cholim Klalith, Jerusalem, Israel.
Although refractory IDA represents only a small fraction of iron deficiency in the general population, in
patients referred for hematology consultation, i.e. in those already selected by primary physicians as a
problem group, it may represent up to 30% of subjects with IDA .In recent years, there is an increasing
awareness of the importance of celiac disease as a possible cause of IDA refractory to iron therapy,
without other apparent manifestations of malabsorption syndrome. The objectives of the present study
were threefold: (a) To determine the proportion of adult patients with IDA referred for consultation with
anemia refractory to oral iron treatment; (b) To examine the diagnostic value of a 2-hour iron-loading test
in identifying patients with abnormal iron absorption and predicting response to therapy, and; (c) To
determine the cause of refractoriness to iron therapy by serologic screening for celiac disease and a
systematic gastroenterologic evaluation. Over a period of 12 months, 86 subjects with treatment resistant
IDA have been identified. There were 7 children below 16 y and 79 adults of whom 70 were women. We
found no correlation between results of the 2-hour iron-loading test and subsequent response to oral iron
therapy. The cause of IDA was identified in 79 subjects. The most common abnormalities in descending
order were: Atrophic body gastritis (ABG) manifested in very high serum gastrin and antiparietal antibody
titers indistinguishable from pernicious anemia (23%); Helicobacter pylori infection (28%) confirmed by
gastric biopsy and/or a positive urease breath test, presently evaluated for response to antibiotic
treatment; Menorrhagia (17%); Diabetic gastropathy (9%); GI bleeding (8%) and; malabsorption caused
by Celiac disease and Gastroplasty (7%). The high degree of overlap between Helicobacter gastritis and
ABG calls for further studies to explore a possible etiologic role of Helicobacter in the pathogenesis of
ABG. Our findings challenge the "common wisdom" that in young women with a negative GI history
menstrual blood loss is the predominant cause of treatment-resistant IDA and implicate Gastropathic
Sideropenia as the most common underlying anomaly.
PODIUM 62
A SYSTEMATIC VIEW OF THE FECA, FHUA AND FEPA STRUCTURES
Dick van der Helm, University of Victoria
At this time the structures of FecA, FhuA and FepA hav been determined;the former two with and
without ligand.They all consist of a 22-stranded ß-barrel and a N-globular (plug ) domain,with long
extracellular loops and short periplasmic loops.A comparison of the three structures show many
similarities but also dissimilarities.These results are combined with a simultaneous sequence alignment of
many ferric siderophore transport proteins.The alignment results should single out the residues of the
common transport mechanism in this family of proteins in contrast to those involved in binding,because
the comparison involves a diverse set of ferric siderophores.Some of the conclusions which combine both
the structural comparisons and results of the simultaneous sequence alignments are listed below.
1) The cross section of the barrel is elliptical with the longest axis between the bottom of strands 8 and
19 in the three structures. 2) There is no sequence similarity in the extracellular loops in the simultaneous
alignment of many proteins in this family.All (~25) occur well below the apices. 3) The topology of the Nglobular domain is the same in the three proteins,but the structures are only similar.The orientation and
location of the mixed ß- sheet (1,5,6,4) is the same(within 2 Å),and thus recognizes the elliptical barrel.On
the other hand the location and height of the apic es (A,B,C) and switch helices are quite different (515Å). 4) The molecular signaling from the apices,after ligand binding,to the switch helices is quite
different in FecA and FepA.Only two residues at the N-terminus of the helix are unwound in FecA.Further
proof is required to ascertain the signaling process. 5) The location of the plug domain with respect to the
elliptical barrel is the same in the three structures.It involves a three-point attachment,using the covalent
bond between plug and barrel,and strictly conserved Arg’s and Glu’s on the plug domain and the
barrel,respectively,(lock region).This feature is also present in the sequences of heme transporters. 6) A
potential channel for the transfer of ligand from the binding site to the periplasm is apparent.It
requires,however,a movement of the ß 5-6 loop,which contains two strictly conserved Gly residues at i
and i+7.The channel is lined by a strictly conserved Arg on the barrel. 7) Alanine substitutions of
conserved residues in the lock region and ß 5-6 loop gives mutants which show normal binding but
deficient transport. 8) Structural and sequence identity indicates a possible second site for TonB
interaction on the outside of the barrel.This also present in the sequences of lactoferrin and transferrin
transporters. 9) The FecA structure shows the principle of bipartite gating,allowing only the transport of
the bound ligand. 10) It seems possible to separate and distinguish the binding of ligand from the
common transport process in the proteins of this family.
PODIUM 63
STRUCTURAL STUDIES OF THE ESCHERICHIA COLI PERIPLASMIC BINDING PROTEIN FHUD
AND ITS COMPLEXES WITH SEVERAL HYDROXAMATE SIDEROPHORES
H.J.Vogel, University of Calgary
Iron is an absolute requirement for all living cells, including nearly all bacteria, with the possible
exception of Lactobacilli. However bacteria face the same problems as eukaryotes, namely that iron is
relatively inaccessible and that it has a propensity to generate toxic radicals. Many bacteria overcome
these two problems by synthesizing small iron-chelating compounds - the siderophores. These small
peptide-based compounds bind iron with great avidity, such that they can release this metal ion from the
iron-hydroxides in the environment or from host proteins, such as transferrin or hemoglobin, in the case of
pathogenic -disease causing -bacteria. There are at least three different classes of siderophores that
differ in the chemical entities responsible for iron binding. Bacteria such as E. coli are opportunistic in the
sense that they can take up many different types of siderophores even though they only synthesize a
limited spectrum themselves. E.coli only produces one catecholate siderophore, but it can take up a
number of hydroxamate ferric-siderophore complexes, whereas the latter siderophores are typically
secreted by fungi. Some siderophores, eg ferrioxamine, are used clinically for iron-detoxification
The uptake of iron-siderophore complexes in Gram-negative bacteria is mediated by a series of
proteins that guide them through the bacterial outer membrane, across the periplasmic space and
through the cytoplasmic membrane into the cytoplasm of the bacterial cell. The protein FhuD is
responsible for picking up all hydroxamate siderophores in the periplasmic space of E. coli. It
subsequently presents them to the transporter proteins in the cytoplasmic membrane which are
responsible for the intracellular uptake. This is markedly different from the outer membrane siderophore
receptors which are specific for different hydroxamate siderophores, indeed E.coli possesses four outer
membrane receptors and only one FhuD protein. Using X-ray crystallography we have solved the
structures of FhuD complexed with three different hydroxamate siderophores as well as with the antibiotic
albomycin. One of the complex structures solved is of FhuD with Desferal, which is in clinical use. In
addition the structure of the apo-form of the protein is being refined currently. In this presentation the
structure of FhuD will be discussed and compared to that of other periplasmic proteins involved in the
uptake of carbohydrates, amino acids, zinc etc. The various complex structures of FhuD show how one
protein is able to recognize seemingly widely divergent ferric-hydroxamate compounds. The unique
complex with albomycin reveals how in the future other Trojan horse antibiotic compounds may be
designed. Finally structural differences between the apo and holo forms of the protein will be described,
and an emerging picture about the interaction of FhuD with the cytoplasmic membrane transporter protein
will be presented. (supported by CIHR).
PODIUM 64
MECHANISM OF ACTION OF A SMALL RNA REGULATOR OF INTRACELLULAR IRON
METABOLISM IN BACTERIA
Eric Massé, Freddie Escorcia, Paula Wilderman, Urs Ochsner, Michael Vasil, David Fitzgerald, Peter
Fitzgerald, and Susan Gottesman, Laboratory of Molecular Biology, National Cancer Institute, Bethesda,
MD and University of Colorado Health Sciences Center, Denver, CO
A small RNA, FerA (previously called RyhB) was found in a genome-wide search for small RNAs
in E. coli. It is repressed by the Fe uptake repressor, Fur, in E. coli. When iron is limiting and repression
is lifted, FerA negatively regulates the expression of Fe-containing proteins of the TCA cycle, including
succinate dehydrogenase, aconitase, and fumarase, as well as SodB, an Fe-containing superoxide
dismutase, and ferritins, by pairing with the messages for these genes. Other targets of FerA action have
also been identified by microarray analysis. Thus, FerA action explains much of the previously observed
positive regulation of iron storage and iron use proteins by Fur and Fe. Positive regulation of iron storage
proteins and non-essential Fe-containing proteins has been seen in many other bacteria, as well, and
FerA homologues are present in the genomes of Salmonella, Klebsiella, Yersinia, and Vibrio. While no
small RNAs with similar sequences are found in more distantly related bacteria, in at least one,
Pseudomonas aeruginosa, two small RNAs have been identified that are repressed by Fur and
participate in regulation of the expression of genes involved in iron storage (e.g. bacterioferritin) and
response to oxidative stress (e.g. catalase). Thus, when iron is limiting, bacteria use small RNAs to
redirect cellular iron usage and storage patterns. FerA both represses translation of target genes, by
pairing with the mRNA in the region near the ribosome binding site, and targets the message for
degradation. This depends on an RNA chaperone, Hfq, as do the action of many other small RNAs.
Degradation of both the target message and FerA itself requires RNAseE. This system allows rapid
response to Fe depletion and rapid recovery when Fe is returned to cells.
PODIUM 65
HEMOPHORE DEPENDENT HEME ACQUISITION SYSTEMS IN GRAM NEGATIVE BACTERIA
Cécile Wandersman
Iron ions are essential for many metabolic pathways. Yet, iron is not readily available due to its low
solubility in the presence of oxygen and to its tight association to iron carrier proteins or to heme in
hemoproteins.
Therefore, iron assimilation is an essential function during microbial infection and it represents a
potential drug target.
Heme which is a major iron source is uptaken in Gram-negative bacteria by two principal pathways. One
involves the direct contact between heme or heme-containing proteins and specific bacterial cell surface
receptors. The second requires the secretion of hemophores, a family of proteins discovered in our
laboratory in 1994. These proteins present in several Gram negative bacteria such as Serratia
marcescens, Pseudomonas aeruginosa, Pseudomonas fluorescens, Yersinia pestis and Yersinia
enterocolitica, capture free heme or extract heme from heme carrier proteins, owing to their higher affinity
for heme, and return it to hemophore-specific outer membrane receptors. The S. marcescens hemophore
dependent heme acquisition system consists of the iron regulated has operon encoding HasR, the
hemophore-specific outer membrane receptor, HasA, the hemophore, HasD and HasE , the specific
inner membrane hemophore secretion proteins. The last gene of the has operon, hasB encodes a TonB
homolog. The 3D structure of the holo-hemophore and alanine mutagenesis have demonstrated that
heme iron atom is ligated by tyrosine 75 and histidine 32. Both heme-free and heme-loaded HasA bind
to HasR to the same or overlapping site with the same apparent Kd (5nM). We found that the binding of
HasA to HasR involves two β sheets located on the same side of HasA and we propose that this double
binding distorts the protein allowing heme transfer to the receptor. The activity of HasR is dependent on
a protein complex comprising the inner membrane proteins ExbB, ExbD, and TonB. This is a property
shared with several other outer membrane iron receptors whose 3D structures have been elucidated
showing an N-terminal domain closing the receptor pore and exposed to the periplasm, where it can
make contact with TonB. Whereas heme is uptaken as a whole through heme receptors, hemophores
are not transported and have to be stripped off at the cell surface: only the heme moiety being uptaken.
This implies the break of the very high affinity bond between the carrier protein and the prosthetic group
and heme transfer to the outer membrane receptor which has a lower affinity for this ligand. It is not
known whether this very first step requires TonB since heme striping and heme uptake processes were
never dissociated.
We showed that heme-hemophore uptake requires higher TonB-ExbBD complex level than free heme
uptake. This demonstrates that heme striping from the hemophore is TonB dependent. Emptyhemophore release from the receptor is concomittent with heme transfer from the hemophore to the
receptor. Thus, we propose a model in which the TonB-ExbBD dependent heme striping from the
hemophore induced a hemophore and or receptor conformational change leading to its drop off from the
receptor.. The step of heme discharge from heme carrier proteins is vital in many cellular functions but
poorly understood.
Two regulatory genes hasI and hasS are located upstream to the has operon in an iron regulated
transcription unit, and encode respectively a sigma factor and an anti-sigma factor. The binding of
heme-loaded HasA to HasR induces the has operon. Heme alone does not induce. This demonstrates
that the inducer and the transported substrate (heme) are different molecules. Since the inducer is a
molecule secrete by the bacteria, it can be considered as a new type of quorum sensing molecule.
PODIUM 66
MECHANISM AND REGULATION OF IRON-SULFUR CLUSTER BIOSYNTHESIS IN BACTERIA
Patricia Dos Santos, Deborah C Johnson and Dennis R. Dean
Department of Biochemistry, Virginia Tech, Blacksburg Virginia, 24060
Iron-sulfur clusters (Fe-S) clusters are known to have diverse biological roles involving: electron transfer,
the control protein structure, environmental sensing, modulation of gene regulation, and generation of
radicals. Such functional diversity reflects the chemical versatility of iron and sulfur, a feature that has led
to the suggestion that pre-biotic iron-sulfur complexes could have played an essential role in the
emergence of life on earth. The continuing importance of Fe-S clusters in today’s biological world can be
appreciated from the perspective that the three major processes required to sustain life on earth –
nitrogen fixation, photosynthesis and respiration – all involve the obligate participation of proteins that
contain Fe-S clusters. In spite of their rather simple structures, the biological assembly of Fe-S clusters is
a complex process involving the participation of a variety of proteins including a sulfur activation protein,
scaffold proteins, molecular chaperones, and a ferredoxin. In this overview lecture contributions from
several laboratories involving the identification of these proteins, their currently proposed functions, and
the regulation of their expression will be discussed.
PODIUM 67
A ROLE FOR MOLECULAR CHAPERONES IN FE/S CENTER BIOGENESIS
Elizabeth Craiga, Brenda Schilkea, William Waltera, Rafal Dutkiewiczb and Jaroslaw Marszalekb
a
Department of Biochemistry, 433 Babcock Drive, University of Wisconsin, Madison, Wisconsin 53706
(USA) bDepartment of Molecular and Cellular Biology, Faculty of Biotechnology, University of Gdansk,
80-822 Gdansk, Kladki 24 (Poland)
Mitochondria of the yeast S. cerevisiae contain a specialized system of molecular chaperones, including
the Hsp70 Ssq1 and the J-type cochaperone Jac1, which play an important role in the biogenesis of
Fe/S centers. In addition, mitochondria contain an abundant Hsp70 chaperone (Ssc1) involved in bulk
protein translocation and folding (1). Although the presence of a chaperone machinery involved in Fe/S
assembly has been conserved from bacteria to the mitochondria of eukaryotes, a recent phylogenetic
analysis suggested that both mitochondrial Hsp70s are more closely related to DnaK, the general Hsp70
chaperone of bacteria, than Hsc66, the Hsp70 involved in Fe/S center assembly (2). This phylogenetic
analysis leads to the prediction that the biochemical properties of Ssq1 might resemble those of DnaK
more than those of Hsc66. Therefore, we have begun a biochemical analysis of the Ssq1:Jac1 system to
better understand the role of these chaperones in Fe/S center biogenesis.
All Hsp70s bind nucleotide, but the stability of the interaction varies widely. DnaK binds nucleotide tightly
and requires a nucleotide release factor. GrpE, in vivo. Hsc66 binds nucleotide very transiently and has
no such requirement. Ssq1 binds nucleotide tightly, and shares the mitochondrial nucleotide release
factor Mge1 with Ssc1. In addition, the interaction between purified Mge1 and Ssq1 is ATP-sensitive, as
is its interaction with Ssc1 and the GrpE:DnaK interaction.
IscU has been shown to interact with Hsc66 as a client (substrate) protein (3). The Isu1 protein of yeast
mitochondria, which is highly homologous to IscU, interacts with Ssq1, suggesting important
fundamental similarities between Ssq1 and Hsc66 function. Thus, while Ssq1 shows similarities with
DnaK, its substrate specificity, and thus critical biological functions, are very likely to be analogous to
Hsc66. Thus the biological role of bacterial and mitochondrial chaperones in Fe/S center biogenesis are
likely to be similar.
(1) Craig, E and J. Marszalek, (2002) A specialized mitochondrial molecular chaperone system: A
role in formation of Fe/S centers, Cellular and Molecular Life Sciences. 59:1658-1665.
(2) Huynen M. et al (2001). The phylogenetic distribution of frataxin indicates a role in iron-sulfur cluster
protein assembly. Human Molecular Genetics, 10: 2463-2468.
(3) Hoff, K et al (2002)Hsc66 substrate specificity is directed towards a discrete region of the Fe/S cluster
template IscU. JBC 277:27353-9
PODIUM 68
FACTORS AFFECTING FE-S CLUSTER ASSEMBLY/DISASSEMBLY IN IRON REGULATORY
PROTEIN 1
W. E. Walden‡, A. Roy‡, N. Solodovnikova‡, and W. E. Antholine¶, ‡Department of Microbiology and
Immunology, University of Illinois at Chicago, Chicago, IL 60612 ¶Biophysics Research Institute, Medical
College of Wisconsin, Milwaukee, WI 53226
Iron regulatory protein 1 is interconverted between an IRE-binding protein (IRE-BP) and cytosolic
aconitase (c-acon) through the assembly and disassembly of a [4Fe-4S] cluster. Interconversion of IREBP with c-acon occurs in the cytosol of animal cells and is central to iron responsive regulation of gene
expression. High intracellular iron promotes Fe-S cluster assembly in IRP1 while low intracellular iron
promotes cluster disassembly. We have developed a model system in the yeast Saccharomyces
cerevisiae, which allows us to investigate cluster assembly/disassembly in IRP1. When expressed in
yeast, the interconversion of IRP1 between IRE-BP and c-acon is dynamic. Fe-S cluster disassembly in cacon in yeast grown aerobically is initiated by reactive oxygen species generated during normal
metabolism. Cluster disassembly initiated in this way proceeds through a [3Fe-4S] cluster intermediate.
The 3Fe intermediate appears to be subsequently converted to apo-IRP1, as opposed to undergoing
cluster repair and conversion back to [4Fe-4S] c-acon. Apo-IRP1 isolated from yeast cells is readily
converted to c-acon by incubation with iron alone, suggesting that apo-IRP1 can retain sulfide without
iron. This supports a model whereby reassembly of an Fe-S cluster in IRP1, converting it to c-acon, can
occur in response to changes in the chelatable cytosolic iron pool without going through the
mitochondrion-requiring de novo cluster assembly pathway. To investigate this further, we have isolated a
number of mutant yeast strains that are defective for conversion of IRP1 to c-acon. One such mutant
strain carries a mutation in an uncharacterized essential gene that we call CFD1 (for cytosolic Fe-S
cluster deficient 1). CFD1 encodes an ~30 kDa Mr protein that has sequence similarity to P-loop
ATPases. Mutation of CFD1 results in severely reduced activity of cytosolic Fe-S proteins, but does not
inhibit mitochondrial aconitase activity. Our results suggest that Cfd1p is a novel cytosolic Fe-S cluster
assembly factor. Further analysis of CFD1 and other yeast mutants that are defective for IRP1 to c-acon
conversion promises to shed important insight into the molecular mechanism of cytosolic Fe-S cluster
assembly and the regulation of IRP1 by iron. (Supported by grant DK47281 from the NIH.)
PODIUM 69
MITOCHONDRIAL FERRITIN: FUNCTIONAL AND EXPRESSION STUDIES.
S. Levi (1), B. Corsi (1), A. Cozzi (1), P. Santambrogio (1), A. Campanella (1), F. Sanvito (2), S.
Olivieri (2), G. Biasiotto (3), P. Arosio (3).
(1) Department of Biological and Technological Research and (2) Department of Pathology,
IRCCS H. San Raffaele, Milano, 20132 Italy; (3) Section of Chemistry, Faculty of Medicine,
University of Brescia, 25100 Italy.
Mitochondrial ferritin (MtF) is encoded by an intronless gene as a precursor protein with a long Nterminal extension that acts as an efficient mitochondrial targeting sequence. The sequence of the
processed peptide overlaps that of the full H ferritin with 70% identity, including the key residues
for ferroxidase activity (1). MtF homologs have been found in humans, primates and rodents.
Previous experiments showed that MtF expressed in HeLa cells is as active as the cytosolic
ferritin in taking up iron and that it reduces cytosolic iron availability (2). These findings and the
identification of MtF in iron-loaded mitochondria of sideroblastic anemia suggested that MtF might
protect mitochondria from the toxicity of local iron excess (3). Iron is considered a pro-oxidant
agent and we considered that MtF might have a broader function in protecting mitochondria from
oxidative damage. To test this hypothesis, we exposed transfectant HeLa cell clones
overexpressing MtF to various oxidative stresses and then analyzed cell viability and mitochondria
functionality. We first used H2O2 and a derivative of doxorubicin, doxoaglycone, as oxidants and
antimycin to block respiratory functionality. The results showed that under conditions that reduce
viability of untransfected Hela cells (which do not express MtF) the transfectant cells are more
viable and that, after cell treatment with antimycin, the mitochondrial aconitase activity is reduced
by about 30% in untrasfected cells, while remain unaltered in MtF expressing cells. The data
support a cytoprotective role for MtF, likely by sequestering mitochondrial free iron. To clarify the
functional role of MtF it is important to define its expression pattern. Present and previous data
showed that, unlike the ubiquitous H and L ferritins, MtF has a restricted tissue expression that is
not related to iron content. These tests have been extended by analysing expression patterns in
mice using a sensitive RT-PCR method. These studies indicated high levels of MtF mRNA in
testis, and lower levels in kidney, heart, brain and thymus. Next we elicited antibodies for the
mature recombinant mouse MtF. These antibodies did not cross-react with cytosolic ferritins. A
systematic analysis of the organs so far has shown the expression of MtF protein in testis
spermatides and interstitial cells, including the testosterone-producing Leydig cells, in pancreatic
Islet of Langherans and in neuronal cells of brain and spinal cord. In addition, MtF protein was
detected in mouse embryo at different stages of development from day E12.5 to E17.5. Using a
sensitive ELISA assay we found levels of MtF in the testis comparable to that of cytosolic L-ferritin
in tissue not specialized in iron storage. The results are in good agreement with those so far
obtained on human tissues that showed high level of MtF protein in spermatozoa and in ironloaded mitochondria of subjects with sideroblastic anemia. In conclusion, the results indicate that
MtF has a restricted tissue specific expression, not correlated with the iron storage function, and
possibly related to tissue mitochondria density or functionality. The protein seems to have an
active role in protecting the organelles from oxidative damage caused by excess iron.
Supported by grant n° GP0075Y01 from Telethon ( to S.L.)
(1) Drysdale J, Arosio P, Invernizzi R, Cazzola M, Volz A, Corsi B, Biasiotto G, Levi S.
Mitochondrial ferritin: a new player in iron metabolism. Blood Cells Mol Dis. 2002 NovDec;29(3):376-83.
(2) Corsi B, Cozzi A, Arosio P, Drysdale J, Santambrogio P, Campanella A, Biasiotto G, Albertini
A, Levi S. Human mitochondrial ferritin expressed in HeLa cells incorporates iron and affects
cellular iron metabolism. J Biol Chem. 2002 Jun 21;277(25):22430-7.
(3) Cazzola M, Invernizzi R, Bergamaschi G, Levi S, Corsi B, Travaglino E, Rolandi V, Biasiotto
G, Drysdale J, Arosio P. Mitochondrial ferritin expression in erythroid cells from patients with
sideroblastic anemia. Blood. 2003 Mar 1;101(5):1996-2000.
PODIUM 70
TARGETED DISRUPTION OF THE MURINE X-LINED SIDEROBLASTIC ANEMIA WITH ATAXIA
GENE, Abc7
C. Pondarré, D. Campagna, B. Antiochos, S. Clarke, E. Greer, R. Eisenstein, and M.D. Fleming,
Department of Pathology, Children's Hospital, Boston, MA and Department of Nutritional Sciences,
University of Wisconsin, Madison, WI, USA
Mitochondria play a unique role in iron metabolism. The early and late steps of porphyrin biosynthesis
occur in mitochondria and culminate in the incorporation of iron into protoporphyrin IX to form heme.
Mitochondria are also the site of the biosynthesis of iron-sulfur (Fe-S) clusters, some of which must be
exported to the cytoplasm for use by several cytoplasmic proteins, including iron regulatory protein 1
(Irp1). An ATP-binding cassette half transporter called Atm1 in the yeast S. cerevisiae has been
implicated in the maturation of cytoplasmic Fe-S proteins. Atm1 is thought in some manner to participate
the mitochondrial to cytoplasmic transfer of nascent Fe-S clusters, but it is unclear exactly what it
transports. Several studies have shown that one functionally orthologous protein in humans is ABC7. An
unusual inherited syndromic form of sideroblastic anemia, X-linked sideroblastic anemia with ataxia
(XLSA/A), is due to mild partial loss of function mutations in ABC7.
In order to further explore the functional consequences of loss of Abc7 in mammalian cells, we have
created a conditionally targeted Abc7 allele that rearranges to an inactive form in the presence of CRErecombinase transgenes, whose expression can be driven by ubiquitous and tissue-specific promoters.
Mice homo- and hemizygous for the conditional allele are viable, fertile and have no phenotype. Global
deletion of the Abc7 by a CRE transgene expressed early in embryogenesis results in early postimplantation lethality. Preferential deletion in the hematopoietic system using an inducible CRE
transgene results in a transient siderocytic anemia that is rapidly followed by death due to pancytopenia,
indicating that Abc7 is essential for hematopoiesis. Deletion in the liver gives rise to a mild,
morphologically distinctive iron overload phenotype that is associated with abnormalities of Irp1 RNA
binding activity.
In sum, our studies support the finding that ABC7 partial loss of function mutations are the cause of
XLSA/A and demonstrate multiple lethal phenotypes in animals lacking the protein entirely or in specific
tissues. Viable tissue-specific deletions of the gene should provide useful in exploring the relationship
between mitochondrial Fe-S biogenesis and cellular Fe metabolism.
PODIUM 71
GATED PORES IN FERRITIN: STRUCTURE AND CHELATOR TARGETS
Elizabeth C. Theil and Xiaofeng Liu
CHORI (Children's Hospital Oakland Research Institute). Oakland, CA 94609
The iron concentrated in ferritin is used for protein synthesis, which is exaggerated in liver after acute
blood loss or red cells during hemoglobin synthesis. Ferritin structure is unusually symmetrical and very
complex (1, 2), as if every feature has been honed by evolution to pack the multiple functions of catalysis,
mineralization and ion transport into the most efficient form. Recent studies have defined 8 gated pores in
ferritin that control access between the ferritin mineral, reductants and chelators. A large (30-fold)
increase in chelating the ferritin iron mineral, induced by NADH/FMN reduction, was associated with
selective unfolding of the helix-loop-helix region at the pores (3). Sets of 3 subunits form the pores seen
gated or mostly closed in crystals of wild type ferritin and open/unfolded in crystals of protein with Leu/Pro
substitution (3). Pore residues were defined by designed, site-directed mutation (SDM) based on three
criteria: phylogenetic conservation, location near the ferritin pores in 3D space and “orphan” function. The
SDM strategy was completely successful (10/10) in increasing the rate of iron removal from the ferritin
mineral (4). Residues which gate the ferritin pores are the hydrophobic pair Leu 134 (137)/Leu 110 (114),
the ion pair Arg 72(76)/ Asp 122(126) and the (C/D) loop of < 10 amino acids. Very low concentrations of
urea or guanidine (0.1-1mM) partly open the pore gates in wild type ferritin (intial rate increased 2-fold,
with multiphase Fe removal kinetics similar to Leu/Val substitutions (5). In contrast the gates are fully
opened (4-5 fold increase initial rate) with 1 M urea, which has linear, monophasic rates of iron removal
similar to the Leu/Pro substitution. Global ferritin structure is stable to 6M urea and heat to 85 degC. The
pores gates are very heat sensitive and were unfolded /melted at 53 degC, judged by CD spectroscopy;
in 1mM urea, unfolding/opening occurred at a lower temperature (43 degC). When the ferritin pore gates
2+
3+
are open, both Fe (bipyridyl) or Fe (desferal) chelators readily remove iron from the ferritin mineral (5).
The gated ferritin pores, which are closed by Leu/Leu and Arg/Asp subunit triples at the 3-fold axid, open
with “stiochiometric” amounts of chaotropes and temperatures that have no affect on the main protein
structure. Control of the reactions between the ferritin iron mineral and chelator/reductant by the highly
evolved, conserved structures that create the pore gates in ferritin are a model of principles for chelating
mineralized iron in other cellular locations such as lysosomes. Defining the properties of the gates which
control reactions between reductants, chelators and ferric iron in the cell is crucial to understanding
turnover of mineralized iron in cells and chelator efficacy during iron overload. Part support: NIH DK
20251.
REFERENCES:
(1) Theil, E.C (2001) in Handbook of Metalloproteins, eds. Messerschmidt, A., Huber, R., Poulos, T., and
Weighardt, K. (John Wiley & Sons, Chichester) pp. 771-781.
(2) Chasteen, N. D., Harrison, P.M. (1999) J. Struct. Biol.126:182.
(3) Takagi, H., Shi, D., Hall, Y., Allewell, N. M. & Theil, E. C. (1998) J. Biol. Chem. 273, 18685.
(4) Jin, W., Takagi, H., Pancorbo, N. M. & Theil, E. C. (2001) Biochemistry 40, 7525-7532.
(5) Liu, X., Jin, W, and Theil, E.C. (2003) Proc. Nat’l Acad. Sci. USA (in press).
PODIUM 72
CHEMOPREVENTIVE AGENTS AND XENOBIOTICS ACTIVATE THE MURINE FERRITIN H GENE
VIA A NRF2 DEPENDENT MECHANISM
EC Pietsch1,3, JY Chan4, FM Torti1,3, and SV Torti2,3
Department of Cancer Biology1, Biochemistry2, and the Comprehensive Cancer Center3, Wake Forest
University School of Medicine, Winston-Salem, North Carolina 27157; Department of Pathology4,
University of California, Irvine, California 92697
Chemoprevention is defined as the use of natural or synthetic compounds to block or suppress
carcinogenesis. Activation of phase II detoxification enzymes, such as glutathione-S-transferase, and
antioxidant enzymes, such as manganese superoxide dismutase and NAD(P)H:quinone oxidoreductase,
constitutes one mechanism of cancer chemoprevention. We hypothesize that the ubiquitously expressed
iron storage protein ferritin, a polymer consisting of 24 subunits of the H and L subunit type, may play a
crucial role in the chemopreventive response due to its ability to store iron in a bioavailable non-toxic form
and protect against oxidative stress.
Activation of phase II detoxification and antioxidant enzymes by chemopreventive agents has
been described to occur by two independent transcriptional mechanisms which are mediated by cisacting DNA enhancer elements known as the electrophile/antioxidant (EpRE/ARE) or xenobiotic (XRE)
responsive element. Transcriptional activation by these two mechanisms is not fully understood, but
evidence suggests that these mechanisms are dependent on two transcription factors known as NF-E2
related factor 2 (Nrf2) and the aromatic hydrocarbon receptor, respectively. Previously, we have
genetically defined an EpRE/ARE in the 5’ promoter region of the murine ferritin H gene that regulates
activation of ferritin H in response to oxidative stress. In addition, examination of 4.8 kb of the murine
ferritin H 5’ promoter region revealed the presence of 5 putative XRE sequences.
Our observation that ferritin H and L mRNA as well as ferritin H protein were induced by
prototypical chemopreventive agents (dithiolethiones) as well as by novel chemopreventive agents
(oxathiolene oxides) suggested that ferritin may function as a component of the chemopreventive
response. To explore the role of ferritin activation by chemopreventive agents via the EpRE/ARE and the
putative XRE sequences we performed genetic experiments utilizing ferritin H-reporter gene constructs.
Transient transfection of these constructs into NIH3T3 cells revealed that the EpRE/ARE mediates
induction of ferritin H in response to dithiolethiones. β-naphtoflavone (β-NF), a polycyclic aromatic
hydrocarbon, was used to assess the role of the XRE sequences. Deletion analysis demonstrated that
the XRE sequences are non-functional in mediating induction of ferritin H in response to β-NF. Rather,
this response was mediated by the EpRE/ARE. Treatment of Nrf2 wildtype and Nrf2 knockout primary
mouse embryo fibroblasts with β-NF and the dithiolethiones oltipraz [5-(2-pyrazinyl)-4-methyl-1,2-dithiole3-thione] and D3T [1,2-dithiole-3-thione] demonstrated that the transcription factor Nrf2 was necessary for
basal as well as dithiolethione and β-NF inducible expression of ferritin H. Electrophoretic mobility shift
assays demonstrated that Nrf2 binds to components of the ferritin H EpRE/ARE. Cotransfection of ferritin
H-reporter gene constructs with a Nrf2 dominant negative expression plasmid demonstrated that Nrf2
does indeed regulate expression of ferritin H via the EpRE/ARE. Additionally, evidence suggests that
ferritin L is similarly regulated.
Collectively, these results provide a mechanistic link between ferritin expression and
chemoprevention.
PODIUM 73
A NEW PATHWAY FOR MINERAL CORE FORMATION IN MAMMALIAN APOFERRITIN. THE ROLE
OF HYDROGEN PEROXIDE
*N. D. Chasteen‡, G. Zhao‡, P. Arosio§, S. Levi¶, C. Janus-Chandler‡, and F. Bou-Abdallah‡
‡
Department of Chemistry, University of New Hampshire, Durham, NH 03824, USA, and §Chemistry
Section, Faculty of Medicine, University of Brescia, 25123 Brescia, Italy, and ¶DIBIT, Protein Engineering
Unit, IRCCS-H. San Raffaele, Via Olgettina 58, 20132 Milano, Italy.
The reaction pathways by which the ferritins sequester and store iron as a stable FeOOH(s) mineral core
have been investigated for many years but their relative importance in the core mineralization have been
incompletely defined. In the present study, core formation in recombinant H- and L-subunit homopolymer
and heteropolymer ferritins and several site-directed H-subunit variants was investigated to determine the
role of H2O2 in this process at various levels of iron flux into the protein. Variants employed were: A2,
nucleation site variant (E61A, E64A, E67A); 222, ferroxidase center variant (E62K, H65G and also K86Q)
and S1, ferroxidase plus nucleation sites variant (E61A, E62K, E64A, H65G, E67A, D42A and also
K86Q). Stopped-flow absorption spectrometry, UV spectrometry and electrode oximetry revealed that the
mineral core forms by at least three pathways, not two as previously thought and that H2O2 is the oxidant
for Fe(II) in the third pathway. The three pathways correspond to the ferroxidase, mineral surface, and
the Fe(II) + H2O2 detoxification reactions, respectively:
2Fe2+ + O2 + 4H2O → 2FeOOH(core) + H2O2 + 4H+
4Fe2+ + O2 + 6H2O → 4FeOOH(core) + 8H+
2Fe2+ + H2O2 + 2H2O → 2FeOOH(core) + 4H+
(1)
(2)
(3)
The H-subunit catalyzed ferroxidase reaction 1 occurs at all levels of iron loading of the protein but
decreases with increasing iron added (48 to 800 Fe(II)/protein). The mineral surface mechanism
(equation 2) begins to grow in importance when 200 Fe(II) are added to the protein in a single addition,
indicating that an incipient core of ~200 Fe(III) is the amount required to sustain reaction 2. This reaction
becomes the dominant at 800 Fe(II)/protein whereas reaction 3 occurs largely at intermediate iron
loadings of 100 to 500 Fe(II)/protein. Some of the H2O2 produced at the ferroxidase center through
reaction 1 is consumed in the detoxification reaction 3; the 2/1 Fe(II)/H2O2 stoichiometry of reaction 3
minimizes hydroxyl radical production during mineralization, a finding confirmed by electron paramagnetic
resonance (EPR) spin-trapping experiments. Human L-chain ferritin and H-chain variants A2, 222, and
S1 lacking functional nucleation and/or ferroxidase sites deposit their iron largely through the autocatalytic
mineral surface reaction 2. H2O2 is shown to be an intermediate product of dioxygen reduction in L-chain
as well as in H-chain and H-chain variant ferritins, indicating that H2O2 is probably produced at the
surface of the growing mineral where it reacts with further Fe(II) to continue building the core.
*This work was supported by Grant R01 GM20194 from the National Institute of General Medical
Sciences (N.D.C.), by the Italian Ministry of the University and Research (MURST) Cofin-2000-01 (P.A.)
and by CNR Agenzia 2000 (P.A.).
PODIUM 74
NUCLEAR DISTRIBUTION AND FUNCTION OF FERRITIN
N. Surguladze1, K.J. Thompson3, M.G. Fried2 and J.R. Connor1, Penn State University, M.S. Hershey
Med. Center, 1Department of Neuroscience and Anatomy, 2Department of Biochemistry and Molecular
Biology, 3Harvard School of Public Health, Boston MA.
The iron storage protein, ferritin plays a key role in iron metabolism. Its ability to sequester iron gives
ferritin the dual function of iron detoxification and iron reserve. Most vertebrate ferritin occurs as hollow,
spherical assembles of 24 protein subunits: H (heavy, 21 kDa) and L (light 19 kDa), which are present in
varying ratios in different tissues. H-ferritin is involved in rapid uptake and release of iron due to its
ferroxidase activity, whereas L-ferritin, which does not have significant ferroxidase activity, is associated
with slow uptake and long-term iron storage. Ferritin is traditionally considered a cytoplasmic iron-storage
protein, but recent reports indicate, that it is also found in cell nuclei. The predominant form of nuclear
ferritin is H-ferritin. Using western blotting analysis with various polyclonal and monoclonal antibodies
ferritin was detected in whole nuclear extract, in the nuclear matrix, and the nucleoli fraction of rat and pig
liver tissue, as well as an SW 1088 astrocytoma cell line. The data indicate that in nuclei and in subnuclear fractions ferritin has a higher MW than cytoplasmic ferritin and that this state is stable in the
presence of high concentrations of SDS and 2-mercaptoethanol.
The interaction of ferritin with plasmid DNA was analyzed by electrophoresis mobility shift assay (EMSA).
No sequence-specificity was detected for any type of ferritin and no difference in the affinity of ferritin for
different DNA topoisomers was found. A study of kinetic interaction with various ferritin (recombinant Hferritin, ferroxidase mutant, apo ferritin, liver ferritin) was performed with super helical DNA (pUC 19). This
analysis revealed a time dependent nicking of DNA, which is also dependent on the type of ferritin that is
used. Incubation of liver ferritin with DNA produced a higher quantity of nicked DNA than other forms of
ferritin. Nicking of DNA was prevented by incubation with EDTA/EGTA and glycerol. This finding
implicates iron that was associated with ferritin as responsible for the DNA nicking. These studies expand
our previous observation that indicated that the role of nuclear ferritin is to protect DNA. This line of
research expands the function of ferritin to include genomic stability.
Supported by NIH grant DK54289.
PODIUM 75
EXPLORING THE BIOLOGICAL FUNCTION OF FERRITINS AND PROTEINS OF IRON METABOLISM
IN HELA CELLS BY THE USE OF SMALL INTERFERING RNAS.
P. Arosio, A. Cozzi, S. Levi, B. Corsi, G. Biasiotto, G.M. Gerardi, P. Santambrogio, A. Campanella, I.
Zanella
Department of Biological and Technological Research, IRCCS H. San Raffaele, 20132 Milano, Italy;
Dipartimento Materno Infantile e Tecnologie Biomediche, University of Brescia, Viale Europa 11, 25123
Brescia, Italy;
We have previously analysed the biological functions of ferritins in HeLa cells by producing transfectant
clones harbouring cDNAs controlled by the inducible tetracycline promoter (1). They showed that the
primary effect of H-ferritin upregulation is a reduction of cellular iron availability, which causes slower rate
of proliferation and increased resistance to oxidative damage by H2O2. In addition they showed that high
levels of H-ferritin reduce the apoptotic effect of TNF-alpha with a mechanism that does not seem to
involve iron availability (2). In contrast, L-ferritin upregulation did not show significant effects on cellular
iron availability, while increased cell proliferation rate. The down-regulation of the genes should produce
the opposite effects and confirm the biological significance of the findings. In principle this is now possible
by the use of siRNA technology, which is based on the transfection with short double strand (ds) RNAs
that guide the sequence-specific degradation of complementary-target RNAs. Ferritins are good models
to test this technology, since their mRNAs are abundant and protein expression is regulated at a posttranscriptional level. First we produced by in vitro transcription 4 ds RNAs of 20 nt complementary to
different regions of the ferritin transcripts. Ferritin suppression was quantified using specific ELISA
assays. This showed that the four ds-siRNAs for H-ferritin reduced the target protein level to 10-20% of
the control after 2 days of transfection. Similarly, the 4 siRNAs for L-ferritin reduced the target protein
level to 20-40% of background. We chose the most effective siRNAs for further studies. When used
alone, the transfection with H-siRNA resulted in a 4-fold repression of H-ferritin, which was accompanied
by a 3-fold increase in L-ferritin, a strong repression of transferrin receptor 1 and a down-regulation of
IRPs activity. The co-transfection with L-siRNA resulted in a suppression of both ferritin types, and an
even more evident down-regulation of TrR1. In contrast, the transfection with L-siRNA alone did not
modify either of the parameters of iron availability. The data confirm that H ferritin level regulates cellular
iron availability, while L-ferritin assists the function of H-ferritin. In addition we found that H-siRNA
increased the rate of cellular proliferation and decreased the resistance to oxidative damage induced by
H2O2, consistent with the results of H-ferritin upregulation. In contrast, L-siRNA reduced by about 50%
the rate of cell proliferation, and this effect was even increased by the co-transfection with H-siRNA. The
data confirm the observation that L-ferritin level is positively related to cell proliferation, with a mechanism
that remains to be studied. By cloning the DNA sequences into the pSilencing I vector that contain the
pol III U6 promoter, we were able to select 26 clones that express constitutively H-siRNA with a
suppression of ferritin level ranging from 27 to 95%. Analysis of the clones is in progress. In addition we
plan to present data on the effect of transferrin receptor down-regulation of ferritin synthesis. In
conclusion, present data show that siRNAs are highly effective in suppressing ferritins and other proteins
of iron metabolism, and indicate that, although H-ferritin is a key regulator of iron availability, cell remain
viable even when its level is very low.
(1) Cozzi A, Corsi B, Levi S, Santambrogio P, Albertini A, Arosio P. (2000) Overexpression of wild type
and mutated human ferritin H-chain in HeLa cells: in vivo role of ferritin ferroxidase activity. J Biol
Chem. 275: 25122-25129
(2) Cozzi A, Levi S, Corsi B, Santambrogio P, Campanella A, Gerardi GM, Arosio P. (2003) Role of iron
and ferritin in TNFa-induced apoptosis in HeLa cells. FEBS Lett. in press
PODIUM 76
RECENT DEVELOPMENTS WITH NON TRANSFERRIN BOUND IRON MEASUREMENT
R C Hider, Z D Liu, D Devanur, D Y Liu, King’s College London
The past decade has witnessed an increasing interest in non transferrin bound iron (NTBI) and its
possible association with an abnormal body distribution of iron and direct toxicity as a result of the
production of reactive oxygen species. The detection of NTBI is a difficult task and methods based on
liquid chromatography, colorimetric and fluorimetric assays, atomic absorption, and the generation of free
radicals have been investigated. We have directly compared a number of these methods.
Recently evidence has been presented which suggests that NTBI exists as oligomeric iron oxide either in
free solution or bound to albumin. We have compared the influence of several iron chelating reagents on
the mobilisation of oligomeric iron in the presence of physiological levels of citrate and albumin. Studies
include, nitrilotriacetic acid, oxalic acid, deferiprone, desferioxamine, bathophenantroline disulphonate
and fluorescent hydroxypyridinones. These mobilisation studies have also been investigated in the
presence of vitamin C. It is essential that scavengers utilised for the mobilisation of NTBI do not interfere
with transferrin-bound iron. This phenomenon has been systematically investigated using acrylamideurea gels to separate the four major transferrin iron complexes.
We conclude from these studies that fluorescent iron scavenging molecules possess the greatest
potential for NTBI measurement.
PODIUM 77
BIOCHEMICAL AND BIOLOGICAL CHARACTERIZATION OF MUTATED L-FERRITIN
INVOLVED IN NEUROFERRITINOPATHY, A RARE NEURODEGENERATIVE DISORDER
P. Santambrogio (1), A. Cozzi (1), B. Corsi (1), A. Campanella (1), P. Arosio (2), S. Levi (1).
(1) Department of Biological and Technological Research, IRCCS H. San Raffaele, Milano, 20132
Italy; (2) Section of Chemistry, Faculty of Medicine, University of Brescia, 25100 Italy.
Neuroferritinopathy is a recently discovered disorder with autosomic dominant transmission
associated to a single base insertion in the gene for ferritin L-chain (1). This modifies the 20 Cterminal residues of the peptide. The mutation apparently causes an abnormal regulation of iron
homeostasis in the basal ganglia of the brain and a reduction in serum ferritin levels. Of the two
subunits that compose human cytosolic ferritins, the L-one is considered the less active, since it
lacks of a ferroxidase center, and its upregulation in the hereditary hyperferritinemia cararact
syndrome, also dominant, has not evident effect on iron metabolism. Therefore we reasoned that
the characterization of this disorder might unveil functions of L-ferritin so far undetected. To clarify
the molecular basis of the neurological disorders we first produced the recombinant mutant
protein, which we named Ln. When expressed in E.coli, most of it (60-70%) was recovered in the
insoluble cellular fraction, while the remaining 30% was present as assembled protein shells.
Renaturation studies of the insoluble fraction indicated that the protein alone has a low tendency
to assemble into ferritin shells, while heteropolymers could be obtained by co-renaturing it with H
and L ferritin chains. The purified soluble Ln-ferritin was unable to incorporate iron in vitro and
seemed to undergo to a rapid and partial proteolytic degradation. The data are consistent with
disruption of the interactions along the 4-fold symmetry axes caused by the mutation, which have
a key role in the ferritin assembly pathway. In addition, they suggest that the C-terminus may be
exposed to outer surface, instead of being accommodated in the cavity as in the wild type protein.
To study le biological function of the mutant, we produced a HeLa clone that expresses Ln under
the control of the inducible tetracycline promoter. We found that in these cells the Ln peptide co55
assembled with the endogenous H and L, making heteropolymers functional in Fe incorporation.
However, the cells respond to the Ln production with an about 2-fold up-regulation of H and L
ferritins and a significant down-regulation of TfR1. This finding indicates that Ln perturbs cellular
iron regulation and increases its availability. The study of the cells is complicated by the presence
of the endogenous L-ferritin, which is hardly separate from Ln mutant. To try to simplify it, we
developed siRNAs targeted to the 5’UTR of the transcript, which is specific to the endogenous
ferritins. Preliminary results indicate that it is effective in reducing L-ferritin, and the cells so
treated show a remarkably abnormal incorporation of 55Fe cellular. In conclusion, the L-ferritin
mutation seems to produce a protein that interferes with the cellular regulation of iron metabolism.
The study of this rare pathology may be relevant for the clarification of the iron involvement in
neuro-degenerative disorders.
(1) Crompton DE, Chinnery PF, Fey C, Curtis AR, Morris CM, Kierstan J, Burt A, Young F,
Coulthard A, Curtis A, Ince PG, Bates D, Jackson MJ, Burn J. Neuroferritinopathy: a window on
the role of iron in neurodegeneration. Blood Cells Mol Dis. 2002 Nov-Dec;29(3):522-31.
PODIUM 78
FUNCTIONAL ANALYSIS OF THE FHUA AND FECA TRANSPORT PROTEINS
V. Braun, M. Braun, F. Endriß, H. Killmann, S. Mahren, M. Ogierman, A. Sauter, Microbiology /
Membranephysiology, University of Tuebingen, Germany.
FhuA and FecA serve as active transport proteins for ferrichrome and ferric citrate, respectively, across
the outer membrane of Escherichia coli. FhuA and FecA are multifunctional proteins since FecA is also
involved in a signaling cascade that initiates transcription of the ferric citrate transport genes, and FhuA
also transports the antibiotics albomycin and rifamycin CGP 4832, microcin J25, and serves as receptor
for colicin M and the phages T1, T5, Φ80, and UC-1. The crystal structures reveal for both proteins two
structural domains, a ß-barrel composed of 22 antiparallel ß-strands and a globular domain that
completely closes the channel formed by the ß-barrel. Binding of ferrichrome to FhuA and of ferric citrate
to FecA cause large structural changes in the proteins which, however, do not open the channels. This is
thought to occur upon interaction of the transport proteins with the TonB protein that is inserted with the
N-proximal end in the cytoplasmic membrane, whereas most part of the protein is located in the
periplasm. Within the cytoplasmic membrane, TonB interacts with two other proteins, ExbB and ExbD,
and all three proteins are required for energy transduction between the cytoplasmic membrane and the
outer membrane transport proteins. The source of energy to open the channels is the proton motive force
across the cytoplasmic membrane which is thought to induce an “energized conformation” in TonB which
is required to open the channels when TonB interacts with the transport proteins. The binding site of
TonB at the transport proteins is the so-called TonB box, previously demonstrated by genetic and
biochemical means to interact with TonB. Data will be presented which show that the TonB box of FecA is
not located close to the N-terminus as is the case for all the other outer membrane transport proteins.
Cystine disulfide bridges formed in vivo between introduced cysteine residues indicate that the TonB box
of FecA (residues 80-84) is exposed to the periplasm and interacts with residues 160, 162 and 163 of
TonB. The flexible N-proximal end of FecA interacts in the periplasm also with the FecR protein, a signal
transducing protein across the cytoplasmic membrane that conveys transcription initiation of the ferric
citrate transport proteins when ferric citrate binds to FecA. This additional function of FecA and interaction
with FecR moved the TonB box from the N-terminus to residues 80-84. Based on the crystal structures of
FhuA and FecA, additional mutations were introduced into the globular domains, the surfaces of contact
between the globular domains and the ß-barrels, and into cell surface-exposed loops. The functional
consequences of these mutations will be reported. Most mutations that affect FhuA function reduce or
abolish all FhuA activities. There are only a few mutations which affect a single activity and leave the
others untouched. Large changes in the N-proximal region of the globular domain of FhuA show little
influence on FhuA activity as long as the TonB box is still intact. Deletion of the two surface loops of FecA
which upon binding of ferric citrate close the access to the ferric citrate binding site abolish FecA activity
and signal transduction. Deletion of FhuA surface loops affects the various functions differently. The
crystal structures serve as a useful guide for mutational analysis of functions but not all predictions
derived from the crystal structures turn out to be true.
PODIUM 79
GLOBAL IRON-DEPENDENT GENE REGULATION IN ESCHERICHIA COLI: A NEW MECHANISM
FOR IRON HOMEOSTASIS*
S. C. Andrews1, J. P. McHugh1, H. Abdul-Tehrani2, D. A. Svistunenko3, R. K. Poole2, C. E. Cooper3, F.
Rodríguez-Quiñones1
1
School of Animal & Microbial Sciences, University of Reading, Reading, RG6 6AJ, UK; 2Department of
Molecular Biology & Biotechnology, The University of Sheffield, Sheffield, S10 2TN, UK;
3
Department of Biological Sciences, University of Essex, Colchester, Essex, CO4 3SQ, UK
Organisms generally respond to iron deficiency by increasing their capacity to take up iron and by
consuming intracellular iron stores. Escherichia coli, in which iron metabolism is particularly well
understood, contains at least seven iron-acquisition systems encoded by 35 iron-repressed genes, as
well as at least two iron-induced, iron-storage proteins. This Fe-dependent regulation is mediated by a
transcriptional repressor, Fur (ferric uptake regulation), which also controls genes involved in other
processes such as the TCA cycle, pathogenicity and redox-stress resistance. We have performed a
macroarray-based global analysis of iron- and Fur-dependent gene expression in E. coli. The
transcription profile of the wildtype, grown in rich broth, was compared with those of both the wildtype
grown with an Fe2+ chelator (200 μ M 2,2´-dipyridyl) and a fur mutant grown without chelator. Samples
were taken at OD650nm 1.0, corresponding to late-exponential or early post-exponential phase. No major
growth differences were observed for the three experimental conditions. Since members of the Fur
modulon are regulated by iron and Fur in conjunction, only those genes that were ≥twofold regulated by
both dip and the fur mutation were considered further. Accordingly, 106 genes were found to be
regulated by the Fe2+-Fur complex of which 58 were repressed and 48 induced. These genes fall into
three major categories: iron metabolism; energy production; and miscellaneous/unknown. In addition to
most of the known Fe-uptake genes in E. coli, 14 ‘unknown’ genes with probable functions in iron
acquisition were also identified. These were initially recognized either because of their chelator- and furdependent expression or by their chromosomal co-location with such genes. It’s speculated that these
genes could specify four new iron-uptake pathways for E. coli.
Interestingly, a large group of ‘energy metabolism’ genes was found to be iron and Fur induced.
Many of these genes encode iron-rich respiratory complexes that together account for 144 iron atoms per
protein. This iron- and Fur-dependent regulation appears to represent a novel iron-homeostatic
mechanism whereby the synthesis of many iron-containing proteins is repressed under iron-restricted
conditions. This mechanism thus accounts for the low iron contents of fur mutants and explains how E.
coli can modulate its iron requirements. Analysis of 55Fe-labeled E. coli proteins from whole-cell extracts
revealed a marked decrease in iron-protein composition for fur mutants, and visible and EPR
spectroscopy showed major reductions in cytochromes b and d levels, and in iron-sulfur cluster contents
for the chelator-treated wildtype and/or fur mutant, correlating well with the array and quantitative RT-PCR
data. In combination, the results provide compelling evidence for the regulation of intracellular iron
consumption by the Fe2+-Fur complex.
*
This work was supported by the BBSRC through a PhD studentship to JM and project grants to SCA and
to RKP, by the Wellcome Trust through a grant to CEC and by the Iranian Government through a PhD
studentship to HAT.
PODIUM 80
FHUF– THE FIRST EXAMPLE OF A SIDEROPHORE-REDUCTASE
Berthold F. Matzanke1£,Klaus Hantke2 and Stefan Anemüller3£
1
Isotopenlabor TNF,£) Universität zu Lübeck, Ratzeburger Allee 160, D-23538 Lü and eck,
Germany;2Mikrobiologie/Membranphysiologie, Universität Tübingen, D-72076 Tübingen Germany and 3
Institut für Biochemie. e-mail: matzanke@physik.uni-luebeck.de
Fe3+ siderophores are an important iron source for bacteria. In fact, it is known, that ferric iron complexed
by the siderophore is reduced inside the cell (1, 2, 3) and the deferrated siderophore is in most cases
rapidly excreted (4). Siderophores have a much lower affinity for Fe2+ than for Fe3+ and the kinetics for
ligand exchange for high-spin Fe2+ are much faster than for Fe3+ (5). Therefore, reduction of ferric
siderophores accompanied by ligand exchange is an excellent mechanism for intracellular iron release.
However, against the expectations of many scientists no specific Fe3+ siderophore reductases could be
identified so far (1).
The fhuF mutant of E. coli showed reduced growth on plates with ferrioxamine B as iron source, although
no defect in siderophore uptake experiments was observed. Moreover, these growth conditions lead to a
derepression of various Fur dependent iron transport systems (6-8). The isolated protein was
characterized by Mössbauer spectroscopy uncovering an unusual [2Fe-2S]-protein (9).
In the present study the redox potential E1/2 of FhuF was determined by EPR redox titration. E1/2 is 310±25mV vs NHE (definition). According to the Nernst equation, the effective range for a
thermodynamically favorable redox reaction is ±59mV with respect to the corresponding reduction
potential. If, in addition, the uncertainty of the experimental data is taken into account it is obvious that
reduction of coprogen, ferrichrome and to lesser extent of ferrioxamine B is achievable,. In an in-vitro
Mössbauer spectroscopic analysis we could prove, in fact, reduction of the ferrichrome type siderophore
ferricrocin and re-oxidation of FhuF. Finally, removal of iron from various siderophores has been
monitored by extraction of Fe3+ siderophores from cells of fhuF mutants and parent strains of E.coli.
These experiments provide evidence for a significant decrease of intracellular reductive iron-removal from
a variety of hydroxamate-type siderophore complexes in vivo, i.e. in fhuF mutants To our knowledge, this
report presents the first experimental evidence for a siderophore-specific reductase in microorganisms.
(1) Fontecave, M., Coves, J. and Pierre, J. L. (1994) Biometals, 7, 3-8
(2) Matzanke, B.F., Berner, I., Bill, E., Trautwein, A.X., Winkelmann, G. 1991 BiolMetals 4, 181-185
(3) Matzanke, B.F., Müller, G.I. and Trautwein, A.X. (1989) Eur.J.Biochem. 183, 371-379
(4) Hartmann, A., Fiedler, H.P. and Braun, V. (1979) Eur. J. Biochem. 99, 517-24.
(5) Wilkins, R.G., Kinetics and Mechanisms of Reactions of Transition Metal Complexes (1991) VCH
Weinheim, Germany 2nd ed.
(6) Sauer, M., Hantke, K., and Braun, V. (1987) J. Bacteriol. 169, 2044–2049.
(7) Rohrbach, M.R., Braun, V., and Köster, W. (1995) J. Bacteriol. 177, 7186-7193
(8) Zheng M, Wang X, Doan B, Lewis KA, Schneider TD, Storz G. (2001) J. Bacteriol. 183, 4571-4579
(9) K.Müller, B.F. Matzanke, V. Schüneman, A.X. TRautwein, K.Hantke (1998) Eur. J. Biochem. 258,
1001-1008
PODIUM 81
SALMOCHELINS ARE THE FIRST SUGAR CONTAINING SIDEROPHORES AND MAJOR IRON
CHELATORS OF SALMONELLA ENTERICA
G. Winkelmann1, K. Hantke2, G. Nicholson3, and W. Rabsch4,
1
Mikrobiologie/Biotechnologie, 2Mikrobiologie/Membranphysiologie, 3Institut für Organische Chemie,
Universität Tübingen, Auf der Morgenstelle 28, Tübingen, Germany and 4Robert Koch Institut, Bereich
Wernigerode, Germany
Salmochelins have been identified as the major siderophores of Salmonella enterica. The siderophores
were purified from culture supernatants of S. enterica strains and certain pathogenic E. coli strains by ion
exchange chromatography (DE52 cellulose), gel filtration (Biogel P2) and preparative HPLC (C18
reversed phase). Mass spectrometry using FTICR-MS/MS and GC-MS as well as genetic data revealed
two tentative structures of Salmochelin S1 and S2, possessing alternate 2,3-dihydroxybenzoylserine and
glucose units:
OH
OH
CO
NH
HO
CH2
CO
[ Glucose ]
O
CH2
OH
OH
OH
OH
CO
CO
NH
NH
[ Glucose ]
CO
S1
O
CH2
COOH
S2
An iroBC mutant of S. enterica was unable to synthesise salmochelin which indicated that the iro genes
are required for salmochelin synthesis. Growth promotion and transport studies showed that the IroN
protein, encoded by genes of the iroA locus is the cognate outer membrane receptor protein for ferric
salmochelins
References:
Hantke, K., Nicholson, G., Rabsch, W. and Winkelmann, G. (2003) Proc. Natl. Acad. Sci. U.S.A. (in
press).
PODIUM 82
NOVEL FEATURES OF GENE REGULATION FOR IRON NUTRITION AND UPTAKE IN THE
SYMBIOTIC BACTERIA RHIZOBIUM.
Andrew W. B. Johnston, Jonathan D. Todd, Margaret Wexler, Kay H. Yeoman. School of Biological
Sciences, University of East Anglia, Norwich NR4 7TJ, U. K.
α-Proteobacteria known as “rhizobia” induce root nodules on legumes, where they reduce (fix) N2 to NH3,
a process of huge agronomic and ecological importance. Iron is very important for rhizobia, since
nitrogenase and many ancillary proteins required for N2 fixation are Fe-proteins. Our recent studies show
that in free-living Rhizobium leguminosarum (Rl), which nodulates peas, clover and beans, the
mechanisms of Fe uptake and of Fe-dependent gene regulation differ markedly from those in (eg.)
Pseudomonas and E. coli.
Rl has a close homologue of Fur, the “global” Fe-responsive transcriptional regulator of many bacteria
and cloned Rl fur corrects a Fur- mutant of E. coli (1). Importantly, though, Fur- mutants are unaffected in
regulating many Rl operons involved in Fe uptake, which are normally expressed at high levels only in
Fe-depleted conditions. These include those for import (fhuCDB and fhuAF) and synthesis (vbsADL,
vbsGSO, vbsC) of the native Rl siderophore vicibactin (VB), the tonB gene, the hmuPSTUV operon which
specifies haem uptake and two operons that specify Fbp-like, Fe2+ ABC transporters. (NB; hitherto, haem
uptake and Fbp systems were thought to be confined to pathogens. Thus, another unusual feature of Rl
is its wide range of Fe sources.
If rhizobial Fur is not the key Fe-responsive transcriptional regulator, what is? We showed (2) that
mutations in a gene, rirA, caused constitutive expression of all known Fe-responsive Rl operons. RirA has
no sequence similarity to Fur or DtxR, but modeling and biochemical experiments indicate that RirA is a
DNA-binding protein, but its recognition sites are unknown. There are very close RirA homologues in
other genera, but only in very closely related rhizobia (Sinorhizobium and Mesorhizobium), the plant
pathogen Agrobacterium and the mammalian pathogen Brucella. Interestingly, in all these cases, their
rirA-like gene abuts genes involved in Fe uptake or storage.
Proteomics of R. leguminosarum now show that RirA- mutants fail to regulate >40 genes, some being
present at higher and some at lower levels in RirA mutants, compared to wild type. Some of these are
connected with Fe metabolism, but not directly in Fe uptake. Thus, directly or indirectly, RirA may have
“taken over” from Fur as the major Fe-responsive regulator in Rhizobium and, maybe, in some of nearrelatives, eg. Brucella.
Significantly, all available deduced proteomes of eubacteria contain a less closely related version of RirA.
None of these other “ghost-RirA” proteins has a ratified, regulatory (or any other) function. But, all have a
C-terminal cysteine cluster that in Rl RirA is needed for its regulatory function and which may be involved
in metal-binding. Rl itself also has a second, ghost version of RirA in its deduced proteome, suggesting
that RirA evolved into a specialized Fe-responsive regulator after gene duplication. We are now trying to
ascertain a role for the possible progenitor of the RirA in some taxonomically diverse bacterial genera.
In addition to the “global” action of RirA, we identified a “local” effector of Fe-dependent gene regulation in
Rl. This is RpoI, a σ factor that resembles PvdS of Pseudomonas, which initiates in vivo and in vitro
transcription of two Rl vbs operons involved in siderophore synthesis. Transcription of rpoI itself is
enhanced in cells grown in low-Fe media, this being mediated, in part, by a likely repressor-binding site
(for RirA?) near the rpoI promoter. However, expression of RpoI is also affected by mutations in a region
3’ of the rpoI promoter possibly through a novel form of post-transcriptional regulation.
An overall scheme for the Fe regulon of Rhizobium and, perhaps, other α-proteobacteria will be
presented, showing that this large group of bacteria may respond to Fe availability in a way that differs
significantly from that of some well-studied γ-proteobacteria.
(1) Wexler, M et al., (2003) Microbiology (in press). (2) Todd, JD et al. (2002). Microbiology 148:
4059-4071.
PODIUM 83
A NOVEL GENE REQUIRED FOR VACUOLAR ACIDIFICATION IDENTIFIED THROUGH A
SACCHAROMYCES CEREVISIAE GENOME WIDE ANALYSIS OF IRON-DEPENDENT GROWTH
J Kaplan, DM Ward, SL Shiflett and SR Davis-Kaplan.
Division of Immunology and Cell Biology, Department of Pathology, School of Medicine, University of
Utah, Salt Lake City, Utah 84132
A genome wide screen in the budding yeast a S. cerevisiae examined the ability of 4,792
homozygous viable deletion strains to grow on low iron media. Strains that were unable to grow on ironrestricted media were defective in either the high affinity iron transport system (FET3, FTR1) or the iron
sensing transcription factor, AFT1 or in genes required for the assembly of the transport system. We
discovered that a deletion in CWH36, a pioneer gene, results in lack of growth on iron-limited medium.
Further phenotypes associated with deletion of CWH36 include: cell wall defects as shown by increased
sensitivity to Congo red and Calcofluor white, distorted vacuole morphology and altered FM4-64
trafficking to the vacuole. We determined that the ∆cwh36 strain has a defect in iron transport resulting
from a copper-deficient apoFet3p present on the cell surface. The multiple phenotypes shown in ∆cwh36
are shared by mutants defective in vacuolar acidification. The ∆cwh36 strain has defective vacuolar
acidification, as shown using the viral dyes LysoSensor Green and Quinacrine. An epitope tagged
Cwh36p was localized predominantly to the vacuolar membrane in addition to smaller vesicular
compartments. These data suggest that CWH36 encodes a membrane protein required for the assembly
+
or activity of the vacuolar H /ATPase. These results again show the importance of vesicular pH in the
copper loading of apoFet3p and the usefulness of a genome-wide screen in defining the physiological
role of unknown genes. (This work was supported by a grant from the National Institute of Health (USANIDDK-DK30534).
PODIUM 84
THE UNIQUE PROCESS HEME CRYSTALLIZATION IS A METHOD OF IRON SEQUESTRATION IN
PLASMODIUM THAT IS VULNERABLE TO CHEMOTHERAPY BY THE QUINOLINES
D. Sullivan
The Malaria Research Institute, W. Harry Feinstone Department of Molecular Microbiology and
Immunology, Bloomberg School of Public Health, Johns Hopkins University
The intraerythrocytic Plasmodium parasite invades a host cell containing 20 mM heme iron in
hemoglobin. Most of the erythrocyte cytosol is ingested via an acidic lysosome-like organelle where
hemoglobin is efficiently degraded as a source of amino acids. The Plasmodium parasite lacks heme
oxygenase activity and also must make its own heme while in the erythrocyte, instead of scavenging both
host heme iron or heme. The toxic reactive heme in the acidic oxygen-rich digestive vacuole is
crystallized into the molecule called hemozoin which is the target of the antimalarial quinolines. The
covalent linkage of the side chain propionate carboxylate group to the adjacent heme iron forms the head
to tail dimer crystal structurally defined by Bohle and colleagues. The exact mechanism of intracellular
formation is still being investigated although lipids and Plasmodium histidine-rich proteins are able to
initiate crystal formation. The P. falciparum derived Histidine-rich protein II, which binds heme and
initiates haemozoin formation, is present in the digestive vacuole. This protein has a 34% histidine amino
acid content and is also the basis for the rapid diagnostic test for malaria infection. PfHRP II and PfHRP
III are sufficient, but not necessary for haemozoin formation as a laboratory clone lacking both still makes
the heme crystals. PfHRP II and III bind copper, zinc and nickel much more than oxidized or reduced iron.
The hemozoin crystals are identical to 
-hematin made chemically under acidic conditions. The
morphology of hemozoin from mammalian and avian Plasmodium species differs by Field Emmission
Inlens Scanning Electron Microscopy. The Reduvid bug and the trematode Schistosoma also make βhematin.
Crystallization of heme into hemozoin is the target of the antimalarial quinolines like chloroquine
or quinidine. Both intraerythrocytic zinc protoporphyrin IX and the quinolines drugs can inhibit
crystallization by binding to hemozoin. Zinc protoporphyrin IX and quinoline binding is saturable, specific,
pH and time dependent, although only quinoline binding is reversible.
Characterization of both the macromolecular formation of heme crystals and their inhibition by
available antimalarials is important in further delineating both the mechanism of action and the
mechanism of drug resistance for the Plasmodium parasites that cause malaria.
PODIUM 85
MUTATIONAL ANALYSIS OF FERRITIN AND FERROPORTIN GENES IN PATIENTS WITH
UNEXPLAINED HYPERFERRITINEMIA. AN INTERIM REPORT
Laura Cremonesi,1 Barbara Foglieni,1 Francesca Ferrari,1 Nadia Soriani,1 Gaetano Bergamaschi,2
Daniela Caprino,3 José Antonio García Erce,4 Silvia Fargion,5 Renzo Galanello,6 Maurizio Longinotti,7
Sonia Levi, 1 Maurizio Ferrari,1 Paolo Arosio,8 Mario Cazzola.2 1IRCCS H S. Raffaele, Milan, Italy;
2
University of Pavia & IRCCS Policlinico S. Matteo, Pavia, Italy; 3Istituto Gaslini, Genova, Italy; 4Hospital
Miguel Servet, Zaragoza, Spain; 5University of Milan, Italy; 6University of Cagliari, Italy; 7University of
Sassari, Italy; 8University of Brescia, Italy.
A large body of evidence indicates that the level of serum ferritin parallels the concentration of
storage iron within the body, regardless of the cell type in which it is stored. This relationship of serum
ferritin to body iron stores, however, is altered in inflammatory states and liver disease, conditions that
can disproportionately elevate the circulating protein. In addition, elevated serum ferritin levels may also
be a result of translational pathophysiology. Hereditary hyperferritinemia/cataract syndrome (HHCS)
arises from various point mutations or deletions within the iron-responsive element (IRE) in the 5’-UTR of
the L-ferritin mRNA that results in increased efficiency of L-ferritin translation. It is currently unclear
whether a translation genetic disorder of H ferritin synthesis exists.
As of December 31, 2002, we studied 50 consecutive subjects referred to us for unexplained familial or constitutional - hyperferritinemia. In all cases the following diagnoses had been previously
excluded: increased body mass index, parenchymal iron overload, inflammation, and liver disease. In
order to detect mutations in ferritin and ferroportin genes we employed denaturing HPLC (DHPLC), a fast
and automated technique for the screening of DNA variations with potential for large-scale application.
This approach was first applied to the screening of L-ferritin IRE. The DHPLC system unambiguously
identified all the 12 accessible mutations, including the difficult C-G transversions, and singled out 22
abnormal patterns (22 individuals belonging to 11 families) in the scanning of DNA samples from the
above 50 subjects. All 22 abnormal patterns were found to reflect mutations in the L-ferritin IRE
sequence, including three not yet reported (C36G, A37G, and A37T). Most of these patients had cataract,
but two adult patients had no evidence at all of lens opacities despite their elevated serum ferritin levels.
At least one of these patients definitely had a de novo mutation. Scanning of 610 DNA samples from
subjects genotyped for HFE-related genetic hemochromatosis led to the identification of four new
mutations all outside the IRE structure (C10T, C16T, C90T, del-T156) whose significance is unclear.
DHPLC analysis of the 5’-UTR of the H-ferritin mRNA did not show any abnormal pattern in the 50
patients studied. Scanning of 586 DNA samples from subjects genotyped for HFE-related genetic
hemochromatosis allowed to us identify two new mutations in the 5’-UTR of the H-ferritin mRNA -G14T
and – C28G from CAGUG. None of them, however, appears to be a gain-of-function mutation, so that
their role in determining hyperferritinemia is uncertain.
In a family with autosomal dominant hyperferritinemia (402-7500 ng/mL), where the proband
showed selective iron accumulation in Kupffer cells on liver biopsy, sequence analysis of the ferroportin
gene (SLC11A3) singled out the heterozygous Val162 del, which can be detected by DHPLC analysis. In
this genetic reticuloendothelial iron overload, serum ferritin levels are directly related to age, but 10-20
times higher than normal.
The remaining 24 patients, most of whom had mild hyperferritinemia (400-800 ng/mL) showed no
mutations in the 5’-UTR of the L and H-ferritin mRNA, and no mutations in the ferroportin gene. Some of
these had evidence on early-onset cataract.
Our interim conclusions are as following: a) patients with unexplained familial hyperferritinemia,
cataract and serum ferritin levels in the order of 800-2,000 ng/mL are likely to have mutations in the Lferritin IRE, which can be easily detected by DHPLC; b) clearly age-dependent elevated ferritin levels are
found in families with genetic reticuloendothelial iron overload; c) mutations in the H-ferritin IRE are
unlikely to be responsible for unexplained hyperferritinemias; d) patients with mild hyperferritinemias
(400-800 ng/mL) are unlikely to have mutations in the ferritin or ferroportin genes despite the evidence of
a genetic basis.
PODIUM 86
A STUDY OF 492 FRENCH CENTENARIANS SUGGESTS THAT LONGEVITY IS NOT AFFECTED IN
CARRIERS OF THE HAEMOCHROMATOSIS GENE C282Y MUTATION
H. Coppin1, M. Bensaid1, S. Fruchon1, N. Borot1, H. Blanché2, M.P. Roth1
1
CNRS UPR 2163, CHU Purpan, Toulouse, and 2 Fondation Jean Dausset - CEPH, Paris, France
Hereditary haemochromatosis (HH) is a common autosomal recessive disorder of iron metabolism. Most
individuals with HH are homozygous for a C282Y mutation in the HFE gene, which is very frequent in
populations of Northern-European origin. One in 5-10 individuals of Caucasian descent indeed is C282Y
carrier. In populations surveys, slightly but significantly higher values for serum iron and transferrin
saturation, and a lower frequency of iron-deficiency anaemia, have been found in persons who are
heterozygous for the C282Y mutation. Iron accelerates free radical generation, which leads to
inflammation, mutagenesis and atherosclerosis, as well as bacterial growth. Therefore, genotypes that
increase transport and storage iron levels are likely to be associated with an increased risk for many
common diseases, including neoplasic, atherosclerotic, infectious and inflammatory conditions. Several
studies support this hypothesis. In 1995, Nelson and coll. compared the health histories of 1950 parents
of haemochromatosis patients (who would be mostly heterozygotes) to those of 1656 parents of the
patients’ spouses. Heterozygosity for haemochromatosis was associated with an increased risk for
colorectal neoplasia, haematological malignancy, and diabetes in men, and with an increased risk for
colonic adenoma and stomach cancer in women. More recently, two studies have shown that
cardiovascular death was associated with the presence of C282Y, although there is currently no
consensus about the possible association of C282Y with the development of coronary heart disease.
In this study, we tested the hypothesis that individuals heterozygous for the C282Y mutation would be
underrepresented in a centenarian population because many would have died previously from
cardiovascular diseases or cancer presumably more frequent in C282Y heterozygotes. Samples were
from 492 French centenarians over 99 years old (80 male, 412 female ; mean age 103.1 years) and 492
controls matched on sex and geographic origin (mean age 51.2 years). The HFE gene was amplified by
PCR and the C282Y mutation was detected by denaturing high performance liquid chromatography on a
TM
WAVE DNA Fragment Analysis System (Transgenomic, Crewe, UK).
There were 44 heterozygotes in the group of French centenarians and 42 among matched controls, a
difference that is not statistically significant, even when controlling for gender. These results suggest that
complications thought to be associated with heterozygosity for C282Y such as carcinomas or
cardiovascular diseases have not depleted this population of C282Y heterozygotes. Of further interest,
two C282Y homozygotes were found among the centenarians and one among the controls. All were
female. None had been treated by phlebotomy and none had been diagnosed with haemochromatosis.
Finding such individuals still alive without treatment at over 99 years of age argues against a complete
clinical penetrance in homozygotes for the C282Y mutation. Distributions of the genotypes in the control
and in the centenarian groups were in agreement with Hardy-Weinberg equilibrium, which further
demonstrates that there was no selection against either homozygotes or heterozygotes for the C282Y
mutation among centenarians.
This study on a large series of French centenarians and matched controls thus does not support the
suggestion that life expectancy is reduced with heterozygosity for C282Y, nor that longevity is
compromised by C282Y homozygosity.
PODIUM 87
DIGENIC ALTERATION (HFE/HAMP) ASSOCIATED WITH ADULT HEMOCHROMATOSIS
PHENOTYPE
S.Jacolot, G.Le Gac, I.Quéré, C.Mura, C.Férec, INSERM EMI 0115 Brest, Université de Bretagne
Occidentale Brest, Etablissement Français du sang Brest and CHU de Brest
Background: Hereditary Hemochromatosis (HH) is a genetically heterogeneous disease. Feder and al
identified the most common and the most prevalent form of hereditary iron disorder (HH1, OMIM 235200)
in 1996. Since other type of HH was described: Juvenile or type 2 HH (OMIM 602390), type 3 HH (OMIM
604250) associated with mutation of TFR2 and type 4 HH (OMIM 606069) associated with ferroportin.
The hepcidin antimicrobial peptide (HAMP, OMIM 606464) was described to play an important role in iron
metabolism and recently Roetto and al reported that HAMP mutations in two Greek families were
associated with a phenotype of severe HH classified as juvenile. Methods: We studied HAMP gene in 31
patients with HH phenotype and one or no mutation in HFE gene and 100 blood marrow donors as
control. We used D-HPLC analysis to scan the HAMP coding sequence in our subjects and sequencing
the patients presenting an alteration profile. Results: We identified two novel mutations (175C>G and
212G>A) in HAMP in four patients already carrier of one mutation at the HFE locus. Discussion: We
report for the first time a digenic inheritance in Hereditary Hemochromatosis between HFE and HAMP
gene. These data shed new light on the complexity of molecular basis of Hemochromatosis.
PODIUM 88
COMPARISON OF SCREENING SERUM FERRITIN CONCENTRATIONS AND TRANSFERRIN
SATURATIONS IN HFE WILD-TYPE AFRICAN-AMERICAN AND CAUCASIAN HEIRS PARTICIPANTS
F.W. Dawkins, B.G. Mellen, D.M. Reboussin, C.E. McLaren, P. Sholinsky, R.T. Acton, P.C. Adams, J.C.
Barton, G.D. McLaren, E.L. Harris, N. Press, M. Speechley, E. Thomson, J.H. Eckfeldt, V.R. Gordeuk,
Center for Sickle Cell Disease, Howard University, Washington, DC, USA; Department of Public Health
Sciences, Wake Forest University School of Medicine, Winston-Salem, NC, USA; Department of
Medicine, University of California, Irvine, Orange, CA, USA; Division of Epidemiology and Clinical
Applications, NHLBI, and ELSI Research Program, NHGRI, Bethesda, MD, USA; Medical Health Care
Group, VA Long Beach Healthcare System, Long Beach, CA, USA; Southern Iron Disorders Center,
Birmingham, AL, USA; Department of Medicine, London Health Sciences Centre, London, ON, Canada;
Departments of Microbiology, Medicine and Epidemiology and International Health, University of Alabama
at Birmingham, Birmingham, AL, USA; Kaiser Permanente Center for Health Research, Portland, OR,
USA; Department of Epidemiology and Biostatistics, University of Western Ontario, London, ON, Canada;
Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN, USA.
Background: Previous population-based studies suggest that serum ferritin concentrations are higher
among African Americans than Caucasians. It is known that HFE mutations influence iron stores. We
hypothesized that screening serum ferritin concentrations are significantly higher in HFE wild-type African
Americans than HFE wild-type Caucasians.
Objectives: To compare screening serum ferritin concentrations and transferrin saturations in HFE wildtype African-American and Caucasian HEIRS participants.
Methods: The goal of the HEIRS study is to screen 100,000 primary care participants in five Field
Centers in the United States and Canada for the C282Y and H63D mutations of the HFE gene, serum
ferritin, and transferrin saturation. This analysis is based on approximately the first 50,000 participants, of
whom 24% were African Americans and 51% were Caucasians. We excluded participants who were not
African-American or Caucasian and participants with C282Y or H63D. We then compared natural logtransformed serum ferritin concentrations and transferrin saturations in wild-type African Americans and
wild-type Caucasians within age (25-44 years, 45-64 years, 65+ years) and sex groups using multiple
linear regression models that adjusted for Field Center.
Results: Interim data were available on 3970 African-American men, 7144 African-American women
6047 Caucasian men, and 9338 Caucasian women, all HFE wild-type. Serum ferritin concentrations
were significantly higher (significance level alpha=0.004, using Bonferroni's correction) for AfricanAmerican males in each age category and for African-American females in each category except 25-44
years. For example, in the 45-64 years category, geometric mean serum ferritin concentrations were 189
versus 148 μg/L in African-American versus Caucasian men, and 90 versus 67 μg/L in women.
Conversely, mean transferrin saturations were slightly (1 to 4%) lower in African Americans; differences
were statistically significant for all age and sex categories except men 22-44 and 45-64 years.
Conclusions: Assuming 1 μg/L serum ferritin represents 8 mg of storage iron and that all other influences
are equal, the above geometric mean level of serum ferritin in African-American men aged 45-64 years
represents iron stores that are 328 mg higher than in the Caucasian men aged 45-64 years; this
difference is 184 mg for women of the same age group. Although hepatocellular dysfunction and
inflammation, which also influence serum ferritin concentration, might not have been fully balanced
between the two samples, our results suggest that differences in basal iron metabolism or iron intake may
exist between African Americans and Caucasians.
PODIUM 89
DETERMINANTS OF IRON ACCUMULATION IN THE HFE KNOCKOUT MOUSE MODEL OF
HEREDITARY HEMOCHROMATOSIS
R.E. Fleming1,3, K.A. Ahmad1, J.R. Ahmann1, B. Roshan1, M.C. Migas1, R.S. Britton2, B.R. Bacon2, A.
Waheed3, W.S. Sly3. 1Pediatrics; 2Internal Medicine and 3Edward A. Doisy Department of Biochemistry &
Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO.
Introduction: The molecular basis for the increased intestinal iron absorption in hereditary
hemochromatosis (HH) remains unclear. Studies examining the expression of intestinal iron transport
genes in HH patients and in murine models of HH have reported discrepant results.
Objective: To reconcile these discrepancies, we characterized the effects of age, strain and gender on the
manifestations of HH in Hfe knockout mice.
Methods: We compared wild-type and Hfe knockout mice on three backgrounds: AKR, C57BL/6, and
C3H. Hepatic and splenic non-heme iron concentrations were measured at multiple time points from 2
weeks to 10 weeks of age. Real-time RT-PCR was utilized to quantify duodenal DMT1, ferroportin1 and
DcytB mRNA content at selected time points.
Results: Hfe knockout mice of each strain exhibited a transient hepatic loading phase, characterized by
increasing hepatic iron concentrations, followed by a prolonged plateau phase, during which elevations in
hepatic iron persisted but did not progress. The loading phase was completed by 4 weeks of age in
C57BL/6 and C3H Hfe knockout mice. By contrast, AKR Hfe knockout mice demonstrated increasing
hepatic iron concentrations through the first 6 weeks, and attained significantly higher plateau levels.
Over the first 6 weeks, the AKR Hfe knockout mice also demonstrated splenic iron sparing. Female Hfe
knockout mice had modestly higher hepatic iron concentrations than did male mice. During the plateau
phase (8 weeks), duodenal expression of DMT1, ferroportin1, and DcytB mRNAs in Hfe knockout mice
were comparable to those in wild-type mice of like strain and gender. However, during the loading phase
(4 weeks) AKR knockout mice demonstrated greater duodenal expression of DMT1 (3.6-fold, P=0.02),
ferroportin1 (2.6-fold, P=0.03), and DcytB (3.6-fold, P=0.005) mRNAs than did wild-type mice.
Conclusions: The effect of loss of Hfe on hepatic iron loading is greatest early in postnatal life in the
mouse. During the loading phase, AKR Hfe knockout mice demonstrate increased duodenal expression
of genes involved in dietary iron absorption. The dependency on age, strain, and gender of the observed
changes may explain discrepancies on the reported consequences of loss of Hfe in murine models of HH.
PODIUM 90
RISK OF HEPATOCELLULLAR CARCINOMA IN PATIENTS WITH HEREDITARY
HEMOCHROMATOSIS AND THEIRE FIRST-DEGREE RELATIVES
Hultcrantz R1,Elmberg M1, Ekbom A1, Brandt L1, Olsson S3, Olsson R3, Lindgren S4,
Lööf L5, Stål P6, Wallerstedt S3,Almer S7, Sandberg-Gertzén H8 and Askling J1
Dept. of Medicine at Karolinska Hospital1, Sahlgrenska University Hospital3, Malmö
University Hospital4, Uppsala Hospital 5, Huddinge Hospital6, Linköping Hospital7 and
Örebro Hospital8
INTRODUCTION
A high risk of intra-hepatic malignancy in patients with Hereditary Hemochromatosis (HH) is well
established; however the results concerning non-hepato-biliary cancers are conflicting. Furthermore,
cancer risk in individuals heterozygous for the C282Y mutation, known to have marginal to moderate iron
load, is unknown. In order to assess the cancer risk in manifest or sub-clinical iron overload, we
performed a population-based cohort study of Swedish patients with HH and of their first-degree relatives
using nation-wide, population-based, high-quality health and census registers.
METHODS
The cohort of patients with HH was assembled from three sources: 1) In the population-based Swedish
inpatient register, all individuals with HH as a discharge diagnosis (according to International
Classification of Disease) 1964-1999 were identified. 2) In the nation-wide Cause of Death Register
19XX-1999, all deaths of individuals with HH coded as the main cause of death were identified. 3) In a
regional and population-based register of HH, 344 individuals were identified.
First-degree relatives were identified by linking the National Registration Number of each patient to the
Nation-wide Multi-generation register. Patients and their first-degree relatives were all followed for cancer
occurrence using standardised incidence ratio (SIR), i.e., the ratio of the observed to the expected
number of cancers, as a measure of relative risk. Expected numbers were calculated by multiplying sex-,
age-, and calendar period-specific cancer incidence rates from the general population with the
corresponding person-years of follow-up. SIR’s were calculated for cancer overall, for selected
gastrointestinal cancers, and for liver-cancer.
RESULTS OBTAINED
We identified 1,847 patients with HH and 5,973 first-degree relatives. 62 intra-hepatic malignancies and
128 non-hepato-biliary cancers were identified among the patients, which corresponded to a 20-fold risk
of liver cancer (SIR 21.0, 95% CI 16-22), but only a marginally increased risk for non-hepato-biliary
cancer (SIR 1.2, 95% CI 1.0-1.4). After ten years of follow-up, the absolute risk of liver-cancer was 6% in
male and 1.5% in female patients, respectively.
21 intra-hepatic and 508 non-hepato-biliary cancers and were found among the first-degree relatives of
patients, corresponding to a marginally increased risk of liver cancer was observed (SIR 1.5, 95% CI 1.02.4, with a different histopathological distribution than among the patients) but a non-elevated risk for the
combined group of non-hepato-biliary cancers (SIR 1.0, 95% CI 0.9-1.1).
CONCLUSION:
We confirm the existence of an elevated risk for primary liver-cancer among patients with HH. Although
our relative risk estimates are compartatively low, the absolute risk in HH remains a (sex-specific)
concern. (Heterozygous) first-degree relatives had a marginal and, clinically, irrelevantly increased risk for
liver cancer, the nature of which argues against causality. Importantly, neither patients nor their firstdegree relatives were at increased risk for any other gastrointestinal cancers.
PODIUM 91
HEMOCHROMATOSIS SUBJECTS AS ALLOGENEIC BLOOD DONORS: A PROSPECTIVE STUDY
Susan F. Leitman, Janet N. Browning, Yu Ying Yau, Glorice Mason, Harvey G. Klein, Cathy ConryCantilena, Charles D. Bolan. Department of Transfusion Medicine, Warren Grant Magnuson Clinical
Center, National Institutes of Health, Bethesda, Maryland, USA
Persons with hemochromatosis constitute a plentiful and willing source of blood for transfusion. However,
regulatory issues in the United States have previously restricted the ability to use blood from individuals
with hemochromatosis for allogeneic transfusion, and the potential impact of allowing subjects with
hemochromatosis to serve as blood donors has been unclear. Recent changes in policy allowed us to
establish and evaluate a program for treating persons with hemochromatosis in a donor center, and to
make their blood available for allogeneic transfusion. Subjects with hereditary hemochromatosis and
clinical evidence of iron overload were recruited via letters to area physicians. Phlebotomy therapy was
performed in the Department of Transfusion Medicine of an academic clinical research center, and was
provided free of charge regardless of whether subjects met criteria for allogeneic blood donation.
Decreases in the red cell MCV and serum iron parameters were used to guide therapy. A fingerstick
hemoglobin of 12.5 g/dL was used as the threshold for performing phlebotomy. A computerized database
followed laboratory values and informed medical staff when preset endpoints were achieved. A total of
100 subjects were consecutively enrolled between January 2000 and July 2002: 80% were homozygous
for the HFE mutation C282Y, 75% met eligibility criteria for allogeneic donation, and 53% were previously
untreated. A median of 20 weekly or biweekly phlebotomies (range 7-99) were performed before the MCV
reached the targeted endpoint of 3% below baseline, at which time the ferritin was less than 30 µg/L and
the transferrin saturation less than 30%. The median phlebotomy interval necessary to keep the MCV
less than 3% below baseline during maintenance therapy was 10 weeks. With use of the MCV guide, a
pre-phlebotomy finger-stick screening hemoglobin threshold of 12.5 g/dL, and monthly transition
phlebotomy, it was not necessary to induce iron deficiency anemia and deferrals for low hemoglobin were
limited in the donors with hemochromatosis. Most subjects who had been previously treated expressed
dissatisfaction with their prior care. Many had been obligated to make two visits per treatment, one to a
physician’s office for blood sampling and one to a phlebotomy site for therapy, while others had financial
difficulties related to insurance or other difficulties obtaining access to care. Criteria used to guide
previous phlebotomy therapy also varied widely among subjects, with 10% of subjects over3
phlebotomized at referral (median hemoglobin 11.4 g/dL, MCV 77 µm , ferritin 6 µg/L, and transferrin
saturation 5%), and 30% having had prolonged lapses in therapy. All subjects expressed significant
frustration with having their blood discarded. A majority of previously diagnosed subjects had withheld
information concerning their diagnosis while serving as allogeneic donors to receive therapy prior to study
entry; four of these had also withheld a history of deferrable risk factors during screening for donations
made at other centers. In these 4 subjects, their blood was no longer utilized for transfusion after
enrollment in this protocol. No seroconversions for viral infections occurred during the study period. All
three subjects who tested positive for transmissible viral infections at enrollment provided a history of prior
risk factors that precluded allogeneic blood donation. Eighteen months after starting the program,
hemochromatosis donors were contributing 10% of the red cell units collected for allogeneic use at our
center. Subjects expressed great satisfaction that their blood was made available for transfusion and
appreciated their improved access to care. In turn, donor center phlebotomy and nursing staff were
appreciative of the fact that hemochromatosis subjects were more likely to keep appointments for blood
donation (89% vs 75%, p < 0.001) and less likely to be deferred for low hemoglobin levels (2.5% vs 7.5%,
p < 0.001) compared to other blood donors. We conclude that hemochromatosis subjects can safely and
significantly augment the allogeneic blood supply. Adoption of this practice on a national scale can reduce
current blood shortages in the United States.
PODIUM 92
HEPATIC IRON CONCENTRATION AND TOTAL BODY IRON STORES IN GENETIC
HEMOCHROMATOSIS AND THALASSEMIA MAJOR
S. Jacquelinet1, G.M. Brittenham2, E. Angelucci3, C.E. McLaren4, P. Brissot1 (1) Service des Maladies du
Foie et INSERM U-522, University Hospital Pontchaillou, Rennes, France; (2) Department of Pediatrics,
Columbia University College of Physicians and Surgeons, New York, NY, U.S.A.; (3) Unita Operativa di
Ematologia, Pesaro Hospital, Italy; (4) Division of Epidemiology and College of Medicine, University of
California, Irvine, College of Medicine, Irvine, CA, USA.
To characterize quantitatively differences in the sites of storage iron deposition in genetic
hemochromatosis and thalassemia major, we compared studies of 87 patients homozygous for the
C282Y mutation in HFE with those of the 54 patients who had undergone successful allogeneic bone
marrow transplantation for thalassemia described by Angelucci et al. (N Engl J Med 2000;343:327-31).
Before beginning phlebotomy therapy, iron was measured in specimens of liver obtained by percutaneous
biopsy and records were then kept of the amount of blood removed until body iron stores were depleted.
To maintain comparability with the results of Angelucci et al., we excluded patients with cirrhosis (n = 12)
or with liver samples obtained by biopsy weighing less than 1.0 mg, dry weight (n = 55) and calculated the
magnitude of total body iron stores similarly. In brief, total body iron stores were calculated from the total
amount of blood removed, assuming that each gram of hemoglobin contains 3.4 mg of iron, with
adjustment for any change in the concentration of circulating hemoglobin, assuming a blood volume of
61.9 mL/kg for women and of 62.4 mL/kg for men. In women with regular menses, an iron loss of 0.5 mg
per day was assumed during each month of menstruation. Because of uncertainty about the quantitative
extent of variation in dietary iron absorption both within and between patients, no adjustment was made
for an increase in iron absorption during phlebotomy but sensitivity analyses were carried out to estimate
potential effects. The Figure shows the results of linear regression analysis between the initial hepatic
iron concentration and calculated total body iron stores (i) in our 20 patients with genetic
hemochromatosis and liver samples that were at least 1.0 mg in dry weight (filled circles) and (2) in the
corresponding 25 patients with thalassemia major after successful transplantation (open circles)
described by Angelucci et al.
300
250
Total Body
Iron Stores
(mg Fe/kg)
Thalassemia
major
P<0.0001
200
Genetic
hemochromatosis
150
100
50
0
0
10
20
30
Hepatic Iron
(mg Fe/g liver, dry weight)
For the patients with genetic hemochromatosis, the estimated slope of the regression line was
significantly different from zero (t=5.3, 18 df, P<0.0001) but the estimated intercept was not significantly
different from zero (t=-0.02, 18 df, P=9.98). The estimated slope of the regression line for the patients
with genetic hemochromatosis was significantly less than that for patients with thalassemia major (F=26.1,
1 and 41 df, P<0.0001). In a further regression analysis with the assumption that hepatic iron stores were
reduced to zero with phlebotomy therapy, variation in the hepatic iron concentration in the patients with
genetic hemochromatosis accounted for more than 90% of the variation in body iron stores and was not
appreciably affected by proposed models of the increase in iron absorption during phlebotomy. Overall,
this relationship could be expressed as
Total body iron stores = 5.2 x Hepatic iron concentration
[Genetic hemochromatosis]
while the corresponding relation found by Angelucci and colleagues was
Total body iron stores = 10.6 x Hepatic iron concentration
[Thalassemia major]
with body iron stores expressed as mg/kg body weight and hepatic iron as mg/g liver, dry weight. These
results provide a quantitative estimate of the magnitude of the difference in the distribution of excess iron
between the liver and extrahepatic sites in the two conditions. For a given hepatic iron concentration, the
body iron excess in thalassemia major is about twice that in genetic hemochromatosis. (European Grant
QLT-2001-00444).
PODIUM 93
IRON AND THE RESPONSE TO HYPOXIA
Chair: Patrick Maxwell, Imperial College London
Many aspects of complex organisms such as ourselves are devoted to supplying oxygen to every cell.
Hypoxia-Inducible Factor-1 (HIF-1) provides a transcription control system which responds to changes in
oxygen and regulates diverse processes including erythropoiesis, angiogenesis, and metabolism. HIF-1 is
also activated by iron chelators and regulates genes involved in iron metabolism. Intense interest has
centred on how HIF-1 is regulated. As discussed in this session, in the presence of oxygen recently
identified nonheme-Fe(II)-dependent enzymes hydroxylate specific prolyl residues leading to destruction
of HIF alpha, and hydroxylate an asparaginyl residue which prevents transactivator recruitment. These
enzymes provide "oxygen-sensors" controlling HIF-1 which are also sensitive to iron.
PODIUM 94
No abstract available at time of printing.
PODIUM 95
FE(II)-DEPENDENT DIOXYGENASES IN THE MAMMALIAN HYPOXIC RESPONSE PATHWAY
Richard K. Bruick, Ph.D., Assistant Professor, Department of Biochemistry, University of Texas
Southwestern Medical Center
The ability to sense and respond to changes in oxygen availability is critical for many developmental,
physiological and pathophysiological processes including angiogenesis, cerebral and myocardial
ischemia and tumorigenesis. In mammalian cells, exposure to a low oxygen environment triggers an
evolutionarily conserved hypoxic response pathway centered on the regulated expression of the hypoxiainducible transcription factor (HIF). HIF is selectively targeted for degradation under normal oxygen
conditions (normoxia) by a ubiquitin-ligase complex that recognizes HIF via hydroxylated proline residues.
A conserved family of Fe(II)-dependent HIF prolyl hydroxylase enzymes perform this post-translational
modification. Prolyl-hydroxylation is blocked under hypoxic conditions allowing for HIF accumulation. Full
HIF activity is further dependent upon induction of its carboxy-terminal transactivation domain (C-TAD).
Under normoxic conditions, association of the C-TAD with coactivators such as CBP/p300 is blocked by
hydroxylation of an asparagine residue. Suppression of this modification under hypoxic conditions allows
HIF to recruit the larger transcriptional apparatus necessary to transcribe downstream target genes. A
second Fe(II)-dependent hydroxylase mediates this additional oxygen-dependent mode of HIF regulation.
Because these key regulatory enzymes utilize molecular oxygen as a substrate, they are prime
candidates for the elusive O2-sensor in the hypoxic response pathway as well as attractive targets for
therapeutic intervention.
PODIUM 96
STRUCTURAL AND MECHANISTIC STUDIES ON THE DIOXYGEN DEPENDENT MODIFICATION OF
HYPOXIA INDUCIBLE FACTOR
Christopher J. Schofield, Kirsty S. Hewitson, Jonathan Elkins, Luke A. McNeill, Jürgen F. Seibel, Imre
Schlemminger, The Oxford Centre for Molecular Sciences and the Dyson Perrins Laboratory, University
of Oxford, South Parks Road, Oxford OX1 3QY, UK; Christopher W. Pugh, Peter J. Ratcliffe, Cellular
Physiology Group, Henry Wellcome Building of Genomic Medicine, Roosevelt Drive,
Oxford OX3 7BN, UK; Patrick Maxwell, Renal Medicine Section, Faculty of Medicine, Imperial College of
Science, Technology & Medicine, Hammersmith Hospital, Du Cane Road, London W12 ONN.
In hypoxic cells, activation of the HIF1 transcriptional cascade directs a series of responses that
enhance oxygen delivery/limit oxygen demand [1]. Activation of HIF in cancer and ischaemic/hypoxic
vascular diseases has indicated a central role in human pathology [1]. The transcriptional complex is
composed of an alpha/beta-heterodimer; HIF-alpha is a constitutive nuclear protein that dimerises with
oxygen regulated HIF- alpha subunits [2]. The activity of HIF is regulated by Fe(II) and 2OG dependent
dioxygenases that catalyse hydroxylation of specific HIF-1alpha residues. In normoxia, 4-hydroxylation of
human HIF-1 alpha at Pro402 or Pro564, catalysed by HIF prolyl hydroxylase isoforms (PHD1-3), [3,4]
enables HIF-1 alpha recognition by the von Hippel-Lindau (VHL) ubiquitin ligase complex leading to its
proteasomal destruction [5,6]. In a complementary mechanism FIH catalyses hydroxylation of HIF-1alpha
Asn803 [7,8] which blocks interaction with the transcriptional co-activator p300. In hypoxia, lack of
hydroxylase activity enables HIF-alpha to escape destruction and become transcriptionally active.
Inhibition of HIF hydroxylases by Fe(II) chelators, other transition metals, and 2OG analogues activates
the HIF transcriptional cascade even in normoxia. The HIF hydroxylases therefore provide a focus for
understanding cellular responses to hypoxia and a target for therapeutic manipulation.
The lecture will include a description of recent structural and mechanistic studies on the HIF
hydroxylases and related 2OG oxygenases including those involved in other signalling pathways, primary
metabolism, and secondary metabolism in microorganisms and plants. In the case of the HIF
hydroxylases, crystal structures of the asparagine hydroxylase (Factor Inhibiting HIF, FIH) complexed
with Fe(II), 2-oxoglutarate cosubstrate and CAD fragments have enlightened the structural basis of HIF
modification [9-11]. CAD binding to FIH occurs via an induced fit process at two distinct interaction sites.
At the hydroxylation site CAD adopts a loop conformation, contrasting with a helical conformation for the
same residues when bound to p300. Asn803 of CAD is buried and precisely orientated in the active site
such that hydroxylation occurs at its alpha-carbon. Together with structures with the inhibitors Zn(II) and
N-oxaloylglycine, analysis of the FIH:CAD complexes have assisted in the design of hydroxylase
inhibitors. Conserved structural motifs within FIH imply it is one of an extended family of Fe(II)
oxygenases involved in gene regulation.
References
[1] Semenza, G. L. (2000) Genes Dev. 14, 1983-1991; [2] Semenza, G. L. (1999) Annu. Rev. Cell Dev.
Biol. 15, 551-578; [3]Epstein, A. C. R., Gleadle, J. M., McNeill, L. A., Hewitson, K. S., O'Rourke, J., Mole,
D. R., Mukherji, M., Metzen, E., Wilson, M. A., Dhanda, A., Tian, Y.-M., Masson, N., Hamilton, D. L.,
Jaakkola, P., Barstead, R., Hodgkin, J., Maxwell, P. H., Pugh, C. W., Schofield, C. J., and Ratcliffe, P. J.
(2001) Cell 107, 43-54; [4] Bruick, R. K., and McKnight, S. L. (2001) Science 294, 1337-1340
[5]Jaakkola, P., Mole, D. R., Tian, Y.-M., Wilson, M. I., Gielbert, J., Gaskell, S. J., von Kriegsheim, A.,
Hebestreit, H. F., Mukherji, M., Schofield, C. J., Maxwell, P. H., Pugh, C. W., and Ratcliffe, P. J. (2001)
Science 292, 468-472; [6] Ivan, M., Kondo, K., Yang, H., Kim, W., Valiando, J., Ohh, M., Salic, A., Asara,
J. M., Lane, W. S., and Kaelin Jr., W. G. (2001) Science 292, 464-468; [7] Hewitson, K. S., McNeill, L. A.,
Riordan, M. V., Tian, Y.-M., Bullock, A. N., Welford, R. W., Elkins, J. M., Oldham, N. J., Bhattacharya, S.,
Gleadle, J. M., Ratcliffe, P. J., Pugh, C. W., and Schofield, C. J. (2002) J. Biol. Chem. 277, 26351-26355;
[8] Lando, D., Peet, D. J., Gorman, J. J., Whelan, D. A., Whitelaw, M. L., and Bruick, R. K.
(2002) Genes Dev. 16, 1466-1471; [9] Dann CE, Bruick RK, Deisenhofer J (2002) PNAS 99, 1535115356
[10] Elkins JM, Hewitson KS, McNeill LA, Seibel JF, Schlemminger I, Pugh CW, Ratcliffe PJ, Schofield CJ
(2003) J. Biol. Chem 278, 1802-1806; [11] Lee C, Kim SJ, Jeong DG, Lee SM, Ryu SE (2003) J. Biol.
Chem 278, 7558-7563
Acknowledgements: This work was supported by The Wellcome Trust, MRC, BBSRC, EPSRC and EU.
I.S. was supported by a German DAAD fellowship. We thank all the colleagues who have made
contributions to our work on the hypoxic response.
PODIUM 97
CONGENITAL DISORDER OF OXYGEN-SENSING: CLINICAL MANIFESTATIONS OF THE
HOMOZYGOUS CHUVASH POLYCYTHEMIA VHL MUTATION
Victor R. Gordeuk, M.D., Adelina I. Sergueeva, M.D., Galina Y. Miasnikova, M.D., Daniel Okhotin, B.S.,
Yaroslav Voloshin, B.S., Katerina Jedlickova, M.S., Josef T. Prchal, M.D., Lydia A. Polyakova, M.D.
Center for Sickle Cell Disease and Department of Medicine, Howard University, Washington, DC (VRG,
YV); Cheboksary Children’s Hospital, Cheboksary, Russia (AIS, DO); Chuvash Republic Clinical Hospital
No. 1, Cheboksary, Russia (GYM, LAP); Department of Medicine, Baylor University, Houston, TX (KJ,
JTP).
Background. In Chuvash polycythemia, the first recognized hereditary disorder of augmented hypoxiasensing, homozygosity for von Hippel-Lindau gene (VHL) arg200trp leads to increased levels of cellular
hypoxia inducible factor-1 (HIF-1) and serum erythropoietin in normoxia. Whether arg200trp
homozygotes develop renal carcinomas, pheochromocytomas and vascular tumors as do heterozygotes
for other germline VHL mutations is not known.
Methods. We studied 96 Chuvash polycythemia patients diagnosed before 1977, 65 spouses, and 79
community members matched by sex, village, birth date, and survival to three years. When possible, we
examined participants and isolated DNA.
Results. Estimated survival to 65 years was ≤31% for Chuvash polycythemia patients versus ≥67% for
spouses and community controls (P≤0.002). Excess mortality in Chuvash polycythemia was related
partially to stroke but not cancer. All 43 Chuvash polycythemia patients tested were arg200trp
homozygotes, while 9 (10.5%) of 86 spouses and controls were heterozygotes. Compared to wild types
and heterozygotes, arg200trp homozygotes had more varicose veins, lower blood pressure, and higher
adjusted serum concentrations of vascular endothelial growth factor (VEGF) and plasminogen activator
inhibitor 1 (PAI-1). Imaging studies in 31 arg200trp homozygotes revealed vertebral or organ
hemangiomas in 17 (55%), kidney, liver or pancreas cysts in 16 (52%), and ischemic or infarctive brain
lesions in 14 (45%), but no spinocerebellar hemangiomas or renal carcinoma.
Conclusion. Homozygosity for VHL arg200trp leads to varicosities, ischemic brain lesions, increased
mortality, and possibly benign hemangiomas and cysts, but not malignancies and vascular tumors
associated with other heterozygous VHL mutations. Increased expression of HIF-1 and VEGF may not
be sufficient to cause tumorigenesis in VHL syndrome.
PODIUM 98
IRON, HFE, HCV GENOTYPE AND HEPATIC MHC CLASS I EXPRESSION IN CHRONIC HEPATITIS
C
EMP Cardoso1,3 MA Duarte2,M de Sousa1,2 (presenter) E Ribeiro2, P Rodrigues1,2, G Porto1,2,4, P
Carvalho5, J Fraga5.
1
Molecular Immunology, Institute for Molecular and Cell Biology (IBMC). 2Department of Molecular
Immunology and Pathology, Abel Salazar Institute for Biomedical Sciences ICBAS), 3Instituto Superior de
Ciências da Saúde-Norte, 4Department of Hematology, Santo António General Hospital, Porto.
5
Gastroenterology, Vila Nova de Gaia Hospital, Portugal.
Elevated markers of iron stores are common in patients with chronic hepatitis C. Growing evidence from
work on gene deficient mice points to an intriguing role for immunological system genes in iron load (1).
Hepatitis C represents a model of viral infection at the crossroads between hepatic iron overload and
immunological responses. The aims of this study were to evaluate hepatic siderosis and relevant
immunological markers, namely CD68, MHC class I, HFE, β2 microglobulin and CD8+ cell numbers
against different HFE and viral genotype backgrounds.
One hundred and thirteen subjects with hepatitis C virus infection, as documented by the presence of
HCV RNA in serum (AmplicorTM, Roche, Switzerland) who had undergone liver biopsy for diagnostic
purposes and staging of their liver disease, were also genotyped for the two main HFE mutations (Vienna
Lab, Austria). Biochemical parameters (serum iron, transferrin saturation, serum ferritin, ALT, etc) were
available in 111 HCV patients. In 29 of these patients, studies for the expression of several immunological
markers such has HFE, MHC-I, 
2m and CD68 were performed using immunohistochemical (APAAP
method) and immunofluorescence techniques in frozen liver biopsies. The quantitative analysis of
distribution of these markers was done with a Leica Qwin software. The number of hepatic CD8+
lymphocytes was also evaluated in formaldehyde-fixed paraffin-embedded liver biopsies.
The allelic frequencies of C282Y and H63D HFE mutations were of 0.0265 and 0.155, respectively. The
differences in the frequencies (albeit lower than in the regional control population - 0.058 and 0.194
respectively, (2)) did not reach statistical significance. In general the results regarding iron markers and
hepatic siderosis confirmed earlier studies showing significantly higher liver siderosis grades in patients
with HFE mutations (3). However, the most original contribution of this study emerged from the
quantitative immunohistochemical analysis of the distribution of HFE and MHC class I. HFE has been
previously reported to be found in Kupffer cells, KC (4). The comparison of the distribution of CD68, a KC
marker and HFE permitted to conclude that not all KC are equally positive for HFE. Interestingly, MHC
2
class I expression was significantly higher (Mean±SD: 173±105 µm ) in patients with the viral genotype
2
3a than in the other genotypes (34±105 µm ) (p=0.007). The present novel finding may represent the
basis for the more favourable response to therapy with α-IFN in patients with the 3a HCV genotype.
1.
2.
3.
4.
Cardoso EM et al, 2002, Blood, 100:4239.
Cardoso C et al, 2001, Eur. J. Hum. Genet., 9:843.
Bonkovsky HL et al, 2002, J. Hepatol. 37:848.
Bastin et al, 1998, Br. J. Haematol., 103:931.
Acknowledgements
EC Grant: QLG1-CT-1999-00665
Funded also by the Calouste Gulbenkian Foundation and the FCT (MGI/49428/2001)
PODIUM 99
INDUCTION OF TRANSFERRIN RECEPTOR BY ETHANOL IN RAT PRIMARY HEPATOCYTE
CULTURE
Y. Suzuki, K. Ikuta, M. Suzuki, T. Otake, H. Saito, Y. Fujimoto, Y. Torimoto, Y. Kohgo, Third Department
of Internal Medicine, Asahikawa Medical College
It is not uncommon for alcoholics to have iron accumulation in the liver which may contribute to the
development of alcoholic liver disease. Recently, we have reported that the expression of transferrin
receptor, which mediates cellular iron uptake, was increased in hepatocytes in patients with alcoholic liver
disease. In order to elucidate the mechanism of the iron accumulation in hepatocytes in alcoholic liver
disease, we examined whether ethanol exposure induced the transferrin receptor expression and
increased the cellular iron uptake. Rat primary hepatocytes were isolated and cultured in the presence of
20 micromol/L iron and 25 mmol/L ethanol. Ethanol exposure to the hepatocytes demonstrated an
approximately 2-fold increase in transferrin receptor expression for 24 hours, shown by Western blot
35
59
analysis and S-methionine metabolic labelling, 19% increase in Fe-transferrin uptake by hepatocytes,
and 20% increase in activity of iron regulatory protein examined by band shift assay. Ethanol exposure
induced the transferrin receptor expression, partially through the activation of iron regulatory protein and
increased the transferrin bound iron uptake in rat hepatocyte cultures. The induction of transferrin
receptor by ethanol might be one of the mechanisms of iron accumulation in the hepatocytes in alcoholic
liver disease.
PODIUM 100
IRON CHELATORS INHIBIT HIV-1 TAT-DEPENDENT TRANSCRIPTION FROM HIV-1 PROMOTER IN
CULTURED CELLS
S. Nekhai1,2, J. Kurantsin-Mills1,3, T. Ammosova1, and V. Gordeuk1,4
1
Center for Sickle Cells Disease, 2Department of Biochemistry and Molecular Biology, 3Department of
Biophysics and Physiology, and 4Department of Medicine, Howard University, Washington, DC 20059
HIV-1 Tat protein activates viral gene expression through promoting transcriptional elongation by RNA
Polymerase II (RNAPII). In this process Tat enhances phosphorylation of the C-terminal domain (CTD) of
RNAPII by activating cell cycle dependent kinases (CDKs) associated with general transcription factors of
the promoter complex. HIV-1 Tat binds to the bulge of transactivation response (TAR) RNA formed at the
non-translated 5’ termini of HIV-1 transcripts. HIV-1 Tat also binds to cyclin T1, a cyclin partner of CDK9,
which in turn interacts with the loop of TAR RNA. This allows CDK9/cyclin T1 to be recruited by Tat to the
HIV-1 promoter. While the role of CDK9/cyclin T1 in Tat-mediated HIV-1 transcription is well established,
our recent data suggest that CDK2 may also be involved in the Tat-induced transcription (1). We recently
reported that CDK2 is part of transcription complex that is required for Tat-dependent transcription, and
that interaction of Tat with CTD and a dynamic association of Tat with CDK2/cyclin E stimulated CTD
phosphorylation by CDK2 (2). Importance of CDK2/cyclin E is also underscored by our earlier findings
that Tat activates HIV-1 transcription at G1 phase of the cell cycle and that this activation required CDK2
activity (3). In the present study we analyzed relative contributions of CDK2 and CDK9 to the regulation of
HIV-1 transcription by selective inhibition of CDK2 or CDK9 in cultured cells. It has been demonstrated
that iron depletion severely deregulated CDK2/cyclin E activity by trapping cyclin E in the cytoplasm of the
treated cells, and by decreasing the protein level of CDK2. We utilized several iron chelators, including
desferrioxamine and 311, to deplete intracellular iron pools in order to disregulate intracellular activity of
CDK2. In addition, we used flavopiridol, which selectively inhibits CDK9. To study the effects of CDK2 or
CDK9 inhibition, we used HeLa MAGI cells that contain an integrated HIV-1 LTR-Lac Z reporter and that
were infected with adenovirus expressing HIV-1 Tat protein to induce HIV-1 transcription. Depletion of
iron disregulated CDK2 activity and inhibited up to 60% of the Tat-induced transcription from HIV-1
promoter in HeLa MAGI cells. Flavopiridol, on the other hand, inhibited up to 40% of Tat-induced
transcription at concentrations below 50 nM, when CDK9 but no CDK2 is selectively inhibited. Our
findings suggest that both CDK2 and CDK9 contributes to the regulation of Tat-induced HIV-1
transcription in HeLa-MAGI cells. Moreover, iron chelators inhibit HIV-1 transcription as efficiently as
flavopiridol.
This work was supported by NIH grant # UH1 HL03679 funded by NHLBI and The Office of Research on
Minority Health.
References
1. Nekhai, S, Zhou, M., Fernandez, A, Lane, W. S., Lamb, N. J. C., Brady, J. and A. Kumar (2002) HIV-1
Tat-associated CTD Kinase, CDK2, Phosphorylates CDK7 and Stimulates Tat-mediated Transcription.
2002. Biochem. J., 364, 649-657
2. Deng, L., Ammosova, T., Pumfery, A., Kashanchi, F., and S. Nekhai (2002). HIV-1 Tat interaction with
RNA polymerase II CTD and a dynamic association with CDK2 induces CTD phosphorylation and
transcription from HIV-1 promoter. J. Biol. Chem. 277: 33922-33929.
3. Nekhai, S., Shukla, R. R., Fernandez, A., Lamb, N., and A. Kumar. (2000) Cell Cycle-Dependent
Stimulation of HIV-I Promoter by Tat-Associated CAK Activator. Virology, 266: 246-256.
POSTER SESSION 1
POSTER 1
IRON STATUS IN DANISH MEN 1984-1994. A COHORT COMPARISON SHOWING INCREASED
IRON STORES AND IRON OVERLOAD DESPITE ABOLITION OF FOOD IRON FORTIFICATION
N. Milman, L. Ovesen, K-E. Byg
Department of Medicine B, Rigshospitalet, University of Copenhagen. Copenhagen County Centre for
Prevention of Disease, Glostrup Hospital, University of Copenhagen. Institute of Food Safety and
Nutrition, Danish Veterinary and Food Administration, Copenhagen, Denmark.
Background and objective: In the period 1954-1986, flour was fortified with 30 mg carbonyl iron per kg.
This mandatory fortification was abolished in 1987; since then, fortified flour has not been available on the
Danish market. The aim of this study was to compare iron status in Danish men before and after
abolishment of iron fortification.
Methods: Iron status (serum ferritin and haemoglobin), was assessed in two population surveys in
Copenhagen County, the first in 1983-1984 comprising 1324 Caucasian men (1024 non-blood-donors,
300 blood donors) and the second in 1993-1994 comprising 1288 Caucasian men (1103 non-donors, 185
donors). The participants were equally distributed in age cohorts of 40, 50, 60 and 70 years.
Results: Non-donors: In the 1984 survey, median serum ferritin values in the four age cohorts were 136,
141, 133, and 111 µg/L, and in the 1994 survey 177, 173, 186 and 148 µg/L, respectively. The difference
was significant in all age groups (p <0.001). There was no significant difference between the two surveys
concerning the prevalence of small iron stores (ferritin 16-32 µg/L), 1.9% vs. 1.2%; depleted iron stores
(ferritin <16 µg/L), 0.5% vs. 0.4%; iron deficiency anaemia (ferritin <13µg/L and haemoglobin <5th
percentile for iron replete men), 0.08% vs. 0.16%. However, from 1984 to 1994, the prevalence of iron
overload (ferritin >300 µg/L) increased from 11.3% to 18.9% (p <0.0001). During the study period there
was an increase in body mass index (p <0.0001), alcohol consumption (p <0.03) and a decrease in the
prevalence of tobacco smoking (p <0.0001), i.e. factors, which tend to increase iron status. There were
positive correlations between ferritin and body mass index both in 1984 (rs = 0.23, p <0.0001) and in 1994
(rs = 0.19, p <0.0001) and between ferritin and alcohol consumption in 1984 (rs = 0.23, p <0.0001) and in
1994 (rs = 0.26, p <0.0001). There was an increase in the use of non-steroid anti-inflammatory drugs
(NSAID) (p <0.0001), and a decrease in the use of vitamin-mineral supplements (p <0.04), both factors,
which tend to decrease iron status. In the 1994 survey, NSAID users had lower median ferritin than nonusers (122 vs. 131 µg/L, p = 0.001). Negative correlations were found between ferritin and coffee + tea
consumption both in 1984 (rs = -0.21, p <0.0001) and in 1994 (rs = 0.16, p <0.0001). In the 1994 survey,
27.4% took ferrous iron supplements, median intake 14 mg/day. Iron status, and the prevalences of iron
depletion and iron overload were not significantly different in iron supplemented and non-supplemented
men.
Blood donors: Median ferritin values in blood donors displayed a significant fall from 1984 to 1994 (103
µg/L vs. 74 µg/L, p <0.02). The prevalence of small iron stores was 8.0% vs. 12.0%; of depleted iron
stores 1.3% vs. 1.2%, and of iron overload 8.3% vs. 7.2%, i.e. not significantly different. The decrease in
ferritin was probably elicited by an increase in the donation frequency from 1984 to 1994 due to a
decreasing number of available donors.
Conclusions: Abolition of food iron fortification reduced the iron content of the average Danish diet by 2.4
mg/10 MJ. The median dietary iron intake in Danish men fell from 17 mg/day in 1985 to 12 mg/day in
1995. However, the carbonyl iron used as fortification had a low bioavailability and obviously the
fortification had no recognizable impact on iron status in men. Despite the abolition of iron fortification,
body iron stores and the prevalence of iron overload in men increased significantly from 1984 to 1994.
The reason appears to be changes in dietary habits with a lower consumption of dairy products and eggs,
which inhibit iron absorption, in combination with a higher consumption of alcohol, meat, and poultry,
containing high-bioavailability haem iron and enhancers of iron absorption. In general, Danish men have
a satisfactory iron status although the high prevalence of iron overload may constitute a health risk.
POSTER 2
BODY IRON STORES AT DIAGNOSIS PREDICT IRON ABSORPTION RATES DURING
MAINTENANCE PHLEBOTOMY IN HEREDITARY HEMOCHROMATOSIS SUBJECTS.
P.D. Phatak, C. Andrews, L. Braggins, RL Sham, Rochester General Hospital and the Mary M Gooley
Hemophilia Center Inc.
Phenotypic expression of iron overload among Hereditary Hemochromatosis (HHC) patients is
variable, even after accounting for HFE genotype. Variable rates of iron absorption necessitate individual
tailoring of phlebotomy frequency during maintenance phlebotomy in order to maintain low normal body
iron stores. Our study population consisted of 103 patients (75 male and 28 female) with HHC who were
enrolled in our treatment center including 65 C282Y homozygotes. We included only individuals who
started maintenance phlebotomy at least 3 years ago, allowing us to compute iron absorption rates. Sixtynine of 103 had liver biopsies and 2/ 69 had hepatic cirrhosis. The mean mobilized iron required to
achieve iron depletion (serum ferritin, SF <25ng/ml) was 6.257g (± 4.605g). Iron stores were somewhat
higher among C282Y homozygotes compared with non-homozygotes (mobilized iron 7.41 vs 4.28 g ; p=
<0 .0001). The frequency of maintenance phlebotomy is titrated to maintain a SF of 25-75. For this
analysis, we excluded the first year on maintenance in order to allow this titration to occur. Iron absorption
rates were calculated using the amount of iron removed by phlebotomy over the subsequent years of
therapy. Study subjects were in maintenance for an average period of 52.66 (±18.27) months beyond the
first year.
Iron absorption rates varied from 0-250.75 mgs per month. Absorption rates were significantly
different between the genders (p= <0.0001). A multivariate linear regression model determined
that phlebotomy mobilized iron was the strongest predictor of iron absorption, followed by gender, SF at
diagnosis, and patient height. In aggregate these four variables accounted for 34.7% of the variance in
monthly absorption rates. HFE genotype was not an independent predictor of iron absorption rate in this
population.
Thus, iron absorption rates among individuals with HHC may vary widely during maintenance
phlebotomy treatments. Our data shows that the degree of body iron overload at diagnosis is a major
predictor of ongoing excessive iron absorption. It is likely that such a vast difference in accumulation rates
is not explainable by differences in iron intake or occult blood loss. Modifier genes are likely to be
involved in modulating iron absorption, resulting in both increased expression at diagnosis and increased
requirement for maintenance phlebotomy. Our data on iron absorption during maintenance phlebotomy
provide an objective measure of phenotypic expression that will be useful in future studies to determine
the role of phenotypic and genotypic modifiers that determine disease expression.
POSTER 3
REVERSE-HYBRIDIZATION ASSAY FOR MULTIPLE MUTATIONS ASSOCIATED WITH
HEREDITARY IRON OVERLOAD
C. Oberkanins, A. Moritz, G. Kriegshäuser, F. Kury
ViennaLab Labordiagnostika GmbH, A-1110 Vienna, Austria.
Inherited iron overload is a heterogenous disorder, including "classical" autosomal recessive hereditary
haemochromatosis (HH), as well as juvenile and autosomal dominant forms of the disease. The most
prevalent variant among Caucasians is autosomal recessive HH due to mutations in the HFE and
transferrin receptor-2 (TFR2) genes. More recently, mutations in the ferroportin (FPN1/SLC11A3/IREG1)
gene were found to be associated with autosomal dominant iron overload. In most cases therapeutic
phlebotomy provides an effective and inexpensive lifelong treatment. DNA testing is now routinely used to
support the diagnosis in patients with abnormal iron parameters, for the presymptomatic identification of
individuals at risk, and its potential for population screening programs is currently under discussion.
We have developed a reverse-hybridization assay (Haemochromatosis StripAssay) for the rapid and
simultaneous detection of 18 known mutations in the HFE (V53M, V59M, H63D, H63H, S65C, Q127H,
P160delC, E168Q, E168X, W169X, C282Y, Q283P), TFR2 (E60X, M172K, Y250X, AVAQ594-597del)
and FPN1 (N144H, V162del) genes. The test is based on multiplex DNA amplification and hybridization to
a teststrip presenting a parallel array of allele-specific oligonucleotide probes for each mutation. The
entire procedure from blood sampling to the identification of mutations requires less than 6 hours, and
hybridization/detection may be carried out manually or essentially automated using existing
instrumentation (e.g. TECAN profiBlot). The test is simple and convenient, requires very small amounts of
samples, and can easily be modified to include additional mutations.
(oberkanins@viennalab.co.at)
POSTER 4
HAEMSCREEN: A WORKPLACE HAEMOCHROMATOSIS SCREENING PROGRAM
M.B. Delatycki, A. Nisselle, A. Wakefield, V. Collins, J. Halliday, S. Metcalfe, M-A. Aitken, D. du Sart, I.
Macciocca, A. Gason, B. Williamson, K. Allen. Murdoch Childrens Research Institute and Genetic Health
Services Victoria, Royal Children’s Hospital, Victoria, Australia
Introduction: Hereditary haemochromatosis (HH) is a common iron overload disease where symptoms
can be prevented by regular venesection. More than 90% of Australians with HH are homozygous for the
C282Y mutation in the HFE gene. About 1:200 Northern Europeans are homozygous for the C282Y
mutation but not all will suffer symptoms. The issue of whether community screening for HH should be
introduced is a matter of considerable debate. By piloting a workplace screening program for the C282Y
mutation amongst well individuals in a multicultural city, we aim to assess test acceptability and
understanding of genetic information for a common, preventable genetic disease.
Methods: Employees were invited to an education session at their workplace and offered screening for
the C282Y mutation by cheek brush DNA sampling followed by real-time PCR melt-curve analysis. There
was time allocated for completion of a questionnaire before providing the DNA sample at the end of the
session. The questionnaire provides information on demographics, knowledge of HH, anxiety and health
status. Four weeks after the results of the DNA testing were known the C282Y homozygotes and age
and sex matched controls (2 per homozygote) were surveyed for the same variables. A cohort of those
who were invited but did not attend the education session were surveyed to determine the reasons for
non-attendance.
Results: 3455 people attended educational sessions and 3377 have been eligible for testing. Of those
eligible 3314 have had testing (98.1%). The attendance rate where this could be determined is 11%.
There is a significant inverse relationship between the attendance rate and the number of employees at
2
workplaces (R =0.46, p<0.0001). C282Y results are available for 2676 of those tested. There have been
11 homozygotes (1:243) and 276 heterozygotes (1:10).
The video education resulted in a good understanding of the cause of haemochromatosis (84% correctly
answered) and how it can be prevented (90%) but poorer understanding of disease penetrance (50%)
and genetic and allelic heterogeneity (65%).
Of the 11 homozygotes (age range 25-59), 7 were male and all but one was of Northern European
extraction. Three of the 11 were already aware of their genetic status and had been previously tested
either because of symptomatology or family history. All of the 8 unaware homozygotes had non-specific
symptoms of lethargy, abdominal pain or joint pain. Five of these had markedly abnormal iron studies
and have commenced a venesection program. Two of those 5 had a family history of haemochromatosis.
Of the remaining 3, 1 was a pre-menopausal female who had a previous history of significant blood loss
and none had a positive family history.
The reason for non-attendance at the sessions (n= 273) was related to practical constraints in 75% (didn’t
know about the program, too busy, unavailable at session times). Other reasons included low iron or
being a blood donor (7%), HH unimportant or hadn’t heard of it (2.5%), concerns about anxiety related to
being found to be C282Y homozygous (1%) and insurance and confidentiality concerns (1%).
Conclusions: - If people attend the education session they almost all have testing.
- People understand the cause and prevention of disease but understand the genetic aspects of
haemochromatosis less well.
- Sixty-two percent (5/8) of those that were unaware of their homozygous state had markedly abnormal
iron studies and are now taking appropriate steps to normalise these parameters.
POSTER 5
THE EFFECT OF CYSTEINE CONTAINING PEPTIDES ON IRON UPTAKE IN Caco-2 CELLS
L.F. Hansen1,2, M. Goodman2, E. Bosa2, A. De Capua2, A.F. Hofmann2, K. Bukhave1
1
Technical University of Denmark, 2University of California, San Diego.
Introduction: Iron deficiency is one of the greatest nutritional problems in the world today affecting
more than 3.5 billion people. Therefore, it is important to understand the mechanisms involved in optimal
iron homeostasis.
The main source of dietary iron is nonheme iron whose absorption is strongly influenced by other
dietary constituents. Muscle protein has long been known to enhance absorption of both nonheme and
heme iron. The enhancing effect of meat on nonheme iron absorption, known as the “meat effect”, is not
well understood, but has been ascribed to the ability of cysteine and cysteine containing peptides to
chelate nonheme iron and thereby facilitate intestinal absorption. It is the aim of the project to synthesize
decapeptides containing four cysteines that would chelate iron in a tetrahedral conformation in order to
test the hypothesis that the peptides solubilize the iron during digestion and enhance iron absorption in
the duodenum and the proximal jejunum.
Methods: The decapeptide was synthesized by manual solid-phase synthesis using 9-fluorenylmethoxycarbonyl (Fmoc)-protected amino acids on a Wang resin. The cyclization of the decapeptide was
carried out at high dilution (10-4 M) with 3-diethoxyphosphoryloxy-1,2,3-benzotriazin-4-(3H)-one (DEPBT).
The peptides were purified by high performance liquid chromatography and structurally identified by
electrospray ionization mass spectrometry as well as two dimensional 1H nuclear magnetic resonance
(NMR) spectroscopy. The coordination of the peptides with iron is currently under investigation by
spectrophotometric monitoring, circular dichroism and NMR spectroscopy.
Influx studies of 55FeCl3 at the physiologically relevant pH 4.6 were carried out using a polarized
monolayer of the human colon carcinoma cell line Caco-2 measuring uptake at the apical side. Both the
linear and the cyclized peptide were analyzed for effects on iron uptake and ascorbic and phytic acids
served as positive and negative controls, respectively.
Results: The preliminary results from in vitro influx studies showed positive effects of both the cyclic
and the linear decapeptide on iron uptake in Caco-2 cells. For the peptides and ascorbic acid the cellular
uptake was ~16%, leaving ~75% in the test solution. Nonspecific binding was small, i.e. 3% with the
peptides and 6% with ascorbic acid. With no modulators added (system control) cellular uptake was 12%,
whereas uptake was <1% when phytic acid was present.
Total iron uptake was calculated from the fractional uptake of radioactive iron and the total amount of
iron added and this value was normalized to cell DNA. Addition of cyclic and linear peptide as well as
ascorbic acid resulted in similar cellular uptakes of 112±10, 96±7, and 102±14 pmol iron per µg DNA
(mean±SD; p>0.05), respectively, and these uptakes were all significantly higher than the system control
(75±12 pmol iron per µg DNA); i.e. p<0.002, p<0.03, and p<0.02, respectively. Cellular uptake for the
negative control, phytic acid, was only 4±1 pmol iron per µg DNA.
Discussion: The capability of the synthesized peptides to significantly enhance iron uptake could be
due to increased iron solubilization. The aqueous solubility of ferric iron is a function of pH and at pH 4.6
ferric iron starts to precipitate and becomes less bioavailable. The addition of the strong chelator, phytic
acid, decreases the bioavailability of iron through a too strong binding, resulting in severely inhibited
uptake. In contrast, ascorbic acid is believed to enhance iron absorption by either reducing or solubilizing
iron. It may be speculated that one of these mechanisms of enhancement also applies for the cysteine
containing peptides.
Conclusion: These results indicate that the synthesized peptides with four cysteine residues are as
efficient as ascorbic acid in enhancing iron uptake by Caco-2 cells under the given conditions. It is still
unknown if this effect reflects iron solubilization or reduction to ferrous iron, which can be transported into
the cell by the divalent metal transporter (DMT1). Further investigations are underway to clarify the exact
mechanism involved in enhanced iron uptake mediated by the peptides.
POSTER 6
THE CURIOUS EFFECTS OF AMINOCARBOXYLIC ACIDS ON CANCER CELL PROLIFERATION
E. Baker, A.C.G. Chua and A. Kicic
Physiology, Biomedical and Chemical Sciences, Faculty of Life and Physical Sciences, University of
Western Australia, Crawley, Western Australia 6009
The concept of iron deprivation using iron chelators to inhibit cancer cell proliferation is being investigated
using a range of classes of chelators. This paper reports on the specific effects on hepatocellular
carcinoma cells (HCC) of the aminocarboxylic acids ethylenediaminetetraacetic acid (EDTA),
diethylenetriaminepentaacetic acid (DTPA) and nitrolotriacetic acid (NTA), compared with the carboxylic
acid, citrate, and desferrioxamine (DFO), which has been shown to have significant activity in some
clinical trials. The effects on cancer cell proliferation, cancer cell progression, iron uptake and toxicity
were measured in rat and human HCC cell lines. The chelator selectivity for HCC compared with
proliferating normal cells and for other types of cancers were also assessed.
Interestingly, the inhibition of HCC proliferation by DTPA was far greater than its analogue EDTA or DFO,
while NTA and citrate had little effect. In addition, while neither DTPA nor EDTA inhibited iron uptake in
the short term (2h), even at 500mM, after 24h, DTPA (50µM) reduced iron uptake to about 10% of the
control, much more than EDTA (approx. 90%) or even DFO (approx. 60%). In contrast, there was no
significant difference between DTPA and DFO in toxicity after 24h incubation at their respective IC50s.
DTPA induced apoptosis in all HCC cell lines. DTPA inhibited melanoma cell proliferation as well as HCC,
but had no effect on breast or prostate cancer cells.
In summary, DTPA caused a highly significant reduction of cancer cell proliferation, which may be related
to its strong inhibition of iron uptake. This suggests that DTPA is a unique aminocarboxylate chelator and
is selective in its activity to hepatoma and melanoma cells.
Thus, DTPA may have potential in short-term chemotherapy, in some cancers, but is not active in breast
or prostate cancers. It has already been used clinically in the short-term treatment of thalassaemia. DTPA
may also have potential as a molecular probe to investigate the differences in iron metabolism between
different types of cancer cells.
POSTER 7
COMBINATION OF DEFEROXAMINE AND DEFERIPRONE INDUCES SYNERGISTIC INHIBITION OF
METALLOENZYMES WHICH CAN BE PREVENTED WITH LARGER HYDROXYPYRIDINONES
R. Kayyali1, R.C.Hider2, J.B.Porter1, 1Department of Haematology, University College London Medical
School, London, UK. 2Department of Pharmacy, King’s College London, London, UK.
Combining a bidentate hydroxypyridinone (HPO) ligand with deferoxamine (DFO) has been proposed as
a strategy to increase the clinical efficiency of iron chelation therapy. This relies on the principle of the low
molecular weight bidentate chelator accessing iron pools unavailable to the larger DFO molecules and
subsequently ‘shuttling’ the chelated iron to a DFO ‘sink’. However, mixed ligand combinations may not
only enhance the efficiency of chelation therapy, but also the toxicity. Thus bidentate chelators, when
coupled with a hexadentate sink, could remove essential metal co-factors from enzymes, thereby
inhibiting their activity. We have shown previously that HPO iron chelators inhibit non-heme iron
containing enzymes at a rate dependent on their size, lipid solubility and metal affinity. In this study, we
have examined the effect of these variables on metalloenzyme inhibition when HPO ligands are used in
combination with DFO. To do this, we have investigated the inhibition rates of key iron enzymes, 5lipoxygenase (5-LO) and ribonucleotide reductase (RR) as well as the zinc-containing enzymes,
phospholipase C (PL-C) and alkaline phosphatase (ALP). The inhibition of RR was monitored indirectly,
by measuring tritiated thymidine incorporation into DNA. 5-LO inhibition was examined
spectrophotometrically by measuring the rate of linoleic hydroperoxide formation by soybean
lipoxygenase. The inhibition rate of both PL-C and ALP was also measured spectrophotometrically by
measuring the rate of hydrolysis of their different substrates into p-nitrophenol, which is chromogenic.
Results using isobol plot analysis show that combining deferiprone, the clinically available HPO, with DFO
has a synergistic inhibitory effect on 5-LO. This is particularly marked at clinically relevant concentrations
of deferiprone and DFO. Thus at 10uM DFO and 30uM deferiprone, 50% inhibition is seen at 15 minutes
incubation. Increased inhibition with DFO could be completely abrogated using a bulky HPO with larger
dimensions than deferiprone (CP358), or a hydrophilic chelator with much lower distribution coefficient
than deferiprone (CP40). An additive rather than synergistic effect was observed when an HPO with a
higher affinity for iron than deferiprone (CP502) was combined with DFO. Unlike 5-LO inhibition, all HPOs
examined acted synergistically with DFO to inhibit DNA synthesis. However, the concentration of each
HPO ligand required to produce the same inhibition was substantially different. Thus in combination with
DFO 3uM, 40% DNA synthesis inhibition was seen at 15uM IBE CP502, 30uM IBE deferiprone and
160uM IBE CP358. DNA synthesis inhibition was particularly marked at clinically relevant concentrations
of CP502 (the HPO chelator investigated with the highest affinity for iron) and DFO, ~50% inhibition is
observed at 30uM (10uM IBE) CP502 and 10uM DFO. Although zinc depletion may be a key mechanism
for apoptosis induction by iron chelators, we found no evidence of increased inhibition of the zinc
enzymes, PL-C (from Bacillus cereus) and ALP at clinically relevant combinations of deferiprone and
DFO. However, combining any of the HPOs, at clinically relevant concentrations, with the zinc chelator
NNNN-tetrakis(2-pyridylmethyl)ethylene diamine) (TPEN) caused an identical synergistic inhibitory effect
on PL-C but not ALP, the difference being due to TPEN being a more potent inhibitor of ALP than PL-C
hence overcoming the need for a shuttler. In conclusion, inhibition of iron containing enzymes, but not
zinc containing enzymes could present a potential toxicity mechanism for combination iron chelation
therapy. Varying the physiochemical properties of the HPO ligands, either by increasing their
hydrophilicity or by increasing their molecular dimensions can be exploited to minimise the inhibition of
such enzymes.
POSTER 8
EXPRESSION OF IRON TRANSPORTER PROTEINS IN THE RAT COLON
Kelly Johnston, Christina Soromani and Paul Sharp
Guildford, GU2 7XH, UK.
Dietary iron is absorbed in the duodenum via the concerted action of two iron transport proteins. DMT1
located in the apical membrane of enterocytes is responsible for uptake from the diet, whereas IREG1 at
the basolateral pole mediates iron efflux. The movement of iron across enterocyte membranes requires
reduction of the ingested ferric iron to the bioavailable ferrous form. This is achieved in the intestinal
lumen by a combination of endogenous (Dcytb) and exogenous (dietary components such as ascorbate)
reducing mechanisms. However, despite favourable uptake conditions, only 10% of dietary iron is
absorbed in the duodenum meaning that 90% reaches distal small intestine and colon. In the colon
further significant amounts of ferrous iron can be liberated as a consequence of gut flora metabolism of
the intestinal contents and potentially this iron source is also available for absorption. Uptake of iron in
the colon would presumably require the presence of DMT1 and IREG1 and therefore, as a first step in
investigating whether colonic iron is bioavailable we have determined the expression of these transport
proteins in rat colon.
The entire colon was removed from male Wistar rats (250 g) following cervical dislocation and separated
into proximal (10 cm nearest to the caecum) and distal (10 cm nearest to the rectum) sections. The
mucosa was separated from the underlying musculature and used to prepare total plasma membrane
proteins, which were subjected to western blotting using transporter specific antibodies.
DMT1 (IRE) and IREG1 proteins were highly expressed in duodenum. In addition, significant levels of
these transporters were also evident in proximal but not distal colon. Duodenal DMT1 (non-IRE)
expression was lower than the IRE-containing isoform. Interestingly, DMT1 (non-IRE) expression in the
proximal and distal colon was similar to levels seen in the duodenum. The expression of DMT1 (IRE) and
IREG1 in the proximal colon suggests that under certain physiological conditions, this tissue could be
capable of absorbing dietary iron. The distal colon lacks this absorptive capacity. However, the
expression of DMT1 (non-IRE) in this tissue points to an as yet unidentified role for this isoform of the
transporter in colonic physiology.
This work was funded by the Biotechnology and Biological Sciences Research Council (grant number
90/D17146).
POSTER 9
DHPLC SCANNING OF A POPULATION AT RISK OF HEMOCHROMATOSIS: IDENTIFICATIONS OF
NEW MUTATIONS OF HFE AND HEPCIDIN GENES
Giorgio Biasiotto, Silvana Belloli, Giuseppina. Ruggeri, Isabella Zanella, Gianmario Gerardi, Alberto
Albertini, Paolo Arosio. Dipartimento Materno Infantile e Tecnologie Biomediche, University of Brescia,
Viale Europa 11, 25123 Brescia, Italy
It is now known that the alteration of various genes contribute to systemic iron overload and genetic
hemochromatosis. They include HFE, which is the most common, and ferroportin-1, transferrin receptor 2,
a gene on chromosome 1, and, more identified recently, Hepcidin. The transmission of these disorders,
except ferroportin1 is recessive, and the subjects with heterozygous mutations are asymptomatic.
However, the association between different mutations may explain the phenotypic heterogeneity of
hereditary hemochromatosis and its incomplete penetrance. Thus, the identification of the mutations of
protein possibly involved in iron regulation may be important for the diagnosis of hemochromatosis.
Denaturing-HPLC (DHPLC) is a recently developed technique for the rapid and economical DNA
scanning of large populations, and it recognises with high accuracy the presence of heterozygous known
and unknown mutations. We have applied DHPLC for the study of DNA variations in HFE and hepcidin
genes. Methods. DNA fragments covering HFE exon 2, exon 3 and exon 4 were amplified by specific
primers. Exon 1 and exon 5 and 6 were not analysed because no significant mutations have been
identified in them, so far. In addition, hepcidin exon 1, 2 and 3, which cover the entire gene, were PCR
amplified. The amplicons were analysed on DHPLC at near-melting temperatures to identify point
mutations. As controls we used samples with H63D (exon2), C282Y (exon 4) mutations. Controls from the
other exons were not available, and samples were run at multiple temperatures. HFE C282Y and H63D
were identified also by restriction analysis, all other mutations were identified by DNA sequencing.
Results. DHPLC was used to scan 657 DNA samples from subjects who presented for genetic
hemochromatosis genotyping who had serum ferritin levels above 400 ug/L. In the population we
identified 36 C282Y homozygous, 22 compound heterozygous C282Y/H63D, and 23 H63D homozygous.
Allele frequency for C282Y was13.7%, for H63D was 16.0%. We found a complete agreement between
DHPLC and restriction analysis in all samples, with no false negative or positive results. In addition we
found 14 heterozygous for the S65C mutation, of which 2 were in associations with C282Y-/+ and two
with H63D-/+. We also identified 2 new HFE substitutions: R66C in exon 2, and R224G in exon 4. They
were heterozygous and not associated with other HFE mutations. Three new HFE polymorphisms were
identified; they interest condons 30, 232 and 289 but do modify the encoded amino acid. Mutations in the
hepcidin gene are rare but do occur: we identified two different substitutions in intron 2 in four subjects
and, more important, the substitution Gly71 into Asp. We found 4 subjects heterozygous for this mutation,
and interestingly, two of them were also HFE-C282Y+/+, and two siblings were HFE-C282Y-/+. The
substitution is potentially relevant, since it carries an acidic group in the basic protein, and Gly71 is
located between two couples of cysteines that are probably structurally important. Conclusions. DHPLC is
a powerful and useful technique for hereditary hemochromatosis genotyping. It detected two new HFE
mutations (R66C and R224G) and three new HFE polymorphisms. More important, it allowed identifying a
new rare mutation of hepcidin gene, the clinical significance of which remains to be studied
Totale 657
Alele frequency
C282Y = 13.7%
H63D = 16%
S65D = 1.07% (but familiarity)
HFE
C282Y +/+ = 36
C282Y -/+ = 108
F C282Y = 13.69%
H63D +/+= 23
H63D -/+= 164
(compound C282Y+H63D = 22)
S65D -/+ = 14
(compound 282 +65 = 2)
(compound 63+65 =2)
R66C -/+ = 1
L30L -/+= 1 CT
R224G -/+ = 1
L289L -/+ = 1 G1011C
P232P -/+ =1 C696T
Hepcidin
Ex3 G71D -/+ = 4
Intr 2 G7A -/+ = 3
Intr 2 G56A -/+ = 1
(compound Hep G71D -/+ + HFE C282Y-/+ =2
compound Hep G71D -/+ + HFE C282Y+/+ =2
POSTER 10
ZINC STIMULATES THE PROMOTER ACTIVITY OF THE DIVALENT METAL TRANSPORTER (DMT1)
GENE IN HUMAN INTESTINAL CACO-2 CELLS
1
Jason Tennant, 2Henry Bayele, 1Deborah Johnson, 2Nita Solanky, 2Surjit Kaila Srai and 1Paul Sharp
1
Guildford, GU2 7XH and 2Department of Biochemistry & Molecular Biology, Royal Free and University
College London Medical School, London, NW3 2PF.
We have previously demonstrated that exposure of the human intestinal Caco-2 cell line to zinc
stimulates iron uptake via an increase in DMT1 transporter expression (Yamaji et al., 2001). The cellular
mechanisms involved in the up regulation of DMT1 by zinc are still unclear, though one possibility is that
activation occurs via interaction with putative metal response elements (MRE) residing in the 5’ promoter
region of the DMT1 gene (Lee et al., 1998). To test this possibility, we have investigated the effect of zinc
on the activity of the DMT1 promoter using a reporter gene assay.
Caco-2 cells were seeded at a density of 6x103 cells/cm2 into 24-well plates. After three days cells were
transfected using the CaPO4 method with pGL3 plasmid (Promega, UK) that contained 1.6kb of the DMT1
promoter cloned in front of a luciferase reporter gene. Three days following transfection, cells were
exposed to zinc (100µM) for 24 hours and luciferase activity in cell lysates was measured by
luminescence.
Zinc stimulation of Caco-2 cells transfected with the DMT1 promoter construct, significantly increased
luciferase activity compared with unstimulated cells (control 928 ± 79 a.u. vs +Zn 4702± 348 a.u. n=6,
p<0.0001, Student’ unpaired t-test). This suggests that zinc may promote the absorption of iron by Caco2 cell monolayers by activating a transcription factor that can interact with a specific consensus sequence
within the DMT1 promoter. The nature of the transcription factor is unknown but the zinc-inducible MTF-1
that binds to MRE sequences in several genes (Andrews, 2001) is a possible candidate. Further studies
are underway in our laboratory to further identify the mechanisms involved in this response.
This work was funded by BBSRC grant (90/D13400).
References:
Andrews GK (2001) Biometals 14, 223-227.
Lee PL et al., (1998) Blood Cells Mol. Dis. 24, 199-215.
Yamaji S et al., (2001) FEBS Lett. 507, 137-141.
POSTER 11
THE ANTIMICROBIAL PEPTIDE HEPCIDIN DECREASES IRON UPTAKE BY HUMAN INTESTINAL
CACO-2 CELLS.
1
Sachie Yamaji, 1Bala Ramesh, 2Paul Sharp and 1Surjit Kaila Srai
1
Department of Biochemistry & Molecular Biology, Royal Free and University College London Medical
School, London, NW3 2PF and 2Centre for Nutrition and Food Safety, School of Biomedical and Life
Sciences, University of Surrey, Guildford, GU2 7XH.
Hepcidin is a 25 amino acid peptide produced in the liver whose expression is increased by iron loading
and decreased in iron deficiency (Pigeon et al., 2001). This site of synthesis and the dramatic regulation
by iron has led to the suggestion that hepcidin might be the master controller of iron metabolism, relaying
information about the status of the body iron stores to the intestine and regulating absorption accordingly.
The mode of action of hepcidin is still unclear. In this study we have utilised the Caco-2 cell model of
human intestinal epithelial cells to investigate possibility that hepcidin might interact directly with the
epithelium.
Cells were cultured in Transwell plates for 21 days. For the final 24 hours of the culture period, human
synthetic hepcidin (30µg/ml) was added to the basolateral medium. At the end of the incubation period
55
cells were either used to measure Fe transport across the Caco-2 cell monolayers, processed for
Western blotting for the iron transport proteins DMT1 and IREG1, or used as a source of RNA to
determine changes in transporter expression by Real-Time RT-PCR.
Following exposure to hepcidin, iron uptake across the apical membrane of Caco-2 cells was significantly
decreased (control 453.3 ± 47.0 pmol/cm2/h; +hepcidin 268.3 ± 63.8 pmol/cm2/h, means ± S.E.M. n=6,
p=0.04 Student’s unpaired t-test). Efflux across the basolateral membrane was unaffected by hepcidin
treatment. In agreement with the transport data, the expression of the apical membrane transporter
DMT1 was decreased by hepcidin treatment at both the protein and mRNA level, whereas expression of
IREG1, the basolateral efflux protein, was unaffected.
Taken together, our data demonstrate that hepcidin can interact directly with intestinal epithelial cells and
that its mode of action appears to be via regulation of the apical DMT1 transporter.
Reference:
Pigeon C et al. J. Biol. Chem. 276, 7811-7819.
POSTER 12
HEMOCHROMATOSIS PATIENTS AS VOLUNTARY BLOOD DONORS
PC Adams, TE Power. University of Western Ontario, London, Ontario, Canada
This study was designed to investigate hemochromatosis patient's suitability as blood donors as well as
their perceptions and experience with the current public donation system. The difference in quality of life
between hemochromatosis patients who currently donate and those who do not was also measured.
Participants were gathered from a list of current hemochromatosis patients of Dr. Paul Adams (N =120),
as well as individuals who were members of the Canadian Hemochromatosis Society (N = 1000) and had
indicated in their memberships that they themselves had the disease. Of those mailed a survey 803 were
returned completed. The sample consisted of 57 % males with a mean age of 57.44 (SD = 12.73; 19-87
yrs). It was found that 20% (160 ) of the respondents have donated blood since their diagnosis, however,
only 12% of the respondents indicated that they use voluntary blood donation as a means of maintaining
their iron levels. Additionally, it should be noted that only 8% (64) are new donors since having been
diagnosed with hemochromatosis.
It was also found that a number of barriers exist for hemochromatosis patients who desire to donate their
blood rather than undergo therapeutic phlebotomy. Forty percent of the respondents indicated that they
had been refused from voluntary donation. Despite the fact that in May 2001, the Canadian Blood
Services in collaboration with the Canadian Hemochromatosis Society, began a promotion campaign to
encourage hemochromatosis patients to become voluntary blood donors, our study found that 15% of the
respondents had been refused from the voluntary blood donation service due to the diagnosis of
hemochromatosis. Additionally, of the 433 participants who indicated they would be willing to donate their
blood to a voluntary blood service , 44% indicated that the only limiting factor was if they knew that the
blood agency would accept the donation from a hemochromatosis patient.
With respect to quality of life, it was found that individuals who donate blood were generally healthier with
respect to physical functioning, and bodily pain, however, these findings may indicate those
hemochromatosis patients who are healthier are better able to donate at public blood banks, rather than
that voluntary blood donation has an affect on the donors physical functioning over medicalized
phlebotomy.
These study findings suggest that although there may be other medical factors limiting individuals from
donating, hemochromatosis patients are interested in having their phlebotomized blood used for blood
donation purposes. They also suggest that more public information campaigns need to target the staff at
voluntary blood banks, the medical professions as well as the hemochromatosis patients themselves in
order to educate the public about the suitability of hemochromatosis patients as blood donors.
POSTER 13
COMPARATIVE STUDY OF THE BIOLOGICAL PROPERTIES OF THE HYDROXYPYRIDINONES
CP411 AND CP20 IN NORMAL AND TRANSFORMED RAT HEPATOCYTE CULTURES
F. Gaboriau1, N. Rakba1, P. Loyer1, N. Pasdeloup1, R.C. Hider2, P. Brissot1 and G. Lescoat1
1
Inserm UR522, Régulations des Equilibres Fonctionnels du Foie Normal et Pathologique, Centre
Hospitalier Pontchaillou, Rennes, France,
2
Department of Pharmacy, King’s College, London, UK.
Abstract
Aim : The present study analyzes the iron mobilization, the cytoprotective and the antiproliferative effects
of the lipophilic hydroxypyridinone CP411, in comparison with the hydrophilic chelator CP20 or
deferiprone used in the treatment of iron overload. The cell uptake of these two chelators and their
inhibiting effect of the Fenton’s reaction were compared in order to analyze their biological properties.
Methods : Iron chelator cell uptake were evaluated by mass spectrometry in primary hepatocyte and Fao
cell line cultures. This method was also used to investigate the stability of the chelators in solution as well
as their scavenging and chelating effects against the hydroxyl radical generated from the Fenton’s
reaction.
The iron mobilization and the cytoprotective effects of the chelators were evaluated in primary rat
55
hepatocyte cultures by measuring respectively Fe and LDH release in the culture medium. The
antiproliferative effect of the chelators was studied using the rat hepatoma Fao cell line by measuring
thymidine incorporation and DNA content by flow cytometry to evaluate DNA synthesis.
Results : In solution, the inhibiting effects of these two chelators against the hydroxyl radical formation
were shown to be equivalent. In contrast, we observed that CP411 entered the hepatocytes and the Fao
cells respectively 4 and 13 times more than CP20. CP411 was 2.5 times more effective than CP20 to
mobilize iron from pre-loaded hepatocytes. Pre-treatment of the hepatocytes with CP20 or CP411
decreased the toxic effect of iron and CP411 was 1.6 more effective than CP20. In the Fao cell line, a
dose-dependent decrease of DNA synthesis, correlated to an accumulation of cells in S phase, was
observed in the presence of CP411, whereas CP20 was without effect. CP411 effect was inhibited by
simultaneous addition of iron with the chelator while addition of Zn or Cu remained inefficient. The
inhibitory effect of CP411 was reversible since 24 hrs after removal of the chelator, DNA replication
reached the control level. The antiproliferative effect of CP411 was correlated to an increase of AST
release in the culture medium.
Conclusions : CP411 is more efficient than CP20 to protect the hepatocyte from the toxic effect of iron
load and to inhibit tumor cell proliferation. Its higher efficiency may result from its better cell uptake since
equimolar solutions of the two chelators in an acellular system exhibit the same ability to inhibit the
Fenton’s reaction.
POSTER 14
A USER PERSPECTIVE ON GENETIC SCREENING FOR HEMOCHROMATOSIS. PRELIMINARY
RESULTS OF THE PSYCHOLOGICAL EFFECTS ON HFE GENOTYPING AND LEVEL OF
INFORMATION IN A POPULATION OF DANISH MEN
P. Pedersen*, P. Elsass¤, K.Husum ¤, and N. Milman§
#
Department of Clinical Biochemistry, Naestved Hospital, Naestved, § Department of Medicine B,
Rigshospitalet, University of Copenhagen, ¤ Institute of Psychology, University of Copenhagen,
Copenhagen, Denmark
Background: Screening for hemochromatosis gives possibilities for qualifying a documentation of the
psychological effects of the information given. Approximately one third of the population is carrier of one
or more HFE mutations. Hereditary hemochromatosis is characterized by iron loading, which may result in
severe organ damage if untreated. Therefore, genetic screening for hemochromatosis has been
suggested to identify subjects prior to clinical presentation of disease. The aim was to evaluate the
psychological reaction to different levels of information.
Methods: Randomly selected men aged 30-51 participated in a two-step screening. A total of 6,020
complete HFE genotypes were obtained and biochemical information was available in 1,453 (24 %) of
which additional data were available for 1,295 (22 %) individuals who had completed their questionnaire.
An initial genetic screening for HFE mutations (845 G→A, 187C→G and 193A→T) was performed. In the
first group, participants with HFE mutation(s) received genetic information and an invitation to a
biochemical screening. The second group were invited for simultaneous genetic and biochemical
screening. The first group (n = 944) received two letters of information, first genetic information and
subsequently the biochemical results. The second group (n = 519) received genetic and biochemical
information simultaneously. According to the genetic information given the participants were grouped as;
'high risk' (homozygous 845 G→A, compound heterozygous 845 G→A/187 C→G), 'unknown risk'
(homozygous 187 C→G and 193 A→T, compound heterozygous 845 G→A/193 A→T and 187 C→G/193
A→T) and 'no risk' (heterozygous individuals) based on the current knowledge available at that time.
Questionnaires about the understanding of and reaction on information given were mailed to all
participants at the same time as the information of their screening result. Most questions were graded in a
five-point Lickert-scale.
Results: 1,294 questionnaires were completed (response rate 43 %). In a five-point scale 99% rated
much or very much satisfied with participating in the screening program; and 86% were much or very
much satisfied with the level of information given. The 'high risk' group had a significant tendency for
being surprised of the information given and had more adverse psychological reactions, e.g. worried,
depressed and anxious. However, no ratings were in the lowest end of the Lickert-scale.
Dividing the three groups of 'high risk',' uncertain risk' and 'no risk' into levels of information, the group
receiving genetic information rated their psychological reactions significant more negative in 7 questions
than the group receiving both genetic and biochemical information. This difference was more pronounced
in the group of 'uncertain risk'.
Conclusions: This study supplements the discussion in relation to the ethic dilemmas of screening
programs with empirical knowledge of a user perspective. In general we found positive reactions among
the users of our screening program. Individuals receiving both genetic and biochemical information were
more satisfied than the individuals receiving only genetic information. Although the differences are small
they demonstrate the importance of giving the most detailed information to the user.
However our results are preliminary and open for discussion. The response rate was incomplete and the
population comprise only individuals with HFE mutation(s). With these precautions our conclusion is not
generalizable to all genetic screening programs but do show the importance of including the biochemical
information on iron status in the screening for hemochromatosis.
POSTER 15
INTESTINAL ABSORPTION OF IRON FROM HEME IN BELGRADE RATS:
A REQUIREMENT FOR DMT1 AND ACIDIFICATION OF CYTOPLASMIC VESICLES
Adrian West, Nancy C. Andrews1 and Phillip S. Oates. Physiology, School of Biomedical and Chemical
Sciences, The University of Western Australia, 35 Stirling Highway, Crawley WA, 6009. Western Australia
and 1Department of Pediatrics (Hematology/Oncology), Children’s Hospital, Enders 720, 320 Longwood
Avenue, Howard Hughes Medical Institute, Boston, Ma 02115, USA.
Heme is an important dietary component, typically making a larger contribution to body iron than nonheme iron. Absorption of iron from heme is thought to involve the receptor mediated endocytosis of intact
heme, heme catabolism within vesicles and transport of the liberated iron to the cytoplasm. However, the
specific mechanisms of this process are largely unknown. This study aims to investigate the relationship
between uptake of luminal heme, function of the H+/Fe2+ symporter divalent metal transporter 1 (DMT1),
and acidification of vesicles in the absorption of iron from heme. For this we used Belgrade rats, which
have a functional mutation (G185R) in the DMT1 gene, and NH4Cl, which is known to reduce DMT1
function in reticulocytes by neutralizing intracellular H+ gradients.
Physiological quantities (15µg) of radio-labelled 59Fe as hemoglobin or Fe2+:ascorbate were diluted in
either NaCl or NH4Cl solutions and given as a gavage to Wistar (Wi), heterozygous (+/b), and
homozygous (b/b) Belgrade rats. Absorption was measured by immediately counting the radioactivity in a
whole body counter, and re-counting after 4.5 days. Luminal heme uptake was assessed by incubating
gut sacs in vivo with hemin chloride for 45 minutes, washing the sac, and then reacting the heme in the
tissue with 3-3’DAB to produce electron dense precipitates. The tissue was then viewed under a
transmission electron microscope.
Both +/b and b/b rats had impaired heme-iron absorption, but only b/b rats had decreased Fe2+
absorption. Heme was located within vesicles in the apical cytoplasm of the enterocyte, and the
abundance of heme-containing vesicles was similar amongst all of the groups. NH4Cl significantly
reduced absorption of heme iron in all cases by up to 64%, while non-heme iron absorption was
unaffected. NH4Cl did not have any effect on the absorption of glucose and leucine.
We conclude that in Belgrade rats, impaired heme-iron absorption is not due to an inability of the
enterocyte to internalize luminal heme. Optimal absorption of iron from heme requires two functional
DMT1 alleles but only one for non-heme iron, suggesting that DMT1 is rate limiting for heme-iron
absorption. This infers that there are two distinct sites of DMT1 function for heme and non-heme iron
absorption – in vesicles, and on the microvillus membrane respectively. NH4Cl causes a specific
decrease in heme-iron absorption, confirming the role of vesicular DMT1 in this process.
POSTER 16
MITOCHONDRIAL MnSOD Val POLYMORPHISM AFFECT PHENOTYPIC EXPRESSION OF
L. Valenti P. Dongiovanni, A.L. Fracanzani, E. Fatta, A. Maraschi, G. Fiorelli, S. Fargion, University of
Milan.
Background: Only 1-33% of C282Y homozygous subjects display clinical features of hereditary
hemochromatosis (HHC). Inherited factors seem to play a role, and TNFalpha and haptoglobin
polymorphisms have been reported to influence the phenotypic expression of the disease. However,
these data were not confirmed in different ethnic groups. Recently, a polymorphism of mitochondrial
MnSOD, involved in ROS detoxification, has been linked to progression of alcoholic liver disease. Aim: to
determine whether the Ala9Val polymorphism in the mitochondrial targeting sequence of MnSOD, by
affecting ROS detoxification, influences the phenotypic expression of HHC. Patients and Methods:137
consecutive patients with HHC (39% with cirrhosis) were considered. MnSOD polymorphism was
evaluated by PCR and restriction fragment length polymorphism analysis. Results: Subjects carrying the
Val allele, associated with a lower enzyme activity, were less represented in patients compared to
controls (P=0.039), and despite comparable iron overload had a higher prevalence of liver cirrhosis (8/30,
27% for Ala/Ala, 35/89, 39% for Ala/Val, and 7/10, 70% for Val/Val subjects; P=0.022 for trend) and
extrahepatic organ involvement, including diabetes, cardiomyopathy, hypogonadism, arthritis and
osteoporosis (7/30, 23% for Ala/Ala, 39/86, 45% for Ala/Val, and 5/10, 50% for Val/Val; P=0.020 for
trend). At multivariate analysis which considered age, sex, the presence of alcohol abuse, chronic
HBV/HCV viral hepatitis, the grams of iron removed to reach depletion, and homozygosity for the C282Y
HFE mutation, the presence of a Val allele was significantly associated with cirrhosis (OR 2.43 per Val
allele; 95% CI 1.1-5.43). Cirrhosis was also associated with the degree of iron overload (OR 1.24 for each
g) and chronic viral hepatitis (OR 4.22), Conclusion: MnSOD polymorphism, possibly influencing ROS
metabolism, seems to affect the phenotypic expression of HHC.
POSTER 17
FERRITIN AND IRP2 EXPRESSION ARE ALTERED BY IRON OVERLOAD (HYPERTRANSFUSION)
IN SICKLE CELL DISEASE AND ß-THALASSEMIA
†‡W Hagar, †Z Jenkins, †H Johansson, *P Harmatz, †‡EP Vichinsky, †EC Theil, †CHORI (Children’s
Hospital Oakland Research Institute) ‡Departments of Hematology/Oncology and *Gastroenterology,
Children’s Hospital and Research Center at Oakland
Introduction: Clinical consequences of iron overload from transfusions appear to differ between sickle cell
disease and ß-thalassemia. An example is the recent observations on organ damage which could relate
to the increased mineralization of iron and the increased release of hydrogen peroxide release during
mineralization of iron by the H-subunit of ferritin. To investigate whether ferritin protein subunit
composition differs in sickle cell anemia and β-thalassemia, we examined the concentration of liver ferritin
and H/L subunits in liver tissue from seven sickle cell, five ß-thalassemia (thalassemia major or
hemoglobin E/ß-thalassemia) patients (without hepatitis C), with two normal liver autopsy samples as
controls. In addition, mRNA concentrations were analyzed for the ferritin L-subunit (FTL), the ferritin Hsubunit (FTH), iron regulatory protein 1 (IRP1), and 2 (IRP2).
Methods: Liver ferritin subunits were resolved in SDS gels, developed with sheep IgG antibodies to
human heart ferritin and peroxidase-coupled swine with anti-goat IgG, chemiluminescence detection and
quantification by phophorimaging, standardized with human liver ferritin. Differences between groups
were analyzed by the Wilcoxon Rank sum test. Quantitative RT-PCR was carried out using GAPDH as a
tissue standard and a specific internal RNA for mRNA analyzed, as a standard for the RT-PCR reaction.
CDNAs were labeled with 33P, resolved by polyacrylamide gel electrophoresis and analyzed by
phophorimaging. Differences between each group were analyzed by the Student’s T test.
Results: Liver ferritin H/L subunit ratios decreased in SCD (0.21 ± 0.06) significantly in contrast to ßthalassemia 0.34 ± 0.12 or controls, 0.62 ± 0.5. Although there was no significant change in liver ferritin
H/L subunit ratio ß-thalassemia, total ferritin increased as in Sickle Cell disease: Total ferritin was 4.7 ±
1.4 , 5.8 ± 1.8 and 1.4 ± 0.2 mg/100 mg protein for Sickle Cell, ß-thalassemia and controls, respectively.
No significant differences were observed in mRNA concentration of FTL, FTH or IRP1, although IRP2
mRNA increased significantly (P < 0.01) in both Sickle Cell Disease and ß-thalassemia, 19.9±12.3,
15.4±9.9, and 2 attamoles/100 ng total RNA for SCD, ß-thalassemia and control, respectively.
Conclusions: The significant decrease in liver ferritin H/L subunit expression in Sickle Cell Disease during
transfusional iron overload contrasts with the lack of change in ß-thalassemia and coincides and
differences in general organ damage that may relate to hydrogen peroxide production during H-ferritin
subunit function. Postranscriptional mechanisms for changes in ferritin expression are suggested by
differences between ferritin mRNA and protein expression Supported in part by NIH grants HL07951(WH), DK-057778(PH,EPV), DK-20251(ECT), M01RR01271 and HL-20985(PH,ZJ, HJ, EPV, and
ECT).
POSTER 18
CHANGES IN IRON AND FERRITIN CONCENTRATIONS IN MICE HETEROZYGOUS FOR βGLOBIN DELETION (β-THALASSEMIA INTERMEDIA MODEL) OR A HUMAN HBS TRANSGENE
(SICKLE CELL TRAIT MODEL)
K. B. Hadley1, E. C. Theil1, C. Bowlus2, and C. Vogler3
1
CHORI (Children's Hospital Oakland Research Institute). Oakland, CA 94609, 2U. C. Davis,
Sacramento, CA 3Cardinal Glennon Children’s Hospital Saint Louis MO, 63110.
Iron overload occurs from treatments of β-Thalassemia (βhal) and Sickle Cell Disease (SCD), with varying
clinical impacts for the two diseases. Transgenic mouse models of β-thal, and SCD recapitulate the
anemia of the respective human diseases. In order to characterize the effects of dietary iron, β-thal and
SCD on ferritin protein and tissue-soluble Fe, weanling C57BL/6 (control, +/+) mice, or mice
heterozygous for deletion of the mouse β-globin gene(∆β/+) or the same mice heterozygous for a human
βs transgene (βs/+) were assigned to one of two dietary treatments (n=4 to 6); standard rodent chow
containing 160 ppm Fe (control diet) or 300 ppm Fe (high iron diet), as ferrous carbonate, for three
months. Ferritin and tissue iron was measured by Western blot and ICP-MS, respectively and normalized
to +/+ mice fed control diet. Liver L-ferritin protein in ∆β/+ mice was 2.5 times greater than +/+ mouse
levels. Liver iron concentration in βs/+ mice was significantly greater than control mice (4.62 + 0.52 and
2.69 + 0.042 µmol/g wet tissue, respectively. P < 0.05). Histology examination showed iron staining
present in hepatocytes of βs/+ mice and in both hepatocytes and Kupffer cells in ∆β m ice fe d the high
iron diet. Regardless of diet, iron concentration in the hearts of βs/+ mice was greater than +/+ mice (P <
0.05). In these same tissues, the H/L ratio of ferritin subunits decreased significantly on the high iron diet
(P < 0.05). These results demonstrate that the heterozygous deletion alone or with expression of βs in
mice induces differential changes in iron homeostasis. In the case of βs/+ mice, the data support the
prediction of Hershko et al. (Scand. J. Haem. 29:304 1982) that iron absorption is changed in humans
with Sickle Cell Trait. This work was supported by: NIH T32-HL 07951, RO1-HL-l56169 and R01-DK
58882.
POSTER 19
RISK FACTORS FOR HEPATIC IRON LOADING IN HFE COMPOUND HETEROZYGOTES
Enrico Rossi 1, Ee Mun Lim 1, Bastiaan De Boer 2, Gary P Jeffrey 3.
Departments of Clinical Biochemistry 1 and Anatomical Pathology 2 PathCentre, Department of Medicine
University of Western Australia 3, Nedlands, Western Australia, 6009.
Inheritance of the compound heterozygous HFE genotype [CY/HD] is considered to be a risk factor for
developing iron loading, however the degree of clinical penetrance is poorly understood.
Patients [190 in total] were initially referred to the Hepatology Clinic at a tertiary hospital for
further assessment of biochemical iron overload, either with or without abnormal liver function tests. The
HFE gene was genotyped for C282Y and H63D mutations and the present study relates to all patients
with the CY/HD genotype. Full clinical evaluations were performed, including the clinical phenotype of
iron overload and presence of predisposing risk factors. Excessive alcohol use was defined as exceeding
60 g/day for more than seven years. We also determined whether detection of the CY/HD genotype
altered subsequent patient management. Liver biopsy histology, including degree of fibrosis, grading of
iron staining and severity of any steatosis was re-staged by a single pathologist.
The CY/HD genotype was present in 19/190 [10%] patients and 126/190 [66%] were C282Y
homozygous. All 19 CY/HD patients had liver biopsy data with quantitative liver iron determinations. Of
the four CY/HD patients with significant histological liver damage, three had concomitant hepatitis C virus
infection and one had autoimmune hepatitis. Grading of iron staining ranged between 0 and 3 on a scale
of 4.
Mild iron overload, defined as an hepatic iron between 30 and 100 mmol/kg dry weight, was
present in 18/19 CY/HD patients, the remaining patient had a normal liver iron [23 mmol/kg dry weight].
The mean hepatic iron concentration was 54 mmol/kg dry weight, [range 23-96]. An hepatic iron index
[concentration divided by the patients age] exceeding 1.9 is proposed as diagnostic of the fully expressed
phenotype and none of the 19 CY/HD patients exceeded this threshold. The maximum hepatic iron index
was 1.9, with two patients recording indices of 1.8.
Other risk factors predisposing for the development of iron overload were documented with the
following frequencies: excessive alcohol use in eight patients, obesity in six, hepatitis C virus infection in
three, diabetes mellitus type 2 in two, multiple transfusions for multiple myeloma in one and autoimmune
hepatitis in one. At least one of these risk factors was present in 17 patients and five patients had more
than one. Detection of the CY/HD genotype did not alter subsequent management of any of the patients
and the cost of HFE testing could be reduced by excluding the H63D mutation.
In conclusion, all 19 CY/HD patients showed mild iron overload as defined by hepatic iron
concentration and further risk factors predisposing to the development of iron overload were present in 17
patients.
POSTER 20
TRANSFERRIN RECEPTOR 2 GENE MUTATION IN JAPANESE NON-HFE HEMOCHROMATOSIS
M. Yano1, H. Hayashi2, S. Wakusawa2, F. Sanae2, A. Hattori2, T. Matsumoto3, T. Ikeda3, K. Togawa4, M.
Kawanaka4, G. Yamada4
1 Gastroenterology, Nagoya University School of Medicine, 2 Department of Medicine, Faculty of
Pharmaceutical Sciences of Hokuriku University, 3 Department of Internal Medicine, Yokosuka Kyousai
Hospital, 4 Center for Liver Diseases, Kawasaki Hospital, Kawasaki Medical School
Genetic background of non-HFE hemochromatosis remained unresolved in 15 % of Caucasian patients
and in almost all Asian patients. Recent reports suggest transferrin receptor 2 (TfR2) may be a candidate
for the third subtype of hemochromatosis. Therefore, we investigated the HFE and TfR2 genes of 11
sporadic and 7 familiar cases of Japanese hemochromatosis. DNA samples were extracted from
peripheral blood cells and the HFE and TfR2 genes were amplified by PCR. C282Y and H63D were
tested by RFLP, followed by final direct sequence. Five hot spots of the TfR2 gene were tested by either
RFLP (exon 6) or direct sequences (exons 2, 4, 16, and 17). No mutation of C282Y or H63D was found
in the HFE gene of our patients. Three mutations were identified in the TfR2 gene studied. Three patients
in one family were homozygous for an AVAQ 594-597 deletion that was originally reported in Italian
patients with familiar hemochromatosis. This mutation was not detected in 100 control individuals. Two
other mutations (251C-T and 1797C-T) were each found in an allele of one patient, and considered a
polymorphism because there was either no relation to the clinical manifestation or a high frequency in
normal subjects. Our study indicates that the TfR2 gene is a candidate responsible for hemochromatosis
in Japan.
POSTER 21
HYPERFERRITINEMIA, UNRELATED TO THE C282Y HOMOZYGOTE STATE, IS ASSOCIATED WITH
LOWER LIVER IRON CONCENTRATIONS COMPARED WITH C282Y HOMOZYGOTES
PC Adams, D Thorburn. London Health Sciences Centre, London, On, Canada.
Background: Unlike genetic hemochromatosis (GH), hyperferritinemia is frequently associated with the
non-C282Y homozygous state. Whether liver iron accumulation occurs in these individuals at a
comparable rate to C282Y homozygotes is untested.
Aims: To compare the accumulation of liver iron in C282Y homozygotes and non-C282Y patients with
hyperferritinemia.
Methods: 43 non-C282Y homozygotes (18 WT/WT, 11 C282Y/WT, 4 C282Y/H63D, 3 H63/H63D,
7H63D/WT) who had undergone liver biopsy to assess liver iron concentrations (LIC) were compared with
a cohort of 108 C282Y homozygotes with regard to ferritin, serum iron (Fe), transferrin saturation and LIC.
Results: Non-C282Y homozygotes have significantly lower serum Fe, transferrin saturations, LIC, hepatic
iron indices and less liver fibrosis while ferritin and transaminases are comparable.
[Mean (SD)]
C282Y Homozygotes
(n = 108)
Age (Yrs)
Serum Fe (µmol/l)
T Saturation (%)
Ferritin (ng/ml)
AST (U/l)
ALT (U/l)
LIC (µmol/g)
Hepatic Iron Index
Fibrosis [n(%)]
Cirrhosis [n(%)]
50.6 ± 13.9
34.0 ± 8.4
77.0 ± 17.4
1588 ± 1424
57 ± 92
64 ± 78
257.1 ± 172.1
5.3 ± 3.5
22 (20)
28 (26)
Non-C282Y
Homozygotes
(n = 43)
52.2 ± 15.7
27.9 ± 10.9
57.6 ± 26.2
1619 ± 1775
67 ± 63
60 ± 42
121.2 ± 164.6
2.7 ± 5.4
4 (9)
5 (12)
P value
0.376
0.002
<0.001
0.949
0.176
0.501
<0.001
<0.001
0.007
Conclusions: In non-C282Y homozygotes with iron overload, hyperferritinemia is out of proportion to the
whole body iron stores assessed biochemically and by LIC and hepatic iron index.
POSTER 22
SERUM IRON, IRON-BINDING CAPACITY, AND TRANSFERRIN SATURATION:
AN INTERNATIONAL ROUND-ROBIN COMPARISON STUDY
H.M. Blanck1, C. Pfeiffer2, S.P. Caudill2, M. Reyes1, E. Gunter2, G. Imperatore3, O. W. van Assendelft4, S.
Strider2, T. Dearth2; 1Division of Nutrition and Physical Activity, National Center for Chronic Disease
Prevention and Health Promotion (NCCDPHP) Centers for Disease Control and Prevention (CDC);
2
Division of Laboratory Sciences, National Center for Environmental Health, CDC; 3Division of Diabetes
Translation, NCCDPHP, CDC; 4Scientific Resources Program, National Center for Infectious Diseases,
CDC, Atlanta, GA 30341
Introduction: The most widely used biochemical marker of body iron status is transferrin saturation (TS),
which can be calculated as [serum iron (Fe) / total iron-binding capacity (TIBC)] x 100 or as [serum Fe /
serum Fe + unsaturated iron-binding capacity (UIBC)] x 100. TS is recommended as the first step in
detecting iron overload in patients suspected of having hemochromatosis and is also used to evaluate
patients with anemia. However, information on interlaboratory variation and methodological differences
for serum Fe, TIBC, and UIBC is limited.
Methods: Twenty-five laboratories (22 U.S., 3 international) participated in a 3-day analysis of 3 iron
concentration pools: low (near iron deficiency), medium (borderline elevated iron storage) and high (iron
overload). The laboratories used 7 different methods. Methods differed as to the chromogen (ferrine or
ferrozine) and whether protein or copper correction were used. Results were analyzed for laboratory and
method imprecision. In addition, TS values were categorized and compared by whether TIBC or UIBC
was performed.
Results: The among-laboratory coefficient of variations (CVs) were 8.6%, 9.8%, and 3.1% for serum Fe
for the low, medium, and high pools, 3.1%, 17.2%, and 3.9% for TIBC and 4.9%, 13.0%, and 43.9% for
UIBC. The two laboratories using both copper correction and true protein removal had much lower TIBC
concentrations in the medium pool than the others. It could be speculated that some protein interference
in the medium pool was removed by these laboratories. The average among-run and within-run serum
iron and TIBC CVs were <2%. The among-run CV was low for the low UIBC pool (2.5%) but was higher
for the medium pool (13.2%) and much higher for the UIBC high pool (33.8%). The within-run CVs for the
low and medium UIBC pools were <5%; however, the within-run CV for the high pool was 28%. No
statistically significant differences were found among iron assay method types using a two-way ANOVA.
Most (90%) of the low and 100% of the high pool samples were categorized appropriately; however, the
medium pool TS values varied, 23% of TS values indicated normal iron (TS = 16-45%), 69% of TS values
indicated borderline iron elevation (>45-60%), and 8% indicated iron overload (>60%).
Discussion: High precision for serum Fe was demonstrated by the among-laboratory CVs for serum iron
of <10%. None of the serum Fe results differed significantly by laboratory or type of measurement. The
among-laboratory CVs for TIBC were low (< 4%), with the exception of the medium pool (17%). Analysis
of the medium data show that the values for each laboratory were consistent from vial-to-vial and day-today. This was true for the laboratories producing the lowest as well as highest results and indicates that
medium pool variability was not due to a lack of homogeneity. Because UIBC levels are low for high
serum Fe levels and low UIBC levels are close to the limit of detection, the among-laboratory CV for UIBC
increased from the low pool (<5%), through the medium pool (13%) to the high pool (44%). Additionally,
the within-run CV for the UIBC high pool was high compared to that found for TIBC. Therefore, we found
that iron-binding capacity results appeared to differ by whether TIBC or UIBC was performed and possibly
by whether true protein removal was performed. Both may affect the utility of TS in classifying body iron
status.
Conclusions: More research is needed to understand potential differences between iron-binding capacity
methods used for TS. As laboratory technology for iron status improves, for example to lower UIBC CVs
at all iron concentrations, there may be an increased level of physician confidence in ordering and
interpreting iron status test results in patients with anemia or suspected hemochromatosis.
POSTER 23
A NEWLY IDENTIFIED ISOFORM OF DMT1 WITH A 31 RESIDUE AMINO TERMINAL EXTENSION
(EXON 1A) EXHIBITS NORMAL DIVALENT METAL TRANSPORT DURING REGULATED ECTOPIC
EXPRESSION
M.D. Garrick, H.-C. Kuo, L. Zhao, F. Vargas, X. Zhou, J.A. Roth, L.M. Garrick
Departments of Biochemistry, Pharmacology & Toxicology, Pediatrics and Medicine, SUNY, Buffalo, NY,
USA
Divalent Metal Transporter (DMT1, also SLC11A2, DCT1 or Nramp2) is responsible for absorption of
ferrous iron in the duodenum and exit of iron from endosomes during the transferrin cycle. It also may
transport multiple other divalent metal ions. Four isoforms exist for DMT1 mRNA and the encoded protein:
The 3’ UTR may contain or lack an iron responsive element (IRE so +/-IRE) and distinct C-terminal
peptides are encoded by the +IRE and -IRE mRNAs. A newly discovered first exon (1A – Hubert &
Hentze 2002 PNAS 99:12345) encodes a 31 residue amino terminal extension for rat DMT1; while the
alternative exon 1B is not translated, so its N-terminus is encoded by exon 2. One suspects that the 4
protein isoforms – 1A+IRE, 1A-IRE, 2+IRE and 2-IRE – localize differently and have different functions,
given the nearly ubiquitous distribution of DMT1 protein among tissues and the multiple potential
functions. We have therefore compared the rat 1A+IRE and 2-IRE isoforms during regulated ectopic
expression. HEK293-F cells carrying a TetR:Hyg element were transfected with pDEST31 carrying
either a 1A+IRE or 2-IRE construct (1A+IRE with or without FLAG® at the N-terminus and 2-IRE with the
54
2+
tag) and geneticin resistant clones were selected. We initially studied Mn uptake for convenience but
59
2+
data on Fe uptake are comparable. Uptake of Mn maximally increased ~20 fold in response to addition
of doxycycline with half maximal response at ~1 nM doxycycline as reported for the TetR system. Uptake
was essentially maximal from 50 nM to 10 µM doxycycline with doxycycline toxicity occurring at the top of
this range. Doxycycline-stimulated Mn uptake was linear with time for 20 min but increased less than
linearly over longer periods. Uptake exhibits a pH optimum at ~5.5 and dependence on incubation
temperature and Mn concentration. Immunofluorescent staining using antibodies specific for the 1A exon,
FLAG®, or the -IRE or +IRE species on cells grown in the presence or absence of doxycycline confirmed
an increase in expression after doxycycline treatment and revealed that DMT1 is in the plasma
membrane and cytosolic vesicles whether endogenously or ectopically expressed. Immunoblotting also
detected the stimulation of expression by doxycycline and revealed that some DMT1 protein isoforms
have distinct apparent sizes after electrophoresis. Our data indicate that the 1A+IRE isoform of DMT1 has
very similar functional properties and pattern of distribution when compared to the 2-IRE species in
HEK293-F cells.
POSTER 24
ASSOCIATION OF INCREASED IRON STATUS WITH MYOCARDIAL PATHOLOGY IN SICKLE CELL
DISEASE
Paul Kple-Faget, Victor R. Gordeuk, Oswaldo Castro.
Department of Medicine, University of Bouake, Bouake, Cote d’Ivoire (PKF)
Center for Sickle Cell Disease, Howard University, Washington, DC. (PKF, VRG, OC)
The contribution of excess parenchymal iron to tissue damage in multiply transfused sickle cell disease
(SCD) patients is not entirely clear. To determine if iron overload is associated with hepatic and
myocardial pathology in patients with SCD, we examined the histological reports of 31 adult Howard
University Hospital patients who came to full necropsy between 1979 and 2001. There were 14 males
and 17 females with a median age of 29 years (range: 20-54). Their hemoglobin types were SS (n = 26),
SC (n = 3), and S-β thalassemia (n = 2). Diagnoses contributing to death included thromboembolism or
fat embolism (n = 22), infection (n = 15), pulmonary hypertension (n = 8), liver cirrhosis (n = 4) and AIDS
(n = 3). Severe hemosiderosis or hemosiderosis involving all hepatocytes was reported in seven patients
and we classified them as having marked hepatocellular iron-loading. In the remaining patients,
hemosiderosis was reported to be moderate or unqualified (n = 8), mild (n = 1), or absent or not reported
(n = 15). Three of seven patients with marked hepatocellular iron loading had cirrhosis versus one of the
other 24 patients (P = 0.028, Fisher exact test). The pathologist reported positive staining for myocardial
iron in three of the seven patients with marked hepatocellular iron-loading and in one of the other patients
(P = 0.028). The findings of myocardial interstitial fibrosis and/or myocardial cell degeneration were
reported in three of four patients whose myocardium stained positively for iron versus five of the other 27
patients (P = 0.043). Our results suggest that iron-loading in SCD contributes to substantial organ
toxicity, including cirrhosis and cardiomyopathy.
POSTER 25
A NOVEL FERROPORTIN MUTATION (G490D) IS ASSOCIATED WITH AUTOSOMAL
DOMINANT IRON OVERLOAD
A.M. JOUANOLLE(1), C. HALIMI(2), P. FERGELOT(1), A.S. de LAJARTHE-THIROUARD(3), J.
ROCHETTE(4), O. LORÉAL(5), V. DAVID(1), P. BRISSOT(5)
(1)
Laboratoire de Génétique Moléculaire et Hormonologie and UMR-CNRS 6061, University
Hospital Pontchaillou, Rennes, (2) Service de Médecine Interne et Hépato-Gastroentérologie,
Centre Hospitalier de Senlis, (3) Département d’Anatomie Pathologique, University Hospital
Pontchaillou, Rennes, (4) Service de Génétique Médicale and UPRES EA 2629, University
Hospital, Amiens (5) Service des Maladies du Foie and INSERM U-522, University Hospital
Pontchaillou, Rennes. France.
BACKGROUND. Classical hemochromatosis is typically autosomal recessive, the majority being
due to homozygosity for the C282Y mutation in the HFE-1 gene. It affects mainly individuals of
Caucasian descent and is associated with progressive iron overload. Characteristic features
include increased transferrin saturation and iron deposition in hepatocytes. AIM. We report a
novel ferroportin mutation in a family with non HFE-1 related iron overload. RESULTS. An Asian
family with three affected members (mother and two daughters from two different fathers)
demonstrated unusual features of iron overload, namely: i) autosomal dominant inheritance
pattern, ii) a major increase in serum ferritin while transferrin saturation was moderately elevated,
iii) iron deposition was predominantly present in Kupffer cells (versus hepatocytes),iv) poor
tolerance to venesection therapy. Given these atypical features we decided to investigate
SLC11A3 gene which was recently implicated in similar cases. SLC11A3 encodes ferroportin, a
transmembrane protein involved in the iron egress from apical duodenocytes and macrophages.
A novel mutation in exon 8 of SLC11A3 gene, resulting in the replacement of glycine 490 with
aspartate (G490D) in ferroportin protein, was found. All affected subjects were heterozygous for
the mutation. In contrast to the previously described mutations which were located in the Nterminal region of ferroportin, G490D was located in the C-terminal part of the molecule.
CONCLUSION. This novel mutation further emphasizes the key role of ferroportin in iron
metabolism and should alert clinicians to search for ferroportin mutations in patients with non
HFE-1 related iron overload of dominant inheritance. (European Grant QLT-2001-00444).
POSTER 26
LABILE PLASMA IRON IN IRON OVERLOAD: REDOX ACTIVITY AND SUSCEPTIBILITY TO
CHELATION1
Breno P. Esposito2, William Breuer2, Pornpan Sirunkapracha3, Pensri Pootrakul3 , Chaim Hershko4 and Z.
Ioav Cabantchik 2
2
Department of Biological Chemistry, Institute of Life Sciences, Hebrew University of Jerusalem,
Jerusalem 91904, Israel, 3Thalassemia Research Center, Institute of Science and Technology for
Research and Development, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand,
4
Department of Hematology, Shaare Zedek Medical Center, Jerusalem, Israel
Iron overload conditions, such as thalassemia and hereditary hemochromatosis (HH), are characterized
by the appearance of high levels of serum iron, transferrin saturation and ferritin. However, it is the
presence of Non-Transferrin Bound Iron (NTBI) forms in the plasma that is believed to be responsible for
catalyzing the formation of reactive oxygen species in the circulation, the accumulation of oxidation
products and tissue iron overload (reviewed by Breuer et al. 2000). To test this hypothesis we assessed
the redox active component of NTBI, which we refer as LPI, labile plasma iron, in the plasma of normal, βthalassemic and hemochromatotic patients. LPI levels in plasma or serum were determined with the aid
of the fluorogenic probe DHR (dihydrorhodamine 123) by monitoring the time dependent generation of
reactive oxidant species (ROS) generated by ascorbate and blocked by iron chelators. The assay is
considered specific for labile iron since: a. it uses physiological concentrations of ascorbate, b. it involves
no manipulation of the serum sample that might lead to extraction of iron from transferring and c. the
signal is blocked by specific iron chelators which neither bind heme-bound iron nor inhibit peroxidases.
The results indicated that LPI was essentially absent from sera of normal individuals but was present in
the majority of sera of 57 β-thalassemic patients at levels of 1-14 µM (mean value of 2.6 ± 0.8 µM). The
LPI levels correlated significantly (r=0.88) with those of NTBI measured either as MDCI (mobilizerdependent chelatable iron, as described by Breuer et al. 2001b) or DCI (deferrioxamine-chelatable iron,
as described by Breuer et al. 2001a) but showed 30-40% higher sensitivity. Importantly, standard oral
treatment of patients with the iron chelator deferiprone (L1) raised the plasma NTBI levels (measured as
DCI and MDCI) due to mobilization of iron from tissues into the circulation. However, L1 administration did
not lead to an increase in LPI levels, indicating that L1-chelated iron appearing in the plasma had
essentially no redox activity prompted by ascorbate. On the contrary, oral deferiprone treatment
effectively reduced the LPI that was already present in the patients prior to drug treatment, to virtually
undetectable levels (< 0.5 µM). Thus ascorbate driven generation of oxidizing species provides a
versatile means for analyzing the redox capacity of labile iron present in native plasma/serum samples
and associated with NTBI. Moreover, the approach used in this work enables the assessment of LPI
susceptibility to in vivo or in vitro chelation and the potential of LPI to cause tissue damage, as found in
iron overload conditions.
This work was carried out in part by contract with Aferrix sponsored by the Israeli Ministry of Industry and
Resources and by grants from the European Community 5th Framework and Apotex Inc., Ont. Canada.
References:
Breuer et al. 2000 Transfus Sci.23:185-92. Review
Breuer et al. 2001 a. Blood: 97:792-798
Breuer et al. 2001b. Anal Biochem., 299:194-202.
POSTER 27
THE EFFECT OF MATERNAL DIETARY IRON DEFICIENCY ON DUODENAL IRON TRANSPORT IN
NEONATES
N.Solanky*, L.Gambling§, H.J.McArdle§, S.K.S.Srai*
* Biochemistry and Molecular Biology, Royal Free and UCL Medical School, London, UK
§
The Rowett Research Institute, Aberdeen, UK
We have previously demonstrated that maternal dietary iron deficiency causes in an up-regulation of
placental transferrin receptor 1 (TfR1) and divalent metal transporter 1 (DMT1) +ire isoform mRNA but no
change in IREG1 mRNA expression (1). We have also shown increased expression of the copper
oxidase involved in iron efflux from placenta (2). How the mother adapts in terms of uptake across the
duodenum, and whether the fetus or the newborn neonate shows similar adaptation to iron deficiency has
not been determined.
Female Hooded Lister rats were weaned at 19 days and placed on a control diet for 2 weeks, then on
either a normal (50 mg/kg), or decreased (7.5 mg/kg) iron content diet for 4 weeks prior to mating. The
rats were maintained on the same diet throughout pregnancy. After birth all pups were nursed to control
diet dams. Duodenal biopsies, taken after stunning and cervical dislocation, were used to determine in
59
vitro Fe uptake (µg Fe uptake/g wet weight) at birth, 6 and 15 weeks after birth.
At birth a significant increase in duodenal ferric iron uptake was observed in the pups (n=8, control 36.64
+/-5.85, iron deficient 114.28 +/-18.62 P=0.002), as well as in the dams (n=8, control 7.054 +/-0.88, iron
deficient 39.56 +/-8.43 P=0.005) raised on an iron deficient diet. This is interesting, given the duodenum
in pups has not been exposed to a low iron diet. Nursing with control diet dams decreased the elevated
iron uptake in pups of iron deficient mothers to that of control levels (n=8, control 848.12 +/-227.7, iron
deficient 631.11 +/-250.6 p=0.532 at 6 weeks, and n=8, control 0.033 +/-0.019, iron deficient 0.005 +/0.012 p=0.972 at 15 weeks). These changes correlated to liver iron levels (µg/g dry weight) which were
reduced in the iron deficient pups at birth (control 2640 +/-312, iron deficient 1713 +/-168 p = 0.029) and
were restored to control values in 6 week old pups (control 1197 +/-130, iron deficient 1151 +/-112 ns)
Iron deficiency during pregnancy caused decreased liver iron levels and an increase in duodenal iron
uptake in dams. This was mirrored by increased placental DMT1 and TFR1 expression in these animals.
Newborn pups from these iron deficient mothers showed decreased liver iron levels, but interestingly,
also showed increased duodenal iron uptake although they were not subjected to a low iron diet. This
confirms the hypothesis that iron uptake is controlled systemically.
This work is supported by the European Union.
References:
Gambling L, Danzeisen R, Gair S, Lea RG, Charania Z, Solanky N, Joory KD, Srai SK, McArdle HJ.
Effect of iron deficiency on placental transfer of iron and expression of iron transport proteins in vivo and
in vitro. Biochem J. 2001 Jun 15; 356(Pt 3):883-9.
Danzeisen R, Ponnambalam S, Lea RG, Page K, Gambling L, McArdle HJ. The effect of ceruloplasmin
on iron release from placental (BeWo) cells; evidence for an endogenous Cu oxidase. Placenta. 2000
Nov; 21(8):805-12.
POSTER 28
ALTERED EXPRESSION OF HFE IN RESPONSE TO A RANGE OF STRESSORS IN MICROGLIA
CELLS
S.Y. Lee, J.R. Connor, Neuroscience & Anatomy, Penn State University College of Medicine, Hershey,
PA 17033
A recent immunohistochemical study suggests that the Hfe (Hereditary Hemochromatosis candidate
gene) protein is expressed on microglia that are associated with neuritic plaques in Alzheimer’s Disease.
The expression of the Hfe protein may influence iron uptake by these cells. Furthermore, expression of
the mutant form of Hfe on these cells could have an undesired effect on iron accumulation by these cells
and alter their function. Because Hfe was not seen on microglia cells that were not associated with
neuritic plaques, we undertook two studies. First, we sought to identify factors that would influence the
expression of Hfe and secondly identify the effect of expression of the mutant form of Hfe on the cell. We
first examined two microglial cell lines, BV2 (mouse) and HAPI (rat). Hfe was detected by RT-PCR in the
BV2 cells but not in the HAPI cells. BV2 cells were exposed to a panel of stress inducing factors such as
LPS, serum withdrawl, tumor necrosis factor α, β-amyloid, iron, hydrogen peroxide. A toxicity profile was
established for each factor and the expression of Hfe monitored on cells using sub-toxic concentrations
by western blot analysis. Cells transfected with mouse FLAG-tagged Hfe were used to identify the
position of native mouse Hfe on the western blots. The expression of Hfe was correlated with that of
Transferrin receptor, ferritin and DMT1. The data were compared to β-actin expression which served as
an internal control. Hfe expression was not influenced by any of the factors except hydrogen peroxide.
Many of the factors influenced the expression of transferrin receptor, ferritin and DMT1 indicating that Hfe
regulation is independent of these proteins. The effect of hydrogen peroxide on Hfe was to decrease
expression of this protein. The decrease in Hfe occurred at the post-transcriptional level. Intracellular
iron status was not affected by the change in Hfe expression as determined by the calcein assay. In
addition to hydrogen peroxide, menadione was used to determine if the type of oxidative stress influenced
Hfe expression. While H2O2 is a nucleophilic, non-radical oxidant, menadione causes catalytic oxidative
stress, generating superoxide anion and other radicals through redox cycling. The results of this study
suggest that Hfe is tightly regulated and its expression is not influenced by exposure to most toxic
substances. However, we cannot rule out that Hfe expression is already at maximal levels in this cell
culture model. Studies are underway to determine if the presence of the Hfe mutation influences the
toxicity profile for these cells.
(Supported by: Alzheimer’s Association)
POSTER 29
A RARE CASE OF JUVENILE HEMOCHROMATOSIS ASSOCIATED WITH TURNER’S
SYNDROME AND SEVERE LYMPHOPENIA SUCCESSFULLY TREATED WITH A
COMBINATION OF PHLEBOTOMY AND GROWTH HORMONE (GH) THERAPY
G Porto1,2, C Bacelar3, E Cruz1,2, B Porto2, M de Sousa2, B Justiça3, H Pessegueiro Miranda4
1. Hematology, Santo António General Hospital, Porto, Portugal; 2. Molecular Pathology, Abel
Salazar Institute for the Biomedical Sciences, Porto, Portugal; 3. Endocrinology, Santo António
General Hospital, Porto, Portugal; 4. Internal Medicine, Santo António General Hospital, Porto,
Portugal
Case report: CMGP, female, 16 years old, was refered for primary amenorrhea and short stature
(High=137cm; Bone Age=10 years) with a history of alcohol ingestion (40g/day) since she was 5
years old. She presented with hepatomegaly, abnormal liver function tests (AST=189U/L;
ALT=285U/L) and some clinical features characteristic of Turner’s Syndrome: sexual infantilism,
thick neck, scutiform thorax, wide apart nipples and gonadal agenesia. The 46,X,X(q-) karyotype
confirmed the diagnosis. Gonadothropic hormones weren’t raised as expected. A severe iron
overload with hepatic fibrosis was diagnostic of juvenile hemochromatosis and explained the
hypopituitarism. There were no signs of cardiac disfunction or diabetes. The HFE mutations
C282Y and H63D were absent. No other family members were affected. She had at diagnosis an
absolute lymphopenia (1x106/ml) mainly due to low CD8+ T lymphocyte numbers (0,16x106/ml),
with a CD4/CD8 ratio>4.
Results of treatment: She was successfully treated with a combination of intensive phlebotomy
treatment, recombinant GH (1 IU/KgBW/w) and estrogens (5µg/Kg/w). Depletion of iron stores
was achieved after 136 weekly phlebotomies (20,8g of iron removed in three years). A Stage IV
(Tanner) sexual development was acquired in 2,5 years. The final expected stature was reached
(High=152cm) with a maximum growth rate during the first year (9cm/year). Liver enzymes
normalized at the end of intensive treatment (AST=20U/L; ALT=17U/L). Total lymphopenia was
maintained along treatment, but the relative proportions of CD4+ and CD8+ cells were changed
with correction of the CD4/CD8 ratio to normal values (average=2). This effect was mainly due to
reconstitution of the CD8+CD28+ subpopulation.
Follow-up study: The patient is presently in maintenance therapy (trimestral phlebotomy). The
iron parameters (serum iron, transferrin, serum ferritin), and the number of lymphocytes and
lymphocyte subpopulations (CD4+, CD8+, CD8+CD28+; CD8+CD28-) have been analysed
serially during a period of five years, providing a unique opportunity of analysing the interactive
effects of GH on iron mobilization, growth and lymphocyte reconstitution.
Acknowledgements: grants from the Gulbenkian Foundation and Portuguese Foundation for
Science and Technology (FCT): POCTI/32986/1999
POSTER 30
HETEROZYGOSITY FOR HEMOCHROMATOSIS IS ASSOCIATED WITH LUNG BUT NOT HEAD AND
NECK CANCERS
J. Rodriguez-Paris, M. Smith, E. Spencer, R.M. Robertson, G. Mills, J. McLarty, J. Glass, Feist-Weiller
Cancer Center, Louisiana State University Health Sciences Center, Shreveport, LA.
Hereditary hemochromatosis (HH) is a common, autosomal recessive disorder of iron metabolism caused
by a mutation in the HFE gene on chromosome 6. The mutation causes an increase in iron absorption,
which results in excess deposition of iron in the parenchymal cells of multiple organs resulting in diabetes,
liver failure, cardiac dysfunction, arthritis, and hypogonadism. In the United States approximately 10% of
the Caucasian population are carriers for the disease. HH heterozygotes have slightly increased iron
absorption, which may result in increased total body iron. Recent studies suggest that HH heterozygotes
may have an increased risk for developing cancer and heart disease. As the generation of active oxygen
species is implicated as causal in lung cancer and head neck cancers and as iron catalyzes the
generation of active oxygen species, we did a case-control study to determine if HH heterozygotes carry
an increased risk for either cancer type. More than 90% of hereditary hemochromatosis patients have a
single missense mutation, causing an amino acid substitution of cysteine to tyrosine at residue 282
(C282Y) in the HFE protein. We screened for the C282Y mutation in 176 Caucasian patients diagnosed
with lung (87) or head and neck (83) cancer and 391 matched controls. All the patients were smokers.
Genomic DNA was isolated from whole blood and the C282Y mutation was detected by allele-specific
PCR. Of the 176 cancer patients 25, or 14.2%, were heterozygotes compared to only 10.0% of the 391
controls. The prevalence of the C282Y mutation was significantly higher among female patients, 17.7%
(p=0.01), than in female controls, 9.6%. Adjusted for age, gender and smoking, females with the C282Y
mutation were more likely to have lung cancer than female with no mutation (adjusted OR=2.5, 95% CI
0.96 to 6.48) and showed an earlier age of onset of lung cancer than female patients with wild type HFE,
51.2 years vs. 57.1 years respectively. Interestingly, the family history of cancer in first-degree relatives of
HH heterozygotes appeared to be increased. The increased risk of cancer was present only in female
heterozygotes with lung cancer and was not present in females with head and neck cancer or males with
either cancer type. The causes of the significantly increased risk of lung cancer with the heterozygous
state among females are being investigated.
POSTER 31
Withdrawn
POSTER 32
ANTIMALARIAL ACTIVITY OF IRON CHELATORS OF THE AROYLHYDRAZONE CLASS
A.Walcourt, M. Loyevsky *D. B. Lovejoy, V. R. Gordeuk, and *D.R. Richardson
Center for Sickle Cell Disease, Howard University, Washington DC; * The Heart Research Institute,
Sydney, New South Wales, Australia
Iron is an essential element for the growth of rapidly proliferating Plasmodium falciparum
parasites. The development of anti-malarial drugs with novel mechanism of action is vital in the face of
the emergence of malaria parasites that have become resistant to conventional therapies. Ironwithholding using desferrioxamine (DFO) and a number of other iron chelators can be exploited as an
alternative strategy to the use of conventional antimalarials in arresting the parasite growth. DFO, the
only iron chelator approved in most countries for use in humans for the treatment of iron overload, also
displays antimalarial activities, but has drawbacks. This agent is costly, causes toxic side effects, and
requires continuous infusion, an inappropriate mode of administration in most facilities treating malaria in
developing countries. Because the aroylhydrazones have high antiproliferative activity and high iron
binding efficiency, we investigated their antimalarial activity.
Anti-malarial activities of five new aroylhydrazone iron chelators were studied on the chloroquinesensitive, 3D7, and the chloroquine-resistant, 7G8, clones of Plasmodium falciparum in vitro. We tested
antimalarial activity of five aroylhydrazone chelators, 311 (2-hydroxy-1-naphthylaldehyde isonicotinoyl
hydrazone), N4mT(2-hydroxy-1-naphthylaldehyde-4-methyl-3- thiosemicarbazone), N4pT(2-hydroxy-1naphthylaldehyde-4-phenyl-3- thiosemicarbazone), NT (2-hydroxy-1-naphthylaldehyde
thiosemicarbazone), and dpT (di-2-pyridyl-thiosemicarbazone), which have been synthesized in the
laboratory of Dr. D. R. Richardson. Parasites were cultured continuously in washed human erythrocytes
+
+
(A or O ) using the classical method of Trager and Jensen. In vitro antimalarial activity was determined
using the incorporation of [3H]hypoxanthine into the nucleic acids of erythrocytic parasites in a standard
48-hr assay.
In a series of experiments, we compared the effects of DFO, and of the aroylhydrazones on the
chloroquine-sensitive and the chloroquine-resistant strains of P. falciparum using the above test. The
aroylhydrazone chelators were 2.0-6.3 fold more active than DFO against the chloroquine-sensitive strain
(P ≤ 0.040), and 1.4-6.9 fold more active than DFO against the chloroquine-resistant strain (P ≤ 0.046).
Iron chelators deprive parasites of metabolic iron, thus arresting parasite growth and inducing
permanent damage to parasites. Therefore, an effective antimalarial iron chelator would have the ability
to cross lipid membranes well, and would selectively bind iron as compared to other trace metals.
Previous studies demonstrated that most of the aroylhydrazone iron chelators satisfy these criteria.
To determine if there is a positive correlation between the antiproliferative and antimalarial
activities of aroylhydrazones, we looked at the results of the present study in the light of those of
previously published investigations. The analyses show that antiproliferative effects of aroylhydrazones
311, N4mT, and NT on K562 erythroleukemia cells, SK-Mel-28 melanoma cells, and MCF-7 breast
cancer and neuroepithelioma cells do parallel the antimalarial effects produced by the same compounds
on chloroquine-sensitive and –resistant malaria parasites. Interestingly, the most active antiproliferative
agent, 311 was also found to be one of the most efficient antimalarials. Comparisons of the IC50s of the
aroylhydrazones for the chloroquine-resistant or –sensitive strains of malaria in the present study and the
IC50s for four mammalian cell lines previously published suggested that the aroylhydrazone N4mT might
have some margin of safety as an antimalarial. The potential of these agents as anticancer drugs
together with their substantial antimalarial activity warrant further investigation of these compounds as
antimalarials in animal models.
POSTER 33
THE ROLE OF IRON IN RESTLESS LEGS SYNDROME
C. J. Earley, R. P. Allen, J. Connor, J. Beard, P. Barker, A. Horska.
Department of Neurology, Johns Hopkins University, Baltimore MD, Department of Neuroscience &
Anatomy, and Department of Nutrition, Penn State University Hershey PA.
Restless Legs Syndrome (RLS) is a sensory-motor disorder with a prevalence of about 5-10% in the
general population. The diagnosis of RLS is based on the presence of clinical symptoms. The primary
feature is an urge to move the legs, frequently associated with uncomfortable or painful sensations in the
legs. The symptoms occur when the patient attempts to rest (sitting or lying) and are relieved with
movement or walking. The symptoms have a marked circadian pattern becoming worse at night.
Symptom severity varies widely, but when pronounced carries significant morbidity. For the more severely
affected patients, the reported accompanying agony of the sensations reoccurs with unremitting
regularity. Total daily sleep times are often reduced to 5 hours or less, with marked reduction of sleep
efficiency. While much of the clinical intervention for RLS has focused on the dopaminergic system, there
is a growing and compelling body of evidence that suggests a significant role for iron. Iron deficiency has
been recognized as a significant contributing cause of RLS for almost 50 years. A strong negative
correlation between serum ferritin levels and RLS symptom severity has also been noted. RLS symptoms
are reportedly relieved by high-dose, intravenous iron therapy for periods of up to 3-9 months, even
though the majority of these individuals had normal iron status prior to treatment. Additionally, oral iron
supplementation improves symptoms in some RLS patients. There are a number of secondary causes of
RLS such as pregnancy that support the claim of a causative role of iron deficiency in RLS. Our studies
have focused specifically on iron insufficiency in the brain in RLS. Cerebrospinal fluid (CSF) levels of
ferritin, iron, and transferrin support the theory that brain iron is deficient in RLS. A 65% decrease in CSF
ferritin, as well as a three-fold increase in CSF transferrin was reported in RLS patients when compared
to controls, despite normal serum levels of ferritin and transferrin in both populations. Using MRI to
quantify iron concentrations in 11 different brain areas, we have found a consistent and significant
reduction in iron concentration in the substantia nigra in patients with RLS compared to controls. We
have previously reported a correlation between iron concentration in the substantia nigra and RLS
severity: decreasing iron concentration is associated with increasing severity. We recently completed a
histopathological analysis of 7 RLS brains that supported the MRI findings that the substantia nigra in
RLS is iron deficient and have implicated misregulation of the transferrin receptor as a possible cause of
the iron deficiency. Overall the findings suggest a central role for brain iron in the pathophysiology of
idiopathic RLS.
POSTER 34
BIOAVAILABILITY OF FERRITIN IRON IN HEALTH AND DISEASE
Elizabeth C. Theil1, John L. Beard2, Bo Lonnerdal 3 Kevin Hadley! , Penni Hicks3, Shannon Kelleher3 and
Laura Murray-Kolb2
1
CHORI (Children's Hospital Oakland Research Institute). Oakland, CA 94609, 2 Dept. Nutrition. Sci..,
The Pennsylvania State University State College, PA 16803 3 Dept. Nutrition, Univ. of California.
Davis, CA 95616
Iron is present as many chemical forms in the diet. Recent studies of iron absorption in humans indicate
that multiple receptor/transport proteins are involved such as DMT-1, ferroportin (IREG1, MTP1),
hephaestin and possibly lipocalin. In bacteria > 25 molecules are used for iron uptake, many selective for
a specific chemical form of the iron, e.g. ferrous salts, ferric citrate, transferrin, ferric enterobactin, etc.
Ferritin, a form of iron found in foods exemplified by soybeans, is also bioavailable. (In soybeans, ferritin
accounts for much of the iron in the seed). When iron deficient rats were fed pure ferritin, soybeans or
FeSO4 with equal iron concentrations, normal hematocrits were observed in 28 days with all the iron
sources, indicating similar bioavailability for all the forms of iron (1, 2). In a group of American women with
borderline iron deficiency, the mean absorption of 59Fe from meals of soybean muffins or soup, based on
appearance of 59Fe in RBC after 28 days, was 27% compared to 61% for a FeSO4 reference dose (3),
higher than the uptake in American males replete for iron (4), possibly due to differences in iron status. In
whole body studies of fasting American women with varied, but normal iron status, administered 59Fe–
ferritin in apple juice varied with mean iron absorption from ferritin of 24 % compared to 24% for FeSO4.
Such data emphasize the bioavailability of iron from ferritin. The data also suggest that absorption of iron
from different chemical forms of dietary iron may be sensitive to the physiological state. Support for such
an idea is the effect of increased dietary iron on tissue iron or ferritin in murine models of Sickle Cell Trait
and Thalassemia (see Hadley et al. Boron 2003) and indicate that diets which consider more completely
the form of iron, such as ferritin, home or organic or inorganic complexes of Fe, can play a role in
nutritional genomics and health in the future. Part support: NIH HL56169.
REFERENCES:
(1) Beard et al. J. Nutr. 1996)126:154.
(2) Theil, E.C., Burton, J.W., and Beard, J.L. (1997) Eur. J. Clin. Nutr.. Suppl. 4:S28.
(3). Murray-Kolb et al. Am. J. Clin. Nutr 77:180.
(4) Lynch et al. Am. J. Clin. Nutr. (1984) 40:42.
POSTER 35
A NOVEL MUTATION IN FERROPORTIN1 IS ASSOCIATED WITH HAEMOCHROMATOSIS IN A
PATIENT FROM THE SOLOMON ISLANDS
D.F. Wallace1, K.E. Arden1, J.L. Dixon2, L. Summerville1, J.W. Searle4, G.J. Anderson2,5, G.A. Ramm3,5,
L.W. Powell2,5, V.N. Subramaniam1,5,6, 1Membrane Transport Laboratory, 2Iron Metabolism Laboratory,
3
Hepatic Fibrosis Group, The Queensland Institute of Medical Research, 4Departments of Pathology,
5
Medicine and 6Biochemistry, The University of Queensland, Brisbane, Queensland, Australia.
A severe form of iron overload with the clinico-pathological features of haemochromatosis has been
previously described in the Solomon Islands (Eason et al. 1990). The disorder was shown to have an
autosomal dominant inheritance and was unlinked to HFE. The genetic basis of the disorder has not been
identified. The disorder has similarities to type 4 haemochromatosis. Type 4 haemochromatosis is an
autosomal recessive disorder caused by mutations in the iron export protein ferroportin1 and is
associated with iron accumulation in reticuloendothelial cells and hepatocytes. We hypothesised that
mutations in ferroportin1 were the cause of Solomon Islands haemochromatosis.
A Solomon Islands man presented to our centre at age 48 years with a cardiac murmur and
hepatomegaly. Biochemical evaluation showed an elevated alanine aminotransferase level of 82 U/L, a
serum iron concentration of 40 µmol/L, transferrin saturation of 80% and serum ferritin concentration of
2,937 µg/L. He was negative for the two common mutations of HFE, C282Y and H63D. Liver biopsy
showed grade 4 iron stores predominantly in the parenchymal cells with significant iron deposits in the
Kupffer cells and portal tract macrophages. The hepatic iron concentration was 363 µmol/g and the
hepatic iron index was 7.6. In addition, portal fibrosis was observed but no cirrhosis was evident. The
patient was born in the Solomon Islands and both parents and grandparents were of Melanesian Solomon Islands extraction. He is not directly related to the family studied by Eason et al., however, a
more distant ancestral connection cannot be ruled out.
DNA was isolated from the peripheral blood of the Solomon Islands patient. The entire coding region and
splice sites of the ferroportin1 gene were sequenced. A control group comprising 100 Australian
individuals was also studied. Unfortunately no samples were available from family members, the pedigree
studied by Eason et al. or a Solomon Islands control group. Amino-terminal myc-tagged cDNA constructs
of wild type and the mutant ferroportin1 were created and transfected into the human intestinal cell line
Hutu80. The localisation of wild type and mutant ferroportin1 was studied by immunofluorescence.
DNA sequencing revealed that the Solomon Islands patient was heterozygous for a novel missense
mutation (N144T) in exon 5 of the ferroportin1 gene. A novel restriction endonuclease-based assay was
developed which identifies both the N144T and the previously described N144H mutations. The N144T
mutation was not detected in the control group. Transient transfection of wild type and N144T ferroportin1
revealed that the mutant protein is correctly localised to the cell surface and intracellular compartments.
This is the first identified mutation associated with haemochromatosis in the Solomon Islands population.
Whether this is the cause of iron overload in the pedigree studied by Eason et al. remains to be
determined. We also describe a novel restriction endonuclease-based assay which could help in the
screening and diagnosis of patients with iron overload in the Solomon Islands and other populations.
Surface localisation of the mutant protein indicates that the defect may lie in dysfunction of the iron
exporting ability of the protein instead of improper trafficking.
Eason et al. 1990, Aust NZ J Med 20:226-30
POSTER 36
EFFECT OF IRON STATUS ON THE ABSORPTION OF IRON BY HFE KNOCKOUT MOUSE MODEL
OF HEREDITARY HAEMOCHROMATOSIS
D. Trinder1, S. Drake1, K. Lowes1, A.G.C. Chua1, J.K. Olynyk1 and E.H. Morgan2.
1
School of Medicine and Pharmacology and 2Physiology, School of Life and Physical Sciences, University
of Western Australia, Fremantle, Western Australia, Australia.
Background: Hereditary hemochromatosis (HH) is a common disorder of iron (Fe) metabolism which is
characterised by increased Fe absorption and liver Fe overload. Most individuals with HH are
homozygous for a mutation (C282Y) in the HFE gene. We have shown previously that Hfe senses the
body Fe levels by regulating the uptake of transferrin-bound Fe from the plasma by the duodenum, and
this function is impaired in Hfe knockout (KO) mice. The aim of this study was to determine how impaired
sensing of body Fe levels affects the regulation of Fe absorption by Hfe KO mice.
Methods: Hfe KO and C57Bl/6 control mice were fed either a Fe deficient, basal or Fe loaded diet to alter
body Fe levels. Fe absorption was measured in vivo by injecting 59Fe ascorbate (20 nmol in 20µl) directly
into the duodenum. After 30 mins the duodenum was removed, washed and radioactivity in the
duodenum, whole body and liver was measured. Duodenal iron transporters DMT1 and ferroportin mRNA
levels were measured by semi-quantitative RT-PCR.
Results: Plasma Fe and liver non-heme Fe concentrations were significantly increased in Hfe KO mice
compared with control mice fed a basal and Fe loaded but not Fe deficient diet. Fe absorption was
inversely related to Fe levels in Hfe KO and control mice and was enhanced significantly in Hfe KO mice
compared with control mice on a basal diet (27.2+3.2% dose versus 10.4+1.8%; mean + SEM, n=5;
p<0.01) but not a Fe deficient or Fe loaded diet. The increase was due a significant rise in both the
uptake of Fe from the intestinal lumen (46.6+2.1% versus 29.8+5.1%; p<0.01) and transfer of Fe to the
body (58.2+5.7% versus 35.0+2.5%; p<0.001). This lead to a significant increase in liver Fe levels in Hfe
KO mice compared with control mice when expressed either as a percentage of 59Fe dose or percentage
of 59Fe absorbed (6.4 + 0.8% versus 0.6 + 0.2; p<0.001). DMT1 and ferroportin mRNA expression was
also inversely related to body iron stores and was increased in Hfe KO mice compared with control mice
fed a basal but not Fe deficient or loaded diet.
Conclusions: Fe absorption (uptake and transfer) and expression of DMT1 and ferroportin by Hfe KO and
control mice was inversely regulated by iron status. Impaired Fe sensing in Hfe KO mice leads to
increased expression of DMT1 and ferroportin, increased Fe absorption (uptake and transfer) and liver Fe
deposition providing an explanation of how Fe overload develops in HH.
POSTER 37
TRANSPORT OF Fe(III) BY INTESTINAL IEC-6 CELLS IS INDEPENDENT OF DMT1
Carla Thomas and Phillip S. Oates. Physiology, School of Biomedical and Chemical Sciences, The
University of Western Australia, 35 Stirling Highway, Crawley WA, 6009. Western Australia.
Introduction: Iron absorption occurs by duodenal villus enterocytes. Luminal ferric Fe(III) iron is reduced
to the ferrous Fe(II) state before uptake by divalent metal transporter (DMT1). Release involves IREG-1
by a poorly understood process. Regulation of this process occurs within dividing cells of the duodenal
crypts. This is thought to involve acquisition of transferrin-bound iron by the activity of transferrin
receptors (TfR) 1 & 2 and HFE protein in combination with TfR1. There is also evidence in vitro of Fe(III)
uptake using an integrin-dependent pathway (IDP) that is independent of DMT1, but whether it functions
to absorb iron or to regulate this process is unclear. Methods: Transport of Fe(II) and Fe(III) was studied
in the rat intestinal enterocyte cell line-6 during stationary and proliferative states. This was done in the
presence of blocking antibodies to IREG-1 and with inhibitors of microtubule formation, colchicine and
vinblastine. Uptake of Fe(II) versus Fe(III) was compared with different pH’s, temperatures and overexpression of DMT1. Results: Tiron a chelator of Fe(III) completely blocked uptake of Fe(III). Ferrozine a
chelator of Fe(II) had no effect on Fe(III) uptake. Blocking IREG-1 reduced uptake of iron from Fe(II) and
Fe(III) by 57% and 95%, respectively. Uptake of Fe(II) doubled when the pH was lowered from 7.5 to 5.5
o
and with over-expression of DMT1, but Fe(III) uptake was unchanged. Compared to 37 C, uptake of
o
Fe(II) and from Fe(III) at 4 C was 85% and 5% of that at pH5.5 respectively. Colchicine and vinblastine
had no effect on Fe(II) uptake but reduced uptake from Fe(III). Compared with the stationary state,
proliferation did not affect DMT1 expression or Fe(II) uptake, but doubled uptake of iron from Fe(III).
Discussion: IEC-6 cells transport Fe(III) without reduction at the cell surface. Because the Fe(III) transport
pathway increases with proliferation, is independent of DMT1 and requires energy we propose that it may
function in the crypt region where it is involved in the uptake of transferrin-bound iron.
POSTER 38
IREG-1 MODULATES THE UPTAKE OF IRON AT THE APICAL MEMBRANE OF ENTEROCYTES
Carla Thomas and Phillip S. Oates. Physiology, School of Biomedical and Chemical Sciences, The
University of Western Australia, 35 Stirling Highway, Crawley WA, 6009. Western Australia.
Background & Aims: Absorption of non-heme iron occurs mainly in the duodenum. It involves the
divalent metal transporter (DMT1) in the uptake of ferrous iron Fe(II) and the basolateral transporter IREG1 in its release. Whether IREG-1 functions in this process at other sites in the enterocyte is unknown. In
this study the effect of a blocking antibody to IREG-1 on the uptake and release of iron was evaluated in
enterocyte-like cells: IEC-6 and Caco-2 and freshly isolated duodenal enterocytes from rats. IREG-1
expression was studied in microvillus membrane preparations from rats with different levels of iron stores.
Methods: Uptake of 1µM Fe(II) iron and its efflux by the cells was studied over in time in the presence or
absence of the antibody. Expression of IREG-1 and DMT1 was determined by Western blot analysis of
duodenal mucosa and enriched microvillus membranes obtained from rats with altered iron stores.
Immunohistochemical detection of IREG-1 was performed in frozen section of duodenum. Results: In all
cell types studied the antibody significantly reduced (p<0.05) uptake of Fe(II) by 40-60%, but had no effect
on the efflux of iron. In Caco-2 cells Fe(II) uptake was reduced only when the antibody was in contact with
the apical membrane. There was no efflux of iron across the apical membrane in the presence or absence
of the IREG-1 antibody. IREG-1 and DMT1 protein were enriched in microvillus membranes. IREG-1
expression was increased by reducing cellular iron as was DMT1. IREG-1 expression was seen along the
brush border as well as the basal cytoplasm and membranes of enterocytes in frozen sections.
Conclusions: In addition to efflux, IREG-1 functions in the uptake of iron at the apical membrane possibly
by modulating the activity of DMT1.
POSTER 39
THE REGULATION OF INTESTINAL IRON ABSORPTION. DO THE CRYPTS PLAY AN IMPORTANT
ROLE?
D M Frazer and G J Anderson. Joint Clinical Sciences Program, The Queensland Institute of Medical
Research and The University of Queensland, PO Royal Brisbane Hospital, Brisbane, Queensland 4029
Australia.
Introduction: Our understanding of how iron transverses the intestinal epithelium has improved greatly in
recent years with the discovery of several new molecules involved in this process. The molecular
mechanisms regulating absorption are less clear, although many models have been proposed. A key
aspect of all current models involves the programming of crypt cells to absorb appropriate levels of iron
once they have matured and migrated to the villus. This theory is based on classic studies conducted in
the 1960s demonstrating a “lag period” between the stimulus to alter iron absorption and the alteration of
absorption itself. Data emerging from recent studies suggest that this concept should be re-evaluated.
Hypothesis: By examining the older literature in the light of recent advances in the field of iron
metabolism, we propose that the crypts do not play a primary role in the regulation of iron absorption and
that signals to alter absorption have a direct effect on the villus enterocytes.
Evidence:
(1) Some stimuli alter iron absorption within hours rather than days e.g. the acute phase response (APR),
reticulocyte infusion.
(2) Hepcidin has now been shown to be a critical component in the regulation of absorption as: a)
hepcidin expression inversely correlates with iron absorption and transporter expression; b) disrupted
hepcidin expression leads to iron loading in mice and overexpression leads to severe anaemia; c) the
hypoferraemia of the APR corresponds with an increase in hepcidin expression; d) hypoferraemia does
not occur in animals with disrupted hepcidin expression suggesting that hepcidin causes the reduction in
plasma iron; and e) mutations in hepcidin have been found to be lead to some cases of juvenile
haemochromatosis, implying that hepcidin has a more generalised role in the regulation of iron
absorption.
(3) The short time between changes in hepcidin levels and changes in iron absorption is inconsistent with
the 2-3 day period required for enterocyte maturation. The decrease in iron absorption during the APR
occurs within hours of the stimulus implying that hepcidin is directly signaling the mature villus cells.
There is no reason to assume that the mechanism by which hepcidin controls iron absorption during the
APR differs from that invoked by other stimuli. Indeed we have recently shown a close temporal
relationship between hepcidin expression and iron absorption following the switch from a control to an
iron deficient diet.
(4) In retrospect, the older literature often quoted in as support for crypt involvement also provides
evidence for a direct signal to the villus. For example, Charlton et al. (J Clin Invest 44:543-554, 1965)
showed that the decrease in absorption following an intravenous injection of iron was maximal 18 hours
after the dose and a decrease was seen as early as 6 hours. Thus the “lag period” was far shorter than
the 48 to 72 hours required for crypt involvement.
Conclusion: The evidence described above suggests that the major regulator of iron absorption is
hepcidin and that this peptide alters absorption by signaling the mature villus enterocytes. Emerging data
on the regulation of iron absorption in response to a variety of stimuli suggest common regulatory
pathways and cast doubt on the involvement of the crypts in this process.
POSTER 40
INHIBITION OF MACROPHAGE IRON RELEASE BY HFE
Hal Drakesmith, Emma Sweetland, Lisa Schimanski, Chryssie Brown, Jon Edwards, Diana Cowley,
Mubeen Ashraf, Judy Bastin and Alain R.M. Townsend Weatherall Institute of Molecular Medicine,
University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK
Mutations in the gene HFE are associated with hereditary haemochromatosis (HH). Wild-type HFE
protein binds to transferrin receptor-1 (TfR1) in competition with transferrin, and in vitro, HFE reduces
cellular iron by inhibiting transferrin-bound iron uptake. However in vivo, HFE is strongly expressed by
liver macrophages, which are relatively iron deficient in HH. This latter observation suggests,
paradoxically, that expression of wild-type HFE may lead to iron accumulation in these specialised cell
types. In specialised cells such as macrophages which possess iron export mechanisms, iron
accumulation could result from increased iron uptake or reduced iron release.
Here we show that wild-type HFE protein raises cellular iron by inhibiting iron efflux from the
monocyte/macrophage cell line THP-1, and extend these results to macrophages derived from healthy
individuals and HH patients. In addition we find that the HH associated mutant H63D does not inhibit iron
release, despite binding to TfR1 as well as wild-type HFE. Finally we find that the ability of HFE to block
iron release is not competitively inhibited by transferrin. We conclude that HFE has two mutually exclusive
functions, binding to TfR1 in competition with Tf, or inhibition of iron release. We hypothesize that HFE
inhibits iron release by blocking the function of the iron exporter ferroportin, which like HFE is expressed
by cells of the macrophage lineage. We are currently testing this idea.
POSTER 41
HOMOZYGOSITY FOR TRANSFERRIN RECEPTOR 2 Y250X MUTATION INDUCES EARLY IRON
OVERLOAD
A Piperno, A Roetto, S Pelucchi, F Daraio, C Corengia, R Mariani, F Longo, A Piga, G Garozzo, C
Camaschella
Department of Internal Medicine, San Gerardo Hospital, Monza. Department of Clinical and Biological
Sciences and Department of Pediatric Sciences, University of Torino. Civile Hospital, Ragusa, Italy.
Hemochromatosis type 3 is a rare form of genetic iron overload with clinical complications identical to
HFE-related hemochromatosis due to inactivation of transferrin receptor 2 (TFR2) which maps on 7q22.
Five different causal mutations of TFR2 have been described in Italian and Portuguese patients.
We have studied two subjects both originating from Sicily (Ragusa). The first is a 16-year-old boy who
had a clinical diagnosis of hemochromatosis following investigation for asthenia. Hb was 13.6 g/dL and
MCV 86 fL. TS was 100% and serum ferritin 963 µg/L. Serum ALT was slightly increased on a single
occasion. No clinical hemochromatosis-related complication was present. The second is a 3-year-old boy
who came to our attention because of casual discovery of a high serum iron level. Hb was 13.9 g/dL and
MCV 83.7 fL. TS was 80% and serum ferritin 58 g/L. Liver biopsy was not performed but liver iron
concentration (HIC) by SQUID was high, especially considering the young age (933 µg/g wet weight and
524 µg/g wet weight, respectively; normal values <400). Analysis of hemochromatosis associated
mutations using a reverse hybridisation based strip assay that simultaneously detect 11 HFE and one
TFR2 mutation identified in the two probands the same Y250X mutation of TFR2 at the homozygous state
and an H63D heterozygous substitution in HFE. Both probands’ parents were symptomless, apparently
unrelated and had normal iron parameters. They were Y250X heterozygous. Moreover, the probands’
mothers were both H63D homozygous and the father of the second proband was H63D heterozygous.
The oldest patient underwent weekly phlebothomy (6ml/kg). After 16 weekly phlebotomies serum ferritin
was 70
g/Lwet
µg/g
a ndweight).
HIC norm
Our
a lize
results
d (240
indicate that Y250X is the most
common TFR2 mutation and it strengthens the early occurrence of iron overload in homozygotes for
Y250X mutation as compared to HFE hemochromatosis.
POSTER 42
BOTH L FERRITIN IRE AND FERROPORTIN MUTATIONS CAN ACCOUNT FOR ISOLATED
HYPERFERRITINEMIA
G. Hetet, I. Devaux, N. Soufir, B. Grandchamp, C. Beaumont
INSERMU409 and Laboratoire de Biochimie B, Faculté X. Bichat, Paris, France
Elevated serum ferritin values are considered as a good index of increased iron stores, although there are
several conditions that can give rise to increased serum ferritin levels such as cancer, inflammation and
infection. However, patients with isolated hyperferritinemia in the absence of iron overload appear to be a
common finding and a source of concern in clinical departments. The unusual association of both
elevated serum ferritin levels and early onset bilateral cataract in some patients has lead to the discovery
of the hereditary hyperferritinemia-cataract syndrome (HHCS). This syndrome is due to point
mutations/deletions in the iron responsive element (IRE) of the L ferritin gene.
Over the past four years, 52 DNA samples from patients with unexplained elevated serum ferritin levels
ranging between 500 to 4,000 µg/L have been referred to us for molecular diagnosis of HHCS. Presence
of cataract and family history was not always documented. We sequenced the L ferritin exon 1 and
identified 24 samples with a mutation in the IRE. There were two interstitial deletion of the IRE and 22
point mutations. Most of these have already been described in HHCS patients, except mutation U34→C
in the upper ascending part of the stem and G47→A in the descending part. Mutations of the bulge
accounted for about 50 % of the mutations, 8 mutations affecting G32 with all three possible
replacements and 4 mutations affecting C33. Only two nucleotides of the loop were found mutated,
C39→U in three cases and A40→G in four other cases.
For the remaining 28 samples where no IRE mutation was found, we genotyped HFE mutations to rule
out type 1 hemochromatosis. We found the normal frequency of heterozygous C282Y HFE mutations (3
out of 28), an increased frequency of H63D heterozygous (12 out of 28) and one H63D homozygous. We
then sequenced the H ferritin gene, since we previously reported that inactivation of the H ferritin gene at
the heterozygous state in recombinant mice is responsible for increased serum L ferritin in the absence of
iron overload. However, we found no H ferritin mutation. Recently, ferroportin mutations have been found
in patients with dominant hemochromatosis (type 4), characterized by increased iron deposits in Küpffer
cells, elevated serum ferritin levels and normal or moderately elevated transferrin saturation. This
prompted us to sequence ferroportin in the 28 DNA samples from patients with unexplained
hyperferritinemia. We identified three new ferroportin mutations, introducing respectively a D157G, a
Q182H and a G323V replacement, suggesting that the corresponding three patients have dominant type
4 hemochromatosis. D157G and Q182H mutations are located in a putative loop between two
transmembrane domains, in a region where several mutations have been found in human and in zebra
fish. G323V falls into a putative transmembrane domain and might alter protein synthesis or stability.
Although we sequenced the entire ferroportin coding sequence, we did not find any of the previously
reported mutation. This suggests that the mutations we have identified give a milder phenotype which
might have remained undetected if the patients had not developed cataract (probably fortuitously). These
results demonstrate that both L ferritin IRE and ferroportin mutations can account for the association of
increased serum ferritin and normal transferrin saturation. A family history of cataract appears as the most
distinctive feature between HHCS and type 4 hemochromatosis. Additional causes of unexplained
hyperferritinemia remain to be identified.
POSTER 43
ANALYSIS OF THE PROMOTER REGION OF THE HUMAN HFE GENE IN HEMOCHROMATOSIS
PATIENTS
Catherine Mura 1, Gérald Le Gac 2, Sandrine Jacolot 1 and Claude Férec 1,2
1
Laboratoire de génétique moléculaire et génétique épidémiologique, INSERM EMI-U 0115, 2EFS, 46 rue
Félix Le Dantec, 29200 Brest, France
HFE-related hemochromatosis is a common autosomal recessive disorder of iron metabolism that affects
1 in 300 individuals in Caucasian populations. The C282Y mutation, and two variants, H63D and S65C,
are the main cause of the disease; few private mutations have also been described. In most studies, the
analysis of HFE coding sequence failed to characterize 5-10% of the hemochromatosis patients. Indeed,
the promoter region may also be a site for mutation liable to alter HFE expression. Here we analyzed, by
direct sequencing, the -1403/-1 region of the HFE gene in 31 hemochromatosis patients lacking mutation
in the coding region of the gene. Thirteen nucleotide variations have been found, those found more than
once in patients have been also analyzed in a series of control subjects. Three polymorphisms have been
characterized: -467 G/C (C 46.4% and G 53.6% in controls and C 59.5% and G 40.5% in the studied
patients) is out of any regulatory element, and the C282Y mutation is in linkage disequilibrium with the C
allele; -970 G/T that is in complete linkage disequilibrium with the -467 G/C change, is located within a
NF-E2 motif TGTATAGC; –1206 G/C (G 61.4% and C 38.6% in hemochromatosis patients). Ten private
variations in the 5’ flanking region of the HFE gene have been detected only once in hemochromatosis
patients. One of them (-491A/T), identified in a C282Y heterozygous patient, is located in a core GATA
sequence. The hypothesis that it could alter the level of the HFE expression remained to be investigated.
POSTER 44
THE pH DEPENDENCE OF THE RATES OF IRON RELEASE FROM THE N-LOBE OF HUMAN
TRANSFERRIN AND ITS K296E AND K296A MUTANTS
W.R. Harris, P. Xiong, Y. Horimoto, and R.T.A. MacGillivray, Department of Chemistry and Biochemistry,
University of Missouri-St. Louis, St. Louis, MO 63121 and Department of Biochemistry and Molecular
Biology, University of British Columbia, Vancouver, B.C., Canada, V6T 1W5.
The structure of the N-lobe of serum transferrin consists of two distinct domains separated by a
cleft. There is a single high-affinity iron binding site within the cleft, and it appears that the rate of iron
release to low-molecular-mass chelating agents is regulated in part by a slow protein conformational
change that partially opens the cleft to expose the iron to attack. This pathway for iron release follows
saturation kinetics with respect to the low-molecular-mass ligand. For some ligands there is an additional
reaction pathway that follows first order kinetics with respect to the ligand concentration. Also within the
cleft is a short hydrogen bond between Lys 296 and Lys 206. It has been suggested that the loss of this
hydrogen bond due to protonation of both lysines can act as a trigger to prompt the release of iron.
Mutants that lack this K206-K296 hydrogen bond retain iron to lower pH than the wild type protein, and
they release their iron to chelating agents much more slowly. To evaluate the importance of the K296 –
K206 hydrogen bond in the mechanism of iron release, the rates of iron release have been measured as
a function of pH for N-terminal monoferric transferrin (Tf-FeN) as well as the K296E and K296A mutants of
the recombinant transferrin N-lobe half molecule (Tf/2N). The K296E and K296A mutants have been
expressed in baby hamster kidney cells and in Pichia pastoris. Mutations were introduced into Tf/2N by
using the Quickchange method (Stratagene), and the cDNAs cloned into the appropriate expression
vector (pNUT or pPIC9). The recombinant proteins were purified from the culture media by ion-exchange
chromatography with Q-Sepharose and Mono Q resins. The proteins were homogeneous by PAGE, Nterminal sequence analysis and electrospray mass spectroscopy. The K296A mutation abolishes the
K296-K206 hydrogen bond, while the K296E mutation replaces this hydrogen bond by a salt bridge that
was expected to be less sensitive to pH changes. The rates of iron release to pyrophosphate (PPi) have
been measured by following the increase in protein fluorescence that accompanies the removal of the
paramagnetic ferric ion. Rates were measured between pH 7.4 and 6.0 for Tf-FeN, between 6.4 and 5.4
for K296E, and between 6.2 and 5.4 for K296A. The plots of kobs vs [PPi] for Tf-FeN showed a
combination of saturation and first-order pathways over the entire pH range. The mutants showed this
type of behavior at higher pH. At lower pH the range of ligand concentrations that could be studied was
too narrow to clearly define the ligand dependence. At a given [PPi], the plots of log kobs vs pH were
linear for all three proteins, with the rate of iron release increasing as the pH decreases. The slope of log
kobs vs pH was only -0.4 for Tf-FeN, compared with approximately -2.3 for the mutants. Thus contrary to
expectations, the mutants that lack the K296A-K206A hydrogen bond are much more sensitive to
changes in pH. Based on extrapolations, it appears that at pH 7.4 Tf-FeN releases iron to pyrophosphate
about 1000 times faster than either of the mutants. At pH 6 iron release from Tf-FeN is only about 20
times faster than release from the mutants. For Tf-FeN the saturation pathway represents about 40% of
iron release for 50 mM PPi at pH 7.4. The saturation and first-order pathways have a similar pH
dependence, so the relative importance of the two pathways varies only slightly as the pH decreases.
POSTER 45
HLA HAPLOTYPES, CD8+ T CELL NUMBERS AND THE PHENOTYPIC EXPRESSION OF
E Cruz1,2, H Alves3, R Gonçalves3, R Lacerda2; G Melo1, B Justiça1, & G Porto1,2,4
1
Clinical Hematology, Santo António General Hospital, Porto, Portugal. 2Molecular Immunology and
Pathology, Abel Salazar Institute for the Biomedical Sciences, Porto, Portugal. 3Molecular Genetics, North
Histocompatibility Center, Porto, Portugal. 4Molecular Immunology, Institute for Molecular and Cell
Biology, Porto, Portugal.
The phenotypic expression of Hereditary Hemochromatosis (HH) is quite variable in patients
homozygous for the C282Y mutation of HFE. Among other factors, the presence of the ancestral
haplotype carrying the HLA A3B7 has been implicated as a possible modifier of the severity of iron
overload (1-3), but the influence of combined HLA haplotypes or alleles in the clinical expression of
HH is not clarified. To approach this question we reviewed the clinical records from 32 HH patients
(only probands included), all homozygous for the C282Y mutation and analysed the relationship
between clinical expression, combined HLA haplotypes and the immunophenotype. Patients were
divided in two groups according to the clinical presentation: patients in Group I (n=21; 16 males, 5
females; mean age=53±12 years) presented with severe iron overload (mean TBIS=11.5±4 g),
generally with related clinical manifestations; patients in Group II (n=11, 6 males, 5 females; mean
age=44±12 years) were asymptomatic probands detected accidentally after a routine test or in the
context of a population screening for HH, and had in general mild to moderate iron overload (mean
TBIS=4.5±2 g). The average total number of CD8+ T lymphocytes was significantly lower in Group
I (average 0.33±0.16) than in Group II (average 0.56±0.15, p=0.0004). No differences were
observed in CD4+ T lymphocytes. As expected, most of the patients in both groups had the HLA
A*03 allele (17 in Group I; 6 in Group II). In 12 of them (11 from Group I) the A*03 allele was
carried in a HLA A*03B*07 haplotype. Only two patients (both from Group I) were homozygous for
HLA A*03, a frequency lower than expected. The distribution of the other haplotypes (contralateral
to the ones containing HLA A*03) differed markedly between the two groups of patients. HLA A*03
patients from Group I had contralateral haplotypes carrying only the most common HLA A*01,
A*02, A*03 or A*24 alleles, whereas patients from Group II did not show those alleles on the
contralateral haplotype, but had instead the less common alleles A*11, A*23, A*32 and A*33. The
frequency of patients without HLA A*03 containing haplotypes was higher in Group II (4/11) than in
Group I (4/21).
In conclusion, in the present C282Y homozygous patients, a more severe expression of the
disease was associated both with lower diversity in HLA haplotypes and low numbers of CD8+ T
cells. On the contrary, patients diagnosed with mild forms of the disease had a great diversity in
HLA haplotypes and normal to high CD8+ T cells. These results suggest that one or more MHC
linked genes may act as modifiers both of the clinical expression and the lymphocyte abnormalities
described in HH patients.
1.Piperno A et al, Hepatology 1996;24:43. 2.Crawford DH et al. Am J Hum Genet 1995;57:362.
3.Porto et al. Hepatology 1997;25:397.
Acknowledgements: grants from the Gulbenkian Foundation and Portuguese Foundation for Science and
Technology (FCT): POCTI/32986/199
POSTER 46
PLASMA NON-TRANSFERRIN BOUND IRON IS ‘SHUTTLED’ ONTO DEFEROXAMINE BY
DEFERIPRONE BUT NOT BY VITAMIN C
P. Evans 1, R. Kayyali 1, R.C.Hider 2, J.B.Porter 1.
1
2
Department of Haematology,
Department of Pharmacy,
University College, London,
King's College, London,
98, Chenies Mews,
150, Stamford Street,
London WC1E 6HX, UK
London SE1 8WA, UK
Non-transferrin bound iron (NTBI) is a potentially toxic plasma iron fraction found in a variety of iron
overload conditions. Although NTBI forms are largely uncharacterized, NTBI is thought to comprise iron
citrate complexes (and possibly other iron-chelates) either free in the plasma or bound to plasma proteins
such as albumin. In both the free and protein-bound forms, the iron may be polymeric. NTBI has been
implicated as one mechanism which can result in the iron-loading of tissues such as the heart and
anterior pituitary in thalassaemia major (TM). Hence its effective removal by chelators is desirable. We
hypothesized that the different species of NTBI are not equally accessible to chelation by deferoxamine
(DFO). Both deferiprone and ascorbate enhance the rate of urinary iron excretion with DFO but it is not
known whether NTBI is a significant source of this iron. In principle this increased excretion could be
achieved by deferiprone or ascorbate accessing NTBI pools which are less available to DFO and shuttling
them onto the more kinetically stable ferroxamine (FO).
In order to address this question, we have developed methods for stabilising DFO in plasma and for
extracting and quantitating FO using an HPLC system. We have applied this method to compare the
rates of access of 3 different NTBI preparations to chelation by DFO, and the effects of deferiprone and
ascorbate on the kinetics of this process. The three species we have examined are 1) iron-citrate
oligomers 2) iron-citrate oligomers bound to albumin 3) NTBI in thalassaemic serum.
When DFO (10µM) was incubated with iron-citrate complexes (ratio 1:10) for up to 24h, FO formation was
biphasic taking over 20 hours to reach completion. Deferiprone enhanced the rate of the second phase in
a concentration-dependent manner, unlike ascorbate which had little effect. By contrast, when iron citrate
was bound to albumin, and subsequently exposed to DFO, the rate of FO formation was significantly
more rapid. The kinetics were again biphasic but were essentially complete in 4 hours. Deferiprone
shortened this access time to 1 hour, whereas adding ascorbic acid had no effect on the rate. When TM
plasma containing 4 µM NTBI was incubated with DFO, there was again a small proportion of rapidly
chelatable iron but the kinetics of subsequent iron removal were very slow, with less than 2 µM FO
formation at 24h. The addition of deferiprone but not ascorbate resulted in a significant increase in the
rate of FO formation with all NTBI chelated at 24 hours.
It is concluded that DFO rapidly chelates only a fraction of NTBI in TM serum. Removal of the remainder
of plasma NTBI by DFO appears remarkably slow and is not complete after 24h. Deferiprone markedly
enhances the rate of NTBI chelation by DFO from iron-citrate, from iron-citrate albumin complexes and in
TM serum. By contrast, ascorbic acid has no enhancing effects on iron mobilization from either ironcitrate complexes or plasma NTBI at clinically relevant plasma concentrations.
This work is supported by NIH grant DK 57645-01
POSTER 47
HLA-LINKED CD8+ T CELL LYMPHOPENIA IN A FAMILY WITH ATYPICAL HEMOCHROMATOSIS:
EFFECT ON PHENOTYPE
E Cruz1,2, H Alves3, S Tafulo3, A Gartner4, P Rodrigues4, JM Cabeda1, F Dias1, J Vieira5 & G Porto1,2,4
1
Clinical Hematology, Santo António General Hospital, Porto, Portugal. 2Molecular Immunology and
Pathology, Abel Salazar Institute for the Biomedical Sciences, Porto, Portugal. 3Molecular Genetics, North
Histocompatibility Center, Porto, Portugal. 4Molecular Immunology, Institute for Molecular and Cell
Biology, Porto, Portugal. 5Molecular Evolution, Institute for Molecular and Cell Biology, Porto, Portugal.
The HFE C282Y mutation is found in homozygosity in the majority of hereditary hemochromatosis (HH)
patients (1). However, not all HH cases are homozygous and not all homozygous have the same
expression of iron overload. Other factors, eventually genetically regulated, may contribute to this
heterogeneity (2). Abnormally low numbers of CD8+ T cells have been consistently described in
association with a more severe expression of hemochromatosis, suggesting that these cells could act as
modifiers of expression in iron overload (3). The hypothesis that the same genes that modify the HH
phenotype could also regulate CD8+ T cell numbers is very attractive, but has never been demonstrated.
Several studies in humans and animal models have shown that T lymphocyte numbers are under the
genetic control of one or more genes, and some may be localised in the MHC region (4-6). We describe
here a family of hemochromatosis where the proband is heterozygous for the C282Y mutation although
he has the phenotype of severe iron overload (Total Body Iron Stores estimated by quantitative
6
phlebotomy = 13.9g) associated with an absolute CD8+ T cell lymphopenia (0.05 x10 /ml). No other
mutations on HFE or TfR2 known to be associated with HH were found. The HLA haplotypes of the
patient are A*02B*37 and A*24B*07, the first carrying the C282Y mutation. The family study revealed 4
additional affected siblings, also with severe iron overload and CD8+ T cell lymphopenia, all of them
sharing the same HLA haplotypes with the proband. The only C282Y heterozygous sibling who does not
have iron overload, does not share with the proband the HLA haplotype HFEwt A*24B*07 and has high
CD8+ T cell counts (0.64 x106/ml). Two other siblings share in common with the proband only the
A*24B*07 haplotype without C282Y and have low CD8+ cells, but not iron overload. In summary, we
report one hemochromatosis family where the phenotypic expression of iron overload depends on the
presence of one haplotype carrying the C282Y mutation, and another haplotype that segregates with
abnormally low CD8+T cell numbers. This may constitute a candidate family for clarifying the role of MHC
class I genes and lymphocyte numbers as modifiers of expression in hemochromatosis.
1.Feder JN et al. Nat Genet 1996;13(4):399-408. 2.Barton JC et al. Blood Cells Mol Dis 1997;23(1):13545. 3.de Sousa M, Porto G. J Hepatol 1998;28:1-7. 4.Damoiseaux JG et al. J Immunol
1999;163(6):2983-9. 5.Amadori A et al. Nat Med 1995;1(12):1279-83. 6.Hall M A et al. Genes and
Immunity 2000;1:423-27.
Acknowledgements: grants from the Gulbenkian Foundation and Portuguese Foundation for Science and
Technology (FCT): POCTI/32986/1999.
POSTER 48
PREVALENCE AND CLINICAL SIGNIFICANCE OF HFE GENE MUTATIONS IN PATIENTS
UNDERGOING EVALUATION FOR LIVER TRANSPLANTATION
David J. Brandhagen, Marijke Wevers, Shamina Dhillon, Russell Wiesner, Ruud Krom, Walter Kremers,
Mayo Clinic
INTRODUCTION: Hereditary hemochromatosis (HH) is the most common genetic disorder in Caucasians
with a prevalence of 0.5% and a carrier frequency of 10%. Nevertheless, HH is an uncommon indication
for orthotopic liver transplantation (OLT). It is uncertain if HFE gene mutations may influence disease
progression in patients with end-stage liver disease due to causes other than HH. AIM: 1. To compare the
prevalence of HFE gene mutations in patients with end-stage liver disease to a U.S. Caucasian control
population. 2. To determine if the prevalence of HFE gene mutations differs based on the etiology of endstage liver disease. 3. To determine if HFE gene mutations influence the prevalence of pre- and posttransplant survival as well as cardiac and infectious complications. METHODS: From January 2000 to
September 2002, we routinely tested for HFE gene mutations in 395 consecutive patients referred for
OLT. Patient and allograft survival as well as pre- and post-transplant cardiac and infectious
complications were compared for cases (one or two copies of the C282Y mutation or two copies of the
H63D mutation) and controls (no copies of C282Y and zero or one copy of H63D). Cases and controls
were matched for MELD score, diagnosis, age, sex, and race. We also compared the prevalence of HFE
gene mutations among the various diagnostic categories. RESULTS: Except for an increased
prevalence of C282Y homozygotes that was likely due to referral bias, the prevalence of HFE gene
mutations did not differ from that reported for a U.S. Caucasian control population (Beutler et al. Ann
Intern Med. 2000; 133: 329-337). In addition, the prevalence of HFE gene mutations did not differ among
patients with various etiologies of end-stage liver disease including chronic hepatitis C. Complications in
the cases and controls are summarized in the table. Mortality in the C282Y heterozygotes (29%) was
increased compared to nonC282Y heterozygotes (p=0.003). The number of cardiac and infectious
complications did not differ between cases and controls. CONCLUSIONS: 1. There was a statistically
significant increase in mortality in C282Y heterozygotes compared to nonC282Y heterozygotes. 2. The
prevalence of HFE gene mutations in a population of patients with end-stage liver disease referred for
OLT did not differ from the US Caucasian population. 3. The prevalence of cardiac and infectious
complications did not differ in patients with (cases) and without (controls) clinically significant HFE gene
mutations.
Complications
Cases
Controls
Cardiac, pre-tx
9/53 (17%)
8/53 (15%)
Cardiac, post-tx
1/11 (9%)
0/10 (0%)
Infectious, pre-tx
19/53 (36%)
25/53 (47%)
Infectious, post-tx
2/11 (18%)
5/10 (50%)
Number of deaths
11/53 (21%)
6/53 (11%)
POSTER 49
LACK OF AN ASSOCIATION BETWEEN THE HFE C282Y MUTATION AND THE ALPHA 1
ANTITRYPSIN Z ALLELE IN PATIENTS WITH END-STAGE LIVER DISEASE
David Brandhagen, Marijke Wevers, Shamina Dhillon, Russell Wiesner, Ruud Krom, Walter Kremers,
Mayo Clinic
INTRODUCTION: Alpha-1 antitrypsin (A1AT) deficiency and hereditary hemochromatosis (HH) are both
inherited, metabolic disorders which can lead to end-stage liver disease. Most HH heterozygotes do not
develop end-stage liver disease, whereas A1AT heterozygotes may. Previous studies have found an
association between HH (non-HFE confirmed) and A1AT deficiency. AIM: To determine if HFE genotypes
and A1AT phenotypes are correlated in patients with end-stage liver disease. METHODS: From January
2000 to September 2002 we routinely tested for HFE gene mutations and A1AT phenotypes in patients
referred for liver transplantation. We also collected information on age, gender and the etiology of liver
disease for each subject. HFE gene testing was performed on whole blood after amplification by PCR.
Alpha one antitrypsin phenotyping was performed by gel electrophoresis. Results were analyzed by Chisquare and Fisher's exact test. RESULTS: The table compares the prevalence of the A1AT phenotype
with HFE genotypes. There was a decreased prevalence of the Z allele in C282Y heterozygotes (1/38)
but this did not reach statistical significance. There were no other associations between the various HFE
genotypes and A1AT phenotypes. CONCLUSIONS: In patients with end-stage liver disease, there was
not an association between the HFE gene mutations and the Z-allele.
HFE Genotype
A1AT Phenotype
MM
MZ ZZ
SZ
Other
Total
C282Y +/+
2
1
0
0
2
5
C282Y/H63D
3
0
0
0
2
5
C282Y +/30
0
0
1
2
33
H63D +/+
4
0
0
0
1
5
H63D +/68
5
1
3
5
82
W/W
195
20
2
1
28
246
Total
302
26
3
5
40
376
POSTER 50
HETEROZYGOSITY FOR NOVEL HEPCIDIN MUTATIONS MAY MODIFY THE PHENOTYPE OF HFE
C282Y HETEROZYGOTES
Alison T Merryweather-Clarke1, Monique G Zaahl2,3, Estelle Cadet4, Vip Viprakasit1,5, Schalk van der
Merwe6, Dominique Capron7, Adrian Bomford8, Jennifer J Pointon1, Victoria LC Wimhurst1, Louise
Warnich3, Voravarn Tanphaichitr5, Maritha J Koetze9, Jacques Rochette4, Kathryn JH Robson1.
1
MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Headley Way, Oxford
OX3 9DS, UK; 2Division of Human Genetics, Faculty of Health Sciences, University of Stellenbosch,
Tygerberg, South Africa (SA); 3Department of Genetics, University of Stellenbosch, Stellenbosch, SA;
4
Génétique Médicale, Faculté de Médicine, Université Jules Verne de Picardie, Amiens, France;
5
Department of Paediatrics, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand;
6
Department of Internal Medicine, University of Pretoria, SA; 7Hépato-gastro-entérologie, Faculté de
Médicine, Université Jules Verne de Picardie, Amiens, France; 8Institute of Liver Studies, Kings College
Hospital, London, UK; 9Genecare Molecular Genetics, Christiaan Barnard Memorial Hospital, Cape Town,
SA.
Hepcidin antimicrobial peptide (HAMP) is secreted by the liver in response to increased iron
stores, resulting in decreased absorption of dietary iron. Juvenile haemochromatosis patients have been
described who are homozygous for mutations in the hepcidin gene (Roetto et al, 2003). We report here a
spectrum of hepcidin mutations found in patients with varying severity of iron loading, some of whom are
heterozygous for the HFE C282Y mutation, and suggest that the phenotype of HFE heterozygotes may
be modulated by HAMP mutations.
The first patient, BS, is a 65 year old British male who had a triple angioplasty at age 55. He had
a serum ferritin (sF) of 1000µg/l, and is heterozygous for HFE C282Y. DHPLC analysis of HAMP exons 2
& 3 revealed a novel heteroduplex pattern. Sequencing revealed that BS is heterozygous for a missense
mutation in exon 3 of HAMP. BS also has two children who are C282Y homozygotes, both undergoing
venesection, a 35 year old son whose serum ferritin on diagnosis was 1000µg/l and who lacks the HAMP
mutation, and a 39 year old daughter with rheumatoid arthritis who is heterozygous for the HAMP
mutation.
The second patient is from Picardy, MP, aged 46 years, who is also a HFE C282Y heterozygote.
At age 27 he was found to have congestive heart failure, with general fatigue and dyspnoea as the main
symptoms. He also presented with hypogonadotrophic hypogonadism, type I diabetes, skin pigmentation
and hepatomegaly. Transferrin saturation (TSat) was 92%, sF 1645µg/l, serum glucose 10µM. Fibrosis
was detected on liver biopsy, when hepatic iron concentration was 481 µM/g dry liver and HII=17.8.
Removal of 27g of iron over 4 years restored his TSat to 30% and sF to 40µg/l. A total of about 80g iron
has been removed over during 19 years of inconsistent attendance for phlebotomy. Unless at least 0.5g
of iron is removed per month, his TSat goes up to >80% in about 3 months. DHPLC and sequencing
analysis of HAMP revealed heterozygosity for a deletion in exon 2 which also involves a splice site. The
resulting transcript is likely to be extended to 179 codons due to a frameshift, completely disrupting the
active peptide. MP’s mother is aged 86 years with no major clinical problems. She lacks HFE C282Y but
is heterozygous for HFE H63D, and also heterozygous for the HAMP mutation. Her transferrin saturation
12
is 35%, ferritin 52µg/l, normal transaminases, serum glucose 6µM, red cells 4.25x10 /l, Hb13g/dl, MCV
82fl.
Two further pairs of related patients with iron overload from South Africa, three of whom are HFE
C282Y and H63D compound heterozygotes and the other a HFE H63D heterozygote, were found to be
heterozygotes for two different pairs of HAMP mutations and polymorphisms following DHPLC and
sequencing.
Finally, a young Thai haemoglobin E patient who has never been transfused, with an sF of
461µg/l, was found to be heterozygous for a polymorphism in HAMP intron 1.
We propose that the phenotype of C282Y heterozygotes may be modified by heterozygosity for
mutations which disrupt the function of hepcidin in iron homeostasis, with the severity of iron overload
corresponding to the severity of the HAMP mutation.
POSTER 51
MUTATIONS AND POLYMORPHISMS IN TFR2, SLC11A3, HFE AND DCYTB IN IRON OVERLOADED
PATIENTS
Alison Merryweather-Clarke1, Antonella Roetto2, Karen J Livesey1,3, Jennifer J Pointon1, Victoria LC
Wimhurst1, A Robins4, Clara Camaschell2, Kathryn JH Robson1. 1MRC Molecular Haematology Unit,
Weatherall Institute of Molecular Medicine, Headley Way, Oxford OX3 9DS, UK; 2Department of Clinical
and Biological Sciences, University of Torino, Azienda Ospedaliera San Luigi, 10043 Orbassano, Torino,
Italy; 3The Genetics Laboratory, Churchill Hospital, Headington, Oxford OX3 7LJ; 4Department of
Biochemistry, Macclesfield District General Hospital, Macclesfield, Cheshire SK10 3BL.
Over 90% of haemochromatosis patients in the UK are homozygous for the C282Y mutation in
the haemochromatosis gene HFE. In C282Y heterozygotes, compound heterozygosity for the HFE H63D
mutation greatly increases the risk of developing haemochromatosis. More than 74% of HH patients who
are C282Y heterozygotes are also compound heterozygotes for the H63D mutation. Other rare HFE
mutations have been observed, and mostly cause disease when present in the compound heterozygous
state with either C282Y or H63D. All but one of these, S65C, have been reported only in one or two
families. A single point mutation, P160delC, has been identified in a single patient who is wild type for
both of the common HFE mutations, and is the only rare mutation which appears to cause iron overload
on its own. Mutations in TFR2 cause haemochromatosis type 3, and in SLC11A3 cause
haemochromatosis type 4. Mutations in hepcidin and h-ferritin have also been reported to cause iron
overload. Haemochromatosis is a heterogeneous disease, with penetrance of the HFE C282Y mutation
varying considerably. It is likely that other genes act as modifiers of C282Y penetrance, although their
identity is currently unknown.
We are using DHPLC to search for other mutations and polymorphisms which may explain the
phenotype of patients referred for haemochromatosis genotyping, who are not C282Y homozygotes but
have elevated transferrin saturation and serum ferritin levels. In the present study, we report the
identification of mutations and polymorphisms in HFE, SLC11A3, TFR2 and DCYTB, including two novel
missense mutations, one in HFE and one in DCYTB, and a previously reported missense polymorphism
in TFR2.
Where there are clear signs of unexplained iron overload, detailed genetic analysis of genes
involved in iron metabolism such as HFE, TFR2, SLC11A3 and HAMP should be considered.
POSTER 52
HEMOCHROMATOSIS MUTATIONS IN THE GENERAL POPULATION: CROSS-SECTIONAL, 25-85
YEAR TIME-COURSE, AND PENETRANCE EVALUATIONS
R. V. Andersen, A. Tybjærg-Hansen, M. Appleyard, H Birgens, B. G. Nordestgaard, Herlev University
Hospital (R.V.A., H.B., B.G.N.), Rigshospitalet, Copenhagen University Hospital, (R.V.A., A.T.-H.), and
The Copenhagen City Heart Study, Bispebjerg University Hospital (A.T.-H., M.A., B.G.N.), University of
Copenhagen, Denmark.
Background It is unclear whether hereditary hemochromatosis should be screened for, and what advice
doctors should give to those identified with C282Y homozygosity. We therefore examined in individuals
from the general population 1) transferrin saturation above 50% as predictor of C282Y homozygosity, 2)
the disease progression rate in C282Y homozygotes from 25-85 years, and 3) the lifetime penetrance of
C282Y homozygosity.
Methods We genotyped 9174 individuals from the Danish general population. The 23 C282Y
homozygotes identified were matched to two subjects each of the wild type/wild type, H63D/wild type,
H63D/H63D, C282Y/wild type, C282Y/H63D genotypes, with respect to gender, age, and alcohol
consumption.
Results Only C282Y homozygotes had average transferrin saturation above 50%. Positive and negative
predictive values were 3% (95%CI:2%-4%) and 100% (98%-100%), indicating that only 3% of those with
transferrin saturation above 50% were C282Y homozygotes, but that transferrin saturation of 50% or less
practically excluded C282Y homozygosity. Transferrin saturation increased from 50-70% and ferritin from
400-550 µg/L from 25-85 years in C282Y homozygotes. None of the C282Y homozygotes developed
clinical overt hemochromatosis.
Conclusions Individuals in the general population with C282Y homozygosity only demonstrate modest
increases in transferrin saturation and ferritin from 25-85 years, and do not develop hemochromatosis.
Consequently, screening programs for C282Y homozygosity in the general population cannot be
recommended. If C282Y homozygotes nevertheless are identified during such screening programs, and
not because of clinical hemochromatosis, at most they need to be screened for manifestations of
hemochromatosis every 10-20 years.
POSTER 53
INTRAERYTHROCYTIC IRON CHELATION DIMINISHES OXIDATIVE DAMAGE IN BETATHALASSEMIC ERYTHROCYTES
N. Szuber, J. L. Buss1, M. Trudel2, M. D. Scott3 and P. Ponka, Lady Davis Institute for Medical Research,
SMBD Jewish General Hospital and Department of Physiology, McGill University, 3755 Cote Ste.Catherine Road, Montreal, Quebec, Canada H3T 1E2; 1 Current address: Wake Forest University School
of Medicine, Medical Center Blvd., Winston-Salem, NC, USA 27157; 2 Institut de Recherches Cliniques
de Montreal, 110 Avenue des Pins Ouest, Montreal, Quebec, Canada H2W 1R7; 3 Canadian Blood
Services & Department of Pathology, University of British Columbia, 2329 West Mall, Vancouver, British
Columbia, Canada V6T 1Z4.
Under normal physiological circumstances, nature dictates a stringent physical separation between iron
and lipids. Although healthy red blood cells (RBC) contain nearly 20 mM iron, it remains safely concealed
within hemoglobin and is thus excluded from the membrane milieu. However, in pathological conditions,
such as the thalassemias and hemoglobinopathies, the intimate association of excessive redox-active,
non-heme iron with the RBC membrane produces a striking number of membrane defects due to iron’s
catalytic role in generating injurious reactive oxygen species. The inappropriate liaison of iron with vital
membrane components specifically targets oxidative damage to the membrane, triggering the grossly
accelerated autoxidation of its components and playing a critical role in premature cell senescence. Betathalassemia is distinguished by an excess of unstable alpha-hemoglobin subunits from which heme
groups and eventually, toxic non-heme iron, are readily liberated and integrated within membrane
constituents. Because of the iron decompartmentalization characteristic of thalassemic RBCs, chelators
which remove iron from the membrane may be of significant therapeutic benefit. Previous studies have
determined the behaviour of normal human RBCs loaded with purified alpha-hemoglobin chains to be
structurally, functionally and metabolically identical to that of actual human thalassemic RBCs. Our in
vitro studies include the preparation of these model thalassemic RBCs through a process of alphahemoglobin purification and subsequent encapsulation of the isolated subunits into normal RBCs by
osmotic lysis and resealing. Non-heme membrane-bound iron was quantified in ghost membrane
fractions derived from model thalassemic RBCs using the ferrozine assay and hemoglobin oxidation was
measured spectrophotometrically. As anticipated, the resulting erythrocytes manifest severe oxidative
damage due to the presence of unpaired alpha-hemoglobin chains and contain an excess of detectable
membrane-associated iron, confirming their usefulness as in vitro models of the disease. The effects of
various standard iron chelators including desferrioxamine, deferiprone, and pyridoxal isonicotinoyl
hydrazone (PIH) as well as novel compound, pyridoxal ortho-chlorobenzoyl hydrazone (ortho-108), were
then evaluated on membrane iron burden and fundamental cellular oxidative parameters. The PIH series
of chelators shows particular effectiveness in improving cellular oxidation status, as parent (PIH) and
analogue (ortho-108) compounds promoted the most significant decrease in the rate and extent of
hemoglobin oxidation, as well as in the amount of erythrocyte membrane-associated iron. These results
have incited a series of promising in vivo studies currently being undertaken. In order to assess the
physiological effects of these chelators on RBC oxidative status and survival rates in vivo, our present
research methodology involves the use of recipient mice transplanted with homozygous beta-thalassemic
murine bone marrow as animal models of the disease. By chronically administering the selected
chelators via implanted osmotic pumps over a period of 4 weeks, and using in vivo biotinylation to track
thalassemic RBC survival via flow cytometry, it is anticipated that interception of intraerythrocytic iron in
these genuinely pathological RBCs will diminish the toxic deposits of the bioactive metal and attenuate
the ineffective erythropoiesis characteristic of beta-thalassemia. This pharmacological approach would
be expected to enhance mature RBC survival in humans suffering from erythrocytic disorders of oxidative
etiology, thereby decreasing their need for transfusion therapy and, ultimately, improving their quality of
life.
POSTER 54
EVALUATION OF THE PENETRANCE OF HEREDITARY HEMOCHROMATOSIS IN SOUTH
FRANCE
P Aguilar-Martinez, M Giansily, A Lepape*, MC Picot, JF Schved and the members of the register of
Hereditary Hemochromatosis. Montpellier University Hospital, and *CNAM, 34 Montpellier, France
Context: Hereditary Hemochromatosis (HH) is one of the most frequent inherited disorder among
Caucasians and affects individuals in the adulthood. Progressive iron overload and related severe
complications can occur if the condition is not detected early. Discussions about the necessity to
implement systematic screening of the disease in at-risk populations and the way to do it have not yet
reached a consensus. More data seem to be needed, especially in the field of the clinical penetrance of
HFE mutations. On the other hand, registers of inherited disorders seem to be a powerful tool for
monitoring these pathologies and their use was recently recommended by WHO (Bulletin of the WHO,
2001,79 (11)). We are currently setting up a register of hereditary hemochromatosis in a Southern
French area where we had previously determined the frequency of HFE mutations. Here we use the
data obtained during the evaluation of the affected population to calculate the penetrance of HFE
mutations in this area.
Methods: General population data were obtained from the INSEE (last census of the French
population). Data concerning the prevalence of the HFE mutation in the area were published previously
(Aguilar-Martinez et al, Br J Haematol, 2000). The figures of affected patients were collected using
different methods. Main data came from the patients referred to the university hospital of the area where
genotyping is performed. The patients’ medical record allowed us to select those subjects who have a
clinical expression of iron overload. Additional data were provided anonymously by the National Health
Insurance Service. Further confirmation came from the patient’s society and by inclusions in the register
obtained through numerous physicians of the area.
Results and discussion: The population of the area is 2.29 millions of people, and consists of 57.9% of
individuals above 35 years of age. As hemochromatosis rarely develops under 35 years, we calculated
the prevalence of the disease among the 1329591 individuals of more than 35 years.
We previously determined that the observed frequency of the YY genotype (C282Y homozygotes) in the
area was of 1 in 637 subjects [YY frequency (95%CL) extrapolated from the allele frequency of the Y
allele: 1/1098 (1/738-1/1085)]. It is noteworthy that about 25% of the population of the area is not of
European extraction. This can explain the relatively low frequency of YY subjects. Using these data, we
calculated the theoretical number of YY subjects of more than 35 years to be 2087 in the area
[extrapolated number of YY subjects (95%CL) : 1211(737-1802)]. The total number (n) of patients with
an expressed clinical disease was estimated to be higher than 300 (lower figure obtained through the
National Health Insurance Service and corresponding to patients declared to this service with a diagnosis
of HH, knowing that a number of affected subjects are not declare to this service), and up to 450 (figure
obtained from the mixed data of the main laboratories and clinical departments of the area where patients
are diagnosed and followed for their phlebotomies). As we have previously shown that only 81% of the
HH patients in our area have the YY genotype, we can assume that the YY subjects account respectively
for 243 (if n=300) or 365 (if n=450) of the patients expressing a clinical disease. Using these data, the
calculated penetrance for the YY genotype is then of 12% for the lower number of expressed diseases
(n=300), or 17,5% for the higher one (n=450). [Using the extrapolated value for the YY genotype
(95%CL), the figures are: 14%(13.5%-32.9%) for n=300 and 21.5%(20.2%-49.4%) for n=450].
In addition, we previously showed that compound heterozygotes for the C282Y and H63D mutations
(HCCY) accounted for about 8% of the HH patients in the same area. Individuals with this genotype were
determined to be 1 in 85 subjects of the general population of this area. Thus, the calculated number of
compound heterozygotes is 15655 among the age bracket above 35 years. If we assume that 8% of the
symptomatic HH have this genotype, then the number of compound heterozygotes expressing clinically
the disease is respectively of 24 and 36 for n=300 or n=450 patients. Using these data, the calculated
penetrance of this genotype would be of 0.15 to 0.23% respectively.
Conclusion: Although theoretical, these evaluations rely on data obtained from dependable sources.
They show that the clinical penetrance of the YY genotype is higher than 10% and may reach 49% in
our area. In the compound heterozygous state for the C282Y and H63D mutations the clinical
penetrance is higher than 1.5/1000. Data obtained from the in progress register will help to confirm and
refine these preliminary evaluations.
POSTER 55
FEASIBILITY AND ACCEPTABILITY OF TWO SCREENING STRATEGIES FOR
HAEMOCHROMATOSIS: A RANDOMISED CONTROLLED TRIAL
C Patch,* W Rosenberg,** P Roderick. * *Health Care Research Unit, University of Southampton. ** Division
of Inflammation, Infection and Repair, University of Southampton.
Haemochromatosis, a treatable adult-onset condition of progressive iron overload is amenable to
population screening. Population screening had been recommended using biochemical tests for iron
overload. Identification of the HFE gene raised the possibility of considering a DNA based screening test.
Initial enthusiasm for screening has now waned with the recognition that although up to 90% of clinical
disease is associated with the known mutations of the HFE gene the risk of developing disease in those
with mutations is not known. In addition, the natural history of disease in those identified with raised iron
indices is also not yet established. Whilst it would be possible to screen for haemochromatosis utilising a
genetic or a biochemical test, the lack of natural history data predicates against initiating population
screening programmes. However screening trials will needed to identify cohorts with the-at risk genotypes
or early iron overload in whom factors influencing the natural history can be studied. The performance of
the two screening tests is different and it has been considered that genetic testing is less acceptable.
Although the at-risk genotypes are equally distributed between males and females, the penetrance of
haemochromatosis related iron overload is greater in males. There is a consensus internationally that
evaluation of screening programmes should include the acceptability and feasibility of the whole
screening programme including assessment of the uptake of screening and characteristics of the
population being offered and accepting screening. We sought to compare acceptability and feasibility of
genetic and biochemical screening strategies for haemochromatosis.
Design: Randomised controlled trial comparing:
a) Biochemical screening for iron overload on a blood sample taken at the General Practitioner’s surgery,
followed by genetic analysis and clinical assessment in those screened positive.
b) Genetic screening for the at risk genotype on a saliva sample performed at home, followed by
biochemical testing for iron overload and clinical assessment in those screened positive.
Sample: General practice population aged 30-70 stratified by age and sex.
Results: Approximately 3000 individuals have been invited. Uptake of screening was approximately 30%.
The modality of the test did not significantly affect uptake of screening. The factors affecting the
probability of accepting screening were age, gender and social deprivation. Uptake was higher in females
than males, specifically in older females, and in less deprived areas.
Discussion: This study systematically evaluates screening for haemochromatosis in primary care,
specifically the effect of population characteristics and the modality of the screening test on acceptance of
screening. There was no significant difference in the uptake of testing between the biochemical and
genetic test. Further data are being analysed in order to compare anxiety and other psychological
measures. Thus, it would appear that a genetic test offered in this way is as acceptable as a biochemical
test rather than being unacceptable as has been suggested. Males aged between 30 and 50 years, the
key target population have the lowest uptake, this has important implications for the design of treatment
or screening trials.
POSTER 56
MECHANISMS OF HFE-INDUCED REGULATION OF IRON HOMEOSTASIS: INSIGHTS FROM THE
W81A MUTATION
An-Sheng Zhang, MD, Hanqian Carlson, MD, PhD, Paige Davies, BS and Caroline Enns, PhD
(Presented by An-Sheng Zhang, MD)
Type 1 Hereditary hemochromatosis (HH) is an autosomal recessive disorder with the clinical
manifestation of severe iron overload in liver, heart, and pancreas. The HH gene, HFE, encodes an
atypical major histocompatibility complex (MHC) class I-related protein. In the past few years a great deal
of progress have been made towards the elucidating the structural aspects of HFE. In vivo and in vitro
studies have documented that HFE forms complexes with transferrin receptor (TfR) at neutral pH. The
binding site of TfR to HFE overlaps with that to diferric transferrin, which explains the observation that
HFE is able to lower the apparent binding affinity of TfR to diferric transferrin. More importantly,
transfection studies demonstrated that expression of HFE in a variety of cell lines down regulates iron
uptake from diferric transferrin. The downregulation does not result from the alteration of TfR endocytosis
by HFE. How HFE decreases iron levels in the cell still remains unknown. In this study the mechanisms
by which the hereditary hemochromatosis protein, HFE down-regulates transferrin-mediated iron uptake
were examined. Co-immunoprecipitation studies using solubilized cell extracts demonstrated that Tf
competed with HFE for binding to the TfR similar to previous in vitro studies using soluble truncated forms
of HFE and the TfR. At concentrations of transferrin approaching those found in the blood no differences
in transferrin binding to cells were detected consistent with the lower binding constant of HFE for TfR than
transferrin. However, cells expressing HFE still showed a down regulation of transferrin-mediated iron
uptake at concentrations of transferrin sufficient to dissociate HFE from the TfR. These results indicate
that the association of HFE with TfR is not an essential prerequisite for its ability to lower intracellular iron
stores. To test the effect of HFE on lowering intracellular iron levels independently of its association with
TfR, a mutated HFE (W81A) that shows greatly reduced affinity for the TfR was transfected into tTA HeLa
cells. HeLa cells expressing W81AHFE behaved in a similar manner to cells expressing wild type HFE
with respect to decreased intracellular iron levels measured by iron responsive protein gel shift assays
and ferritin levels. The results indicate that HFE can lower intracellular iron levels independently of its
interaction with the TfR.
POSTER 57
INTESTINAL IRON ABSORPTION: THE INTERACTION OF DMT1 WITH PAP7, AN IRON
RESPONSIVE PROTEIN
J. Glass, J. Rodriguez-Paris, Y. Ma, K.-Y. Yeh, M. Yeh, Y. Chen. Feist-Weiller Cancer Center, LSU
Health Sciences Center, Shreveport, LA, USA
The divalent metal transporter 1 (DMT1) is essential for cellular iron uptake both in the intestine and in
erythroid cells. DMT1 is a transmembrane protein that transports Fe(II) and other divalent cations into
cells. Two isoforms exist based on an alternative splice mechanism. The mRNA of isoform I contains an
iron responsive element (IRE) in the 3' UTR and has a unique 18 amino acid C-terminus that is replaced
in the non-IRE isoform II by a 25 amino acid cytoplasmic tail. Isoform I is preferentially expressed in the
intestine with DMT1 on the brush border membrane (BBM) of the duodenum, the site of iron absorption.
In the rat lumen iron causes DMT1 to undergo endocytosis. This behavior suggests that DMT1 interacts
with other cellular proteins both to be primarily expressed on the apical surface and to undergo
transcytosis. The yeast two-hybrid system was used to find proteins that interact with the unique tail of
isoform I. A cDNA fragment coding for the C-terminal cytoplasmic domain of DMT1 (+IRE) (amino acids
512-561) was cloned into pGBKT7 to form the bait, BD-CT512 using the Matchmaker GAL4 yeast twohybrid system 3 (Clontech). The prey was constructed from the insertion of a rat duodenal cDNA library
into pGADT7 to form the AD-library. The AD-library was transfected into the AH109 host previously
transformed with BD-CT512 and grown under high stringency conditions. Multiple colonies were obtained
from which the plasmids were isolated and the cDNAs sequenced. One of the isolated cDNAs encoded a
protein that specifically interacts with DMT1(+IRE) and which has previously been identified as PAP7 by
its interactions with a peripheral-type benzodiazepine receptor (PBR) (Li et al, Mol. Endo. 15, 2211,
2001). PAP7 has also been shown to interact with PKA. Using a specific antibody raised against the
PAP7 protein, a 75 kD protein was located in many tissues including the central nervous system, liver,
kidney, duodenum, jejunum and spleen. In rat duodenum PAP7 was localized in the BBM with DMT1.
With iron feeding both DMT1 and PAP7 translocate: PAP7 moves from the BBM to the basolateral
membrane (BLM) and DMT1 undergoes endocytosis and is found in cytoplasmic vesicles. The interaction
of PAP7 and DMT1 was seen also in BBM vesicles (BBMV) prepared from rat duodenum which were
enriched with both proteins. From solubilized BBMV the immunopreciptate obtained with anti-PAP7
antibodies contained DMT1 and, vice a versa, the immunoprecipitate obtained with anti-DMT1 contained
PAP7. Localization of PAP7 in Caco2 cells by immunohistochemistry was similar to that in rat intestine.
Interaction between DMT1 and PAP7 could be demonstrated also in Caco2 cells both by 1) incubation of
solubilized Caco2 cells with S-tagged PAP7 followed by immunoprecipitation and identification of DMT1
35
by western blotting; and 2) detection of S-methionine labeled PAP7 derived from in vitro translation
following incubation with Caco2 extracts and immunopreciptiation with anti-DMT1. In addition, by western
blot analysis in both Caco2 and K562 cell lines PAP7, which has a nuclear localization signal, could be
found located in the nuclei. The iron status of the cells markedly affected PAP7 protein levels. In both rat
duodenal epithelium and Caco2 cells iron loading markedly decreased PAP7 levels while iron depletion
increased PAP7 protein levels. Iron status also affected the amount of PAP7 localized in the nucleus.
Taken together these findings support that PAP7 interacts with DMT1 in rat duodenum and Caco2 cells
and that in both the duodenum and Caco2 cells PAP7 is localized in the BBM. The translocation of PAP7
from the BBM to the BLM with iron feeding suggests that PAP7 has a role in iron transport. That PAP7
levels are affected by iron status and that PAP7 also localizes to the nucleus suggests that PAP7 may a
regulatory effect in the intestinal iron uptake process.
POSTER 58
EFFECT OF SECONDARY IRON OVERLOAD ON TISSUE SPECIFIC EXPRESSION OF IRON
TRANSPORT MOLECULES
I.Theurl, S. Ludwiczek, M. Seifert, J. Jehle, G. Weiss
Department of Internal Medicine, University Hospital, A-6020 Innsbruck, Austria
Background: Secondary iron a frequent clinical condition found e.g. in subjects with hemoglobinopathias
receiving multiple transfusions. However, the regulatory changes of iron homeostasis under these
conditions are poorly understood.
Methods: We studied C57BL/6 mice receiving intraperitoneal injections of 5 mg iron dextran for up to six
days. The expression of genes involved in iron metabolism and iron transport was analyzed by means of
RT-PCR and Western blot in duodenum, liver, and kidney, tissue iron content was measured by atom
absorption, iron tissue distribution was visualized by pearl blue staining and monitoring of intracellular
iron availability was performed by determination of IRP binding affinity via gel shift assays.
Results: At day 0 we found a positive correlation between HFE expression with tissue mRNA levels of
DMT-1, Ferroportin-1, TfR and IRP activity in duodenum, liver and kidney, respectively, and all these
parameters were negatively associated with the iron content of the respective tissue. Following
experimental iron overload HFE expression increased over time in the duodenum while in liver HFE levels
decreased with prolonged iron accumulation. As a consequence of this TfR, DMT-1 and FP-1 expression
remained high in the duodenum while DMT-1 and TfR decreased in the liver and kidneys. Secondary iron
overload led to progressive iron storage in the liver which was also paralleled by an increase of hepcidin
and FP-1 expression.
Conclusions: Our results show that with secondary iron overload and progressive tissue iron
accumulation the iron uptake molecules in the liver and kidney are down-regulated while FP-1 expression
and presumably iron export are increased which may represent a protective mechanism against
unlimited tissue iron accumulation. However, the iron transport armory in duodenum is up-regulated with
secondary iron overload suggesting that iron sensing to the duodenum is impaired under these conditions
leading to increased iron duodenal absorption under secondary iron overload conditions.
POSTER 59
THE IRON-DEFICIENT PHENOTYPE OF HFE-OVEREXPRESSING CELL LINES IS NOT DUE TO β2MICROGLOBULIN INSUFFICIENCY
J. Wang, G. Chen and K. Pantopoulos
Lady Davis Institute for Medical Research and Department of Medicine, McGill University, Montreal,
Quebec, Canada
HFE is an atypical major histocompatibility complex (MHC) class I type molecule with a critical, but as yet
elusive function in the regulation of systemic iron metabolism. HFE mutations are linked to “type I”
hereditary hemochromatosis, an autosomal recessive disorder of iron overload with high prevalence in
populations of Northern European descent. Most patients of “type I” hemochromatosis are homozygous
for a C282Y point mutation that abrogates the interaction of HFE with β2-microglobulin (β2M), and thus
impairs its proper processing and expression on the cell surface. A second HFE mutation, the H63D
substitution, is also associated with disease. To investigate the function of HFE we have generated
clones of human H1299 lung cancer cells that express wild type, C282Y or H63D HFE under the control
of a tetracycline-inducible promoter. Consistently with earlier observations in other cell lines, the
expression of wild type or H63D, but not C282Y, HFE induces an apparent iron deficient phenotype,
manifested in the activation of iron-regulatory protein and concomitant increase in transferrin receptor
levels and decrease in ferritin content. This phenotype persists in cells expressing wild type HFE following
transfection with a β2M cDNA. While endogenous β2M is sufficient for the presentation of at least a
fraction of chimeric HFE on the cell surface, this effect is stimulated by ~2.8-fold in β2M transfectants. The
co-expression of exogenous β2M does not significantly affect the half-life of HFE. These results suggest
that the apparent iron-deficient phenotype elicited by HFE in various cell lines is not linked to β2M
insufficiency.
POSTER 60
GENETIC HEMOCHROMATOSIS IN ITALY: PRELIMINARY REPORT OF A STUDY ON THE THE
ROLE OF H63D MUTATION ON IRON OVERLOAD
C. Velati, L. Fomiatti, V. Sancassani, G. Garozzo, P. Delbini, M. Sampietro, S. Fargion, G. Fiorelli,
Transfusion Medicine Department of Sondrio and Ragusa, Institute of Internal Medicine University of
Milano, Italy
Introduction.
Increased transferrin saturation and/or serum ferritin have been observed in Italy in approximatively 5% of
subjects at first blood donation. Among those subjects with persistent alteration of iron parameters at
fasting control, C282Y mutation was overrepresented in Northern Italy whereas H63D was in the South,
although the allelic frequencies of HFE mutations were similar in a control group of repeat blood donors in
the North and in the South of the Country, as reported in a previous study.
The H63D mutation is common in the general population, but its role in the pathogenesis of iron overload
remains uncertain. The aim of the present study was to compare the iron parameters and to determine
the iron depletion in repeat blood donors heterozygous for the H63D mutation compared to a population
of blood donors wt/wt for the H63D mutation.
Methods.
37 blood donors heterozygous for the H63D mutation and 25 blood donors wt/wt for the same HFE
mutation, both groups wt/wt for the C282Y, (previously genotyped in the study of HFE mutations
prevalence in italian blood donors), were enrolled. The two groups are similar for number of blood
donations (expressed as iron loss) and for sex distribution. Serum ferritin (SF) was the iron index
recorded at first and second observation.
Results.
The table summarizes the results.
HFE genotype
n. of
cases
37
SF 1° observation
mean ng/ml
(range)
188 (6-856)
SF 2° observation mg iron loss
mean ng/ml
mean (range)
(range)
85 (7-385)
1401 (225-2925)
H63D/wt total
wt/wt total
25
93 (7-531)
71 (17-291)
1179 (225-2700) 24 (11-45)
H63D/wt North
22
112 (6-644)
62 (7-189)
1575 (225-2925) 41 (12-90)
wt/wt North
16
43 (7-97)
42 (17-91)
1392 (225-2700) 28 (14-45)
H63D/wt South 15
299 (16-856)
119 (9-385)
1147 (225-2250) 30 (11-42)
wt/wt South
181 (11-531)
123 (22-291)
800 (225-2025)
9
n months of
observation
mean (range)
37(11-90)
14 (11-17)
Discussion.
These data suggest that subjects with H63D mutation of the HFE gene have, at first observation, a higher
ferritin levels than subjects wt/wt. This seems to be more evident in blood donors of Southern Italy than
in Northern. Blood donation induces significant reduction of the iron stores both in H63D heterozygous
and in wt/wt subjects. Although our observation is preliminary and restricted to a limited number of
subjects, it seems worthwhile to extend the follow-up of blood donors H63D heteroxygotes or even
homozygotes when available, in order to get further insights on the H63D role in iron metabolism.
POSTER 61
HEMOCHROMATOSIS PROTEIN (HFE) AND TUMOR NECROSIS FACTOR RECEPTOR 2 (TNFR2)
CONTRIBUTE TO A COMMON PATHWAY OF IRON REGULATION IN THE GUT
M. J. Chorney, P.N. Meyer, Y. Yoshida, H. Saito, K.A Chorney, and *G. S. Gerhard.
Department of Microbiology and Immunology, Penn State College of Medicine, Hershey, PA, *Department of
Pathology, Dartmouth Medical School, Hanover, NH.
Introduction: To investigate the function of HFE in the gut, we applied histochemical analysis of the intestine to
knockout and wildtype mice challenged with dietary iron in conjunction with a quantitative trait locus (QTL)
analysis of (C57BL/6 x DBA/2) recombinant inbred (RI) mice. Our hypothesis focuses on tumor necrosis factor
(TNF) and HFE as components of a signaling pathway impacting upon iron flux. This hypothesis is based on an
inverse relationship between the level of TNF release by intraepithelial lymphocytes (IELs) and hepatic iron
deposition following dietary iron supplementation reported previously.
Results: QTL analysis of normal RI mouse hepatic and splenic iron levels suggested a role for a gene(s)
mapping to chromosome 4, cM 75.5, which contains the TNF receptor 2 gene (TNFR2). TNFR2 knockout (-/-),
TNFR1-/-, HFE-/-, and wildtype control mice, therefore, were challenged with carbonyl iron-fortified chow for two
weeks in order to extend the above QTL result. HFE-/- mice increased their hepatic iron load above
significantly increased resting iron levels, although splenic iron levels remained unchanged following challenge.
TNF receptor knockouts increased their hepatic iron levels comparable to controls; however, TNFR2-/- mice
demonstrated a significant increase in splenic iron levels above other lines studied. Unlike TNFR1-/- and
wildtype mice which showed Perls’ staining of intestinal epithelial cells (IECs) following iron challenge, HFE-/and TNFR2-/- gut sections reproducibly failed to yield any staining, demonstrating a common defect in iron
retention within the IEC compartment. IELs derived from HFE-/- mice also failed to demonstrate intracellular
TNF staining, confirming a proposed intersection between TNF synthesis, following iron loading, and HFE
expression. Finally, the absence of HFE in the gut failed to result in a qualitative increase in the IEL population
expressing the gamma/delta form of T-cell receptor, comparable to control mice fed increased amounts of ironladen chow.
Discussion: TNF and TNFR2 have now been linked to HFE in contributing to the flux of dietary iron from the
intestine to the blood. The proposed pathway commences with the iron-loading of enterocytes followed by
stimulation of IELs via HFE. Gamma/delta+ IELs may orchestrate the initiation of the TNF response, while
TNFR2 expressed on IECs appears to bind TNF culminating in iron compartmentalization. We further
hypothesize that the membrane expression of HFE, enhanced due to downregulation of transferrin receptor 1,
may lead to the suppression of iron regulated protein 1 (IREG1), thus reducing the rate of iron efflux and
affording the cell the opportunity to sequester iron within ferritin cores.
Conclusion: Enhanced levels of the IEC labile iron pool (LIP) equate to an oxidative stress which is proposed to
be communicated to neighboring IELs through HFE, either directly or indirectly. The net result is the release of
TNF followed by iron compartmentalization mediated through TNFR2 in conjunction with the proposed
downregulation of IREG1. The data support the previously promulgated Piggyback-Sensor model of HFE
function which proposes a novel model of HFE function.
POSTER 62
HEME AND NONHEME IRON ABSORPTION IN HUMANS WITH THE C282Y AND H63D HFE
MUTATIONS ASSOCIATED WITH HEMOCHROMATOSIS
J. R. Hunt and H. Zeng. USDA-ARS Grand Forks Human Nutrition Research Center, Grand Forks, ND
58202, USA.
The iron storage disorder hemochromatosis is commonly associated with homozygous C282Y mutations,
and less frequently with compound heterozygous (C282Y and H63D) mutations in the HFE gene.
Because of the relatively high frequency of HFE C282Y heterozygotes (~ 10%) among populations with
Northern ancestry, the possibility of greater iron absorption by heterozygotes has implications for public
health policies on food iron fortification. Before the specific mutation had been determined, Lynch et al.
(1989) identified heterozygotes as children or siblings of hemochromatosis patients that shared a single
HLA haplotype. These heterozygotes absorbed similar amounts of nonheme iron from a hamburger test
meal, but more than twice as much nonheme iron from the same meal fortified with additional iron and
ascorbic acid. We investigated whether C282Y heterozygotes absorb more iron, by measuring HFE
C282Y and H63D mutations in DNA from buccal smears of 103 subjects in past radioiron absorption
studies. Heme and nonheme iron absorption from experimental diets had been determined by labeling
food with Fe-55 hemoglobin (from rabbit blood) and Fe-59 tracers. Isotope retention had been determined
by measuring erythrocyte incorporation (both isotopes) and whole body scintillation counting (Fe-59 only)
two weeks later. Duplicate DNA samples were genotyped using PCR with primers described by Feder
(1996), digestion with endonuclease restriction enzymes, and electrophoresis to identify the C282Y and
H63D mutations. H63D heterozygotes (n=24) did not differ from wildtypes (with neither mutation) in iron
absorption, after adjusting for the logarithmic association of iron absorption with serum ferritin. Two
compound heterozygotes absorbed heme iron similarly to wildtypes, and absorbed somewhat more
nonheme iron than wildtypes when tested with a high bioavailability diet rich in meat and ascorbic acid,
but not with a low bioavailability diet abundant in phytic acid and tannins. Five C282Y heterozygotes
absorbed no more heme or nonheme iron from a high bioavailability diet. Consistent with published
studies suggesting low phenotypic expression of the C282Y homozygous genotype, these limited data do
not suggest generally greater heme or nonheme iron absorption from common diets by people
heterozygous for the C282Y HFE mutation. We are in the process of prospectively testing heme and
nonheme iron absorption from unfortified and fortified meals by 11 subjects genotyped as C282Y
heterozygotes and their ferritin-matched wildtype controls.
POSTER 63
HIGH HFE C282Y HOMOZYGOTE FREQUENCY IN CAUCASIANS BUT NOT IN HISPANICS,
AFRICAN AMERICANS, OR ASIANS: AN ANALYSIS OF 50,290 PRIMARY CARE PATIENTS IN THE
HEMOCHROMATOSIS AND IRON OVERLOAD SCREENING (HEIRS) STUDY
G.D. McLaren1,2, J.C. Barton3, V.R. Gordeuk4, C.E. McLaren1, P.C. Adams5, D.M. Reboussin6, R.T.
Acton7, E.L. Harris8, F.W. Dawkins4, B.G. Mellen6, P. Sholinsky9, M. Speechley10, and J.H. Eckfeldt11.
1
University of California, Irvine, CA; 2VA Long Beach Healthcare System, Long Beach, CA; 3Southern Iron
Disorders Center, Birmingham, AL; 4Howard University, Washington, D.C.; 5London Health Sciences
Centre, London, Ontario, Canada; 6Wake Forest University School of Medicine, Winston-Salem, NC;
7
University of Alabama at Birmingham, Birmingham, AL; 8Kaiser Permanente Center for Health Research,
Portland, OR; 9National Heart, Lung and Blood Institute, Bethesda, MD; 10University of Western Ontario,
London, Ontario, Canada; 11University of Minnesota, Minneapolis, MN, 55455.
The HEIRS Study is designed to estimate the prevalence of iron overload and hemochromatosis in a
multi-ethnic primary care-based sample of 100,000 adults age 25 years or older at five Field Centers in
the U.S. and Canada. Participants are screened for HFE alleles (C282Y and H63D) and for transferrin
saturation and serum ferritin levels. They are invited to return for a clinical examination if they are C282Y
homozygotes or have the combination of elevated transferrin saturation (>50% for men, >45% for
women) and serum ferritin (>300 µg/L and >200 µg/L). Here, we present an interim analysis of the first
50,290 participants, of whom 50.6% were Caucasian, 24.3% African-American, 11.2% Asian, 10.6%
Hispanic, and 3.3% other or unknown race. The respective C282Y homozygote and heterozygote
frequencies were 0.69% and 12.38% in Caucasians, 0.02% and 2.33% in African Americans, 0% and
0.12% in Asians, and 0.02% and 3.38% in Hispanics. The majority of Hispanic participants were enrolled
at two Field Centers, UC Irvine (63%) and Howard University (25%), and the C282Y genotype
frequencies among Hispanics at the two Centers were comparable. The frequencies of C282Y
homozygosity among Caucasians at four Field Centers with at least 1,000 Caucasian participants
(together representing over 98% of Caucasians enrolled in the overall study) were: 0.58% in Ontario,
Canada; 0.60% at UC Irvine, 0.63% at Kaiser Permanente Center for Health Research; and 0.98% at the
University of Alabama at Birmingham. On initial testing, elevated transferrin saturation and serum ferritin
levels were detected in 49% (95% CI: 42%, 57%) of C282Y homozygotes, 10.8% (8.4%, 13.7%) of
C282Y/H63D combined heterozygotes, 4.7% (3.2%, 6.6%) of H63D homozygotes, 2.5% (2.0%, 3.1%) of
C282Y heterozygotes, 2.0% (1.7%, 2.3%) of H63D heterozygotes, and 1.6% (1.4%, 1.7%) of wild-type
homozygotes. The mean transferrin saturation and serum ferritin values (± 1 SD) in 185 C282Y
homozygotes were 65 ± 24.9% and 500 ± 585 µg/L. Consistent with previous investigations, these data
indicate that C282Y homozygosity is common in Caucasians but uncommon in the other racial/ethnic
groups in the HEIRS Study. Further, the C282Y homozygote frequency of 0.02% in Hispanics at HEIRS
Field Centers is lower than the frequency of 0.41% detected in Hispanics in the San Diego area (Ann
Intern Med. 2000;133:329-337). Geographic area and race/ethnicity represent important sources of
variability in C282Y homozygote frequency that should be considered in designing population testing
strategies for detecting iron overload and hemochromatosis.
POSTER 64
HEPCIDIN-ASSOCIATED HEMOCHROMATOSIS IS A RARE SUBSET OF JUVENILE
HEMOCHROMATOSIS
Roetto A*, Papanikolaou G’, Politou M’, Daraio F*, Girelli D”, Porporato P*, Christakis JI°, Loukopoulos D’,
Camaschella C*. *Department of Clinical and Biological Science, University of Torino, Italy; ‘First Department of
Medicine, University of Athens, School of Medicine, Greece; “Department of Clinical and Experimental Medicine,
University of Verona, Verona, Italy; °Department of Haematology, Theagenio Cancer Center, Thessalonike, Greece
Juvenile hemochromatosis (JH) is a rare form of primary iron overload characterized by early age of onset
and a severe clinical course. JH is a distinct disease compared to HFE-related hemochromatosis from
both the phenotypic and the genetic point of view. The JH locus was identified on chromosome 1q
(Roetto et al, Am J Hum Genet 64: 1389-1393, 1999). Recently, a Greek family with clinical symptoms of
JH unlinked to 1q was identified (Papanikolaou et al, BCMD 68: 168-173, 2002), demonstrating the
genetic heterogeneity of JH. Among possible candidates for this disorders Hepcidin (HEPC, LEAP1) has
been demonstrated to play a major role in iron overload (Nicolas et al, PNAS 98; 8780-8785,2001).
We have sequenced HEPC coding regions, exon/intron boundaries and promoter region and identified
two causal mutations in two unrelated families (the previously characterized 1q-unlinked Greek family and
one Italian family). Linkage analysis revealed linkage to chromosome 19q13, where HEPC gene is
mapped in the Greek family, while the Italian family was uninformative. The three patients with mutated
HEPC show a phenotype identical to the JH (fully saturated transferrin, strikingly increased serum ferritin,
hypogonadism and liver fibrosis in all cases, cardiac disease, diabetes and cirrhosis in one case). In the
Italian family we found that a C->T mutation at position 166 changes the aminoacid arginine encoded at
position 56 to a stop codon (R56X). In the Greek family, a deletion of a G at position 93 causes a
frameshift and changes all the encoded aminoacids after position 32, giving origin to an abnormally
elongated peptide. Both mutants were found at the homozygous state, as expected on the basis of the
consanguinity of the respective families. The same mutations were not found in 50 normal controls, using
SSCP or restriction enzyme analysis.
Individuals heterozygous for the two mutations were symptomless and with normal iron parameters.
Reevaluation of our JH series indicated that 14 unrelated patients (either isolated cases or patients from
uninformative families) had normal hepcidin sequence. Other 12 multiple or consanguineous JH families
were clearly 1q associated.
Our conclusion is that JH is an heterogeneous disease and that hepcidin-associated hemochromatosis is
a rare subset of the juvenile disease. Our data should facilitate the identification of the 1q gene
suggesting that it is hepcidin-related or a component of its signaling pathway.
POSTER 65
A FOLLOW-UP STUDY OF SERUM FERRITIN IN 48 UNTREATED C282Y HOMOZYGOTES
Y Deugnier, AM Jouanolle, F Lainé, C Pithois, M Pouchard, B Lafraise, V David. Service des Maladies du
Foie, Centre de Dépistage de l’Hémochromatose, Centre d’Investigation Clinique, Laboratoire de
Génétique Moléculaire and UMR CNRS 6061, CHU Pontchaillou, 35003 Rennes, France & Centres
d’Examens de Santé de Saint-Brieuc, Rennes et Saint Nazaire, France.
The natural history of C282Y homozygosity remains poorly documented. The present study was aimed at
describing the spontaneous course of serum ferritin (SF) in 48 untreated C282Y homozygotes. Patients
and methods. These 48 subjects (8 males and 40 females) were selected from a survey among the 2000
persons affiliated to the French association of patients with hemochromatosis (7 males and 20 females)
and from a screening program of C282Y homozygosity conducted in 20.000 adults from Brittany, France
(1 male and 20 females). All presented with the following criteria: (i) prooven C282Y homozygosity, (ii)
availability of a biochemical follow-up for at least one year and (iii) absence of venesection therapy prior
to and during follow-up. The mean SF variation per year (∆SF/y) was calculated as the ratio of the
difference in serum ferritin levels (ng/ml) between the end and the beginning of follow-up to the duration
of follow-up (years) Results. Males were aged from 27 to 69 years (mean ± sd : 54.5 ± 13.7) and followed
for 5.4 ± 3.1 years. Females were aged from 33 to 73 years (47 ± 10.4) and followed for 3.8 ± 3.4 years.
The reasons why venesection therapy was not performed were (i) low or normal body iron stores at the
time of diagnosis in 24 subjects (SF < 300 in men and < 200 in women), (ii) delayed diagnosis until the
discovery of the HFE gene in 17 patients, and (iii) rejection of phlebotomies by 9 subjects. Among the 8
C282Y homozygous males, 3, aged 67, 68 and 27 years, had a negative ∆SF/y (- 146, - 109 and – 83
ng/ml/y), of whom the two oldest had a cause of chronic blood loss, and 5 had a positive ∆SF/y ranging
from + 18 to + 241 ng/ml/y. Among the 40 C282Y homozygous females, 11 had a negative ∆SF/y (- 17 ±
38 ng/ml/y) and 29 a positive ∆SF/y (+ 69 ± 111 ng/ml/y). These two groups differed significantly with
respect to age at the begining (42 ± 6 vs 49 ± 11- p = 0.05) and at the end (45 ± 7 vs 53 ± 12 – p = 0.04)
of follow-up and to the frequency of regular blood donation (20% vs 60% - p = 0.04). ∆SF/y was
significantly (p = 0.02) lower in the 28 pre-menopausal (23 ± 55 ng/ml/y) than in the 11 post-menopausal
women (107 ± 165 ng/ml/y). Among the 23 females with low or normal body iron stores at the beginning
of follow-up, 4 presented SF levels exceeding 200 ng/ml at the end of follow-up, of whom 3 became postmenopausal during follow-up. Conclusion. In the absence of blood loss and of blood donation,
progressive increase of serum ferritin levels along time is the rule in C282Y homozygotes, especially in
men and in post-menopausal women.
POSTER 66
NON-INVASIVE LIVER IRON QUANTIFICATION BY SQUID- BIOSUSCEPTO- METRY IN THE
DIAGNOSIS, STAGING, OR FOLLOW-UP IN PATIENTS WITH HEREDITARY HEMOCHROMATOSIS
OR THALASSAEMIA
P. Nielsen, R. Fischer, R. Engelhardt, P. Buggisch1, G. Janka2
Zentrum für Experimentelle Medizin/Zentrum für Frauen- und Kindermedizin,
1
Zentrum für Innere Medizin, 2Zentrum für Frauen- und Kindermedizin
Universitätsklinikum Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, GERMANY
In hereditary hemochromatosis in the post-HFE-gene era, precise diagnostic parameters remain
important to characterize individual iron stores, because indication for therapy and prognosis mainly are
related to the extent of iron loading. In secondary siderosis, esp. thalassaemia major, early and well
adjusted treatment with iron chelators is of crucial importance for the long-term survival, especially in
children. Basis of the adequate treatment are also appropriate diagnostic parameters which are capable
to monitor the range of the individual iron burden. Both in primary and secondary siderosis, the frequently
used serum ferritin often interferes with non-iron related factors such as inflammation and may therefore
produce falsely positive results. As the invasive liver biopsy is not recommended any longer by many
doctors treating patients with hemochromatosis and because repeated biopsies are not suitable in
thalassaemic children, some confusion exists at the moment regarding the criteria for diagnosis or follow
up in iron overload diseases.
In large series of patients suspected for hereditary hemochromatosis (n=659), or with known secondary
siderosis (n=1112), the liver iron concentrations was measured non-invasively using the Hamburg-SQUID
biomagnetometer (BTi-Ferritometer, San Diego, USA; SQUID = super-conducting-quantum-interferencedevice) (1). This truly non-invasive method is sensitive, reliable, fast (online results), and also costeffective when compared to invasive liver biopsy.
In hemochromatosis, SQUID-biosusceptometry is very helpful to i) identify patients with severe iron
overload who are at higher risk in the future to develop (more) severe clinical symptoms and ii) to
characterize patients with rather low liver iron concentration who are in a low risk group and need less
intensive follow-up diagnostic (2). In our opinion, such a technique represents the optimal combination
with the HFE-mutation analysis.
In patients with secondary siderosis, SQUID-biosusceptometry for non-invasive liver iron quantification
was a powerful tool to study different questions (3).: i) definition of a new therapeutic deferrioxamin(DFO)index, in order to prevent overdosing with this chelator, ii) comparing the efficacy of deferiprone in
thalassaemia patients, iii) indication for a therapy by iron chelation or phlebotomy in iron loaded patients
with Blackfan Diamond anemia or in bone-marrow transplanted patients
1.)
2.)
3.)
Fischer, R. (1998) Liver iron susceptometry. In Magnetism in Medicine pp. 286-301. Wiley-VCH
Berlin, New York.
P. Nielsen et al. Blood Cells, Molecules, and Diseases 29(3): 451-458 (2002).
Fischer R et. al. Am J Hematol 60:289-299 (1999).
POSTER 67
EFFECTIVE TREATMENT OF HEREDITARY HEMOCHROMATOSIS WITH DESFERRIOXAMINE IN
SELECTED CASES
P. Nielsen, R. Fischer, R. Engelhardt, P. Buggisch1, G. Janka2
Zentrum für Experimentelle Medizin/Zentrum für Frauen- und Kindermedizin,
1
Zentrum für Innere Medizin, 2Zentrum für Frauen- und Kindermedizin
Universitätsklinikum Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, GERMANY
Hereditary hemochromatosis is a genetic disease, in which food iron absorption is inadequately increased
and not balanced to the body iron stores. Exhaustive phlebotomy is the routine treatment procedure and
has clearly proven its effectiveness and safety. Some patients, however, cannot be treated with weekly
phlebotomy due to a prohibitive clinical situation (e.g. critical liver or heart function), or due to the lack of
appropriate access for the venipuncture. In these cases, subcutaneous desferrioxamine can be an
alternative. The use of DFO in hemochromatosis has been reported in a very early study (Wöhler 1963) in
which it was suggested as a new treatment procedure. Later on, this topic was mentioned only
anecdotally in some reviews (Walker et al.; 1998; Barton et al. 1998). According to Powell, DFO is a poor
substitute for phlebotomy and it is difficult to achieve a negative iron balance with chelation in
hemochromatosis (Powell 1994). The effectiveness of DFO in hemochromatosis in comparison to
phlebotomy has never been studied in the literature so far.
We studied three patients with C282Y-positive hereditary haemochromatosis in which phlebotomy
treatment was not possible due to the serious clinical situation or to the lack of appropriate peripheral
veins. In two patients with severe liver cirrhosis, the treatment with subcutaneous desferrioxamine (DFO)
for 9-11 months improved the clinical situation substantially making regular phlebotomy treatment
possible. As judged by repeated non-invasive measurements of the liver iron concentration by SQUIDbiosusceptometry, the DFO treatment (2 g/d) was similarly effective (liver iron elimination rate: 12 mg/d)
as the normal phlebotomy treatment (5.9 or 14.3 mg/d, respectively) with weekly 500ml-blood removals
(see Figure).
phlebotomy
--- DFO ---
6000
case 1
DFO+
phleb
4000
5000
4000
3000
12.4 mg/d
serum iron
18.6 mg/d
2000
liver iron
1000
2000
14.3 mg/d
serum ferritin
1000
0
0
100
200
300
400
3000
500
600
700
liver iron [mg/g liver w.wt]
serum Ferritin-& Fe [µg/l]
5000
0
800
time [days]
Important is the fact that a clear negative iron balance was achieved using DFO in a low dosage in all
three patients with hereditary hemochromatosis. This demonstrates that DFO is an effective alternative
therapy for hemochromatosis when phlebotomy is not possible.
In the normal patient, however, phlebotomy is simpler, safer, and much more cost-effective compared to
DFO.
POSTER 68
NON-INVASIVE LIVER IRON MEASUREMENT IN PATIENTS WITH 
-THALASSEMIA/HEMOGLOBIN
E DISEASE USING MRI: MONITORING THE EFFECT OF ORAL IRON CHELATION WITH
DEFERIPRONE (L1)
Wanida Chua-anusorn (1), Adam Fleming (1), Paul Clark (1) Pichest Metarrugcheep (2), Pornpan
Sirunkapracha (3) Pensri Pootrakul (3) and Timothy St. Pierre (1)
(1) Biophysics Programme, School of Physics, The University of Western Australia, Crawley, Western
AUSTRALIA, (2) Radiology, Neurological Institute, Bangkok and (3) Thalassemia Research Centre,
Institute of Sciences and Technology for Research & Development, Salaya Campus, Nakornpathom,
Mahidol University, THAILAND
Non-invasive measurement of liver iron concentrations can be achieved through measurement of proton
transverse relaxation rates (R2) using magnetic resonance imaging [1-3]. In this study, the potential for
using the R2-MRI measurement technique to monitor changes in liver iron concentration during chelation
therapy was examined.
R2-MRI and needle biopsy were used to measure liver iron concentrations in 10 βthalassemia/hemoglobin E patients who had not previously undergone chelation therapy. The volunteers
were measured a second time after having been treated with the oral iron chelator deferiprone for a
period of between 12 months and 17 months. MRI liver iron measurements were obtained by measuring
the mean R2 for the largest cross sectional axial slice of the liver using the method outlined in [4].
The mean and standard deviation of liver iron concentrations for the 10 patients measured by R2-MRI
and needle biopsy iron assay were 16.1 ± 4.4 and 19.7 ± 7.0 mg Fe/g dry tissue respectively before
commencement of iron chelation therapy. The corresponding measurements at follow-up were 6.7 ± 6.7
and 7.1 ± 7.8 mg Fe/g dry tissue respectively. The mean fractional change in liver iron concentration for
the 10 patients measured by R2-MRI and needle biopsy iron assay was –61 ± 38 % and –63 ± 31%
respectively. Nine of the patients exhibited decreases in their liver iron concentrations as measured by
both MRI and biopsy iron assay. The one remaining patient exhibited an increase in liver iron
concentration over the period as measured by both MRI and needle biopsy iron assay. The standard
deviation of R2 within the liver image (σR2) is related to the standard deviation of local iron concentrations
in the tissue [5]. The mean change in σR2 for the patients was –56 ± 23% suggesting that the iron
remaining within the liver was more uniformly distributed after the administration of chelation therapy.
These results suggest that R2-MRI has the potential to be used as a clinical monitoring tool in chelation
therapy trials.
[1] Wang ZJ, Haselgrove JC, Martin MB et al J. Magn. Reson. Imaging 15: 395-400 (2002).
[2] Engelhardt, R., J. H. Langkowski, R. Fischer, P. Nielsen, et al. Magn. Reson. Imaging 12 (1994) 9991007.
[3] Clark P, Chua-anusorn, W., St. Pierre, T. et al (2003) BioIron abstract this volume.
[4] Clark PR & St. Pierre TG. Magnetic Resonance imaging, 18 (2000): 431-438.
[5] Clark PR, Chua-anusorn W, St. Pierre TG. Magn. Reson. Med. (2003) in press.
POSTER 69
LIVER IRON MEASUREMENT AND MAPPING USING MAGNETIC RESONANCE IMAGING
Timothy St. Pierre1, Paul Clark1, Wanida Chua-anusorn1, Gary Jeffrey2, John Olynyk2, Erin Robins3, Rob
Lindeman4, Pensri Pootrakul5
(1) School of Physics and (2) School of Medicine, The University of Western Australia, Crawley, WA,
AUSTRALIA (3) SKG Radiology, St John of God Hospital, Subiaco, WA, AUSTRALIA, (4) Department of
Haematology, Prince of Wales Hospital, Sydney, NSW, AUSTRALIA, (5)Thalassemia Research Centre,
Institute of Sciences and Technology for Research & Development, Salaya Campus, Nakornpathom,
Mahidol University, THAILAND
Magnetic resonance imaging can be used for the non-invasive measurement of liver iron concentration
(LIC) in tissue with iron overload via quantitative image measurement of the hydrogen proton transverse
relaxation rate (R2) [1-3]. The potential range of LIC measurement has been further extended by the
development of a bi-exponential R2 imaging technique [4].
The aim of this study was to (i) determine the accuracy and reproducibility of R2 measurements on a set
of phantoms on different MR scanners, (ii) determine the precision of R2 measurements on the liver made
on different scanners, and (iii) determine whether a universal calibration curve could be developed for the
quantification of liver iron concentration.
The accuracy and reproducibility of R2 imaging on aqueous MnCl2 phantoms with a range of R2 values
covering those encountered in human liver has been demonstrated on five 1.5 T whole body imaging
units. The mean relaxivity value for the five scanners was 74.1 s-1 (mM)-1, with a standard deviation of 0.3
s-1 (mM)-1. The coefficient of variation between the five scanners was less than 0.5%.
For the precision measurement study, 10 volunteers (3 healthy, 2 with hemochromatosis, 5 with 
thalassemia) were measured twice each on 2 different scanners a day apart. The precision of interscanner R2 image measurement of the liver was 7.7%, with a non-significant systematic difference
between scanners of 1.2%.
For liver iron calibration curve development, 105 volunteers (32 with hepatitis, 23 with hemochromatosis,
and 50 with 
-thalassemia) who were undergoing liver biopsy during the course of their treatment, were
recruited. The mean R2 in the right-hand side of the liver for slices of maximal cross-section was found to
correlate significantly with needle liver biopsy iron concentration (Spearman rank order correlation
coefficient of 0.97). A universal calibration curve was applicable to all patient groups, and covered the
entire range of liver iron concentrations presented, from 0.3 to 43 mg of iron per gram dry tissue. The
accuracy of the calibration curve over different ranges of LIC is estimated by calculating the standard
deviation of the LIC differences between the calibration and the ranked order data yielding the following
uncertainties and (ranges): ±0.1 (0.3 - 1.8); ±0.3 (1.8 - 5.0); ±0.9 (5 - 20); ±1.1 (20 - 43) mg Fe/ g dry
tissue.
The results of this study demonstrate that hepatic R2 image measurement can be used in a machine
independent manner for the non-invasive measurement of liver iron concentration, from normal liver iron
levels to highly loaded liver iron levels (approx 40 mg Fe/ g dry tissue).
[1] Engelhardt, R., J. H. Langkowski, R. Fischer, P. Nielsen, et al. Magn. Reson. Imaging 12 (1994) 9991007.
[2] Clark PR & St. Pierre TG. Magn. Reson. Imaging, 18 (2000) 431-438.
[3] Clark PR, Chua-anusorn W & St. Pierre TG. Magn. Reson. Med., (2003) in press.
[4] Clark PR, Chua-anusorn & St. Pierre TG. Magn. Reson. Imaging, (2003) in press.
POSTER 70
A STRATEGY TO INCREASE THE DETECTION OF UNDIAGNOSED HFE1 HAEMOCHROMATOSIS
J. Rochette, E.Cadet, A.S.Crépin, S.Arlot, M. Dautréaux, P. Fardellone,
J-P Ducroix, P. Leflon , UPJV, Faculté de Médecine & CHU-Amiens, France
J-M Sueur, Biobanques de Picardie, Amiens, France.R. Feyt,CPAM, Amiens , France
J.J. Rose, P. Harcourt, Institute of Health Sciences, University of Oxford, UK
J., Emery , Department of Public health and Primary care, University of Cambridge, UK. A.T
Merryweather-Clarke,.J., Pointon, KJH Robson,MRC Moleculat Haematology Unit, WIMM, Oxford, UK. D.
Capron,Hépato-Gastroenterologie ,CHU-Amiens, France.
Introduction : Early symptoms of haemochromatosis are often non-specific, leading to a delay in
diagnosis. Over 90% of patients of northern European origin are homozygous for the C282Y mutation in
the HFE gene. It is unclear why the number of C282Y homozygotes in the population does not correlate
with the number of patients with haemochromatosis.The degree of penetrance (1-50%) varies from study
to study and a clear definition is needed.Therefore widespread genetic screening for haemochromatosis
is currently neither practical nor cost-effective.It is important therefore to improve early detection of a
potentially fatal disease that is easily treatable;often haemochromatosis is a chance diagnosis. This study
was designed to see whether a more focused approach to identifying patients with haemochromatosis is
possible.Methods : We have screened healthy controls from Picardy in France, who attended a clinic for a
free health check up (n = 991) and subsets of patients from both primary care (UK) and specialist hospital
clinics (France) presenting with any combination of the following haemochromatosis associated
conditions: diabetes, arthropathy, unexplained fatigue, osteoporosis, cardiac arrythmia, and arthralgia.
We made sure hemochromatosis was not previously diagnosed in patient groups.The study included
clinical observation, questionnaire, serum ferritin and transferrin saturation measurements,
transaminases, serum glucose and HFE H63D and C282Y genotyping. All comparisons were two-tailed.
TM
Statistical analysis was conducted with Statview (Abacus Concepts, Inc, Berkeley, California, USA).
Results : There were 4022 consultations in the primary care group, during which 169 patients were
identified with an index symptom .The allele frequencies in the primary care group of patients were not
significantly different from the asymptomatic. The Y/Y genotype was absent in the 169 patients. In the
secondary care group (n=611), only patient groups presenting with unstable diabetes or disabling fatigue
of a least 6 months often accompagnied with arthralgia and presenting with a raised serum ferritin
concentration showed an enrichment in the haemochromatosis-associated genotype HH/YY, odds ratio
(OR) = 40.1, confidence interval (CI) = 8.0-202.1 and OR = 103, CI = 22.9-469.7, respectively. Over 10%
of patients that fulfil these criteria are either C282Y homozygotes or H63D/C282Y compound
heterozygotes.Ten of the 13 C282Y homozygotes identified via hospital clinics had a serum ferritin
concentration higher than 1000 µg/L, putting them at high risk for cirrhosis, while the serum ferritin
concentration was normal in the two HHYY subjects detected by genotypic screening in the control group.
Discussion : It should be noted that the group of diabetic patients were those who required admission to
hospital for diabetic related complications or stabilisation. It is possible that in patients with more severe
forms of diabetes the HFE genotypes HH/YY, HD/CY and HH/CY are contributing to their disease.Until
now no study has reported the prevalence of haemochromatosis in patients presenting with chronic
fatigue syndrome. A simple strategy of selecting patients who present in hospital clinics with one or more
conditions associated with haemochromatosis, and genotyping only those for the two common HFE
mutations, who have a transferrin saturation of >40% or ferritin >300 µg/L efficiently identifies those
patients with undiagnosed haemochromatosis.Conclusion :This targeted approach increases probability
of diagnosing the full penetrant YY genotypes and hence reduced cost of screening .
POSTER 71
HFE, DCYTB, IREG1 AND HAMP MUTATIONS IN PRIMARY IRON OVERLOAD PATIENTS IN THE
SOUTH AFRICAN POPULATION
Zaahl MG 1,2, Merryweather-Clarke AT3, Pointon JJ3, van der Merwe S4, Warnich L2, Kotze MJ5, Robson
KJH3
1
Division of Human Genetics, Faculty of Health Sciences, University of Stellenbosch, Tygerberg,
2
Department of Genetics, University of Stellenbosch, Stellenbosch, SOUTH AFRICA, 3MRC Molecular
Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UNITED
KINGDOM, 4Department of Internal Medicine, University of Pretoria, Pretoria and 5Genecare Molecular
Genetics (Pty) Ltd, Christiaan Barnard Memorial Hospital, Cape Town, SOUTH AFRICA
Background: Our increasing understanding of the molecular basis of genetic disorders of iron metabolism
in mammalian species is providing new insight into genes involved in mediating intestinal iron absorption.
It has become clear that the control of iron metabolism is interconnected with other cellular pathways and
further analysis of these aspects are likely to provide a better understanding of pathological processes in
which iron may participate.
Aim: To identify the possible involvement of genes involved in primary iron overload including DCYTB,
HAMP, IREG1 and HFE in patients referred for a diagnosis of hemochromatosis who tested negative for
the most frequent HFE mutation C282Y.
Materials and methods: The study population consists of 72 patients with primary iron overload and 50
population-matched control individuals. Polymerase chain reaction (PCR) amplification of genomic DNA
was performed on the coding and promoter regions of DCYTB, HAMP, IREG1 and HFE using intronic
primers. The PCR products were subjected to denaturing high performance liquid chromatography
(dHPLC) analysis and those with variation in wave patterns were subjected to sequencing analysis.
2
Statistically significant differences were determined by the Fisher exact test and/or chi-squared (χ )
analysis with Yates’ correction. A P value smaller than 0.05 was regarded as statistically significant.
Results and Discussion: Mutation analysis of the various genes revealed several variations with 6 novel
mutations in IREG1, 2 in HAMP, 2 in DCYTB and 2 in HFE as well as novel polymorphisms in all the
genes investigated. Several previously described polymorphisms were also detected, including 7 HFE
and 4 IREG1 polymorphisms. Statistically significant differences were observed when comparing the
patient and population-matched control groups for a polymorphism identified in the promoter region of
IREG1 (p<0.003, χ2 = 8.51, 1df).
Conclusion: The identification of several mutations in the investigated genes in 17% of the patient group
indicates the involvment of these genes in iron homeostasis.
This project was approved by the Ethics Review Committee of the University of Stellenbosch.
POSTER 72
POTENTIAL USE OF HEPATIC R2 MAGNETIC RESONANCE IMAGE TEXTURE CHARACTERISTICS
IN THE STAGING OF FIBROSIS IN HEMOCHROMATOSIS
Beau Pontré (1), Bastiaan DeBoer (3), Gary Jeffrey (2), John Olynyk (2),
Wanida Chua-anusorn (1), Paul Clark (1), Timothy St. Pierre (1)
(1) School of Physics, (2) School of Medicine, The University of Western Australia, Crawley, Western
Australia.
(3) Anatomical Pathology, PathCentre, QEII Medical Centre, Nedlands, Western Australia
In iron overload diseases such as hemochromatosis, the excess iron acts to catalyse cell damaging
reactions, resulting in the formation of fibrosis [1]. Severe fibrosis results in a change in the spatial
distribution of tissue iron [2]. Spatial distributions of tissue iron can be measured non-invasively using
magnetic resonance imaging (MRI), as the transverse relaxation rate (R2) measured by MRI is
significantly correlated with hepatic iron concentration (HIC) [2]. R2 maps generated from the MRI
measurements provide a means to visualise the spatial distribution of iron in the liver.
In this study, texture analysis and image classification techniques used in other applications have been
adapted for use to characterise the texture of hepatic R2 images. Texture measures were generated from
gray-tone spatial-dependence (GTSD) matrices [3]. The GTSD matrices, obtained by comparing the
intensities of pixels separated by some distance (d), are assumed to retain all the texture information
contained in the R2 maps. Each measure was calculated for a number of pixel separations for a group of
hemochromatotic subjects (n = 20) and then compared with fibrosis staging. The patients were separated
into two distinct groups based on the Knodell scoring method for fibrosis. A Knodell score of 0 or 1 was
designated as group I (n = 10), and Knodell scores of 2 and above were designated as group II (n = 10).
The Kolmogorov-Smirnov test was used to test the significance of differences in the distributions of the
texture parameters between the two patient groups.
The distribution of the Correlation Means (CM) for Group I was significantly different from that for Group II
at a pixel separation of d = 17mm (10 pixels) (Table 1). Additionally, at the smaller separation of d =
3.4mm (2 pixels), the distribution of the Sum Entropy Ranges (SER) for Group I significantly differed from
that for Group II (Table 1). Neither the CM nor the SER correlate significantly with HIC.
CM
SER
GROUP I
GROUP II
GROUP I
Mean
4.7
2.6
3.0
SD
2.3
1.3
0.6
K-S max diff.
0.80
0.70
K-S p-value
0.003
0.015
n
10
10
10
Table 1 – Statistics for texture measures and fibrosis staging
GROUP II
2.1
0.5
10
The results from this preliminary study suggest that measures of texture from the hepatic R2 image could
potentially be used to stage fibrosis in liver tissue. The relatively low correlations of the texture measures
with HIC indicate that the relationship between the texture measures and fibrosis is dependent on the
spatial distribution of hepatic iron, rather than the mean HIC. Future work will test the relationship of
fibrosis scores with alternate measures of texture from various texture characterisation techniques.
[1]
[2]
[3]
Arthur MJP. Journal of Gastroenterology and Hepatology, 11, (1996): 1124-9.
Clark PR et al. Magnetic Resonance in Medicine, (in press) (2003)
Haralick RM et al. IEEE Trans. Syst. Man and Cybern., SMC-3 (6), (1973): 610-621.
POSTER 73
CELLULAR FORMATION AND MOBILIZATION OF TACHPYRIDINE AND ITS METAL COMPLEX
METABOLITES STUDIED BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY
R. Zhao, R.P. Planalp, M.W. Brechbiel, F.M. Torti, S.V. Torti, Wake Forest University School of Medicine,
Winston-Salem, NC; University of New Hampshire, Durham, NH; Radiation Oncology Branch, NIH,
Bethesda, MD.
Tachpyridine (N,N′,N′′-tris(2-pyridylmethyl)-cis,cis-1,3,5-triaminocyclohexane; Figure), a potent
hexadentate metal chelator, binds divalent metal ions and exerts a cytotoxic effect on cultured cancer
cells. Tachpyridine is currently being evaluated as a potential anti-cancer agent through the RAID and
RAND programs of the NCI. In the present study, a reliable and sensitive ion-pair reversed-phase highperformance liquid chromatography (RP-HPLC) method utilizing gradient elution and UV detection was
developed and validated. Mobile phase A consisted of 25mM citric acid, 10mM SDS, acetonitrile:water
(50:50, v:v); mobile phase B consisted of 25mM citric acid, 3mM sodium dodecylsulfonate (SDS), 3mM
triethylamine, acetonitrile:water (50:50, v:v), in which SDS was used as an anionic ion-pairing reagent.
The elution was initially held at 100% A over 20min, then changed to 100% B over 25min, and returned to
100% A over 8min. Sample preparation was carried out by solid phase extraction using a Waters
Oasis™ HLB cartridge. HPLC separation was performed on a SymmetryShield™ RP18 column with
quantification of analytes using (N-methyl)3-tachpyridine as internal standard. Tachpyridine and its metal
complexes ([Zn(tachpyr)]2+, [Cu(tachpyr)]2+, [Fe(tachpyr)]2+ and its oxidized species [Fe(tachpyr)-ox-n]2+
(n=2, 4, 6) (Figure) were identified in the Hela cell line and tissue culture media. The time course of
formation of these metal complexes in tachpyridine-treated Hela cells and tissue culture media were
determined. In addition, pulse-chase experiments were designed to investigate cellular mobilization of
tachpyridine and metal complexes. The successful application of this HPLC method will enable a clearer
understanding of the cellular metabolism of tachpyr and the relation of chelation chemistry to its cytotoxic
N
H2
C
NR
H2
C
N
2+
NR
N N
M
NR
N
C
H2
N
HN NH HN
R= H, Tachpyr
R= Me, (N-methyl)3-tachpyr
M=Fe, Zn, Cu
2+
N N N
Fe
N NH HN
[Fe(tachpyr-ox-2)]2+
2+
2+
N N N
Fe
N NH N
N N N
Fe
N
N N
activity.
Figure. Molecular structure of tachpyridine, (N-methyl)-tachpyridine, metal complexes (M=Fe, Zn, Cu)
and the oxidized Fe complexes [Fe(tachpyr-ox-n)]2+ (n = 2, 4, 6).
POSTER 74
FERRITIN HEAVY CHAIN LOCALIZES TO THE NUCLEUS AND REPRESSES THE ADULT HUMAN βGLOBIN GENE IN CULTURED CELLS
R. H. Broyles, V. Belegu, B. T., Kurien, C. A. Stewart, Q. N. Pye, W-X. Guo, and R. A. Floyd, University of
Oklahoma Health Sciences Center and Oklahoma Medical Research Foundation, Oklahoma City, OK
73104, U.S.A.
Understanding mechanisms of gene repression is crucial to the goal of targeting specific repressors to
specific genes. A repressor thus delivered could be useful in gene regulation therapy for sickle cell disease,
cancers and other major diseases. Stage-specific combinations of DNA-binding proteins that interact via DNA
looping are thought to mediate gene repression, in developmental and tissue-specific regulation as well as in
carcinogenesis. We hypothesize that such interactions mediate repression of the human adult β-globin gene
in embryonic erythroid cells. Nuclear Ferritin-H (ferritin heavy chain, FH) has been found to repress EKLFactivated human β-globin-reporter constructs in a transfection assay in CV-1 cells (Broyles et al., PNAS 98:
9145-9150, 31 July 2001). Mutation of the –150 CAGTGC motif inhibits in vitro FH binding 20-fold and
abolishes repression in the co-transfection assay, linking binding with function. The dissociation constant
-10
(KD) for K562 cell nuclear FH is approximately 10 M, suggesting very tight binding and very effective
repression. To further study the repression mechanism, we developed an in vitro DNA looping assay to show
interaction between the FH (repressor) binding site at –153/-148 of the β-globin promoter, and the
silencer-binding protein BP1 sites that map to –302/-294 and –553/-527. Partially pure K562 nuclear
extract, enriched in FH and BP1, was reacted with a –610/+20 β-globin promoter, yielding a single EMSA
band. Cutting the DNA-protein complex with Sau 96A at –210/-209 of the β-globin promoter before
electrophoresis, gave a complex that also migrated as a single band. Removing the proteins in this singleband complex yielded DNA that migrated as two bands corresponding to the expected restriction fragments,
confirming that the cut fragments were kept together by interactions of their bound proteins and indicating
looping. EMSA supershift bands with anti-ferritin- or anti-BP1-specific antisera confirmed that the looped
complex contained both FH and BP1. The binding of these proteins to the DNA exhibits cooperativity,
resulting in a very stable complex. Co-transfections in CV-1 cells and chromatin pull-down (ChIP) assays with
K562 cells are being used to confirm these interactions in vivo. Our recent data also show that a GFP-FH
fusion protein made from a transfected recombinant EGFP-C1 plasmid localizes to the nucleus of cultured
CV-1 cells, suggesting FH-gene or FH-protein transfection is potentially applicable to a variety of cell types.
Treatment with FH may also help amerliorate excess free iron that occurs in the iron overload of sickle cell
disease, in many cancers, in vascular disease, and in certain neurodegenerative diseases.
POSTER 75
HISTOPATHOLOGIC FEATURES OF LIVER BIOPSY IN SICKLE CELL PATIENTS WITH IRON
OVERLOAD AND HEPATITIS C
M. Hassan, M.D., S. Hasan, M.D., S. Giday, M.D, M. Elbedawi, M.D, T. Naab, M.D, O. Castro, M.D, A.
Banks, M.D, V. Gordeuk, M.D, D. Smoot, M.D. Howard University Hospital, Washington, D.C.
Objective: To evaluate the effects of HCV infection on hepatic pathology in sickle cell patients with Iron
overload.
Patients and Methods: Between 1992 and 2002, 150 patients with Sickle cell disease were tested for
hepatitis C virus antibody. The prevalence was found to be 35%.This patients were also tested for ferritin.
Twenty of 44 sickle cell patients with ferritin level more than 1000 ng/dl agreed to have liver biopsy at
Howard University Hospital (50% men, 50% women, median age 37 years). All the patients had multiple
transfusions in the past (more than 40 units). The majority of the patients (90%) had Hemoglobin SS and
10% had Hemoglobin SC. None of the patients had iron chelation therapy prior to biopsy. Liver biopsy
results and clinical records were retrospectively reviewed. A blinded pathologist performed the
histopathologic assessments of liver biopsies. Histology including hepatic iron content was graded.
Results: Hemosiderosis was present in all of the biopsy specimens. Fourteen (70%) had 4+ iron
depositions in hepatocytes, one patient had 3+, one patient had 2+ and three patients had only 1+ hepatic
iron deposition. One patient had pigment granules in hepatocytes but no iron stain was performed.
Advanced cirrhosis was noted in three patients, incipient cirrhosis in one patient and bridging fibrosis in
three patients. Patchy sinusoidal fibrosis was observed in eight patients. All the patients with advanced
and incipient cirrhoses had anti HCV antibodies, whereas only two patients with patchy sinusoidal fibrosis
were HCV positive. Only one patient with HCV infection had no evidence of fibrosis. All patients with HCV
infection had 4+ hepatic iron whereas only 61% of patients without HCV infection had 4+-iron deposition,
considering that all received similar number of transfusions.
Conclusion: Our data indicates that patients with sickle cell disease who have hepatitis C virus infection
are at an increased risk of developing severe hepatic iron overload and advanced cirrhosis.
POSTER 76
THE IRON CHELATOR TACHPYRIDINE INDUCES G2/M CELL-CYCLE ARREST AND FUNCTIONS
AS A RADIOSENSITIZER IN p53+/+ AND p53-/- ISOGENIC HUMAN COLORECTAL CARCINOMA
CELL LINES
J. Turner, C. Koumenis, T.E. Kute, R.P. Planalp, M.W. Brechbiel, F.M. Torti, S.V. Torti, Wake Forest Univ.
School of Medicine, Winston-Salem, NC; Univ. of New Hampshire, Durham, NH; Rad. Onc., NIH,
Bethesda, MD.
Iron deprivation has been proposed as a strategy for inhibition of tumor cell growth. Supporting this
approach is the fact that iron is involved in essential biochemical reactions ranging from respiration to
DNA synthesis, and that rapidly proliferating cells such as tumor cells require more iron. We have
described a metal chelator, tachypyridine (N,N',N"-tris(2-pyridylmethyl)-cis,cis-1,3,5-triaminocyclohexane)
that not only inhibits growth of cultured tumor cells, but actively induces apoptosis. Since cell death
induced by tachpyridine is p53-independent, tachpyridine may be useful in targeting tumors with mutant
as well as wild-type p53. In order to study the mechanism of action of tachpyridine, we explored its effect
-/+/+
on the cell cycle. HCT116 p53 and HCT116 p53 human colorectal carcinoma cells differing in p53
status were treated with tachpyridine, stained with propidium iodide, and analyzed by flow cytometry. Cell
cycle distribution of untreated p53+/+ cells was 54% G0/G1, 34% S, and 12% G2/M. The distribution of
p53+/+ cells treated with 10 μM tachpyridine for 24 hours was 30% G0/G1, 30% S, and 40% G2/M.
Untreated HCT116 p53-/- cells were 45% G0/G1, 39% S, and 16% G2/M, whereas the distribution of
tachpyridine treated p53-/- cells resulted in 32% G0/G1, 21% S, and 47% G2/M. Thus, tachpyridine
treatment led to a 2.3 and 1.9-fold increase in the percent of cells in G2/M in p53+/+ and p53-/- cells,
respectively. Treatment with lower doses of tachpyridine and earlier time-points also resulted in significant
G2/M arrest. Since agents that induce G2/M arrest are frequently used as radiosensitizers, we explored
the ability of tachpyridine to function as a radiosensitization agent. HCT116 p53-/- and p53+/+ cells were
pretreated with tachpyridine for 12 hrs and then exposed to ionizing radiation. Using a clonogenic assay,
survival was measured after 9 days. Results indicate that pre-treatment with 7.5 μM tachpyr results in
radiosensitization in combination with 1, 2, and 4 GY. A dose enhancement of 1.3 -1.5 at 10% surviving
fraction was obtained for both cell lines, values within the range of clinically relevant radiosensitizing
agents such as 5-fluorouracil. These results suggest that tachpyridine may be a clinically useful
radiosensitization agent for the treatment of both p53 mutant and p53 wildtype tumors.
POSTER 77
HEPATIC AND SPLENIC IRON OVERLOAD WITHOUT HEMOCHROMATOSIS GENE MUTATIONS IN
DIVERSE ETHNIC GROUPS DEMONSTRATED BY MRI
T K Desai, S Lingam, K Bis, A Shetty. William Beaumont Hospital, Royal Oak, Michigan.
Hepatic iron overload is thought to occur primarily in subjects who are of European origin and
demonstrate hemochromatosis (HFE) gene mutations. We report hepatic iron overload documented by
magnetic resonance imaging (MRI) in patients of Arabic, African, Asian and European origin. All patients
demonstrated high ferritin levels and were negative for the cys282tyr gene mutation for hereditary
hemochromatosis(HFE). Sixteen of the 22 patients demonstrated normal iron saturation. This pattern is
characteristic of hepatic iron overload associated with insulin resistance, but only 6 of the 22 patients
demonstrated insulin resistance.
Methods: MRI exam of the liver was performed using a routine liver protocol with an additional T2* technique
on a 1.5 Tesla(Siemens,Vision) scanner, and the liver and spleen signal were compared to skeletal muscle.
Hepatic and or splenic iron overload was felt to be present if the hepatic and or splenic signal was less than
the skeletal muscle signal. Twenty two patients with hepatic iron overload demonstrated on MRI were
identified, 18 men and 4 women. Five patients were of Arabic origin, 8 patients were of African origin, 2
patients were of Asian origin and 7 patients were of European origin. Mean age was 58±15 years. Mean
hemoglobin was 13.7±2.1 grams/dl. Mean ferritin levels were 1469±2049ng/ml. range(357-8250). Mean
iron saturation was 46%±23% range(18%-100%). Two patients were positive for hepatitis C and 1 patient
was positive for hepatitis B. Transaminase levels were elevated in 14 patients, and 7 of these patients have
been phlebotomized; and ALT and AST levels decreased in all 7 patients. MRI demonstrated splenic iron
overload in 9 patients, implying iron overload of the reticuloendothelial system. Liver biopsy was available in
5 patients and none demonstrated excess iron accumulation within the hepatocytes. Liver biopsy
demonstrated cirrhosis in 3 patients, normal liver in 1 patient, and steatosis in 1 patient. One patient had a
hepatoma.
CONCLUSION: MRI demonstrates iron overload in patients of diverse ethic origin without HFE gene
mutations. The occurrence of splenic as well as hepatic iron overload, and the absence of abnormal
hepatocyte iron staining in 5 patients with a liver biopsy suggests that the iron overload is primarily of the
reticuloendothelial system. MRI exam can be useful in patients with a high ferritin and normal iron saturation.
The clinical significance of this hepatic iron overload remains to be determined.
POSTER 78
STRUCTURE-REACTIVITY RELATIONSHIPS OF IRON COORDINATION AND REDOX PRODUCTS
IN CYTOTOXIC TRIPODAL CHELATORS
Roy P. Planalp,1 Gyungse Park,1 Frances H. Lu,2 Ann Przyborowska,1 Fan Su,1 Grant A. Broker,4 Robin
D. Rogers4, Rong Ma3, Martin W. Brechbiel,2 Frank M. Torti3 and Suzy V. Torti,3 Dept. of Chemistry,1
Univ. of New Hampshire, Durham, NH 03824-3598, Chemistry Section,2 Rad. Oncol. Branch, N.I.H.,
Bethesda, MD; and Wake Forest University School of Medicine, Winston-Salem, NC; The Univ. of
Alabama,4 Tuscaloosa, AL.
Tripodal aminopyridyl compounds (Figure) are effective chelators of the bioavailable metal ions Fe(II),
Fe(III), Cu(II) and Zn(II). One of these, tachpyridine (tachpyr), is currently being evaluated as a potential
anti-cancer agent through the RAID and RAND programs of the NCI. These compounds demonstrate
preferential binding of Fe(II) over Zn(II) and feature an oxidative dehydrogenation process specific to
Fe(II) or Fe(III) chelation. To investigate the role of the donor groups pyridine, imidazole and thiazole, the
complexes [FeIIL]2+(X–)2 (L shown in Figure) were prepared and the spin-state of iron characterized in
aqueous and non-aqueous solution by 1H NMR spectroscopy and Evans’ method. Ni(II) complexes were
also prepared and ligand-field strength toward Ni(II) was assessed with visible spectroscopy. The fieldstrength order was inferred as pyridine > thiazole > imidazole. The role of framework was similarly
studied resulting in an order of tach > tren. To investigate consequences of iron-mediated redox, the
Fe(II) complexes of L-ox-6 (the eventual products of oxidation) were prepared and their aqueous lability
and solid-state structures measured using visible spectroscopy and single-crystal X-ray crystallography,
respectively. [Fe(L-ox-6)]2+ decomposed in aqueous pH 7.4 media (24 h, 37 °C) for all L (Figure) and for
tachpyrMe (structure not shown), but [Fe(trenpyrMe-ox-6)]2+ was entirely inert under these conditions.
Structural data show that [Fe(tachpyrMe-ox-6)]2+ has a pseudo-octahedral coordination geometry (trigonal
twist angle = 48.6(2)°) which is favored by the Fe(II)-low-spin ion, but at a cost of considerable ligand
distortion. Whereas, [Fe(trenpyrMe-ox-6)]2+, likened to the known compound [Fe(trenpyr-ox-6)]2+, should
provide the pseudo-octahedral geometry without ligand distortion; this is consistent with its inertness.
Cytotoxicity of the chelators has been studied by an MTT assay of the response of cultured breast cancer
cells to chelator treatment. The order of increasing toxicity is tachIM < tachthiazole < trenpyr < tachpyr,
as indicated by IC50 values (80 µM ,56 µM, 25µM and 2 µM, respectively). In total, the metal chelation
chemistry of these chelators helps to predict their cytotoxic effects. Further, the aqueous lability of Fe(II)L-ox-6 oxidation products is explained by structural distortions of bound L-ox-6. These findings may be
significant to metal ion complexation and oxidative release of toxic free iron in the cellular metabolism of
tripodal chelators.
R
N(H)(R)
(R)(H)N
N(H)(R)
(R)(H)N
(R)(H)N
-CH2
N
N(H)(R)
tach framework
tren framework
2+
NN
Fe
N NH
N
N
II
2+
oxidation
NN
N
II
NH
[Fe(tachpyr)]2+
Fe
NN
N
-CH2
S
-CH2
N
tachpyr
trenpyr
tachthiazole
N
N
tachIM
POSTER 79
MUTATIONS THAT PREDISPOSE TO BOTH HEMOCHROMATOSIS AND HEREDITARY
HYPERFERRITINEMIA CATARACT SYNDROME IN THE SAME INDIVIDUALS: WHAT FACTORS
CONTROL FERRITIN EXPRESSION?
DG Brooks1 and D Stambolian 2. 1) Division of Medical Genetics, and 2) Departments of Ophthalmology
& Genetics, University of Pennsylvania, Philadelphia, PA 19104.
Hereditary Hyperferritinemia Cataract Syndrome (HHCS) is a heritable cause of hyperferritinemia
that has frequently been misdiagnosed as hemochromaotosis. HHCS is defined by autosomal dominant
inheritance of cataracts and specific mutations of the L-ferritin gene that produce elevated serum ferritin
levels. These mutations are located in, and disrupt function of, the iron responsive element in the 5'
untranslated region of the L-ferritin mRNA. These mutations dis-inhibit L-ferritin translation and L-ferritin
is over produced in most tissues independently of iron stores. Serum levels of ferritin reach 500-2,300
mg/dL in HHCS and are often misinterpreted by clinicians as indicating high iron stores. When HHCS
patients are subjected to phlebotomy therapy, to treat the supposedly elevated iron, they quickly develop
iron deficiency anemia; thus confirming that they do not have elevated iron stores.
It is of interest that we now report that 2 of the 6 unrelated HHCS families referred to our
laboratory have individuals with definitive HHCS and compound heterozygosity for the HFE mutations
C282Y/H63D. The etiology of hyperferritinemia in these individuals poses a diagnostic and therapeutic
dilemma to physicians.
A 36 year old female proband from family 1 was diagnosed with presumptive hemochromatosis
because of persistent hyperferritinemia (1,300 mg/dL) and a C282Y/H63D HFE genotype. However, iron
deficiency anemia developed after only 6 phlebotomy treatments at 1 month intervals. This woman and
her mother had bilateral 'congenital' cataract and the mother had a G32A mutation in the L-ferritin IRE
typical of HHCS.
In the second family, 21 individuals have early-onset cataract including the proband who had a
serum ferritin of 1,500 mg/dL and a G32A mutation of the L-ferritin IRE. However, two brothers with
childhood-onset cataracts were found to have ferritin levels in the range of 4,700 mg/dL. One brother had
a transferrin saturation of 46% with genotypes: C282Y/H63D at the HFE locus and G32A in the L-ferritin
IRE. He was given a presumptive diagnosis of hemochromatosis and prescribed weekly phlebotomy.
After 11 units of blood was removed his hemoglobin was too low to give blood for 3 weeks despite a
ferritin of 4,170. This individual is healthy with no known liver disease, consumes minimal alcohol and
has no other evident cause of hyperferritinemia.
There was no increased iron evident in the proband of family 1 consistent with non-penetrance of
the C282Y/H63D HFE genotype. In this circumstance attributing the increased serum ferritin to HHCS is
logical. In family 2, mild iron accumulation is indicated by tolerance of 11 weekly phlebotomy treatments.
However, the failure of ferritin to decrease below 4,000 at a time of iron-limited erythropoiesis indicates
that this individuals very high ferritin cannot easily be attributed to iron overload. It is unknown why
siblings with HCS have serum ferritin levels that range from 1,500 to greater than 4,000. Ferritin levels
above 4,000 and marked intra-familial variability in ferritin levels are unique in our HHCS case series. It is
likely that factors other than an IRE mutation and iron levels influence ferritin expression in family 2.
Individuals with both HHCS and 2 HFE mutations highlight three clinically significant issues. Ferritin
should not be used in the diagnosis or management of iron overload in individuals with HHCS. Transferrin
saturation should be used as a measure of iron stores and response to phlebotomy therapy in HHCS.
Community physicians appear to be more likely to diagnose hemochromatosis in HHCS patients with
double-mutant HFE genotypes.
POSTER 80
HEPHAESTIN IS A FERROXIDASE
Huijun Chen‡, Zouhair K. Attieh‡†, Trent Su‡, Andrew T. McKie**, Gregory J. Anderson§, and Chris D.
Vulpe‡¶ Department of Nutritional Sciences and Toxicology, University of California, Berkeley, CA 947203104 **Department of Molecular Medicine, King's College, London SE59NU, United Kingdom §Joint
Clinical Sciences Program, The Queensland Institute of Medical Research and the University of
Queensland, Post Office Royal Brisbane Hospital, Brisbane, Queensland 4029, Australia
Sex-linked anemia (sla) mice have a defect in the release of iron from intestinal enterocytes into the
circulation due to a mutation in the heph gene. The gene product, hephaestin (Hp), is predicted to serve
as ferroxidase based on the significant sequence identity to the serum multicopper ferroxidase
ceruloplasmin. We demonstrate oxidase activity using both gel and solution amine oxidase assays in
enterocytes as well as other cell types. We similarly demonstrate ferroxidase activity using a ferrozine
assay. Hephaestin was shown to be responsible for the observed activity using a hephaestin-specific
antibody by immunoblotting, immunoprecipitation and immunodepletion experiments. Surprisingly, the
truncated hephaestin expressed in sla mice still has measurable oxidase activity. This work demonstrates
that Hephaesitn is ferroxidase and suggests that iron oxidation facilitates iron release from enterocytes.
POSTER 81
SYSTEMIC REGULATION OF BASOLATERAL IRON TRANSPORT
Huijun Chen‡, Trent Su‡, Zouhair K. Attieh‡†, Andrew T. McKie**, Gregory J. Anderson§, and Chris D.
Vulpe‡¶ ‡Department of Nutritional Sciences and Toxicology, University of California, Berkeley, CA
94720-3104 **Department of Molecular Medicine, King's College, London SE59NU, United Kingdom §Joint
Clinical Sciences Program, The Queensland Institute of Medical Research and the University of
Queensland, Post Office Royal Brisbane Hospital, Brisbane, Queensland 4029, Australia
Hephaestin is a membrane-bound multi-copper ferroxidase necessary for iron egress from intestinal
enterocytes into the circulation. Mice with sex linked anemia (sla) have a mutant form of hephaestin and a
defect in intestinal basolateral iron transport which results in iron deficiency and anemia. Ireg1
(SLC11A3, also known as ferroportin1 or MTP1) is the putative intestinal basolateral iron transporter. We
compared iron levels and expression of genes involved in iron uptake and storage in sla mice and
C57BL/6J mice fed iron deficient, overload or control diets. Both iron deficient wild-type mice and sla
mice showed increased expression of the basolateral transport molecules Heph and Ireg1 compared to
controls, whereas only iron deficient wild-type mice had increased expression of the brush border
transporter DMT1. Unlike iron deficient mice, sla mouse enterocytes accumulated non-heme iron and
ferritin. These results indicate that Dmt1 can be modulated by the enterocyte iron level, whereas Heph
and Ireg1 expression respond to systemic rather than local signals of iron status. Thus the basolateral
transport step appears to be the primary site at which the small intestine responds to alterations in body
iron requirements.
POSTER 82
CHEMICAL NATURE OF NON-TRANSFERRIN-BOUND IRON
R.W. Evans, A. Zarea, R. Cammack, R.C. Hider, Iron Metabolism Group, King's College London, London,
UK.
In health, plasma iron is tightly bound to transferrin rendering it unavailable to participate in the generation
of harmful free radicals. In iron overload, or in conditions when haematopoiesis is suspended, transferrin
becomes fully iron-saturated and plasma iron is present in other molecular forms, collectively known as
plasma non-transferrin-bound iron (NTBI), at concentrations of between 1-10 μM. Although the exact
nature of plasma NTBI has yet to be defined, it is thought to exist in various forms, namely iron-citrate,
oligomeric iron complexes and albumin-bound iron.
In order to gain further insight into the nature of NTBI, at physiological pH, we have undertaken three
simple studies of iron-citrate complexes prepared at different molar ratios of iron:citrate: ultrafiltration of
iron-citrate complexes in the presence and absence of human albumin, mobilisation of iron from ironcitrate complexes by deferiprone and electron spin resonance (ESR) spectroscopic measurements of
iron-citrate complexes.
Ultrafiltration of a series of iron-citrate solutions, in which the mole ratio of iron:citrate varied from 1:1 to
1:1000, revealed that at ratios of 1:2 and above at least 85% of the iron could permeate membranes with
a molecular weight cut-off of 5K. In the presence of physiological levels of albumin, iron-citrate
complexes remained predominantly protein-bound at molar ratios of iron:citrate up to 1:100. At a molar
ratio of 1:1000 about 40% of the complex remained protein-bound.
The ability of deferipone to sequester citrate-bound iron was observed to be critically dependent on the
molar ratio of iron:citrate. At iron:citrate values of 1:1000 the t1/2 values for mobilisation of iron was < 5
sec but increased to over 600 sec when the ratio was 1:1.
ESR spectra of iron-citrate complexes were indicative of the existence of a mononuclear species at an
iron:citrate ratio of 1:1000. However, at an iron:citrate ratio of 1:100 the spectrum was indicative of the
presence of a different species, most probably the dimer in view of the rapid mobilisation of iron by
deferiprone from the iron-citrate complex at this molar ratio.
The results of our filtration studies demonstrate that with iron:citrate ratios over the range 1:1 to 1:1000
the major species present at pH 7.4 have molecular weights of less than 5K. However, the mobilisation
experiments with deferiprone indicate that the predominant species that exist at ratios between 1:1 and
1:10 are different from those that predominate when citrate is present in greater excess over iron. In view
of the slow rates of mobilisation of iron by deferiprone, the dominant species at the lower ratios is likely to
be a larger oligomeric form (molecular weight 3.5K). At iron:citrate ratios over the range 1:100 to 1:1000,
ESR studies are consistent with the presence of a mixture of the di-iron and mono-iron complexes.
Typical physiological levels of NTBI and citrate, when transferrin is fully iron-saturated, are 5 μM and 100
μM, respectively. Under these conditions the two major species present are predicted to be oligomeric
iron-citrate and dimeric iron-citrate. Although albumin has a high affinity for both these complexes the
resulting ternary complex have yet to be characterised.
We are grateful for financial support from the Medical Research Council.
POSTER 83
DUODENAL IRON CONTENT IS DECREASED DESPITE HEPATIC IRON LOADING IN A MURINE
MODEL OF HEREDITARY HEMOCHROMATOSIS
H.D. Hall1, J.R. Ahmann1, M.C. Migas1, R.S. Britton2, B.R. Bacon2, A. Waheed3, W.S. Sly3, R.E.
Fleming1,3.
1
Pediatrics; 2Internal Medicine and 3Edward A. Doisy Department of Biochemistry & Molecular Biology,
Saint Louis University School of Medicine, St. Louis, MO.
Introduction: HFE-related hereditary hemochromatosis (HH) is an autosomal recessive disorder
characterized by increased intestinal absorption of dietary iron and excess iron deposition in many
tissues. Genes encoding proteins involved in dietary iron absorption are appropriately up-regulated in
patients with iron deficiency, but inappropriately up-regulated in patients with HH. Thus despite systemic
iron loading, the duodenal mucosa in HH functions as if it were iron-deficient.
Objective: We utilized a murine Hfe knockout model of HH to test whether the duodenal iron
concentration or distribution is altered in HH.
Methods: We measured hepatic and duodenal non-heme iron concentrations after an overnight fast in
Hfe knockout and wild-type AKR mice at two ages: 4 weeks (a phase of progressive hepatic iron loading)
and 8 weeks (a plateau phase of stable but elevated hepatic iron). Additionally, duodenal tissue sections
were stained for iron utilizing Perls’ method with diaminobenzidine enhancement, and analyzed by light
microscopy.
Results: At 4 weeks of age, the duodenal iron content (mean +/- SEM) of the Hfe knockout mice was 393
+/- 112 µg/g protein (n=12) and of the wild-type mice was 1644 +/- 256 µg/g protein (n=13), P = 0.002.
Hepatic iron concentrations were 2594 +/- 243 µg/g dry tissue in the Hfe knockout mice compared with
876 +/- 48 µg/g dry tissue in the wild-type mice, P < 0.0001. Modified Perls’ stain of the duodenum
revealed a decrease in the diffuse cytoplasmic iron staining of the villus enterocytes in the Hfe knockout
mice. Duodenal crypt enterocytes had negligible iron staining in both groups. During the plateau phase (8
weeks of age), duodenal iron concentrations in Hfe knockout (1438 +/- 335 µg/g protein, n=10) and wildtype mice (1627 +/- 149 µg/g protein, n=12) were similar.
Conclusions: During the phase of progressive hepatic iron loading, the iron concentration in the duodenal
villus enterocytes of Hfe knockout mice is significantly less than that of wild-type mice. These
observations suggest that the rate of transfer of iron from the absorptive villus enteroctyes into the
circulation in Hfe knockout mice is increased. These data are consistent with the concept that
inappropriate up-regulation of basolateral iron transport contributes to excess dietary iron absorption in
HH.
POSTER 84
ESTIMATION OF ENDOSOMAL pH IN ISOLATED VESICLES AND IN INTACT RETICULOCYTES
FROM BELGRADE RAT STRAINS
J. Abra Watkins. Todd Griffin, Raghu Maramraj, Brian J. Lindenmayer, Erick D. Battle, A. James Williams,
Mary Yeh, Jonathan Glass, and Kwo-Yih Yeh, Department of Medicine, Hematology/Oncology Section,
Feist-Weiller Cancer Center, LSU Health Science Center, 1501 Kings highway, Shreveport, LA 711303932
Acidification of endocytic vesicles is a mechanistically important event involved in the processing
of diferric transferrin and iron absorption via endocytosis by reticulocytes. The Belgrade rat has a
mutation in the DMT1 protein giving rise to a phenotype of iron deficient anemia. Although iron is
transferred from transferrin to the reticulocyte cytosol in this mutant, there appears to be insufficient
acidification to obtain adequate iron dissociation from transferrin as observed in isolated endosomes.
Previous observations on reticulocyte endosomes suggest that minimal acidification is achieved although
significantly coupled ionic fluxes are nearly as robust as in endosomes isolated from rabbit reticulocytes.
Several concerns regarding the relationship between processes occurring in isolated vesicles to events
occurring in intact cells have lead us to attempt to measure the change in pH of endosomes in intact red
cells. The primary technical obstacle is the high concentration of hemoglobin and significant background
emission in red cells which provides for significant fluorescence quenching. Here we compare the
measurements in intact reticulocytes to those observed with isolated endosomes.
White cells were removed from whole blood, and 1 ml of packed reticulocyte/erythrocyte cells
were diluted with 4 ml of PBS to minimize lysis. A solution of FITC or radioactively labeled transferrin (Tf)
and FITC –dextran (10kd or 70kd) was added to 1 ml of the diluted red cells to give final concentrations of
2 or 4 µM Tf (rabbits or rats, respectively) and 5 mg/ml of FITC-Dextran-10 kD or 20mg/ml FITC-Dextran
o
70 kD followed by incubation for 30 min at 37 C with frequent vortexing. The cells were then washed
three times with PBS and incubated in plasma containing 33mg/ml of amidated dextran (10kd or 70kd) on
ice for 30 min with frequent vortexing. The cells were washed three times with PBS and resuspended in
1ml of 0.22 micron filtered PBS before the last wash followed by resuspension in 1ml of filtered PBS.
Flow cytometric measurements were kindly performed on a Becton-Dickson FACS Vantage SE by Ms.
Deborah Dempsey of the LSUHSC Flow Cytometry Facility. Rat reticulocyte endosomal vesicles were
isolated using a modification of the procedure for rabbit reticulocyte endosomes that employs differential
centrifugation and chromatographic methods. Acidificiation (∆pH), membrane potential (∆Ψ), and sodium,
potassium, and chloride fluxes were observed using FITC-Tf as well as permeant and impermeant optical
probes (PBFI, SBFI, SPQ, di-4-Anepps, and Oxonol VI; Molecular Probes), upon the addition of 1mM
ATP.
The vesicles from intact red cells achieve an apparent internal pH of 6.1+ 0.15 for wild type
Wistar rats, 6.5+ 0.3 for conditional wild type (+/?) and heterozygous (+/b), while the homozygous (b/b)
and Sprague-Dawley (SD) strains obtained a pH of 6.75+0.25 respectively. The differences between the
levels of acidification may be due to DMT1 by a variety of mechanisms. A significant caveat to these
results arises because transferrin processing is perturbed under the conditions used to make reasonable
fluorescence measurements. The isolated endosomes achieve an acidic ∆pH of 0.2±0.05, 0.1±0.05, and
alkaline change of 0.1±0.05 for “wild type” (+/?), Sprague-Dawley (SD), heterozygous (+/b), and
homozygous (b/b) strains respectively, while rabbits obtain a ∆pH of 1.0±0.2. The effective changes in ∆Ψ
are 20 to 40mV for the rats and >80mV for rabbits. The similar rates of ionic fluxes between rats and
rabbits compared to the much larger acidification of rabbit endosomes relative to the rat strains, suggest
that a rapid equilibrium for acidification and proton leak is obtained for rats that does not occur in the
rabbit endosomes. The mechanisms leading to this rapid equilibrium could be due to extensive
+
+
deacidification directly or indirectly involving DMT1 or more likely the Na /H exchanger, uncoupling of the
ATPase enzyme per se, or alterations in the activity of ionic transporters due to patterns of
phosphorylation and dephosphorylation.
POSTER 85
DID VIKINGS IMPORT THE HEMOCHROMATOSIS MUTATION TO SCANDINAVIA?
K.S Olsson1, J. Konar2, N Hansson3, E Bygren4, Section of Hematology1, Regional Blood Center2
Sahlgren´s University Hospital, Göteborg , Primary Health Care Centre, Strömsund3, Dep. of Medicine,
Östersund Hospital4 , Sweden.
Hereditary hemochromatosis (HH) is caused by a point mutation of the HFE gene on chromosome 6 in a
person who lived in Northern Europe 60 -80 generations ago. This ancestor carried HLA A3 and is said to
be of Celtic origin. His/her descendants are most prevalent in countries involved in the westward Viking
activity. Recent studies using mtDNA have shown that Vikings took female slaves (wives) from Celtic
Ireland to Iceland. (Helgason et al 2001). Celtic DNA including the C282Y mutation may therefore have
been introduced to Scandinavia through slaves taken by returning Vikings about 30 - 40 generations ago.
Aims of the present study was to compare HLA marker haplotypes on chromosomes from two
Scandinavian HH series: West Coast (WS): n = 340, Central Sweden (CS) n = 395 with those from Celtic
areas, Brittany (BF) n = 609 (Simon M et al 1987). We also wanted to investigate the age of the A1B8C282Y haplotype, considered to be a young marker haplotype resulting from a recent recombination in
the most conserved part of the ancestral haplotype.
Methods: Phenotypic iron tests, HLA haplotypes, HFE genotype tests.
Results: The prevalence of the ancestral haplotype A3 was almost identical between Scandinavia and
Celtic areas: WS: 49 %,CS: 50% and BF: 50% (Values corrected for “extra space” taken by local
founders : WS: A2B15 = 5% , CS: A1B8 = 18%, BF: A2B12= 7% ) . The sparsely populated Central
Sweden had their HLA marker haplotypes spread all over the county but A1B8 was concentrated to the
northern part. Pedigree studies revealed that 15 A1B8 lineages extended over 11 generations ended in
th
the Mid 17 Century in North Trondelag, Norway. Another pedigree comprising 15 families traced for up
to 16 generations ended in the mid 16th century in a small farmer village in a Swedish river valley.
Conclusions: Vikings may well have taken Celtic DNA with the C282Y mutation back to Scandinavia.
There is, however, reason to believe that this mutation already existed here at the time of their return. A
recent recombination, such as the A1B8 haplotype, seems to be older than expected. The present
findings may support the theory that the original mutation may be older than reported and may have
migrated both to Celtic countries and to Scandinavia.
POSTER 86
INTESTINAL IRON ABSORPTION IN THE CACO2 CELL MODEL INVOLVING VESICULAR AND
CYTOPLASMIC TRANSPORT AND COMPETITION BY OTHER METAL IONS
M. Moriya, Cyndie Gilley, M.C. Linder, California State University, Fullerton
Much remains to be learned about the mechanisms and pathways by which iron is absorbed by
enterocytes. We studied aspects of these processes in polarized Caco2 cell monolayers with tight
junctions grown on filters, using 59Fe. Fe was offered at the apical (brush border) side of the monolayers
in the form of Fe(II)-ascorbate. To induce a robust absorption, monolayers were pretreated overnight with
deferoxamine (DFO). Uptake was inversely related to the [H+] of the solution, falling 5-fold from pH 5 to
8, suggesting that DMT1 might be the only transporter involved. At pH 5.5, uptake kinetics obtained from
initial rates indicated an apparent Km for uptake of about 5 uM. Brush border uptake was markedly
inhibited by increasing concentrations of Mn(II), inhibition being virtually complete at 200 uM. However,
inhibition appeared to be non-competitive. Addition of apotransferrin to the basal medium enhanced iron
uptake as well as overall transport about 30%, consistent with previous findings of Jon Glass and Marco
Tulio Nunez, and their colleagues. During transport, 59Fe was associated with two cytoplasmic and one
vesicular component, the latter released with detergent. These were separated in native polyacrylamide
gel electrophoresis. The vesicular component had the same Rf of migration as Fe2-transferrin. The most
consistent cytoplasmic component was ultrafiltrable through a membrane with a 10 kDa cutoff. Evidence
for vesicular transport of Fe during absorption was also obtained with inhibitors of various steps in these
processes. Brefeldin A (20 ug/ml), an inhibitor of exocytosis and Golgi function that promotes
translocation of transferrin receptor (TfR) to the apical membrane (Xia and Shen, Pharm. Res. 18: 191,
2001) slightly decreased basolateral transport and increased cellular 59Fe accumulation. Tyrophostin A8
(AG10; 500 uM), a GTPse inhibitor that also translocates TfR to the apical membrane (Xia and Shen),
considerably enhanced basolateral iron and 59Fe release, but also inhibited brush border uptake (3040%). (The same effect was obtained with or without apotransferrin in the basal medium.) Nocodazole
(100 uM), a disrupter of microtubules, decreased overall transport as well as uptake 30-40%. FSBA [5’(4fluorosulfonylbenzoyl) adenosine; 100-1000 uM), an ATP analog that decreases uptake of transferrin-Fe
(Olusanya et al., Curr. Biol. 11: 896, 2001), also decreased uptake and overall transport of 59Fe by the
monolayers. We conclude that there is more than one way for iron to enter, cross and exit the enterocyte
during absorption by the intestinal mucosa; that this involves both vesicular and cytoplasmic transport;
and that brush border iron uptake as well as basolateral iron release are partly dependent on vesicular
mechanisms.
Supported by PHS Grant RO1 DK 53080
POSTER 87
POTENTIAL ROLE OF SERUM FERRITIN IN SIGNALING REDUCED INTESTINAL IRON
ABSORPTION: STUDIES WITH NORMAL AND HEMOCHROMATOSIS SERUM AND POLARIZED
CACO2 CELL MONOLAYERS
M.C. Linder, R. Malpe, M. Moriya, L. Zhang, California State University, Fullerton
Intestinal iron absorption changes in response to a variety of physiological conditions, including
inflammation or infection and the status of body iron stores. The mechanism by which intestinal cells are
informed to alter their iron absorption remains unclear. We have hypothesized that a glycosylated form of
serum ferritin is that signal, in that (a) levels of serum ferritin are inversely related to rates of iron
absorption, and (b) iron and inflammatory cytokines enhance the synthesis and secretion of this ferritin by
hepatic cells. We propose that this ferritin signals developing enterocytes to reduce their expression
and/or deployment of transporters and/or vesicular iron transport mechanisms, in the brush border and
basolateral membranes, as these cells emerge from the crypts and enter the villi. Polarized monolayers
of (human) Caco2 cells with tight junctions, cultured on collagen in Transwell filters, are an established
model for studying the individual steps in intestinal iron absorption (Alvarez-Hernandez et al., Biochim.
Biophys. Acta 1070: 205, 1991). The monolayers respond like normal intestine to changes in iron
availability, enhancing brush border (apical) uptake as well as basolateral transport of iron in response to
iron deficiency, and vice versa. A portion of the transport into and across these monolayers appears to
be vesicular and is stimulated by the availability of apotransferrin on the “blood” side of the cells. To
begin to test our hypothesis, we pretreated polarized monolayers for 24 h with culture medium containing
from human serum with low and high levels of serum ferritin, obtained from normal subjects (NS) and
those with hemochromatosis (HH). Ferritin concentrations in the HH sera used were 400-1500 ng/ml;
those in NS from 30-230 ng/ml, as determined by ELISA. To prevent differences due to iron,
deferoxamine (DFO; 100 uM) was included during the treatments. To enhance iron absorption that might
be inhibited by serum ferritin, cells were treated with DFO overnight, prior to serum exposure. Uptake of
1 uM Fe(II) from the apical solution, and its overall transport across the basolateral membrane, were
59
monitored with Fe. Monolayers pretreated with 20-50% HH serum (in DMEM) transported 2-4-fold less
59
Fe across the basolateral surface than those pretreated with normal serum (NS). Pretreatment with
100% HH vs. NS also suppressed uptake about 50%. In dose-response studies in which HH serum was
diluted with fetal bovine serum, uptake increased linearly from 4.4 to 16.8 percent of dose with decreasing
proportions of HH serum (from 100% to 50%) corresponding to decreases in serum ferritin from 1500 to
750 ng/ml. Overall transport of 59Fe remained at low levels in monolayers pretreated with serum that had
ferritin concentrations between 1500 to 1175 ng/ml, but increased when it was diluted to 750 ng/ml or
less. (Fetal bovine serum contained no serum ferritin detectable with the human ELISA.) In further
studies, serum was dialyzed into DMEM to remove hepcidin, another potential signaling agent. Dialysis
had no significant effect on iron uptake or overall transport, implying that hepcidin was no involved in the
HH inhibitory effect. Intracellular ferritin from human liver and spleen (at concentrations of 500 ng/ml, and
in the presence of DFO) also failed to alter iron absorption. These findings are consistent with the concept
that a form of serum ferritin signals the enterocyte to suppress iron absorption and suggest that this
occurs by independent reductions in basolateral transfer and brush border uptake of iron, depending
upon the level of serum ferritin to which the cells are exposed.
Supported by PHS Grant RO1 DK 53080
POSTER 88
THE DISEASE DUE TO MUTATIONS OF THE SLC11A3 GENE
A. Pietrangelo, Centre for Hemochromatosis, Department of Internal Medicine, University of Modena and
Reggio Emilia, Modena , Italy.
In 1999, a hereditary disease associated to systemic iron overload in adults and not linked to 6p
was described 1. This clinical entity showed features distinct from classical HFE-hemochromatosis
including autosomal-dominant inheritance, early increase in serum ferritin despite normal transferrin
saturation and predominant iron accumulation in reticuloendothelial (RE) macrophagic cells, suggesting
defective iron-recycling in RE cells1. In 2001, a genome-wide screen was performed in the original
pedigree and evidence of linkage for 2q32 was found2. One compelling candidate gene, SLC11A3
(encoding ferroportin1/IREG1/ MTP1; GenBank accession # NM_014585)3-5 was identified and all
affected patients were heterozygous for a C to A substitution in exon 3 that results in replacement of
alanine 77, a small, hydrophobic, amino acid, with aspartate, a large, negatively charged amino acid,
within the first predicted transmembrane domain 2. A N144H change in the SLC11A3 gene product has
been also reported in a dutch pedigree with an hereditary iron overload disease 6, and, more recently, a
common 3–base pair deletion in exon 5 of SLC11A3 leading to a Val162 deletion has been found in
patients in different ethnic groups 7, 8. Additional families with the disease are being described worldwide.
The SLC11A3 gene encodes for a main iron export protein in mammals,
ferroportin1/IREG1/MTP-1. This multiple transmembrane domain protein is likely to function in placental
materno-fetal iron transfer, in intestinal iron absorption and in release of iron from hepatocytes and
reticuloendothelial macrophages. Although the physiological role of this protein and its regulation by iron
and non-iron stimuli have not been fully understood, we can speculate that the disease is due to a
selective disturbance of iron egress from cells, particularly from RE macrophages. In this context, a lossof-function mutation might cause a mild but significant impairment of iron recycling by RE macrophages,
which normally must process and release a large quantity of iron derived from the lysis of senescent
erythrocytes. A defective iron exit from RE cells may explain the early rise in serum ferritin levels and low
transferrin saturation, the latter due to inability of RE cells to load circulating transferrin with iron. As a
consequence, in subjects with increased erythron demands, iron retention by macrophages could lead to
decreased availability of iron for the hematopoietic system and anemia, as may occur in young female
patients at the time of menstruation or in some patients after aggressive phlebotomy regimen. The clinical
manifestations are milder as compared to expressed HFE hemochromatosis since macrophagic iron
overload is safer than parenchymal iron overload. Mutations in the SLC11A3 gene should be searched in
all subjects with unexplained hyperferritinemia, particularly in the presence of a normal transferrin
saturation.
The increasing number of families with this hereditary iron overload disorder indicates that the
disease is spread worldwide and it may represent the most common hereditary iron overload disorder
beyond classical HFE-associated hemochromatosis.
1.
2.
3.
4.
5.
6.
7.
8.
Pietrangelo A, et al. N Engl J Med 1999;341:725-32.
Montosi G, et al. J Clin Invest 2001;108:619-23.
Abboud S, Haile DJ. J Biol Chem 2000;275:19906-12.
Donovan A, et al. Nature 2000;403:776-81.
McKie AT, et al. Mol Cell 2000;5:299-309.
Njajou OT, et al. Nat Genet 2001;28:213-4.
Devalia V, et al. Blood 2002;100:695-7.
Wallace DF, et al. Blood 2002;100:692-4.
POSTER 89
CONTROLLED STUDY OF CHANGES IN HEMATOLOGIC INDICES, IRON PARAMETERS, AND
OTHER LABORATORY MEASUREMENTS DURING MCV-GUIDED PHLEBOTOMY THERAPY FOR
HEREDITARY HEMOCHROMATOSIS (HH)
Charles D. Bolan, Yu Ying Yau, Glorice Mason, Janet Browning, Susan F. Leitman, Department of
Transfusion Medicine, National Institutes of Health, USA. Collaborators: William Breuer and Z. Ioav
Cabantchik, Department of Biological Chemistry, Hebrew University, Israel.
Phlebotomy therapy remains the only effective treatment for patients with HH. However, a paucity of data
exists to guide the pace of phlebotomy in HH subjects, and no prospective studies have been performed
to evaluate the determinants of maintenance phlebotomy after initial iron depletion. We report data from
100 consecutive patients (pts) enrolled on a protocol evaluating clinical and laboratory responses during
phlebotomy therapy guided by stereotypic changes in the red cell MCV. Subjects received phlebotomy in
the donor center of the Department of Transfusion Medicine of an academic clinical research center,
without charge and irrespective of eligibility for allogeneic blood donation. Induction phlebotomy to
achieve iron depletion was performed every 1 to 4 weeks, depending on subject weight and initial ferritin
levels, until the MCV decreased by 3% below pretreatment baseline. A fingerstick hemoglobin (HGB) >
12.5 g/dL on the day of the visit was used as the threshold for performing phlebotomy. Previously treated
pts, and untreated pts referred before 1999, received maintenance phlebotomy targeted to maintain the
red cell MCV at 3-5% below baseline. Untreated pts referred between January 1999 and December 2001
received maintenance phlebotomy every 12 weeks, with weekly to monthly measurements of MCV,
ferritin, and transferrin saturation (TS) to determine the rate of reaccumulation of iron. Subjects referred
after December 2001 had maintenance phlebotomy therapy performed to maintain the MCV 3-5% below
baseline with a TS% <45. Transferrin saturation was calculated from serum iron and transferrin
measurements. Non-transferrin bound iron (NTBI) was measured in consecutive samples beginning in
January 2001 by Drs Breuer and Cabantchik, Hebrew University, Israel. Median pre-treatment values
included ferritin 1039 (range 65-5248) ng/mL, transferrin saturation (TS) 80 (32-95)%, and MCV 96.3 (90105) cubic microns. A mean of 4 additional induction phlebotomies were performed after ferritin levels
decreased to 50 ng/mL, using the MCV target as a guide to assure iron depletion. Median TS was 11 (322)%, ferritin 16 (5-47) ng/mL, and HGB 12.0 (10.9-13.3) g/dL at the point of transition from induction to
maintenance therapy, as defined by the MCV guide. Nadir HGB levels of 11.7 (10.4-12.6) g/dL occurred
1.5 (0-4) wks after the transition to maintenance therapy. The mean iron removal necessary to maintain a
stable ferritin, MCV and TS during maintenance therapy in 22 C282Y homozygotes was 50+11 ug/kg/d,
was highly correlated with body weight (R=0.74, p<0.0001), and was significantly higher in the
homozygotes than in 5 C282Y/H63D compound heterozygotes, 33+7 ug/kg/d, p<0.003. NTBI was
undetectable, or present at low levels (<0.5 micromolar, n=2) in 190 samples with TS < 45%. NTBI levels
were strongly correlated with increasing TS at TS > 35% (r=0.88, p<0.000001) during all phases of
phlebotomy. NTBI disappeared at the transition to maintenance therapy, remained absent in pts
controlled on a careful MCV-guided maintenance program, and reappeared as early as 12 weeks after
initial iron depletion. Alanine transaminase (ALT) levels decreased significantly in all patients during iron
depletion therapy, including those with normal as well as those with elevated initial ALT values, and
decreased still further during controlled maintenance therapy. Women and older subjects without HFE
gene mutations tolerated initial induction phlebotomy better at 2, rather than 1 week intervals. These data
correspond to a stable maintenance interval of every 7 to 9 weeks for an 85 kg C282Y/C282Y
homozygote, and every 10 to 12 weeks for a compound heterozygote of similar size using 500 mL whole
blood phlebotomy and a HGB of 14 g/dL. More widely spaced phlebotomy intervals are associated with
increasing ferritin, TS, and NTBI levels. Use of the red cell MCV provides an additional parameter to
guide therapy that is reliable, inexpensive, individualized, widely available and suitable for use in the
setting of a blood donor center.
POSTER 90
A VIRAL PROTEIN, HCMV US2, AFFECTS INTRACELLULAR IRON HOMEOSTASIS BY
DEGRADATION OF HFE MOLECULES
S. Vahdati Ben-Arieh, N. Laham and R. Ehrlich, Department of Cell Research and Immunology, Tel Aviv
University, Israel
Hereditary hemochromatosis (HH) is a common genetic disorder that is characterized by inappropriate
control of intestinal iron absorption, resulting in excessive accumulation of iron in the organs, leading to
multi-organ dysfunction. The gene, which is mutated in HH patients, encodes a protein named HFE that
resembles class I major histocompatibility complex (MHC) molecules both in sequence and in threedimensional structure. HFE interacts with the transferrin receptor (TfR) and reduces its affinity to transferrin,
one mechanism by which it possibly affects iron uptake. The structural similarities between HFE and MHC
class I led us to the hypothesis that viral proteins that affect MHC expression and antigen presentation can
also regulate HFE expression and function. Previously, we used recombinant vaccinia viruses expressing
hHFE and various viral proteins to analyze the effects of viral proteins on HFE expression and trafficking.
HCMV US2, dramatically reduced the expression of HFE complexes and free HFE chains. We also found
that US2, but not US11, targets wild type HFE, as well as mutant HFE, for rapid proteasome-mediated
degradation, hence US2 acts independently of HFE conformation. Similar results were obtained with 293
cells that stably over-express hHFE and US2 genes. In contrast, US2 did not have any effect on soluble
HFE expression in 293 cells suggesting that US2-mediated activity is dependent on the cytoplasmic and
transmembrane domain. Furthermore, while the expression of HFE induced TfR and reduced ferritin levels,
the additional expression of US2 in these cells reverted the expression of TfR and ferritin to basal levels.
These results demonstrate that virus encoded proteins may interfere with the expression and function of
proteins involved in iron metabolism and create an environment that supports viral replication. Moreover,
one viral protein, as in the case of HCMV US2, interferes with antigen presentation and thus, immune
responses and in approprriate regulation of iron homeostasis.
POSTER 91
TRAFFICKING AND ASSEMBLY OF HFE COMPLEXES
M. Gleit-Kielmanowicz and R. Ehrlich, Department of Cell Research and Immunology, Tel Aviv University,
Israel.
Iron is one of the key elements for cellular growth and development and is required for DNA synthesis
and transport of oxygen and electrons. Hereditary Hemochromatosis (HH) is a common autosomal
recessive disorder characterized by excessive intestinal iron absorption that leads to iron overload in
many organs. HFE is an MHC class I-like glycoprotein that is mutated in HH. There is evidence that HFE
competes with transferrin for binding to the transferrin receptor, reduces the affinity of transferrin receptor
for transferrin, and affects the endocytosis and recycling of transferrin receptor. The aim of this study is to
further characterize HFE and transferrin receptor interactions and trafficking in mouse cells. The
trafficking and localization of human and mouse HFE have been analyzed in a mouse fibroblast cell line.
Using this cell line expressing human (h)HFE, hβ2m and hTfR we show that hHFE and hTfR co-localize in
the recycling endosomes with Tf. Treatment of the cells with excess iron results in the reduction of
endogenous TfR expression. However, treatment with iron results in Golgi accumulation of both TfR and
HFE, suggesting that HFE may affect TfR localization and trafficking.
While hHFE expression in mouse cells does not inhibit cell growth, over expression of mouse HFE in the
same cells inhibits their growth and proliferation. Therefore, we have analysed the effect of mouse HFE
on TfR trafficking following transient transfection with mouse HFE. By tracking the expression and
trafficking of transfected mouse HFE and endogenous TfR with time we could see that over expression of
mouse HFE leads to increased endogenous TfR expression but to the reduction in transferrin uptake. 48h
following transfection we have detected HFE and TfR in early endosomes, however, 2 weeks later HFE
and TfR have been detected in both early and recycling endosomes. These data suggest that HFE alters
the localization of TfR, either by retaining TfR in the early endosomes, or by interfering with TfR recycling.
POSTER 92
NONINVASIVE MEASUREMENT OF LIVER IRON USING A ROOM TEMPERATURE
SUSCEPTOMETER
S. Kumar1, W.F. Avrin2, D. Hecht1, W. Freeman1, H.S. Trammell III1, A.R. Perry1, B.R. Whitecotton1, T.
McManus1, S. Bennett1, S. Kuhn1
1
Quantum Magnetics, Inc., San Diego, CA; 2Insight Magnetics, San Diego, CA
We have developed a susceptometer that uses room temperature sensors to measure liver-iron stores
noninvasively. Using this susceptometer we have made two sets of measurements on a small number of
human subjects at the Columbia University laboratory of Dr. Gary Brittenham. Most of the subjects had
elevated levels of liver iron. Both the sets of measurements, including the second that was done blind,
demonstrated a good correlation with the liver iron measurements obtained with the same subjects using
an existing SQUID susceptometer. We will describe the room temperature susceptometer and present
the results. This work was supported by the NIDDK under a Small Business Innovation Research
Contract.
POSTER SESSION 2
POSTER 93
GENOTYPING ANALYSIS FOR HEMOCHROMATOSIS MUTATIONS IN AMYOTROPHIC LATERAL
SCLEROSIS
XS. Wang1, Z. Simmons2, JR. Connor1, 1G.M. Leader Family Laboratory for Alzheimer's Disease
Research, Department of Neuroscience & Anatomy, 2Department of Neurology, Penn State College of
Medicine, Hershey, PA 17033.
Abstract Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative syndrome characterized by
adult onset progressive loss of motor neurons in the motor cortex, brain stem and spinal cord causing
muscular wasting, paresis and inevitable death. Despite the identification of a mutation associated with
ALS in superoxide dismutase 1 (SOD1) in some familial cases, the cause of the majority of cases of ALS
is unknown. Furthermore, the prevalence of the SOD1 mutation in patients with ALS is not consistent with
the extent to which oxidative damage is thought to contribute to the disease. This suggests that there are
other pro-oxidative activities that are factors in this disease. SOD1 catalyzes the conversion of superoxide
anion to hydrogen peroxide. Superoxide anion can combine with nitric oxide to form peroxynitrite, which
can interact with reduced metals such as iron. Subsequently, iron can interact with hydrogen peroxide to
form a dangerous hydroxyl radical. Cellular iron management is thus a critical factor in minimizing
oxidative stress. We have reported that iron mismanagement, not necessarily an increase in the amount
of iron, is associated with a number of neurodegenerative diseases. We propose that a mutation in the
Hfe gene, which is associated with iron mismanagement, may be overrepresented in neurodegenerative
diseases. Thus, the following genotyping analysis was carried out by PCR, restriction digestion and gel
electrophoresis. In the analysis of 74 individuals that underwent muscle biopsy in our ALS clinic, 30%
diagnosed with ALS were at least heterozygotic for the Hfe mutation whereas only 6% of those not
diagnosed with ALS had an Hfe mutation. Our findings of a high prevalence of the Hfe mutation in ALS
patients suggest that this could be a significant risk factor for ALS and will be useful in the development of
ALS therapies and in analyzing the efficacy of existing therapies. Further study will be focused on a
prospective genotyping analysis of blood samples from all patients who are seen in our Neuromuscular
Disease Clinic and from normal control subjects (supported by Dean's Feasibility Grant, Penn State
College of Medicine).
POSTER 94
MYNAHS: A NATURAL MODEL OF HUMAN HEREDITARY HEMOCHROMATOSIS
A. Metea, H.G. Hendriksa, P.H.M. Klarenc, G.M. Dorresteina, J.E. van Dijka, J.J.M. Marxb
a
Department of Pathology, Faculty of Veterinary Medicine, Utrecht University, bEijkman-Winkler Institute
for Microbiology, Infectious Diseases and Inflammation, University Medical Center, Utrecht, cDepartment
of Animal Physiology, University of Nijmegen, Nijmegen, The Netherlands
Hereditary hemochromatosis (HH), the most frequent autosomal recessive genetic disorder in
Caucasians, is characterized by inappropriately high absorption and parenchymal deposition of iron and
is caused by mutations in iron-regulating genes. Iron overload is also a very frequent finding in several
animal species, suggesting a genetic predisposition. In one of the most reported species with
susceptibility for iron overload (mynah bird) it was recently shown that the background of this
phenomenon is high uptake and retention of dietary iron. The present work compares susceptible
(mynahs) with non-susceptible avian species (chickens) by evaluating iron uptake at the intestinal
absorptive cell level. Enterocytes from mynahs and chickens were isolated and uptake of Fe(II) and Fe(III)
was studied. It was found that Fe(III) uptake is much lower than Fe(II) uptake. Although the liver iron
(present only in hepatocytes) content was at least 10 fold higher in mynahs compared to chickens,
enterocyte Fe(II) uptake was considerably higher in mynahs. Fe(II) uptake showed saturation at the
studied concentrations in both species. Kinetic studies revealed a 3-fold increase in Vmax for mynahs.
Calculated values for the uptake kinetics of the probable membrane transporter suggest that mynah bird
enterocytes have a significantly higher limiting uptake rate, due to the possible increase in the number of
transporters with respect to chicken enterocytes. The susceptibility of this species is due to intestinal iron
uptake despite hepatic iron accumulation, implicating a ‘mis-sensing’ of body iron similarly to human HH.
We conclude that mynahs are a natural model for human HH.
POSTER 95
IRON AND GLUTATHION HOMEOSTASIS IN NEURONS: ACTIVATION OF AN OXIDATIVE LOOP
THAT RESULTS IN CELL DEATH
Marco T. Núñez, C. Núñez-Millacura, P. Muñoz, V. Tapia, T. Egaña, V. Gallardo and R. B. Maccioni.
Department of Biology and Millennium Institute CBB, University of Chile, Casilla 653, Santiago, Chile.
In studying cell iron homeostasis, we noticed that neuronal cells do not stop accumulating iron with time.
As iron accumulation may lead to oxidative stress and cell damage, we analyzed the interrelations
between iron and glutathion (GSH) homeostasis, in an attempt to understand the interrelations between
iron accumulation and antioxidant defenses operating in neuronal cells.
Methods. Cells used in this study included neuroblastoma (N2A and SHSY5Y) cells, cortex neurons and
hippocampal neurons. Oxidative stress was determined by H2DCFDA (DCF) fluorescence, accumulation
of 4-hydroxy-2-nonenal (HNE)-protein adducts and 8-hydroxy-deoxiguanosine (8-OH-dG) formation (1).
GSH and oxidized GSH, GSH peroxidase, GSH reductase and caspase-3 were determined with
commercial kits (Calbiochem). IRP activity and glutathion transferase (GT) and were determined as
described (1, 2).
Results. Increasing concentrations of iron in the culture medium elicited increasing amounts of
intracellular iron, labile iron pool and oxidative stress. Neurons had both iron regulatory protein (IRP) 1
and IRP2 activities, being IRP1 activity quantitatively predominant. When iron in the culture medium
increased from 1 to 40 µM, IRP2 activity decreased to nil. In contrast, IRP1 activity decreased when iron
increased up to 10-20 µM, and then, unexpectedly, increased to maximal activity. IRP1 activity at iron
55
concentrations above 20 µM was functional since it correlated with increased Fe uptake. The increase
in IRP1 activity was mediated by oxidative-stress, since N-acetyl-L-cysteine and quercetin largely
abolished it. GSH levels in neuronal cells were markedly lower than in other cell types. Increasing cell
iron induced first an increase in GSH levels and then a decrease to critically low levels that correlated
with caspase-3 activation and loss of viability. Increases in cell iron induced the activities of GSH
transferase and GSH peroxidase, but not of GSH reductase. Increased iron load resulted in increased
extracellular GSH.
Discussion and Conclusions. The above results indicate that in neurons iron accumulates and generates
oxidative damage. The oxidative stress induced by iron accumulation produced a paradoxical activation
of IRP1, thus generating a vicious cycle of further iron accumulation. The GSH system presented a limited
capacity to respond to increases in Fe load. The increase in GSH transferase activity found at high cell
iron may underlie the decrease in cell GSH levels, since xenobiotic-GSH conjugates formed by GSH
transferase are exo-transported. These findings support the following chain of events:
↑OxStr
Time
↑GT
↓GSH
↑ Fe
↑IRP1
↑Ox. damage
Apoptosis
Cell death
↑NO
The characteristics of iron and GSH homeostasis described here may underlie some processes
associated with neuronal aging, dysfunction and death.
Biotechnology, and by grants 1010657 from Fondo de Ciencia y Tecnología, Chile.
1. Núñez-Millacura et al. 2002. J. Neurochem. 82: 240-248.
2. Veal et al. 2002. J. Biol. Chem. 277: 35523-35531.
POSTER 96
NUCLEAR FACTOR κB AND NITRIC OXIDE MEDIATE THE IRON-INDUCED ACTIVATION OF IRP1
IN NEUROBLASTOMA SHSY5Y CELLS
V. Gallardo, P. Muñoz, C. Núñez-Millacura, V. Tapia and M.T. Núñez. Departamento de Biología and
Millennium Institute CBB, Universidad de Chile, Casilla 653, Santiago, Chile.
INTRODUCTION. Intracellular iron is a pro-oxidant factor involved in cellular damage. The IRE/IRP
system sustains cellular iron homeostasis regulating the synthesis of transferrin receptor (TfR) and the
iron-storage protein ferritin. We found that neuronal cells do not turn off iron incorporation under high iron
conditions, so these cells accumulate iron in time (1). Moreover, we noticed that IRP1 is active at high
iron concentrations, and that this activity is abolished by antioxidants, which suggest that this induction in
IRP1 activity is mediated by oxidative stress (1). In this work we characterized in SHSY5Y neuroblastoma
cells the signal transduction pathway that links increased cell iron and IRP1 reactivation. Our working
hypothesis was that iron-induced oxidative stress activates nuclear factor κB (NFkB), which activates
nitric oxide synthase(s) (NOS). Nitric oxide (NO) produced by NOS would in turn activate IRP1 by
displacing Fe from the Fe-S core (2).
METHODS. Neuroblastoma SHSY5Y cells were used as test system. IRP1 expression levels and IRP1
nitrosylation were detected by Western blot analysis. IRP activity and the labile iron pool (LIP) were
detected as described (1). NFκB DNA-binding activity was determined by inmunoblotting of NF-κB p65
and p50 sub-units present in nuclear homogenates, and by immunofluorescence detection of nuclear
NFκB p65 protein. NOS protein was detected by Western blot analysis and production of nitric oxide (NO)
with Griess reagent.
RESULTS. Treatment of SHSY5Y cells for 48 hours with increasing iron concentration in the culture
media (1.5 to 80 µM) correlated with increased total cell iron and increased LIP IRP1 protein expression
decreased at high iron concentrations. Nevertheless, IRP1 activity remained high in the 5-80 µM range of
iron supply. Increased NF-κB activity as a function of iron concentration was observed both as nuclear
translocation of the p65 sub-unit and as an increase in nuclear p65 immunofluorescence. A timedependent increase in NO production as a function of iron concentration was also observed. Concurrent
with increased NO production, we found changes in nNOS protein expression levels. Moreover, iron
induced IRP1 nitrosylation.
DISCUSSION AND CONCLUSIONS. We found that iron induced, in a concentration-dependent form,
increases in NFκB activity, NO production and IRP1 nitrosylation. The activation of NFκB was most
probably mediated by iron-induced oxidative stress (3). NFκB could activate the expression of iNOS and
the consequent NO production (4). NO could in turn activate IRP1 (2), even under high iron conditions.
The finding reported her support the following sequence of events:
oxidative stress
Fe
+
-
nNOS +
+
NF-κB
+
-
-
+
NO
IRP1
Fe
+
iNOS
The NFκB-NOS-IRP1 mechanism proposed here explains the reactivation of IRP1 observed at high iron
concentration (1). In addition, these results support the notion that in SH-SY5Y cells iron is an agonist for
NF-κB activation through a redox-mediated mechanism.
1. Núñez-Millacura, C., Tapia, V., Muñoz, P., Maccioni, R.B. and Núñez, M.T. 2002. J. Neurochem. 82:
240-248.2.
2. Drapier,J.C., Hirling, H.,Wietzerbin, J., Kaldy, P., and Kühn, L.C. 1993. EMBO J. 12: 3643-3649.
3. Nanxin, L. and Michael, K. 1999. FASEB J. 13: 1137-1143
4. Ganster Raymond W., Taylor Bradley S., Shao Lifang and Geller D.A. 2001. Proc.Natl. Acad.Sci. 98,
15:8638-8643.
POSTER 97
α-SYNUCLEIN TRANSGENIC MICE SHOWS IRON ACCUMULATION IN BRAIN. FE-SYNUCLEIN
INTERACTION: A POSSIBLE MECHANISM FOR NEURODEGENERATION IN PARKINSON DISEASE
Ruma Raha-Chowdhury1,2, , Piers C Emson2, Maria Spillantini1, and Timothy M Cox3.
1
Cambridge Centre for Brain Repair, University of Cambridge, 2Neurobiology Programme, The Babraham
Institute, Babraham, 3Department of Medicine, University of Cambridge, UK.
Parkinson’s disease (PD) is a common neurodegenerative disease characterized by loss of dopaminergic
neurones in the substantia nigra pars compacta (SNc), and also Lewy bodies in some surviving
neurones. Accumulation of α-synuclein in Lewy bodies and neurites is a pathological hallmark of
Parkinson Disease (PD). Mutations in the α-synuclein are only found in familial PD, suggesting that
altered α-synuclein function can trigger neurodegeneration. The aetiology of PD is unknown, however,
iron is abundant in brain areas that degenerate in this disorder. Accordingly, we examined iron deposition
in human brain and mouse brain tissues. Transgenic mice expressing human α-synuclein (human wild
type, A30P and A53T mutations) were used in this study to investigate the functional role of α-Synuclein
and iron in neurodegeneration. We investigated non-haem iron in mouse brain and liver samples. The
expression of iron-metabolism genes: ferritin, divalent metal transporter1 (DMT1), ferroportin 1 (MTP1),
hephaestin (HEPH), hepcidin, and transferrin receptor (TFR) were determined by in-situ hybridisation and
RT PCR in the brain, duodenum and liver of synuclein mice. Several of these mRNA transcripts were
examined in Parkinson’s disease (PD), Alzheimer’s disease (AD) and control brain samples.
The results showed that with age, the accumulation of iron in the brain of α-synuclein/transgenic mice
was greater than in age-matched controls (108.01 µg/g vs. 207.14 µg/g, in control brain vs in transgenic
mice brain tissues, p<0.001). There was no difference in non-heam iron concentrations in the liver, of
transgenic or control mice (264 µg/g in controls vs. 294 µg/g in transgenic mice). α-synuclein was found
to colocalise with ferritin in globus pallidus, substantia nigra, thalamus and several other brain regions.
Ferritin, DMT1 and MTP1 have higher expression in α-synuclein mice than control mice. Our findings
implicate the activity of iron transporters in the neurodegenerative process in PD. We propose the iron
has a specific role in the pathogenesis of PD and other neurodegenerative diseases.
POSTER 98
LOSS OF PARACELLULAR JUNCTION GENE EXPRESSION AND E-CADHERIN PROMOTER
ACTIVITY FOLLOWING IRON-LOADING OF PRIMARY RAT HEPATOCYTES
J. P. Bilello,1 E. E. Cable,1 and H. C. Isom1,2*
Department of Microbiology and Immunology1 and Department of Pathology,2 Milton S. Hershey Medical
Center, The Penn State College of Medicine, Hershey, Pennsylvania 17033
Iron overload in the liver may occur in the clinical conditions hemochromatosis or transfusion-dependent
thalassemia or by long-term consumption of large amounts of dietary iron. As iron concentrations
increase in the liver, cirrhosis develops, and subsequently, the normal architecture of the liver
deteriorates. The onset of cirrhosis marks an increased risk of hepatocellular carcinoma. The underlying
mechanisms whereby iron loading leads to the pathology of the liver are not understood. Numerous
studies have associated an inverse relationship between the expression levels of E-cadherin and the
incidence and severity of hepatocellular carcinoma. Reduced expression of E-cadherin, as well as other
paracellular junction genes, has been correlated with poor prognosis and reduced survival time of
patients with hepatocellular carcinoma. Therefore, both iron overload and decreased expression of
paracellular junction genes have been associated with hepatocellular carcinoma. However, the
relationship between iron loading and expression of paracellular junction genes has not been determined.
Iron loading of primary rat hepatocytes in culture, in the absence of significant oxidative stress, is best
accomplished using the 3,5,5-trimethylhexanoyl ferrocene (TMH-ferrocene) iron donor. TMH-ferrocene
entry into hepatocytes occurs via passive diffusion and is therefore, independent of transferrin-mediated
iron absorption. Upon examination of cultures treated with TMH-ferrocene for 21 days, we observed that
paracellular junction complex integrity and gap junction-mediated intercellular communication were
compromised. Expression of numerous paracellular junction genes was decreased in primary rat
hepatocytes treated with TMH-ferrocene for 21 days. The mRNA expression of numerous paracellular
junction genes of the Wnt-signaling pathway, such as E-cadherin and α- and β-catenin, decreased in a
time-dependent manner following treatment of primary rat hepatocyte cultures with TMH-ferrocene. Ecadherin protein levels were decreased in iron-loaded primary rat hepatocytes, which was reversible
following removal of TMH-ferrocene from the cultures. The expression of the tight junction protein,
occludin, and the gap junction protein, connexin32, were also decreased in iron-loaded primary rat
hepatocytes, markers of tumorigenesis. Perinuclear localization of β-catenin and the absence of a
proteolytic β-catenin breakdown product were also observed in iron-loaded primary rat hepatocytes. Ecadherin promoter activity was also decreased following treatment of primary rat hepatocyte cultures with
TMH-ferrocene. Baculoviruses infected internal hepatocytes in iron-loaded compared to control long-term
hepatocyte cultures most likely because iron loading reduced expression of paracellular junction complex
genes and hence compromised paracellular junction complex integrity. In addition, treatment of the Huh7
human hepatoma cell line with ferric nitrilotriacetic acid resulted in decreased E-cadherin protein
expression and promoter activity, therefore extending our studies into a human hepatic system. These
studies suggest that in hereditary hemochromatosis, as well as other iron adsorption disorders, iron
loading of hepatocytes may decrease paracellular junction gene expression, predisposing hepatic cells to
pathology induced by additional carcinogenic and viral factors, thus leading to increased risk for
developing hepatocellular carcinoma.
POSTER 99
A LONG-TERM PRIMARY MOUSE HEPATOCYTE CULTURE SYSTEM AS A MODEL OF CHRONIC
IRON OVERLOAD
S. A. Stoehr1 and H. C. Isom1,2
Department of Microbiology and Immunology1 and Department of Pathology,2 Milton S. Hershey Medical
Center, The Penn State College of Medicine, Hershey, Pennsylvania 17033
Increased hepatocellular iron concentration is associated with a variety of iron overload disorders.
Chronic iron deposition in hepatocytes has been shown to lead to tissue damage, fibrosis and organ
failure, and has been associated with an increased risk of hepatocellular carcinoma; however, the
mechanism by which iron overload contributes to tumorigenesis is not fully understood. Several in vivo
transgenic murine models have been developed to study iron overload disorders; however, a long-term
primary mouse hepatocyte culture system to study the mechanism and cytological effects of chronic iron
deposition has not been developed. Our lab has developed a serum-free long-term primary mouse
hepatocyte culture system to study the effects of intracellular iron loading in hepatocytes. In this system,
the cells retained morphological and biochemical characteristics of differentiated hepatocytes, including
liver-specific gene expression, through day 30 post plating. Time course analysis demonstrated that cellcell communication is acquired and maintained in this system; specifically, connexin32 expression and
gap junction plaque formation increased over time in culture, concomitant with increased gap junction
mediated intercellular communication between adjacent hepatocytes. To determine if primary mouse
hepatocytes could be iron-loaded in culture, the cells were treated with 3,5,5-trimethylhexanoyl ferrocene
(TMH-ferrocene), an iron donor that can passively diffuse across the hepatocellular membrane without
prior metabolism. Primary mouse hepatocytes treated with 5, 10, or 15 µM TMH-ferrocene for 21 days
demonstrated increased ferritin protein expression over time in culture, indicative of increased
intracellular iron concentration. In addition, Perls’ Prussian blue staining of TMH-ferrocene-treated
cultures detected an increase in intracellular hemosiderin over the 21-day treatment period. We conclude
that the findings described in this study make it possible for the first time to maintain differentiated primary
mouse hepatocytes with gap junction mediated intercellular communication in long-term culture.
Furthermore, long-term cultured primary mouse hepatocytes treated with TMH-ferrocene represent a
model of chronic hepatocellular iron loading, and may be used to study the pathological effects
associated with various iron overload disorders. Importantly, this system may be extended to study
primary mouse hepatocytes isolated from transgenic mice in order to evaluate specific gene functions of
the iron-loaded liver in vitro.
POSTER 100
OXYGEN REGULATION OF IRON REGULATORY PROTEIN 2: EVIDENCE FOR ROLE OF 2OXOGLUTARATE-DEPENDENT DIOXYGENASE ACTIVITY
E. Hanson, M. Rawlins, E. Leibold, Department of Medicine, Division of Hematology, University of Utah,
Salt Lake City, UT
We have investigated the hypoxic regulation of the RNA-binding protein iron regulatory protein 2 (IRP2).
IRP2 is central in the control of cellular iron homeostasis due to its regulation of mRNAs encoding
proteins involved in iron and energy homeostasis. The stability of IRP2 is inversely related to intracellular
iron concentration due to iron stimulation of its ubiquitination and proteasomal degradation. Our studies
demonstrate that hypoxia (1% oxygen) inhibits IRP2 degradation. To examine the underlying mechanism,
we examined the potential role of 2-oxoglutarate-dependent dioxygenase activity. Prolyl hydroxylases are
members of the 2-oxoglutarate-dependent dioxygenase superfamily and have recently been shown to be
central in oxygen sensing due to their oxygen-dependent hydroxylation of hypoxia inducible factor-1 
(HIF-1
 , a m odifica tion re quire
-oxoglutarate-dependent
dioxygenases
d for its de gra
require
da tion. 2
+2
Fe and oxygen in addition to 2-oxoglutarate for enzymatic activity, whereas they are inhibited by CoCl2.
Similar to hypoxia, CoCl2 and iron chelation results in IRP2 stabilization. Furthermore, the 2-oxoglutaratedependent dioxygenase inhibitor dimethyloxalylglycine (a 2-oxoglutarate analog) blocks IRP2 ironmediated degradation without affecting bulk protein turnover or ubiquitination. Ubiquitination of IRP2 in
vivo is inhibited by hypoxia and dimethyloxalylglycine, thus explaining their effect on the stabilization of
IRP2. In addition, ubiquitination of IRP2 in vitro is stimulated by iron and 2-oxoglutarate. Taken together
these data lead us to propose a model whereby a 2-oxoglutarate-dependent dioxygenase(s) is involved in
the oxygen and iron regulation of IRP2 via modulation of its ubiquitination.
POSTER 101
IRON-MEDIATED OXIDATIVE STRESS SELECTS A SUB-POPULATION OF STRESS-RESISTANT
CELLS IN HUMAN NEUROBLASTOMA SHSY5Y CELLS
C. Núñez-Millacura, P. Muñoz, V. Tapia, V. Gallardo and M. T. Núñez. Department of Biology, Faculty of
Sciences and Millennium Institute CBB, University of Chile.
Introduction. Because of their highly aerobic metabolism, neurons are particularly sensitive to oxidative
stress. Moreover, their postmitotic nature does not allow for iron dilution by cell division. Studying a
variety of neuronal cell types, we found that these cells do not shut-off iron uptake at high iron
concentrations. Since intracellular reactive iron causes oxidative stress and damage, in this work we
characterized the effects of increasing iron loads on iron homeostasis and cell viability of human
neuroblastoma SH-SY5Y cells. Our working hypothesis was that oxidative stress generated by iron
accumulation deregulates the IRE/IRP system, and that this deregulation results in cell damage and
eventual death.
Methods. Iron load model. Human neuroblastoma SHSY5Y cells were cultured for 8 days in regular
medium and then for 48 hours in a low-iron medium supplemented with 1.5 to 80 µM Fe-NTA. IRP1
activity was determined by Electrophoretic Mobility Shift Assay (EMSA). Ferritin (Fn) was determined by
ELISA. Cell iron accumulation was analyzed using 55Fe as a tracer in the culture media. Calcein
quenching determined the labile iron pool (LIP). Cellular viability was determined by MTT reduction and
by retention of DCF. H2DCFDA, a fluorescent marker sensitive to reactive oxygen species (ROS),
evaluated iron-induced oxidative stress. Oxidative damage in proteins was determined identifying 4hydroxy-nonenal (HNE)-histidyl adducts by Western blot analysis and by cytochemistry. DNA oxidative
damage was determined by immunocytochemistry identifying the presence of 8-OH deoxyguanosine (8OHdG).
Results and Discussion. Increasing extracellular iron from 1.5 to 80 µM resulted in intracellular
accumulation of 55Fe and increasing LIP. The cell response capacity to iron load was very limited. Fn
levels increased in the 1-5-5 µM Fe range remaining constant further on. EMSA assays indicated
persistent IRP1 activity over in the 10-80 µM Fe range. Cell viability decreased to 50% at 80 µM Fe. This
loss in viability was abrogated by 0.5 mM di-methylthiourea, indicating the participation of oxidative stress
in cell death. The remnant cells maintained viability up to 500 µM Fe, suggesting the existence of a
stress-resistant cell sub-population. Evaluation of oxidative stress (H2-DCFDA) and protein damage
(HNE) showed that the highest levels of ROS and damage was produced with 5 µM of iron, while 8OHdG levels increased in the 1.5-80 µM Fe range. The attenuation of ROS and protein damage in the
10-80 µM Fe range supports the hypothesis that a stress-resistant cell sub-population is selected.
However, the increase in 8-OHdG indicates that damage to the DNA is cumulative.
Conclusions. Iron accumulation by SHSY5Y neuroblastoma cells produces an oxidative stimulus that
activates IRP1. The activation of IRP1 fosters a vicious cycle of further iron uptake and oxidative stress.
This cycle results in loss of viability in a fraction of the cell population, while another fraction survives by
controlling oxidative oxidative damage to lipids and proteins. These later cells are an interesting model for
the study of the protective changes generated in response to iron overload, as those observed in certain
neurodegenerative disorders.
Biotechnology (CBB), and by grants 1010657 and 2000100 from Fondo National de Ciencia y Tecnología
(FONDECYT), Chile.
POSTER 102
EFFECT OF MANIPULATION OF IRON STORAGE, TRANSPORT OR AVAILABILITY ON MYELIN
COMPOSITION AND BRAIN IRON CONTENT USING 3 DIFFERENT ANIMAL MODELS
J. M. Pasquini1, E. Ortiz1, K.Thompson2, B. Felt3, G. Butkus4, J. Beard5 and J. R Connor4
1
Biological Chemistry Department, School of Pharmacy and Biochemistry, University of Buenos Aires,
2
Harvard School of Public Health, 3Department of Pediatrics, University of Michigan, 4Department of
Neuroscience & Anatomy, Penn State College of Medicine and 5Department of Nutrition, Penn State
University.
A number of observations point to iron as an essential factor in myelination and oligodendrocyte biology.
However, the specific role of iron in myelin production and maintenance remains to be elucidated. This
role could be as an essential co-factor, as a transcriptional or translational regulator (direct or indirect) or
as a metabolic fuel. We introduce two animal models each with a unique defect in iron metabolism to test
the hypothesis that altered iron handling by oligodendrocytes is associated with specific changes in
myelin composition and a third model of iron deficiency that has been used extensively but not specifically
for myelin analyses. One of the animal models is the hypotransferrinemic (hpx) mouse that lacks the
ability to make transferrin due to a splicing defect in transferrin mRNA. This animal represents a model of
compromised ability to mobilize iron within oligodendrocytes and requires injections of transferrin to
remain viable. The other animal model is a heterozygote for a null mutation in H-ferritin the iron storage
protein resulting in oligodendrocytes with a limited capacity to store iron. The third animal model is the
iron deficient rat. The iron content of the myelin was assessed by atomic absorption spectrophotometry.
The myelin iron content did not differ from control in the iron deficient rat model or in the mice
heterozygous for the H-ferritin mutation. However, in the hpx mice, the iron content of the myelin was
almost 4X higher than normal. In the hpx mice the total protein concentration in isolated myelin was
increased by 20%. An increase in total phospholipids (40%) and galactolipids (63%) was also measured
in hpx mice in comparison to controls. The myelin proteins CNPase and PLP and all isoforms of MBP
were significantly higher in the hpx compared to the controls. These observations are strikingly consistent
with previous studies that found an increase in myelin components in rats following intracranial injections
of transferrin. A decrease in H-ferritin expression was associated with a 22% decrease in myelin
proteins, but only the decrease in PLP reached statistical significance among the individual proteins
analyzed. The galactolipids decreased by 20% and the phospholipids decreased by 14%. In the iron
deficient rat model, all myelin components measured were significantly decreased. These data
demonstrate that specific compromises in intracellular iron transport do not diminish myelin production.
The reliance of the hpx mice on transferrin injections for survival appears to impact substantially and
uniformly on myelin production. On the other hand, compromised iron storage capacity has a
predominant effect on lipids in myelin. However, iron deficiency results in a more global decrease in
myelin composition suggesting these changes could result from a loss of metabolic support to the
oligodendrocyte. Supported by HD 39386.
POSTER 103
THE TEMPORAL RELATIONSHIP BETWEEN HEPCIDIN AND FERROPORTIN 1 EXPRESSION IN
THE RAT LIVER AND INTESTINE AFTER LPS ADMINISTRATION: EVIDENCE FOR FPT1 AS THE
TARGET FOR HEPCIDIN
K. Y. Yeh, M. Yeh and J. Glass. Departments of Medicine and Molecular and Cellular Physiology; and
Feist-Weiller Cancer Center; LSUHSC, Shreveport, LA 71130-3932
Recent evidence suggests that hepcidin is the hepatic signaling molecule of hepatic iron stores that
regulates intestinal iron absorption [Nicolas et, PNAS 98:8780, 2001;Pigeon et al., J. Biol. Chem. 276:
7811, 2001]. The mechanism by which hepcidin exerts its effect on intestinal iron absorption remains to
be defined. Intestinal iron absorption is a complex process occurring in several subcellular domains and
involving at least two transporters and other components. Hepcidin could regulate iron absorption by
modulating the expression of any of the molecules involved in the intestinal iron absorption. Critical for
intestinal iron absorption, and hence potential targets of regulation by hepcidin, is the brush border
membrane iron transporter DMT1 and the basolateral membrane (BLM) transporter ferroportin 1 (FPT1)
[Fleming et al., PNAS 95:1148, 1998; Gunshin et al, Nature 388:482, 1997;Abboud and Haile, J. Biol.
Chem. 275:19906, 2000;Donovan et al., Nature 403:776, 2000;McKie et al., Mol Cell 5:299, 2000]. Since
the expression of DMT1 is not altered in USF2 knockout mice, who don’t express hepcidin and who
develop iron overload [Nicolas et, PNAS 98:8780, 2001], FPT1 is a more likely regulatory target. We
hypothesized that if FPT1 is the regulatory target, then changes in the expression of hepcidin would elicit
changes in FPT1 in the same sequential pattern. LPS has been shown to stimulate hepatic expression
of hepcidin but decrease FPT1 expression in the reticuloendothelial system [Pigeon et al., J. Biol. Chem.
276: 7811, 2001; Yang et al., J. Biol. Chem. 277:39786, 2002]. Hence, we used the LPS-stimulated rat
model to determine in parallel the expression of hepcidin and FPT1 expression in liver and intestine. As
iron transport across the BLM also involves hephaestin (Heph), the expression Heph expression was also
induce d a b
determined. In the liver, LPS (ip, 1
g/g)
8 h and at 36 h. The biphasic hepcidin increases were accompanied by concurrent reductions of hepatic
FPT1 mRNA in both phases. In the intestine, LPS induced a decrease of FPT1 mRNA at 6-12 h, at least
4-6 h later than that of hepatic FPT1. The reduced intestinal FPT1 expression did not recover until 36 h;
by 48 h the FPT1 was increased to higher than the normal level. The LPS induced changes in intestinal
FPT1 expression were specific: Heph mRNA showed a steady increase for the first 6 h after LPS and
hepatic and intestinal H-Ft mRNA levels did not change at any time. Protein levels for FPT1 and Heph
paralleled the changes in the corresponding mRNAs. Indirect immunofluorescent staining revealed that
in enterocytes both FPT1 and Heph were mostly localized in the BLM. After LPS the staining intensity of
FPT1 was reduced to very low levels, corresponding to the decrease in mRNA and protein as detected by
Northern and Western blots. No distinguishable increase of Heph staining was seen in LPS-treated
samples. Since LPS stimulated a steady reduction of serum iron to 60% at 6 h and about 40% at 12-24
h, the decrease of serum iron might alter intestinal iron status and decrease FPT1 expression. Dietary
iron ingestion (8 mg iron in 500 mg chow) that increased mucosal H-Ft protein 6 h later exerted no effect
on FPT1 mRNA and protein expression. The iron chelator desferoxamine administered 1 h prior to LPS
enhanced LPS induced increase of hepcidin and did not abolish LPS induced reduction of intestinal FPT1
expression. The close temporal relation between LPS induced changes in hepatic hepcidin and FPT1
suggest autocrine signaling regulation of FPT1 expression by hepcidin in the liver. The delayed response
of intestinal FPT1 expression compared to hepcidin expression is consistent with endocrine signaling
regulation. Changes in cellular iron metabolism could account neither for the LPS-induced biphasic
changes in hepatic hepcidin and FPT1 expression nor the reduction of intestinal FPT1 expression. Heph
is unlikely to be the regulatory target of hepcidin. The present circumstantial evidence supports the
hypothesis that FPT1 is the regulatory target of hepcidin and underscores the need of establishing an
intestinal epithelial cell system that responds to hormonal signals such as hepcidin to define the
mechanisms by which hepcidin regulates intestinal iron absorption.
POSTER 104
LONG TERM FOLLOW-UP OF IRON STATUS IN CHRONIC HEPATITIS C PATIENTS RESPONDERS
TO INTERFERON PLUS RIBAVARIN
S. Jacquelinet(1), E. Boucher(1), B. Turlin(1,2), C Le Lan(1), R Lohro(1), O. Loréal(1) Y. Deugnier(1), D.
Guyader(1), P. Brissot(1)
(1)
Service des Maladies du Foie and Inserm U-522, University Hospital Pontchaillou, Rennes,
(2)
Département d’Anatomie Pathologique, University Hospital Pontchaillou, Rennes, France.
Background. In chronic hepatitis due to HCV (HCV-CH), hepatic iron load has been reported to be
decreased by interferon therapy and, conversely, increased by ribavirin due to its hemolytic effect. The
resulting effect of these combined treatments on iron status has not yet been documented. Aim. To
evaluate the fate of hepatic and serum iron parameters in an homogeneous group of HCV-CH patients
prospectively enrolled in a therapeutic trial and who were long term responders after one year of
bitherapy. Patients and Methods. Study of 18 HCV-CH patients (9 men, 9 women, mean age 44 years),
without alcohol abuse, who had been relapsers after a single course of interferon, and were prospectively
treated for one year by the association interferon alpha-2B (3 Millions IU, subcutaneously, three times a
week) and ribavirin (per os). All these patients remained total responders (i.e. with both normalization of
serum transaminases and undetectable serum HCV-RNA) one year after completion of the bitherapy. The
following iron parameters were determined : i) In the blood: hemoglobin (Hb), iron, transferrin saturation,
ferritin, transferrin soluble receptor (TfsR), at MO (= before treatment), M12(= at the end of the twelve
months of bitherapy), and at M24 (= 12 months after completion of therapy). ii) In the liver : histological
iron scoring (TIS = total iron score ; HIS =hepatocytic iron score) and biochemical hepatic iron
concentration (N < 36 µmol/g dry liver weight), at MO and M24. Mean liver biopsy sizes were similar (M0 :
2.48 mg ; M24: 2.87 mg, ns). Results. i) At the end of therapy (M12): significant serum ferritin increase
(231 µg/l versus 158 µg/l at M0 ; p=0.04) and TfsR increase (1.86 mg/l versus 1.18 mg/l at M0 ;
p<0.0001) ; Hb dropped from 14.1 g/dl to 12.3 g/dl (p = 0.0002). ii) At the end of follow-up (M24) :
significant decrease of serum ferritin as compared to M12 (91 µg/l versus 231 µg/l ; p = 0.002) but also to
M0 (91µg/l versus 158 µg/l, p = 0,0003). TfsR levels decreased as compared to M12 (1.86 mg/l versus
1.25 mg/l ; p = 0.0004). At the hepatic level (M24 vs M0): significant increase of periportal hepatocytic iron
score (HIS : 1.8 versus 0.8 ; p = 0.028) without significant modification of TIS (2.1 versus 1.2 ; ns).
Hepatic iron concentration increased significantly (17 µmol/g dry liver weight versus 13 µmol/g dry liver
weight; p = 0.04). Summary and Conclusions. In the present series, antiviral C bitherapy led, after one
year of follow-up, to a statistically significant increase of hepatocytic iron load, while remaining within the
normal range. This increase might be related to the hemolytic effect of ribavirin with subsequent iron
redistribution to the parenchymal cells. As to serum iron parameters, they must be interpreted with great
caution for the prediction of hepatic iron status, especially owing to the ribavirin-related hemolytic
process. (European Grant QLT-2001-00444).
POSTER 105
IRON AND INSULIN RESISTANCE: EFFECT OF IRON MANIPULATION ON INSULIN RECEPTOR IN
HEPG2 HEPATOCYTES
P. Dongiovanni, L. Valenti, G. Occhino, A.L. Fracanzani, S. Fargion, University of Milan.
Background: mild hepatic iron overload is a frequent clinical feature in patients with NAFLD, characterized
by hepatic insulin resistance. Iron depletion improves insulin sensitivity in patients with diabetes, but the
mechanisms are unclear.
Aim: to define the interaction between iron and insulin sensitivity, we determined the effect of iron
manipulation on insulin receptor expression in a hepatocyte cell line.
Methods: HepG2 cells were cultured in RPMI complete medium. I125-insulin binding was evaluated 48
hours after plating in basal condition and in cells treated for 24 hours with iron (FeAmCit/holo-transferrin),
desferrioxamine and glucose. Insulin receptor expression was evaluated by Western blotting.
Results: the number of binding sites at the saturating insulin concentration of 100 ng/ml was 20000/cell in
basal conditions. Iron depletion, obtained with desferrioxamine 100 microM, increased by two-fold
(P<0.001) the insulin binding at 50 ng/ml, which was the half-maximal binding concentration, while at
saturating concentration the increase was 20% (P<0.05). Iron addition with either FeAmCit (150 microM)
and holo-transferrin (5mg/ml) reduced insulin binding at 50 ng/ml by 30%. The effect was comparable to
that obtained by incubating the cells with a high glucose concentration (33 mM, 44% reduction of insulin
binding), previously reported to induce marked insulin resistance in HepG2. Western blot analysis
showed no difference in the cytosolic expression of insulin receptor in cells treated with iron,
desferrioxamine, and glucose compared to basal conditions.
Conclusion: iron may induce insulin resistance modulating the membrane expression/affinity of insulin
receptor in hepatocytes.
POSTER 106
IRON USE FOR HEME SYNTHESIS IS UNDER CONTROL OF THE YEAST FRATAXIN HOMOLOGUE
(Yfh1)
E. Lesuisse1, R. Santos1, B.F. Matzanke2, J.M. Camadro1 and A. Dancis3, Institut Jacques Monod,
France.
Saccharomyces cerevisiae cells lacking the Yeast Frataxin Homologue (YFH1) gene showed very low
cytochrome content. In ∆yfh1 strains, the level of ferrochelatase (Hem15p) was very low, as a result of
transcriptional repression of HEM15. However, the low amount of Hem15p was not the cause of heme
deficiency in ∆yfh1 cells. Ferrochelatase, a mitochondrial protein, able to mediate insertion of iron or zinc
into the porphrin precursor, made primarily the zinc protoporphyrin product. Zinc protoporphyrin instead of
heme accumulated during growth of yfh1 mutant cells, and furthermore, preferential formation of zinc
protoporphyrin was observed in real time. The method for these studies involved direct presentation of
porphyrin to mitochondria and to ferrochelatase of permeabilized cells with intact architecture, thereby
specifically testing the iron delivery portion of the heme biosynthetic pathway. The studies showed that
yfh1 mutant cells are defective in iron use by ferrochelatase. Mössbauer spectrometry analysis showed
that iron was present as amorphous nano-particles of ferric phosphate in ∆yfh1 mitochondria, which could
explain the unavailability of iron for heme synthesis. A high frequency of suppressor mutations was
observed, and the phenotype of such mutants was characterized by restoration of heme synthesis in the
absence of Yfh1p. Suppressor strains showed a normal cytochrome content, normal respiration, but
remained defective in Fe-S proteins and still accumulated iron into mitochondria although to a lesser
extent. Yfh1p and Hem15p were shown to interact in vitro by Biacore studies. Our results suggest that
Yfh1 mediates iron use by ferrochelatase.
Laboratoire d’Ingénierie des Protéines et Contrôle Métabolique, Institut Jacques Monod, Université
Paris 6/Paris 7, 2 place Jussieu, 75251 Paris cedex 05, France.
2. University of Luebeck, Isotopes Laboratory TNF, Ratzeburger Allee 160, D-23538 Luebeck,
Germany.
3. University of Pennsylvania, Department of Medicine, Division of Hematology/Oncology, BRBII/III
Room 731, 431 Curie Blvd, Philadelphia PA 19104, USA
1.
POSTER 107
IRON DEFICIENCY ATTENUATES BUT DOES NOT ABLATE EXPERIMENTAL PORPHYRIA
CUTANEA TARDA IN MICE HETEROZYGOUS AT THE UROPORPHYRINOGEN DECARBOXYLASE
LOCUS
J.D. Phillips, M.R. Franklin and J.P. Kushner, Departments of Medicine and Pharmacology and
Toxicology, University of Utah School of Medicine, Salt Lake City, Utah, 84132.
Iron plays a central role in the pathogenesis of porphyria cutanea tarda (PCT). Hepatic siderosis
is nearly always present and iron depletion through repeated phlebotomies is effective therapy. We have
developed a three-week murine model of PCT that employs the continuous ingestion of drinking water
supplemented with ∂-aminolevulinic acid (ALA) and a single exposure to a polychlorinated biphenyl (PCB)
and iron dextran on day one. Porphyria is fully developed by three weeks and is characterized by
accumulation of hepatic porphyrins, diminished activity of uroporphyrinogen decarboxylase (URO-D) and
an inhibitory activity, independent of uroporphyrin, in denatured liver cytosol that affects recombinant
human URO-D (rhURO-D).
We have altered this three-week model in order to determine the effect of iron deficiency on the
porphyria inducing properties of ALA and PCB. We utilized C57Bl6 mice that were either wild type at
both alleles of URO-D (URO-D +/+) or heterozygous for a targeted deletion of one allele (URO-D +/-).
Animals of both URO-D genotypes were maintained for 14 to 20 weeks after weaning on normal mouse
chow or an iron free diet (Teklad #80396). Animals were then given an I.P. injection of PCB (Aroclor
1254, 4 mg in 0.4 ml corn oil) and maintained on drinking water supplemented with ALA (2 mg/ml).
Animals were sacrificed after 21 days, the livers removed and the following measurements were made:
iron content (ug/g), porphyrin content (nmol/g), URO-D activity (nmol/mg/h) and inhibitory activity of
denatured cytosol on rhURO-D (%). Results (mean values) in female animals are shown in the following
table. Numbers in parentheses indicate the number of animals per group. Similar data were obtained in
males (data not shown).
Parameter
Iron
Porphyrins
URO-D act.
URO-D Inhib.
Iron
Porphyrins
URO-D act.
URO-D Inhib.
No treatment
URO-D
Normal
genotype Diet
+/+
0.21 (13)
+/+
0.67 (19)
+/+
2.09 (13)
+/+
0.0 (15)
+/0.19 (18)
+/0.79 (18)
+/0.92 (18)
+/0.0 (15)
Iron
Deficient
0.07 (9)
0.36 (7)
2.25 (5)
0.0 (5)
0.04 (20)
0.36 (7)
1.05 (6)
0.0 (6)
ALA, PCB
URO-D
genotype
+/+
+/+
+/+
+/+
+/+/+/+/-
Normal
Diet
0.18 (7)
237.97 (7)
1.50 (7)
21.0 (7)
0.16 (7)
1368.2 (7)
0.59 (7)
36.9 (7)
Iron
Deficient
0.04 (6)
1.65 (6)
2.04 (6)
3.0 (6)
0.03 (6)
466.08 (6)
0.65 (6)
14.7 (6)
These results indicate that the iron deficient diet reduced hepatic iron stores but did not affect any
other parameter in untreated mice of either URO-D genotype. In contrast, iron deficiency ablated the
effects of ALA and PCB on porphyrin accumulation, URO-D activity and inhibitor activity in URO-D +/+
animals. Porphyrin accumulation and inhibitor activity induced by ALA and PCB were attenuated but not
ablated in URO-D +/- animals but there was very little effect on URO-D activity. These results in URO-D
+/+ animals corroborate previous reports indicating that iron is an essential factor in the development of
experimental porphyria. When base-line activity of URO-D is reduced, as in URO-D +/- animals,
however, trace amounts of iron are sufficient to permit induction of the porphyric phenotype even though
the effect of the inducing regiment is little changed. An iron dependant process appears to primarily
affect porphyrin accumulation and inhibitor generation.
POSTER 108
IRON AND INSULIN RESISTANCE: EFFECT OF IRON MANIPULATION ON INSULIN RECEPTOR IN
HEPG2 HEPATOCYTES
P. Dongiovanni, L. Valenti, G. Occhino, A.L. Fracanzani, S. Fargion, University of Milan.
Background: mild hepatic iron overload is a frequent clinical feature in patients with NAFLD, characterized
by hepatic insulin resistance. Iron depletion improves insulin sensitivity in patients with diabetes, but the
mechanisms are unclear.
Aim: to define the interaction between iron and insulin sensitivity, we determined the effect of iron
manipulation on insulin receptor expression in a hepatocyte cell line.
Methods: HepG2 cells were cultured in RPMI complete medium. I125-insulin binding was evaluated 48
hours after plating in basal condition and in cells treated for 24 hours with iron (FeAmCit/holo-transferrin),
desferrioxamine and glucose. Insulin receptor expression was evaluated by Western blotting.
Results: the number of binding sites at the saturating insulin concentration of 100 ng/ml was 20000/cell in
basal conditions. Iron depletion, obtained with desferrioxamine 100 microM, increased by two-fold
(P<0.001) the insulin binding at 50 ng/ml, which was the half-maximal binding concentration, while at
saturating concentration the increase was 20% (P<0.05). Iron addition with either FeAmCit (150 microM)
and holo-transferrin (5mg/ml) reduced insulin binding at 50 ng/ml by 30%. The effect was comparable to
that obtained by incubating the cells with a high glucose concentration (33 mM, 44% reduction of insulin
binding), previously reported to induce marked insulin resistance in HepG2. Western blot analysis
showed no difference in the cytosolic expression of insulin receptor in cells treated with iron,
desferrioxamine, and glucose compared to basal conditions.
Conclusion: iron may induce insulin resistance modulating the membrane expression/affinity of insulin
receptor in hepatocytes.
POSTER 109
THE ROLE OF LABILE IRON POOL IN DIFFERENTIAL SENSITIVITY OF HUMAN JURKAT T CELL
LINES TO HYDROGEN PEROXIDE
Anthie Yiakouvaki, Rex M. Tyrrell and Charareh Pourzand
Department of Pharmacy & Pharmacology, University of Bath, Bath BA2 7AY, United
Kingdom
Exposure of cells to oxidizing agents such as ultraviolet A (UVA, 320-400nm) or hydrogen peroxide
(H2O2) has been shown to cause an immediate increase in intracellular “labile” iron (1,2). This newly
discovered phenomenon might be of crucial importance to oxidative damage to cells, since iron is a
catalyst of biological oxidations. In the present study, two human Jurkat T cell lines with different level of
sensitivity to H2O2, (i.e. parental J16 and H2O2-resistant HJ16) were used as a model to assess the
importance of the intracellular increase in the Labile Iron Pool (LIP) on the increased susceptibility of cells
to H2O2-induced oxidative damage. The cells were exposed to a range of doses of H2O2 (0.05-3mM) and
the level of LIP was monitored by the Calcein fluorescence assay (3). It was shown that the basal level of
LIP in both cell lines is similar (i.e. 3.08±0.59µM and 3.34±0.87µM in J16 and HJ16, respectively).
However, when both cell lines were challenged with different doses of H2O2, the level of LIP in J16 cells
increased in a dose-dependent manner but no significant peroxide-induced increase in the LIP was
observed in the HJ16 cell line, indicating a direct correlation between the lack of “induction” of labile iron
release and the cellular resistance to H2O2. To study to what extent LIP plays a role in the resistance of
cells to H2O2, an attempt was made to correlate the level of H2O2-induced iron release and the extent of
cell damage following oxidative treatment. For this purpose, cells were treated with hemin (as a method of
iron loading) prior to exposure to a range of doses of H2O2. The results illustrated that hemin pretreatment protects J16 cells against H2O2-induced necrotic cell death, but sensitises HJ16 cells to the
cytotoxic effects of H2O2, as measured by quantification of apoptosis and necrosis (Annexin V/ Propidium
Iodide uptake). In addition, the hemin pre-treatment dramatically decreased the H2O2-induced level of
labile iron release in the J16 cells, while increasing the level of H2O2-induced labile iron release in HJ16
cells. The differential response of hemin treatment in both J16 and HJ16 cell lines highlights the
importance of oxidant-induced labile iron release in the increased susceptibility of cells to oxidative
damage.
1.
2.
3.
Pourzand, C., Watkin, R.D., Brown, J. and Tyrrell, R.M., (1999), Proc. Natl. Acad. Sci. USA, 96,
6751-6756.
Breuer, W., Epsztejn, S. and Cabatchik, Z.I., (1996) FEBS Letters, 382(3):304-308
Epsztejn, S., Kakhlon, O., Glickstein, H., Breuer, W. and Cabantchik,
Z.I., (1997), Anal. Biochem., 248, 31-40
POSTER 110
THE EFFECTS OF TRIENTINE ON IRON METABOLISM IN WILSON’S DISEASE
H. Hayashi1, A. Harashima1, S. Wakusawa1, F. Sanae1, M. Yano2, K. Yoshioka2, T. Okada3, H. Mabuchi3
1 Department of Medicine, Faculty of Pharmaceutical Sciences of Hokuriku University, 2
Gastroenterology, Nagoya University School of Medicine, 3 Second Department of Internal Medicine,
Kanazawa University School of Medicine
As ceruloplasmin is a major serum ferroxidase, iron transport may be impaired under conditions of
hypoceruloplasminemia. This study tested the effects of trientine on iron metabolism in male patients
with Wilson’s disease. Two male patients with Wilson’s disease were found to have a compound load of
copper and iron in the liver. Both patients were compound heterozygous for ATP7B mutations, without
C282Y on the HFE gene. Tolerance to phlebotomy under trientine treatment (2,500 mg/day) in these
patients was compared with that of male patients with chronic hepatitis C. Their low basal levels of serum
ceruloplasmin were diagnostic for Wilson’s disease, and copper chelation therapy further reduced these
levels. The post-treatment serum ferroxidase activity was also markedly low. Iron reduction-induced
anemia developed in the two patients when serum ferritin reached 38 and 17 ng/ml, respectively.
Compared to other male patients with chronic hepatitis C, the patients with Wilson’s disease were more
intolerant to iron reduction therapy. The impaired availability of iron in the patients may be due to low
ferroxidase activity, with a further reduction during copper chelation therapy. Two male patients with
Wilson’s disease had a low tolerance to iron reduction therapy during trientine treatment.
POSTER 111
APPROACHES FOR REPRESSING FRATAXIN IN HELA CELLS USING SMALL INTERFERING RNAS
(siRNAs)
I. Zanella, C. De Cato, G. Biasiotto, G.M. Gerardi, F. Taroni, A. Albertini and P. Arosio.
Brescia, and Division of Biochemistry and Genetics Istituto Nazionale Neurologico "Carlo Besta", via
Celoria,11 – 20133 Milano, Italy
Frataxin is a mitochondrial protein defective in Friedreich’s ataxia (FRDA). Its involvement in iron
metabolism is indicated by the finding of iron deposits in the heart of subjects with FRDA and by a
massive iron deposition in the mitochondria of yeast cells deficient the in the frataxin homologue, Yfh1p. It
has been indicated that frataxin acts as an iron binding protein, or that it has a role in the assembly of
iron-sulphur clusters, but its actual role in mitochondrial iron handling is unclear. Yeasts have been largely
used as models for the FRDA, although they differ markedly from mammalian cells in the regulation of
iron homeostasis, and lack of the iron storage proteins, ferritins. Thus, simple, frataxin-deficient,
mammalian cell models may be useful to study the role of the protein in the regulation of cytosolic and
mitochondrial iron. In addition, they can be used to verify whether mitochondrial ferritin (MtF) may protect
mitochondria from the iron excess caused by frataxin deficiency, as it seems to occur in the yeast. We
started exploring the new technology of small interfering RNA (siRNA) to repress frataxin expression in
HeLa cells. To this aim we produced by in vitro transcription 5 double-strand siRNAs complementary to
different regions of frataxin mRNA, which were transfected in the cells. Blotting experiments with specific
antibodies showed that all ds-siRNA repressed frataxin expression down to 10-40% of the controls. A
time curse analysis using the most efficient siRNA showed that the highest repression was obtained at
24-48 h post transfection with a residual Frataxin level of about 10%. Preliminary results indicate that the
artificial frataxin down-regulation causes a significant reduction of cell proliferation rate, while does not
seem to affect the level of cytosolic L ferritin. Work is in progress for a detailed characterization of HeLa
cell frataxin-deficient phenotype and to study the effects of MtF expression on it. In conclusion, present
data show that siRNA is an efficient technology for the transient down-regulation of frataxin. It can be
extended to constitutive or inducible down-regulation by using proper vectors, now in construction.
POSTER 112
THE ROLES OF IRON AND REACTIVE CYSTEINE RESIDUES IN OXIDATIVE STRESS,
APOPTOSIS, AND NEUROTOXICITY CAUSED BY HIV-1 PROTEASE
S.Y. Lee, W.Y. Ong§, T. Andoh, and C.C. Chiueh, LCS, NIMH, NIH, Bethesda, MD 20892, and
§
National University of Singapore, Singapore 119260
It is known that most of the AIDS patients develop neuroAIDS manifested by severe and
progressive brain atrophy leading to cognitive and mental problems. In addition to brain
inflammation, HIV-1-induced brain atrophy may be triggered by the release of toxic viral proteins
such as protease, tat, and gp-120. Hawkins, Sheng, and Chiueh (1999) discovered that not only
the protease inhibitor kynostatin and also antioxidants prevent HIV-1 protease-induced oxidative
stress and neurotoxicity at nanomolar concentrations. We proposed further that protease's
cysteine residues may become reactive via the generation of highly toxic thiyl radicals and/or
iron-sulfur clusters thereby leading to oxidative stress and cell death. Recombinant protease
mutants (C67A and C95A) were provided by David Davis (NCI) to assess the relative role of
these cysteine residues in redox regulation of the enzyme (Davis et al., 1996). In the present
study cell and molecular biological methods were employed to investigate in human brain-derived
cell lines the oxidative stress hypothesis of neuroAIDS (Mollace et al., 2001).
At nanomolar concentrations HIV-1 protease caused neurotoxicity in pyramidal neurons in
hippocampal slice cultures and potentiated serum deprivation-induced apoptosis in cell lines
derived from the human brain such as SH-SY5Y and U87-MG. Experiments with recombinant
cysteine mutants, C67A and C95A, revealed that protease’s neurotoxicity is mediated by the
67
95
reactive cysteine (i.e., Cys >> Cys ). Moreover, HIV-1 protease-induced cytotoxicity is due to
oxidative stress produced by thiyl radicals in the presence of trace amounts of iron because iron
chelators (i.e., sodium salicylate, deferroxamine, and S-nitrosoglutathione) completely blocked
the neurotoxicity caused by the HIV1 protease. Furthermore, HIV-1 protease up-regulated p53,
activated caspase 3, cleaved PARP protein for the promotion of apoptosis.
In conclusion, even though the cysteine residues of HIV-1 protease may become reactive in the
presence of iron and oxygen, the functional role of these cysteine residues may be different
based on the new observations obtained from the recombinant HIV-1 protease mutants, C67A
and C95A. Iron-catalyzed oxidative stress diminished when the cysteine residue of the position
67 rather than 95 was point mutated. In the presence of oxygen, the redox cycling of these
2+
• 2+
•
reactive iron-cysteine sites, [-S-Fe-OH] ↔ [-S-Fe-O ] , generates H2O2, OH, and thiyl radicals
yielding not only lipid peroxidation and also apoptotic cell death. Therefore, the modification of
HIV-1 reactive cysteine residues at the position 67 by iron and oxygen may contribute to the
propagation of the progressive neurotoxicity and brain atrophy in neuroAIDS. For preserving
brain neurons, iron chelators and thiol modifying agents can be added to the antiAIDS therapeutic
cocktail.
(HIV-1 protease mutants such as C67A and C95A were provided by D.A. Davis, NCI;
Corresponding e-mail: <chiueh@nih.gov> )
POSTER 113
NON-HEME INDUCTION OF HO-1 DOES NOT APPARENTLY ALTER CELLULAR IRON LEVELS
A.D Sheftel, S. Kim, P. Ponka, Lady Davis Institute, McGill University, Dept. of Physiology, Montréal
The catabolism of heme is carried out by members of the heme oxygenase (HO) family. The
predominant isozyme in the digestion of erythroid heme is the inducible form, HO-1. The products of
heme catabolism by HO-1 are ferrous iron, biliveridin (subsequently converted to bilirubin), and carbon
monoxide (CO). In addition to its function in the recycling of erythrocyte iron, this microsomal enzyme has
been implicated in a number of cytoprotective mechanisms mainly in the context of oxidant insult. HO-1
can be induced not only by heme, but also by cytokines, heat shock, metals, and other cellular stressors,
especially those that generate reactive oxygen species (ROS). Implicit in all the reports of HO-1
cytoprotection to date are effects on the cellular handling of heme/iron. While bilirubin is an antioxidant,
ferrous iron is known to catalyze the production of hydroxyl radicals through Fenton chemistry. Thus, the
release of iron from heme could be counterproductive in protecting the cell from oxidative stress.
Furthermore, there are a number of non-heme stimulators of HO-1 induction, bringing to question the
source of substrate for this enzyme in these paradigms. In the present study, HO-1 was induced by
either sodium arsenite or hemin in the murine macrophage-like cell line, RAW264.7. Both of these
inducers elicited a transient increase in both the mRNA and protein levels of HO-1, however, only hemin
exposure induced an increase in the synthesis rate of the iron storage protein, ferritin. This increase in
35
ferritin production rate, as measured by S-methionine incorporation, was attenuated by the HO inhibitor,
tin-protoporphyrin IX (SnPP) as well as the cell permeable iron chelator, salicylaldehyde isonicotinoyl
hydrazone (SIH). Additionally, treatment of the cells with hemin was able to elicit a decrease in the
activity of iron regulatory proteins (IRPs) that could be blocked by preincubation with SnPP. Sodium
arsenite had no effect on IRPs. These results suggest that iron released from hemin by HO-1 stimulates
ferritin synthesis via the IRE/IRP system, while an increase in the enzyme via a non-heme inducer does
not lead to iron release from endogenous heme sources.
POSTER 114
MUTATIONS IN THE NUCLEOTIDE BINDING DOMAIN OF THE MITOCHONDRIAL TRANSPORTER
ATM1p AFFECT ITS DIMERIZATION AND FUNCTION
S.K. Reaves, M. Chloupková, D.M. Koeller, Oregon Health and Science University, Portland, OR
An ATM1 deletion in yeast (∆atm1) results in excessive mitochondrial iron accumulation, oxidative
damage to mitochondrial DNA, loss of mitochondrial cytochromes and loss of cytosolic heme- and Fe-Scontaining proteins. ∆atm1 cells exhibit impaired growth on fermentable carbon sources and do not grow
on non-fermentable carbon sources. Atm1p is an ABC (ATP-binding cassette) transporter that exists in
the mitochondrial inner membrane. ABC transporters are typically comprised of two hydrophobic
transmembrane domains (TM) containing multiple transmembrane segments and two hydrophilic
nucleotide binding domains containing the ABC motif. Residues within the TM domains are not highly
conserved and are thought to confer substrate specificity. The nucleotide binding domains (NBDs) drive
substrate translocation across the membrane by coupling ATP hydrolysis to transport. Three highly
conserved motifs within the NBD known as the Walker A, Walker B and signature motifs have been
shown to be important for ATP binding and hydrolysis although specific details of how this drives
substrate transport across the membrane remain to be elucidated. The C-terminal NBD of Atm1p is
within the mitochondrial matrix thereby suggesting it functions as an exporter. Although the substrate for
Atm1p remains unknown, roles in mitochondrial iron export and Fe-S cluster assembly have both been
proposed. METHODS Techniques used for these studies include site-directed mutagenesis, isolation of
mitochondria, yeast complementation assays, co-immunoprecipitations and ATP binding assays.
RESULTS Complementation experiments indicated that the conserved lysine residue of the Walker A
motif, residue 475 in Atm1p, is crucial for function as a K475M substitution resulted in complete loss of
Atm1p function. Functional significance of the Walker B region was examined by changing the glutamate
residue directly adjacent to the Walker B motif to an alanine residue (E598A). Complementation assays
indicated that this mutation also resulted in complete loss of function. Western blot analysis confirmed
that loss of function was not due to lack of expression of Atm1p(K475M) or Atm1p(E598A). ATP-binding
assays indicated that the K475M mutation significantly impaired ATP binding while the E598A mutant had
ATP binding properties similar to wild-type (wt) Atm1p. Co-immunoprecipitation experiments using
isolated mitochondria from strains co-expressing HA and V5 tagged versions of Atm1p revealed that
Atm1p exists as a homodimer. Co-immunoprecipitations also showed that both the Atm1p(K475M) and
Atm1p(E598A) could form dimers with wt Atm1p. However, Atm1p(K475M) did not co-immunoprecipitate
with either Atm1p(E598A) or itself. On the other hand, homodimers of two Atm1p(E598A) molecules
were found to be present. DISCUSSION We have provided evidence that conserved residues within the
Walker A and Walker B motifs are critical for Atm1p function. The sequence of Atm1p suggests that it is a
half-transporter that requires interaction with another half-transporter in order to function. Our work
shows that Atm1p exists as a homodimer. The decision to make Atm1p(K475M) mutants was based on
studies of other ABC transporters that indicated that this conserved Walker A residue is involved in ATP
binding through an interaction with the signature sequence of an opposing ATP binding cassette.
Consequently, the ATP molecule is sandwiched between the two NBDs thereby stabilizing their
association. Our results agree with this model since the Walker A mutant exhibited reduced ATP binding
and an inability to form Walker A/Walker A homodimers. Other studies have indicated that the glutamate
residue adjacent to the Walker B motif is essential for ATP hydrolysis but does not affect ATP binding.
Indeed, ATP binding of the atm1p Walker B mutant was not diminished and we were able to detect the
presence of Walker B/Walker B homodimers. CONCLUSIONS 1) Atm1p exists as a homodimer 2)
Atm1p is an exporter that functions in an ATP-dependent manner 3) ATP stabilizes the structure of Atm1p
homodimers. This work was supported by the National Institutes of Health (PO1 HD08315).
POSTER 115
SERUM FERRITIN AND CARDIOVASCULAR DISEASE: A 17-YEAR FOLLOW-UP STUDY IN
BUSSELTON, WESTERN AUSTRALIA
J. Olynyk, M. Knuiman, M. Divitini, D. Cullen, H. Bartholomew, University of Western Australia and
Department of Gastroenterology, Fremantle Hospital
The association between serum ferritin level and coronary heart disease and stroke events was evaluated
in a long-term prospective study. The cohort consisted of the 1,612 men and women aged 40-89 years
who participated in the 1981 Busselton Health Survey and who were free of cardiovascular disease at
that time. Serum ferritin levels were obtained from serum samples that had been stored frozen since
1981.The outcomes of interest were time to first coronary heart disease (CHD) event (hospital admission
or death) and time to first stroke event. Case-cohort sampling was used to reduce costs, preserve serum
but still allow efficient analysis. Ferritin assays were performed for 217 CHD cases, 118 stroke cases, and
a random sample of 450 of the total cohort. Proportional hazards regression models were used to obtain
age-adjusted and multivariate-adjusted hazard ratios for ferritin level in relation to CHD and stroke. There
was little or no evidence that ferritin level was a risk factor for cardiovascular disease. The hazard ratio for
the highest tertile group compare to the lowest group was 0.96 (95% CI 0.60-1.53) for CHD and 1.43
(95% CI 0.78-2.64) for stroke.
POSTER 116
IRON DEFICIENCY ALTERS THE METABOLISM OF DOPAMINE
J. Weisinger, B. Jones, J. Connor, J. Beard, The Pennsylvania State University
Iron deficiency during growth and development in rats is believed to cause an alteration in brain
concentrations of dopamine and possibly other neurotransmitters (1,2). Studies in human infants suggest
irreversible changes in cognition and behavior if iron deficiency occurs in early life. Little direct evidence
exists, however, regarding the mechanisms whereby iron deficiency may alter the metabolism of
monoamines. We conducted a series of iron chelation studies with pheochromocytoma, PC12, cells to
determine if DA uptake and metabolism were altered with iron removal, and replacement. The amount of
DA transporter (DAT) was reduced by 30%, 55%, and 75% within 12, 18, and 24 hrs of 50 uM DFO
treatment. TfR levels increased by 32%, 56%, and 66% with 25, 50, and 100 uM DFO within a 24 hr
period demonstrating effective iron chelation. Iron repletion with ferric ammonium citrate resulted in
normalization of DAT levels within 24 hours whereas rescue with iron replete media resulted in significant
increases in DAT but not complete normalization. These data demonstrate reversibility of effect of
3
chelation. H-DA uptake studies were conducted to determine if Vmax and KM for the DAT were affected
by iron chelation. Iron chelation with 50 uM DFO for 24 hours resulted in significant, 80% decrements of
uptake of 3H-DA without any change in affinity of the transporter for the ligand or changes in the rate of
3
H-leucine uptake. Blockade with NE transporter inhibitors demonstrated that this effect was specific to
the Na-co transport protein, DAT, that is responsible for moving DA from extracellular to intracellular
spaces. Thy-1 appears to be essential for synapic vesicle formation so Western blot studies were
conducted to determine if this marker of recycling vesicle formation were affected by PC-12 iron status.
Dose response studies demonstrated a reduction in PC12 cellular content of Thy-1with DFO chelation
between 25-100 uM DFO. Interestingly, the amount of DA within the cell was elevated as a result of DFO
chelation of iron suggesting a failure of feedback regulation of tyrosine hydroxylase, an iron dependent
enzyme. The source of this feedback signal is believed to be presynaptic D2R mediated thus we
evaluated D2R levels in these cells. As wilth the DAT and Thy-1, D2R levels decreased in a doseresponse fashion with DFO chelation while cell viability remained normal. These studies, demonstrate for
the first time, a direct effect of cellular depletion of iron on proteins responsible for the re-uptake of DA
into neuronal compartments. The effect might be mediated by transcription factor control as well as signal
cascade modulation of post-translational modification of these proteins. This pattern of responses is
consistent with a role of neuronal iron concentration on DAT and D2R receptor gene expression.
(1)
Beard JL, Erikson, K, Jones BC, Neo-natal iron deficiency results in irreversible changes in
dopamine function in the rat. J Nutr. (in press) 2003
(2)
Beard JL, Erikson, K, Jones BC, Neurobehavioral analysis of developmental iron deficiency in
rats. Behavioural Brain Research. 134: 517-524, 2002
POSTER 117
NON-ALCOHOLIC STEATOSIS IN THE β2M -/- MICE
Pedro Rodrigues1,3, Lucia Lacerda2,4, Daniel Rodrigues2, Elsa Cardoso1 , Clara Sá-Miranda2,4 and Maria
De Sousa1,3.
1
Molecular Immunology and 2Lysosome and Peroxisome Biology Unit from the Institute for Molecular and
Cell Biology (IBMC) 3 Instituo de Ciências Biomédicas Abel Salazar (ICBAS), 4Jacinto Magalhães
Institute for Medical Genetics (IGMJM).
Non-alcoholic steatosis (NAS) is becoming an increasingly represented entity in human hepatology, with
limited experimental counterparts (1). The beta 2 microglobulin deficient (β2m -/-) mouse was originally
created with the hope that it would provide a model for the study of susceptibility and defense against
viral infections. The mice, lacking both MHC class I expression and CD8+ cells proved disappointed for
such immunological studies (2). With the basis on the finding of T lymphocyte populations defects in HH
patients, we demonstrated that 3-3,5 months β2m -/- mice are an excellent model of HH (3). A very recent
study of Mycobacterium in β2m -/- has demonstrated further the additional importance of the iron overload
in infection (4).
C57Bl/6J (B6), (β2m-/-) mice with B6 background were raised at the IBMC animal facilities. All mice used
on this study were female, aged 40 weeks and were maintained on standard diet. The animals were
sacrificed (n=4 per group), the liver removed and processed for histology, iron content and lipid analysis.
Liver non-hem iron was measured by the bathophenanthroline method. Lipid analysis was performed by
thin layer chromatography (TLC). After routine histology, the paraffin or cryostat sections were stained by
haematoxylin-eosin, the Perls’ method was utilized for the iron staining and the Oil-red for lipid staining.
As previously reported, the hepatic non-hem iron content of β2m -/- mice was significantly higher than the
B6 control mice (2098±390 versus 495±37 µg/g dry tissue). The histological analyses confirmed by the
Perls’ method the hepatic iron deposition. Interestingly, the lipid accumulation observed in the 40 week
old β2m -/- mice by Oil-red staining, was analyzed by TLC and found to be specially due to a significant
increase in the hepatic level of triglycerides (20.4±3.2 and 13.4±2.6 mg/g of tissue, for β2m -/- mice and
controls respectively).
In this study we show that 40 week old β2m -/- mice present (naturally) non-alcoholic steatosis, mainly
composed by significantly higher levels of hepatic triglycerides reproducing the steatosis often found in
humans. The present finding represents an additional pointer to roles hitherto unsuspected of MHC class
I itself and/or β2 microglobulin in the regulation of non-immunological systems.
(1) Clark JB, Palmer CJ, Shaw WN. Proc Soc Exp Biol Med 1983, 173:68-75.
(2) Spriggs MK, Koller, BH et al., PNAS 1992 89:6070-6074.
(3) de Sousa M, Reimao R, Lacerda R, Hugo P, Kaufmann SH, Porto G. Immunol Lett 1994; 39:105-11
(4) Schaible UE, Collins HL, Priem F, Kaufmann SH. J Exp Med. 2002; 196, 1507-13
Acknowledgements
EC Grant: QLG1-CT-1999-00665
Funded also by the Calouste Gulbenkian Foundation and the FCT (MGI/49428/2001)
POSTER 118
IRON STATUS AND THE CHUVASH POLYCYTHEMIA MUTATION OF THE VON HIPPEL-LINDAU
(VHL) GENE
Yaroslav Voloshin, Tiffany N. Johnson, Daniel Okhotin, Mark Loyevsky, Victor R. Gordeuk.
Center for Sickle Cell Disease, College of Medicine, Howard University, Washington DC.
Von Hippel-Lindau (VHL) protein consists of 213 amino acids and is the recognition component of
an E3 ubiquitin-protein ligase complex, which mediates proteosomal degradation of HIF-1 alpha under
normoxic conditions. Numerous heterozygous germline mutations in the VHL gene on chromosome
3p25(4) associated with tumor predisposition have been identified. A homozygous missense mutation,
arg200trp, was recently identified as the cause of Chuvash polycythemia, the first recognized hereditary
condition of augumented hypoxia-sensing. This mutation leads to abnormally increased expression of HIF
in normoxia. HIF-1 is a transcription factor that regulates many genes affected by hypoxia, including
transerrin receptor.
Genotyping for arg200trp mutation in VHL was performed in 43 patients with Chuvash
polycytemia, 44 spouses and 42 community controls. All (100%) 43 Chuvash polycythemia patients
tested were homozygotes for the arg200trp mutation. None of the spouses or community controls were
homozygotes for this mutation, while 7 spouse controls (16%) and 2 community control (5%) were
heterozygotes.
Serum concentrations of transferrin receptor and ferritin were determined with enzyme linked
immunosorbent assay kits (Ramco Laboratories, Inc., Stafford, TX). After adjusting for sex, age, and
number of phlebotomies, transferrin receptor concentrations were markedly elevated in arg200trp
homozygotes versus wild types (14.2 mg/L vs. 4.8 mg/L) (p>0.005), while adjusted serum concentrations
of ferritin did not differ significantly according to genotype (64 ug/L vs. 21 ug/L) (p=0.5).
We also calculated the transferrin receptor to ferritin ratio in our participants, which may
accurately reflect iron status even in presence of inflammation. The Chuvash polycythemia homozygotes
had an adjusted ratio of 3.0, while the wild type group had a ratio of 3.7 (p=0.94). The heterozygotes for
arg200trp gene had the ratio of 3.9.
The purpose of this study was to assess transferrin receptor expression and iron status of
patients with Chuvash Polycythemia as reflected in serum levels of transferrin receptor and ferritin. We
conclude that transferrin receptor concentrations are increased in homozygotes of the Chuvash
polycythemia VHL gene mutation, but iron stores as reflected in serum ferritin and transferrin receptor to
ferritin ratio are not increased.
POSTER 119
FERRITIN LOCALIZATION AND IDENTIFICATION IN CEREBELLAR AXONS OF IRP2 KNOCKOUT
MICE BY ELECTRON MICROSCOPY AND ELECTRON ENERGY LOSS SPECTROSCOPY (EELS)
W. Land, S. Smith, J. Juliani, P. Zhang, J. Lefman, M. Kessel, R. Leapman, T. Rouault, S. Subramaniam,
National Institutes of Health
Iron Regulatory Protein 2 (IRP2) knockout mice develop a progressive neurodegenerative disease in
which ferritin is excessively expressed and iron metabolism is misregulated. Significant amounts of ferric
iron can be found in white matter tracts and nuclei throughout mouse brain - especially the cerebellum.
These accumulations precede by many months the development of a movement disorder in adult mice
characterized by ataxia, bradykinesia, and tremor. Neuronal degeneration is preceded by colocalized iron
and ferritin staining within axons.
Increased iron levels are known to be found in many patients with Alzheimer’s disease, Parkinson’s
disease, Multiple Systems Atrophy, and other forms of neurodegenerative disease. Such diseases have
attracted more attention to date due to the significant impact they have upon human health. IRP2
knockout mice serve as an attractive model for studying the onset and progression of neurodegenerative
diseases, particularly the subset of axonopathic forms. By studying ferritin within axons at the EM level
we can further improve our understanding of the nature of these diseases – perhaps even elucidating the
mechanism by which neurodegeneration itself occurs.
By using EM and EELS we were able to localize and identify ferritin molecules within axons of wildtype
and knockout IRP2 mice. Ferritin was also found intracellularly in oligodendrocyte cytoplasm, both on the
interior and exterior invaginations of the myelin sheath. Electron dense ferritin cores appeared as black
circular dots (~10nm) when visualized using varying concentrations of osmium tetroxide (.05%, .1%,
.15%). EELS microanalysis was used to identify these electron dense cores as ferritin cores by showing
that they contained roughly 2000 Fe atoms per core – an amount of iron characteristic only of ferritin. We
show that ferritin can be imaged by electron microscopy and reconstructed in 3-D by electron
tomography.
POSTER 120
GENETIC ANALYSIS OF MITOCHONDRIAL IRON METABOLISM IN YEAST
OS Chen, S Hemenway and J Kaplan
Department of Pathology, School of Medicine, University of Utah, Salt Lake City, UT 84132, USA
Yeast (fungi) are the only eucaryotes that can survive in the absence of mitochondrial respiration,
as they can satisfy their energy requirements through glycolysis. The ability of yeast to survive without
respiration permits the study of eucaryotic genes that affect mitochondrial function. We have exploited
this property of yeast to study mitochondrial iron metabolism. Deletion of genes involved in iron-sulfur
cluster synthesis results in excessive mitochondrial iron accumulation due to an inhibition of mitochondrial
iron export. Whereas deletion of genes that affect mitochondrial respiration or heme biosynthesis do not
lead to mitochondrial iron accumulation. Yeast with a deletion in YFH1, the yeast homologue of
mammalian frataxin, also shows excessive mitochondrial iron accumulation due to an inhibition of
mitochondrial iron export. Loss of Yfh1p affects the activity of iron sulfur proteins without affecting the
synthesis of the apoproteins. These results suggest that Yfh1p plays a role in iron-sulfur cluster
biosynthesis.
While loss of Yfh1p affects iron-sulfur cluster synthesis it is the accumulation of mitochondrial iron
that mediates oxidative damage leading to respiratory deficit. Conditions that prevent mitochondrial
accumulation in cells deficient in Yfh1p preserve respiratory activity. We used a genetic screen to identify
mutants that maintain respiratory activity in ∆yfh1 cells. Disruption in a gene that encodes a peroxisomal
citrate synthase, Cit2p, prevented the loss of respiratory activity in ∆yfh1 cells. Conversely,
overexpression of Cit2p in ∆yfh1 cells resulted in an increase in iron-dependent cell death. These data
confirm that loss of respiratory activity is a consequence of iron-mediated damage, The toxicity of ironcitrate is not restricted to mitochondria, as overexpression of Cit2p in cells with elevated cytosolic iron
also leads to iron-dependent cell death. This data suggest that high levels of cytosolic citrate may
promote iron-mediated damage. (This work was supported by a grant from the National Institute of
Health (USA-NIDDK-52380).
POSTER 121
CONTINUOUS OVEREXPRESSION OF ERYTHROPOIETIN ALTERS IRON METABOLISM IN MICE
A. Monge1, S. Kim2, A. Sheftel2, P. Ponka2, M. Gassmann1,3, 1Institute of Physiology, University of Zurich,
Zurich, Switzerland; 2Lady Davis Institute for Medical Research, SMBD-Jewish General Hospital and
Department of Physiology and Medicine., McGill University, Montreal, Quebec, Canada; 3Institute of
Veterinary Physiology, University of Zurich, Zurich, Switzerland
We developed a transgenic mouse line overexpressing erythropoietin. The animals were generated by
microinjection of the full length human erythropoietin cDNA with the human platelet-derived growth factor
(PDGF) B-chain promoter into pronuclei of fertilized oocytes derived from B6C3 hybrid mice (Ruschitzka
et al PNAS 97: 11609-13, 2000). The human erythropoietin level in plasma was ~ 12 fold higher than
seen in wildtype animals. The transgenic animals suffer from polycythemia, reaching a hematocrit of
about 85% in adulthood with a 75% increase of the total blood volume. In this study we investigated the
effect of chronically overexpressed erythropoietin on iron metabolism in these mice. Serum iron, serum
ferritin and transferrin saturation were decreased in transgenic mice and the amount of iron in the spleen
was considerably decreased. The liver iron content did not differ from wildtype animals. Gastrointestinal
iron absorption in transgenic mice was significantly increased compared to wildtype animals. The mice
were parenterally loaded with iron to increase the amounts of iron in the stores and this treatment led to
approximately a 50% decrease in iron absorption in both wildtype and transgenic animals. Since a
considerable part of erythropoiesis in mice takes place in the spleen, splenectomy was performed to
manipulate the putative erythroid regulator of iron absorption. Three weeks after splenectomy iron
absorption in transgenic animals was reduced approximately 50%. This finding strongly suggests that
erythropoietin–overrexpressing mice retain their ability to downregulate iron absorption. The mice
overexpressing erythropoietin are a valuable model for investigating the relationship between iron
homeostasis and erythropoiesis in vivo.
POSTER 122
THE EFFECT OF TRANSFERRIN ON T CELL DIFFERENTIATION, A STUDY USING THE
HYPOTRANSFERRINEMIC MOUSE MODEL
MF Macedo1,2, M Correia-Neves1, R Ned3, NC Andrews4, M De Sousa1,2
1
IBMC, Porto, Portugal; 2ICBAS, Porto, Portugal; 3Harvard Medical School, Children’s Hospital, Boston,
USA; 4Harvard Medical School, Children’s Hospital and Howard Hughes Medical Institute, Boston, USA.
A mouse model of hereditary hypotransferrinemia (Trfhpx) has provided interesting data to better
understand different aspects of iron metabolism (Andrews NC, 2000). Although a straight correlation
between iron metabolism and the immune system has been extensively reported, little is known about the
role of iron and iron related proteins on T cell differentiation. In this report we make use of Trfhpx mice to
study the role of transferrin on thymocyte differentiation.
Thymic populations from Trfhpx and control mice (wild type and Trfhpx heterozygous mice) were
examined at 12 and 16 weeks of age, for the following cell markers: CD3, CD4, CD8, CD44 and CD25.
Trfhpx mice have a thymic cellularity significantly lower than the control mice. Despite this reduced
cellularity no differences were observed on the relative proportion of the four main thymic populations:
CD3-CD4-CD8- (TN), CD4+CD8+ (DP), CD4+CD8- (CD4SP), CD4-CD8+ (CD8SP). Taking into account
that iron is necessary for cell division, we investigated thymocyte division in these mice. Studies with
Bromodeoxyuridine (BrdU) incorporation in vivo indicated that cell division is not altered in Trfhpx mice
thymus. An effect was found, however, after a more detailed analysis of the TN thymocytes. The
proportion of the CD44-CD25-, the most mature cells among the TN thymocytes, is manifestly diminished
in Trfhpx mice compared to control mice. This result is supported by earlier results of Brekelmans in 1994
showing inhibition of proliferation and differentiation of the more mature stage of the TN thymocytes, by
anti-transferrin receptor antibody treatment of fetal thymus organ cultures.
Additionally a peculiar expression of the transferrin receptor (CD71) was noted, in the most immature and
the intermediate Trfhpx thymic populations compared to control mice.
The present results strongly suggest that transferrin is of importance in the regulation of thymic cellularity
by controlling the intermediate to late stages of TN T cell differentiation, providing an additional item to
delineating significant reciprocal interactions between the immune system and iron metabolism.
Andrews NC (2000) Iron homeostasis: insights from genetics and animal models. Nat Rev Genet
1(3):208-217
Brekelmans P, van Soest P, Voerman J, Platenburg PP, Leenenn PJ, van Ewijk W (1994) Transferrin
receptor expression as a marker of immature cycling thymocytes in the mouse. Cell Immunol 159(2):331339
POSTER 123
EFFECT OF BROAD SPECTRUM PULSED LIGHT ON HUMAN SERUM TRANSFERRIN
(1)
JSC Smith(1,2,3), RW Evans(3), A Patel(3), J More(2), J Rott(2) and B Gorinsky(1). School of Crystallography,
(2)
Birkbeck College, University of London, Malet Street, London, WC1E 7HX. Bio Products Laboratory,
(3)
Dagger Lane, Elstree, Herts. WD6 3BX. GKT Department of Biomolecular Sciences, Guy’s Campus,
London, SE1 9RT.
Therapeutic proteins manufactured from materials derived from human or animal sources require virus
reduction steps before regulatory approval is granted. Broad spectrum pulsed light (BSPL) has been used
in wastewater decontamination and as a terminal sterilization technology and is now being examined as a
virus inactivation technology for therapeutic proteins. We have used human serum transferrin (HST), as
a model to investigate the possible effects of BSPL on protein structure and function. HST is capable of
reversibly binding two ferric ions (Fe3+), one in the N-lobe and one in the C-lobe. It has been shown that
UVB (290-320nm) irradiation of diferric transferrin solutions induces iron release accompanied by an
alteration of the protein. BSPL incorporates wavelengths from 200 – 1100nm.
Iron-free and iron loaded HST were subjected to different intensities of BSPL and numbers of exposures.
Structural and functional integrity were assessed by SDS PAGE, visible spectrophotometry and 6M urea
gel electrophoresis which resolves HST into four forms: iron free, C-terminal monoferric, N-terminal
monoferric and diferric transferrin.
As BSPL intensity and number of exposures increased, a broadening of the bands in the gels were
observed caused by an increase in heterogeneity of the sample. The difference in net charge on the
protein is consistent with deamidation of asparagine and glutamine residues. 6M urea gel electrophoresis
revealed that the iron binding properties of the N-terminal site were altered upon increased exposure to
BSPL as there was a loss of diferric protein and a concomitant gain of the C-terminal monoferric.
3+
Although all irradiated iron-free samples retained the ability to bind Fe in solution, 6M urea gel
electrophoresis revealed a similar banding pattern to the irradiated diferric samples.
Our results suggest that residues directly or indirectly involved in the N-terminal iron-binding site may
have been affected by the irradiation and lead to an irreversible change in the protein. These
observations indicate that more studies are necessary to assess the efficacy and safety of BSPL as a
method for viral inactivation of proteins.
We would like to thank the BBSRC and the Department of Health for their financial support and
PurePulse Technologies, Inc., for use of their BSPL irradiation equipment.
POSTER 124
REDUCED RATE OF TRANSFERRIN CYCLING CAUSES ANEMIA OF hbd/hbd MICE
A.-S. Zhang, P. Ponka. Department of Cell & Developmental Biology, OHSU, Portland, OR, USA and
Lady Davis Institute, Jewish General Hospital and Departments of Physiology and Medicine, McGill
University, Montreal, QC, Canada.
Hemoglobin-deficit mouse mutant (hbd) is characterized by a hypochromic microcytic anemia that is
inherited in an autosomal recessive manner. The defective gene product is specific to hematopoietic
cells since the anemia can be cured by transplanting normal marrow into the hbd mouse. Data from other
laboratories has shown that hbd anemia is not caused by iron deficiency because serum iron levels,
transferrin saturation and serum ferritin levels are normal. In this study we investigated whether erythroid
cells from hbd homozygotes exhibit a defect in iron metabolism. We confirmed previous reports that, as
59
59
compared to wild-type (+/+) reticulocytes, in hbd/hbd reticulocytes both Fe uptake from Fe-transferrin
59
and incorporation of Fe-incorporation into heme were significantly (40%) inhibited. Importantly, reduced
heme synthesis in hbd reticulocytes can be restored to levels seen in +/+ reticulocytes by incubating hbd
reticulocytes with ferric-salicylaldehyde isonicotinoyl hydrazone, a membrane permeable iron carrier
which bypasses the transferrin-receptor pathway. This finding indicates that hbd/hbd reticulocytes have
no defect in the enzymes involved in heme biosynthesis. We also found that reticulocytes from +/+ and
hbd mice take up ferrous ion (59Fe2+) at identical rates, indicating that in hbd mice the endosomal iron
transporter DMT1 is functionally normal. We examined the rate of transferrin cycle and found that the
rates of both transferrin internalization and transferrin externalization were significantly decreased in
reticulocytes from hbd/hbd mice. Both parameters were decreased by about 40%, the extent of decrease
seen for iron uptake and heme synthesis. Furthermore, when endosomes were pulse-labeled with 59Fetransferrin, in hbd/hbd reticulocytes 59Fe incorporation into heme was about 40% inhibited, suggesting
decreased mobility of endosomes within hbd/hbd reticulocytes. Taken together, our results support a
conclusion that the hbd anemia results from the reduced rate of transferrin cycling in immature erythroid
cells.
POSTER 125
DEVELOPMENT OF A MURINE FERRITIN H TRANSGENIC MODEL
J. Wilkinson IV, S.V. Torti, F.M. Torti. Wake Forest University School of Medicine, Department of Cancer
Biology (JWIV, FMT) and Department of Biochemistry (SVT).
Free or labile iron is a central mediator of oxidative stress. Ferritin, the primary cellular iron storage
protein, is thought to buffer free iron flux and moderate the generation of oxygen free radicals. The role of
ferritin in this critical process has been demonstrated in cellular model systems, but has not been directly
tested in vivo. To assess the function of ferritin in vivo, we designed a transgenic ferritin H (FerH)
construct with the following characteristics: 1) it is bi-cistronic, expressing both FerH and enhanced green
fluorescent protein (EGFP), 2) it is doxycycline (Dox) regulated, requiring a permissive Dox condition and
a tetracycline transactivator transgene for expression, 3) the iron regulatory element in the FerH
transgene is mutated, enabling it to escape from translational control. This construct was first tested in
vitro following transfection into Hela cells; results demonstrated that both FerH and EGFP transgenes are
highly expressed and Dox regulated. Transgenic mice were then generated through pronuclear injection.
FerH/EGFP founder lines, after being bred with a CMV regulated tTA transgenic mouse line, are being
evaluated for ferritin H and EGFP transgene expression in a variety of tissues. EGFP is readily visualized
in the mesentery and skeletal muscle; ferritin H expression is increased approximately 6 fold in skeletal
muscle. We report preliminary findings in a novel model system for the study of diseases which involve
iron and oxidative stress.
POSTER 126
DEFECTIVE YOLK SAC ERYTHROPOIESIS IN HIF-1∀-NULL MICE: A ROLE OF IRON
Y.D. Pastore, V. Divoky, E. Liu, P. Ponka, G.L. Semenza, J.T. Prchal, Medicine and Pediatric
Hematology/Oncology, Baylor College of Medicine, Houston, TX, USA; Palacky University, Olomouc,
Czech Republic; McGill University, Montreal, QC, Canada; The John Hopkins School of Medicine,
Baltimore, MD, USA
Hypoxia-inducible factor-1 (HIF-1) regulates the expression of an array of genes including erythropoietin
(EPO), vascular endothelial growth factor (VEGF), transferrin and its receptor (TfR); however, its role in
the erythropoiesis remains to be defined. In Hif1∀-/- mice, targeted disruption of the HIF1∀ gene
encoding the O2-regulated HIF-1∀ subunit causes cardiac malformations, vascular regression, and death
by embryonic day (ED) 10.5. In order to understand the role of HIF-1∀ in erythropoiesis, we studied the
yolk sac (YS) erythroid progenitors from HIF1∀-/-, HIF1∀+/-, and wild-type (WT) littermate embryos.
Hematopoietic progenitors from isolated YS at ED 9-9.5 were analyzed by in vitro culture in presence of
interleukin-3 (IL-3), IL-6, Epo, stem cell factor and granulocyte-macrophage colony stimulating factor
(GM-CSF). The numbers and the size of the erythroid colonies (CFU-E and BFU-E) from the YS of
HIF1∀-/- embryos were decreased, and had a marked defect of hemoglobinization compared to erythroid
colonies from WT and HIF1∀+/- YS. Neither VEGF nor high levels of Epo added to the cultures fully
rescued these defects. Some of the differences might be related to a developmental arrest in the mutant
embryos, as the number of somites in HIF1∀-/- embryos was significantly lower compared to the
HIF1∀+/- and WT embryos (12, 20, 22 respectively). However, we hypothesized that the defective
hemoglobinization may be due to abnormalities in iron metabolism, e.g. down-regulation of TfR. We
cultured YS cells in the presence of salicylaldehyde isonicotinoyl hydrazone saturated with iron (Fe-SIH)
which transports iron into cells independently of the TfR. In the presence of 100 micromolar Fe-SIH, we
observed a significant increase in the numbers of erythroid colonies derived from YS of WT and, to a
lesser degree, HIF1∀+/- embryos. Although there was no increase in the number of colonies from the YS
of HIF1∀-/- embryos, the size of the erythroid colonies and the degree of hemoglobinization was markedly
improved. These results demonstrate defects in YS erythroid colony formation and hemoglobinization in
HIF1∀-/- embryos and suggest that the latter may be associated with a disturbance of iron metabolism.
POSTER 127
IRON METABOLISM AND EARLY HYPOXIC SIGNALING IN HUMAN CANCER CELLS
J. Eckard, X. Huang, K. Frenkel, New York University School of Medicine
Hypoxia is an important condition in solid tumor biology. Hypoxic regions of tumors display greater
invasiveness and decreased sensitivity to traditional cancer therapies. Patients with highly hypoxic
tumors have been shown to have a shorter survival rates and overall poorer prognosis than patients with
less hypoxic tumors. Hypoxic signaling is transmitted by a transcription factor hypoxia inducible factor α
(HIFα) that is responsible for hypoxia-induced gene regulation. Under normal oxygen conditions, or
normoxia, interactions between HIFα and a class of cytosolic prolyl-hydroxylase proteins (PrH) lead to the
destabilization of HIFα and prevention of gene up-regulation. During hypoxia, or lowered oxygen levels,
HIFα no longer interacts with PrH leading to the stabilization of the transcription factor and HIFα-mediated
gene up-regulation.
The mechanism blocking the interaction of HIFα with PrH is not fully understood. Many investigators
have assumed the decrease in oxygen during hypoxia is the singular reason for the inhibition of the PrH
reaction. However, it is shown that the addition of iron clelators to normoxic cells potentiate powerful
hypoxic responses, underscoring the importance of iron in this mechanism.
Hypoxia changes cellular iron metabolism by altering the function of iron responsive proteins (IRP),
which control the translation of constitutively present ferritin and transferrin receptor mRNAs. The initial
reaction of IRP to hypoxia imitates the response to elevated iron conditions, which is to increase ferritin
translation. The increases in ferritin in the absence of elevated iron levels would cause a reduction in the
chelatable, or cellular low molecular weight (LMW) iron concentrations in cells. LMW iron represents the
portion of the cellular iron pool that is chelatable, bioavailable for uptake, and catalytically reactive. LMW
iron is the important component of the total iron pool in regards to iron-catalyzed biological reactions,
such as with PrH and oxidant formation via the Fenton-reaction. A hypoxia-induced reduction in LMW
iron could mimic effects of iron chelators; thus, initiating hypoxic signaling.
Our hypothesis is that hypoxic exposures cause decreases in LMW iron concentrations and contribute
to the induction of HIFα signaling. Our primary objective is to determine changes in LMW iron levels that
occur during the course of hypoxia, and to correlate them to other hypoxic signaling events. To
corroborate our hypothesis we are testing the ability of iron supplementation, previously shown to
increase LMW iron pools, and other treatments to alter the cell responses to hypoxia. We are also
examining iron levels at various points of a hypoxic exposure to determine any variations in iron
metabolism events.
Using a novel fluorescent method, we are able to accurately measure LMW iron to levels as low as
0.02 µM. Preliminary data has shown that a 30-minute hypoxic exposure (2% 02), leads to a potentially
significant decreases in LMW iron in both human hepatocarcinoma (HepG2) and human breast
carcinoma (MCF-7) cell lines (50% decrease, p<0.01; 45% p < 0.05, respectively). Iron supplementation
increased LMW iron in normoxic cells and minimized the reduction of LMW iron caused by hypoxia.
Potentially significant changes in ferritin levels were also observed in both cell lines, following a 30-minute
exposure.
Our study suggests that iron metabolism may be affected during hypoxia. Further investigation could
provide evidence supporting the role of iron in hypoxic signaling, and offer insights to more effective
cancer therapies.
POSTER 128
CERULOPLASMIN AND HEPHAESTIN HAVE OVERLAPPING REGULATORY ROLES IN RETINAL
IRON HOMEOSTASIS
J.L. Dunaief1, P. Hahn1, R.W. Wong1, L. Chen1, T. Dentchev1, and Z. L. Harris2
1: F.M. Kirby Center for Molecular Ophthalmology,Scheie Eye Institute,University of Pennsylvania
2: Department of Pediatric Anesthesiology, Johns Hopkins University
Corresponding author: jdunaief@mail.med.upenn.edu
Purpose: Ceruloplasmin and hephaestin are ferroxidases important for the export of iron from cells to
plasma. The purpose of this study was to investigate the roles of ceruloplasmin and hephaestin in
regulating retinal iron transport.
Methods: Mice deficient in ceruloplasmin (cp) were generated and crossed with hephaestin-deficient (sla)
mice to generate cp-/-sla-/Y mice. Iron levels in these retinas (cp-/-, sla-/Y, and cp-/-sla-/Y) were
compared to wild-type by the enhanced Perl’s stain and indirectly by immunolabeling retinas for ferritins.
The effects of ferroxidase deficiency on iron regulated proteins, ferroportin and transferrin receptor, were
also studied by immunohistochemistry. Ultrastructural changes were examined by electron microscopy.
Ceruloplasmin and hephaestin in normal retinas were localized by immunohistochemistry, Western
analysis, and RT-PCR.
Results: Increases in iron were observed in the retinal pigment epithelium (RPE) of cp-/-sla-/Y retinas
only (5-9 months old, n=5), with corresponding increases in ferritins. Both H- and L-ferritin were also
increased in retinas of cp-/-sla-/Y mice. These increases were accompanied by increased ferroportin and
decreased transferrin receptor. These changes appear to be age-dependent, as 4 week cp-/-sla-/Y
retinas were Perl’s stain negative. At the ultrastructural level, the RPE of 5 month cp-/-sla- eyes only was
highly vacuolated and contained electron-dense siderosomes. Both ceruloplasmin and hephaestin are
expressed in the retina.
Conclusions: Ceruloplasmin and hephaestin serve overlapping functions in the regulation of ocular iron,
such that combined deficiency results in age-dependent accumulation of iron in the RPE and retina with
alterations in iron regulated proteins. Increased iron in the RPE results in vacuolization, possibly as a
result of iron induced oxidative damage. Iron accumulates in the RPE in some age-related macular
degeneration eyes (P. Hahn et al., submitted), and the cp-/-sla- mouse may provide a model of agerelated macular degeneration and/or other retinal diseases.
POSTER 129
ETHANOL INCREASES HEPATOCYTE IRON AND UROPORPHYRIA IN HFE KNOCKOUT MICE
P.R. Sinclair, N. Gorman, H.W. Trask, W.J. Bement, J. Szakacs, G.H. Elder, D. Balestra, J. F. Sinclair and
G.S. Gerhard. VA Medical Center, White River Junction, VT; Depts of Biochemistry,
Pharmacology/Toxicology, Medicine, Pathology, Dartmouth Medical School, Hanover, NH; Dept of
Pathology, University of Utah Medical School, Salt Lake City, UT, USA; Dept of Medical Biochemistry,
University of Wales Medical School, Heath Park, Wales, UK.
Porphyria cutanea tarda (PCT) is a liver disease characterized by excess hepatic uroporphyrin
(URO) accumulation associated with excess hepatic iron. It is often associated with mutations in the
hereditary hemochromatosis gene (HFE), particularly homozygosity for the C282Y mutation. The most
common and effective treatment is phlebotomy indicating the importance of iron. The exact role of iron in
the disease is unknown. Consumption of alcoholic beverages is the most common risk factor.
Previous attempts to develop an animal model that more closely mimics the alcohol-mediated
disease were unsuccessful. We treated Hfe knockout and wild-type 129 mice continuously with 10 or 15 %
ethanol in their drinking water for 6-7 months. By 4 months, URO was detected in the urine of Hfe(-/-) mice.
In Hfe(-/-), but not wild-type mice, the ethanol treatment increased hepatic URO, hepatic non-heme iron,
hepatic 5-aminolevulinate synthase activity and decreased uroporphyrinogen decarboxylase (UROD).
Although stainable hepatocyte iron was detected in untreated Hfe(-/-) mice, ethanol treatment greatly
increased the diffuse iron staining in hepatocytes, but only in the Hfe(-/-) mice. There was no evidence of
liver injury in ethanol-treated mice. A time course study of ethanol treatment indicated that the ethanolinduced increase in hepatocyte iron preceded URO accumulation. Wild-type mice also became
uroporphyric but only when treated with both ethanol and high doses of iron dextran.
In conclusion, this study indicates that the effect of ethanol to cause hepatic accumulation of
URO in mice is mediated by its effects on hepatic iron metabolism, and is associated with increases in
hepatic 5-aminolevulinate synthase. Ethanol-treated Hfe(-/-) mice appear to be an excellent model to study
the effect of ethanol on hepatic diseases in which iron has been implicated, especially PCT.
This work was supported by funds from the Department of Veterans Affairs and by grants from the
National Institutes of Health ES06263 (PRS) and AG14731 (GG).
POSTER 130
DROSOPHILA IRON REGULATORY PROTEINS ARE ACTIVATED BY CYTOSOLIC SUPEROXIDE
F. Missirlis, J. Hu, T. Rouault and J. P. Phillips, NICHD/NIH and University of Guelph, Canada
Iron regulatory protein-1 (IRP-1) exerts its dual function in modulation of iron homeostasis through the
reciprocal use or dissasembly of its cubane iron sulfur cluster [4Fe-4S]; the holoprotein functions as a
cytosolic aconitase whereas the apoprotein is an RNA-binding translational regulator. Iron levels, but also
oxidative stress dramatically affect IRP-1’s [4Fe-4S] stability and therefore determine its function. Despite
previous knowledge that [4Fe-4S]s are specifically inactivated by superoxide (O2˙), the question whether
IRP-1 [4Fe-4S] reacts with O2˙ has not yet been addressed. Loss of the cluster is predicted to enhance
binding of IRP-1 to mRNA iron responsive elements (IREs). We use Drosophila genetics to demonstrate
that animals bearing a mutation on the cytosolic superoxide dismutase gene (Sod1) acquire more IREbinding activity and exhibit decreased cytosolic aconitase activity. These results were confirmed using
transgene-derived double stranded RNA as a means to reduce Sod1 levels in whole flies. Conversely,
animals in which mitochondrial superoxide dismutase (Sod2) expression was silenced by RNA
interference are identical to wild-type controls regarding IRP-1 activation. However, in such flies
mitochondrial aconitase activity is dramatically decreased. We conclude that O2˙ metabolism is
compartmentalized, that the superoxide dismutases are required for the integrity of iron-sulfur cluster
proteins and that specifically IRP-1 is responsive to O2˙ levels in vivo.
POSTER 131
DISTURBANCES OF IRON REGULATORY AND TRANSPORT SYSTEMS IN THE PATHOGENESIS
OF CYSTEAMINE-INDUCED DUODENAL ULCERATION IN RATS: A NEW ROLE FOR IRON?
T. Khomenko, S. Szabo, X.M. Deng, H. Ishikawa, *G.J. Anderson, G.D. McLaren. VA Medical Center,
Long Beach, CA; University of California, Irvine, CA, USA; *Queensland Institute of Medical Research,
Brisbane, Australia
Introduction: Cysteamine (β-mercaptoethanolamine) induces perforating ulcers in the first 0.5-1.0 cm of
the proximal duodenum within 24-48 hr in rats. This aminothiol (HS-CH2-CH2-NH2) also has the ability to
reduce transition metals and generates H2O2, according to the following reactions: (1) RSH + Mn
→1/2RSSR + Mn-1 + H+; (2) O2 + Mn-1→ O2- + Mn and (3) O2- + 2 H+ →H2O2. Reduced metals then can
react with H2O2 through the Fenton reaction and produce reactive oxygen intermediates (Mn-1 + H2O2 →
Mn +OH-), which induce oxidative stress and tissue damage. Most intestinal iron absorption occurs in the
first part of the duodenum, which also has a higher mucosal iron content than other parts of the small
intestine. We hypothesized that cysteamine may increase the local availability of ferrous iron for
absorption in the duodenum and promote iron uptake by the duodenal mucosal epithelium through
modulation of the activity of IRP1, an important intracellular regulator of TfR1, DMT1 and ferritin
expression. The resulting increase in mucosal iron may contribute to localized tissue damage. Methods:
Groups of rats were given cysteamine-HCl (25mg/100g) by gavage as a single dose and killed 1, 2 or 6 hr
later, or 12, 24 or 48 hr after 3 doses (at 4 hr intervals). Other groups were pretreated (30 min prior to
cysteamine) with either saline, FeSO4 (20mg/100g), FeCl3 (20.5mg/100g) by gavage or desferrioxamine
(DFO, 30mg/100g) subcutaneously, or were placed on an iron-deficient diet for 6 weeks. IRP1 activity
was assessed by electrophoretic mobility shift assay using a rat ferritin IRE probe. mRNA levels were
measured by RNase protection assay and protein by Western blotting. Results: A 6-10 fold increase in
IRP1 activation was observed in the duodenal mucosa from 0.5 to 12 hr after cysteamine. The expression
of TfR1 in duodenal mucosa was also elevated (2.2 fold), but ferritin levels remained unchanged during
duodenal ulceration. Cysteamine increased DMT1 mRNA expression at 0.5 hr and DMT1 protein (IRE
form) at 2 hr. The iron concentration in duodenal mucosa increased by 25-33% from 0.5 - 12 hr after
cysteamine. Pretreatment of rats with ferric or ferrous iron aggravated cysteamine-induced ulcer size up
to 3 fold. Iron depletion by DFO decreased the number of duodenal ulcers by 38% (p=0.04). The irondeficient diet diminished the plasma iron concentration by 32% and the concentration in the duodenal
mucosa by 34%, and this was accompanied by a 6.6 fold (p=0.004) decrease in duodenal ulcer size.
Discussion: The protection of duodenal mucosa by DFO or an iron-deficient diet from the ulcerogenic
action of cysteamine, and the aggravation of duodenal ulceration after pretreatment of rats with ferric or
ferrous iron, suggest a role for iron in cysteamine-induced ulceration. These results may explain the
localization of mucosal injury to the duodenum, where most iron absorption takes place. Thus, the
duodenal ulcerogen cysteamine may increase the iron concentration within the duodenal mucosa by
reducing Fe3+ to Fe2+ and disrupting the post-transcriptional regulation of IRE-containing mRNA
expression, thereby promoting increased iron uptake. The resulting activation of the Fenton reaction may
then lead to tissue damage. Conclusions: 1) The duodenal ulcerogen cysteamine increases IRP1 activity
and expression of TfR1 and DMT1 in duodenal mucosa. 2) The duodenal iron concentration was also
increased, without a concomitant increase in ferritin expression, suggesting an increased cytosolic iron
fraction in these cells and increased susceptibility to oxidative stress. 3) Cysteamine-induced iron
disturbances in intestinal mucosal epithelium may explain the localization of ulcers to the proximal
duodenum. 4) These data, along with the demonstration that iron treatment enhanced and iron deficiency
reduced ulcer formation, implicate iron in the pathogenesis of duodenal ulcers.
POSTER 132
A GENOME SCREEN FOR MODIFIER GENES INFLUENCING IRON ACCUMULATION IN A MURINE
MODEL OF HEREDITARY HEMOCHROMATOSIS
1
M. Bensaid, 1S. Fruchon, 1C. Mazères, 2S. Bahram, 1N. Borot, 1H. Coppin, 1M.P. Roth
1
UPCM-CNRS, UPR 2163, CHU Purpan, Toulouse, and 2Centre de Recherche d'Immunologie et
d'Hématologie, Strasbourg, France
Hereditary hemochromatosis is a highly prevalent genetic disorder characterised by excessive iron
absorption and accumulation in parenchymal organs. Most patients are homozygous for a single mutation
(C282Y) in the HFE protein. A significant proportion of the population is homozygous for this mutation and
therefore genetically predisposed to iron loading. Many epidemiological arguments in favour of
developing population screening programs for hemochromatosis have been advanced, but this issue still
raises questions. The most significant problem with this strategy is that penetrance associated with
homozygosity for the C282Y mutation is not complete. Different factors may contribute to the observed
phenotypic variability, most notably gender, physiological and pathological factors which influence body
iron stores, agents harmful to the liver, and genetic factors. The knowledge of the genes able to modulate
the iron load in humans is essential as it will allow better understanding of the mechanisms that influence
penetrance and severity of hemochromatosis. In hemochromatosis patients, however, complex
interactions between exogenous and genetic factors determining the level of iron overload hamper the
search for modifier genes by usual genome-wide scanning approaches. Use of an animal model allows to
concentrate on genetic factors and, therefore, to increase the study power.
Hfe knockout mice develop, starting from the fourth week of age, an iron overload that mimics
hemochromatosis. Our group has, by successive backcrosses, obtained Hfe knockout mice on two
distinct genetic backgrounds, C57BL/6 et DBA/2, and pointed very significant differences in the severity of
hepatic iron loading and in the mRNA expression of three genes involved in intestinal iron absorption
(Dcytb, DMT1 et FPN1) between the two strains of mice. These results demonstrate the involvement of
genetic factors modulating the intensity of iron loading in response to the disruption of the HFE gene.
-/In order to localise these modifier genes, we generated 1000 (C57BL/6 x DBA/2) F2 Hfe hybrids that
were assessed for serum iron parameters and hepatic iron loading. F2 mice, obtained by crossing
brothers and sisters F1, are all genetically different and also present variable degrees of iron loading.
Comparison of the hepatic iron distribution between parental C57BL/6 Hfe-/- and DBA/2 Hfe-/- mice,
(C57BL/6 x DBA/2) F1 and F2 Hfe-/- hybrids, shows that about 50% of the phenotypic variance of the F2
progeny can be ascribed to additional genetic variance and therefore confirms that liver iron content is
influenced by genetic loci. We selected the 300 F2 mice with the most extreme liver iron concentrations,
i.e. the lowest 15% and the highest 15% of the population. These 300 F2 mice were subjected to a
genome-wide linkage analysis, using 145 microsatellite markers distributed on all autosomes and on the
chromosome X. These markers cover 92.5% of the genome with a spacing of 10 cM or less. This work
pinpoints several chromosomal regions contributing to the variability in iron storage. The identification of
the modifier genes contained in these regions is of great practical importance because their human
homologues may potentially be involved in the variable susceptibility to iron loading observed in
individuals homozygous for the HFE C282Y mutation.
POSTER 133
MOUSE MODELS FOR IRP DEFICIENCY: CRE/LOX-ASSISTED TARGETING OF THE IRON
REGULATORY PROTEIN 1 AND 2 GENES
B. Galy, D. Ferring, M. Muckenthaler, B. Minana, O.Bell and M.W. Hentze
European Molecular Biology Laboratory, Heidelberg, Germany.
Cellular iron homeostasis is maintained by coordinated regulation of the expression of genes
involved in iron acquisition, utilisation, and storage. In mammals, many aspects of this regulation occur at
the post-transcriptional level through the interaction of two trans-regulators, the Iron Regulatory Proteins 1
and 2 (IRP1 and 2), with cis-regulatory sequences called Iron Responsive Elements (IRE) present in
untranslated regions of mRNAs.
To determine the role of the IRP/IRE regulatory system in body iron homeostasis we have generated
mouse lines with targeted mutagenesis of the IRP1 or the IRP2 loci. For both genes, so-called “promoter
trap constructs” were inserted into an early intron to direct the interruption of the respective open reading
frames near the amino-terminus. The targeting vectors were designed to facilitate conditional inactivation
of the IRP1 and IRP2 genes using the Cre-Lox system.
We present the analysis of animals with global, constitutive inactivation of IRP1 and IRP2, respectively.
Insertion of the promoter trap cassette leads to the complete loss of IRP2 expression in homozygous
mutant mice. By contrast, the identical cassette creates a hypomorphic IRP1 allele with a reduction of the
protein level down to 5 % of the wild-type (WT).
IRP2-deficient mice until 2 months of age display no gross phenotypic abnormalities. Hematocrit, serum
ferritin and haemoglobin levels are unchanged. “IronChip” microarray analysis reveals limited, defined
changes in the abundance of several mRNAs excluding the ferritin mRNAs. Interestingly, IRP2 appears to
exert a differential effect on the translation of L versus H-chain ferritin, a finding that has escaped
detection in cellular model systems: western-blot analysis identifies increased ferritin L-chain but not Hchain levels in the liver of IRP2-deficient mice. A similar differential augmentation of ferritin L-chain
expression was found in the proximal part of the duodenum. Prussian blue staining shows iron deposition
in the duodenal mucosa which is not associated with any down-regulation of the basolateral iron
transporter ferroportin.
In contrast to the IRP2 knock-out, which is inherited in mendelian proportions, intercrosses of
heterozygotes bearing the IRP1 mutation generate only 7 % of pups that are mutant homozygotes. This
disproportion suggests that a strong reduction in the level of the IRP1 may interfere with early
developmental stages. A second line arising from an independent embryonic stem cells clone is currently
under development in order to confirm this observation.
Both mouse models are currently subjected to various stimuli known to challenge iron homeostatic
networks to determine the respective contributions of IRP1 and IRP2 to these adaptive processes.
Bruno GALY
EMBL, Gene Expression Programme, Heidleberg, Germany
e-mail: galy@embl.de
Fax: (49)6221387518
POSTER 134
ENHANCED SPLENOMEGALY AND SEVERE LIVER INFLAMMATION IN
HAPTOGLOBIN/HEMOPEXIN DOUBLE NULL MICE AFTER ACUTE HEMOLYSIS
E. Tolosano1, S. Fagoonee1, E. Hirsch1, F. G. Berger2, H. Baumann3, L. Silengo1 and F. Altruda1
1
Department of Genetics, Biology and Biochemistry, University of Turin, Turin, Italy
2
Department of Biological Sciences, University of South Carolina, Columbia, South Carolina 29208
3
Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York 14263
Various pathologic conditions, such as hemorrhage, hemolysis and cell injury, are characterized by the
release of large amounts of heme, which is highly toxic because heme-iron participates in the Fenton
reaction, thus producing free radicals. Among plasma protective proteins against heme-mediated
oxidative damage haptoglobin (Hp) and hemopexin (Hx) are thought to play a crucial role.
Hp and Hx, by binding with high affinity hemoglobin (Hb) and heme respectively, exert an anti-oxidant
action by preventing heme-catalyzed free radicals production. Analysis of knockout mice has shown that
the role of Hp and Hx becomes crucial after intravascular hemolysis, that is, when high amounts of Hb
and heme are released into plasma. Moreover, analysis of single null mice has also suggested that the
HpHx system is redundant because when one protein is missing, the other is up-regulated, probably to
better counteract the toxic effects of Hb/heme overload.
In order to evaluate the physiological relevance of the HpHx system and the principal targets of its action,
we generated HpHx double knockout (dKO) mice and analyzed them under basal conditions and after
acute hemolysis, obtained by intraperitoneal injection of phenylhydrazine (PHZ). Whereas HpHx dKO
mice displayed no obvious alteration in phenotype under basal conditions, non-lethal hemolytic stress in
these animals led to pronounced splenomegaly. Spleen/body weight ratio was significantly higher in
HpHx dKO mice than in wild-type and single null mice 1 and 3 days after PHZ treatment. Histological
analysis showed that spleen enlargement in size was accompanied by stronger red blood cells
accumulation in HpHx dKO mice than in wild-type and single null mice with a consequent increase of the
splenic red pulp and a proportional reduction of the white pulp. The reticulo-endothelial system of the
spleen, analyzed by evaluating the expression of the heme-degrading enzyme heme oxygenase-1 (HO1), was similar in HpHx dKO and control mice.
On the other hand, the state of activation of liver reticulo-endothelial system was higher in HpHx dKO
mice than in wild-type and single null mice. From day 1 to day 3 after PHZ, about 60-70% of Kupffer cells
expressed HO-1 in wild-type mice and single null mice, whereas almost 100% of Kupffer cells expressed
HO-1 in HpHx dKO mice. Moreover, the liver of HpHx dKO mice presented important signs of
inflammation 7 days after hemolysis compared to other genotypes. The liver of HpHx dKO mice revealed
several inflammatory foci with pronounced macrophage infiltration, necrotic areas and, in some cases,
extensive fibrosis. Therefore, the simultaneous lack of Hp and Hx affected the reticulo-endothelial system
after acute hemolysis resulting in a state of severe hepatic injury.
These data demonstrated that the HpHx system is important to protect from excessive splenomegaly and
liver injury after a non-lethal hemolytic stress, that is a hemolytic stimulus that does not compromize
kidney function. Splenomegaly after intravascular hemolysis is mainly due to vascular obstruction and
occlusion due to clumping of hemolyzed erythrocyte membranes. This phenomenon could be potentiated
by free plasma Hb and heme which are able to induce the expression of adhesion molecules on
endothelial cells, thus promoting the interaction between blood cells and vessels. Moreover, heme is a
potent inducer of inflammation in vivo as it induces increased vascular permeability and leukocyte
recruitment. Hence, in HpHx dKO mice, free plasma Hb and heme could possibly potentiate vasoocclusion thus promoting splenomegaly.
Free plasma Hb and heme could also contribute to liver damage other than account for the increased
number of HO-1 expressing Kupffer cells in HpHx dKO mice. However, several other factors could
contribute to hepatic injury. Firstly, the state of activation of Kupffer cells has been associated to liver
injury as these cells produce pro-inflammatory cytokines. Secondly, the overexpression of HO-1 could
worsen the damage, probably by increasing toxic iron. Therefore, in HpHx dKO mice, unbound Hb and
heme, overexpression of HO-1 and the state of activation of Kupffer cells might contribute towards the
high level of liver inflammation and fibrosis.
We conclude that Hp and Hx are important protective proteins against heme-mediated oxidative stress as
they protect the spleen from excessive enlargement and the liver from inflammation and fibrosis under the
pathological conditions characterized by hemolysis. Moreover, either of the two proteins can alone control
spleen and liver homeostasis. Interestingly, in humans, a variant of the Hp gene has been reported and
the Hp2 allele has been associated with an increased risk of inflammatory complications. Our data
support these observations indicating that plasma levels of Hp and Hx can influence the inflammatory
status in patients suffering from hemolytic disorders.
POSTER 135
ANTHRACYCLINES, IRON, AND CARDIOTOXICITY: EVIDENCE THAT DOXORUBICIN UNDERGOES
SUICIDE DEGRADATION TO PREVENT OXIDATIVE DAMAGE INDUCED BY HYPERVALENT HEME
G. Minotti, P. Menna, E. Salvatorelli, R. Giampietro, *A. Cartoni, Department of Drug Sciences, University
of Chieti School of Medicine, and *Department of Chemistry, Menarini Ricerche S.p.A., Pomezia (Rome),
Italy
Clinical use of the anticancer anthracycline doxorubicin (DOX) is limited by cardiotoxicity. This is
attributed to one-electron reduction of the quinone moiety in ring C of DOX, yielding a semiquinone which
converts molecular oxygen to O2⋅- and H2O2. Attention has long been paid to reactions between H2O2
and low mol wt nonheme iron, resulting in formation of strong oxidants like hydroxyl radicals, ferryl ions,
or equivalent iron-oxygen complexes. However, attempts to mitigate the cardiotoxicity of DOX with
antioxidants have been unsuccessful in clinical studies. Whether iron and DOX conspire in inducing
oxidative damage is, therefore, controversial. Quinone-derived H2O2 can react also with myoglobin, the
most abundant source of heme iron in the heart. This reaction generates a hypervalent species
(ferrylmyoglobin, MbIV) whose iron-oxo moiety (FeIV=O; Eo' = 1V) may contribute to cardiotoxicity by
oxidizing polyunsaturated fatty acids and other biomolecules, similar to what observed with hydroxyl
radical or the FeIV=O moiety of Compound I and II of H2O2-activated peroxidases. We show that DOX is
a janus molecule which not only forms MbIV but also reduces it to less harmful MbIII, according to
reaction pathways that can help to reappraise the role of iron in anthracycline-induced cardiotoxicity.
MbIV
O
O
OH
OH
was formed by reacting horse heart MbIII with two-fold H2O2.
C
B
A
D
OH
Computer-assisted detection of electronic spectra showed that
OH
DOX reduced MbIV to MbIII with a second order rate constant
O CH3 O
O
DOX
(k2) of 1.3 ± 0.3 x 103 M-1 s-1. Standard antioxidants like ascorO
H3 C
NH2
bate or GSH reduced MbIV with lower k2, as they dissipated in
HO
reactions with globin-centered radicals rather than with FeIV=O.
Out of many possible reducing residues in the anthracycline molecule (like the primary alcohol at C-14, the methoxy group at C-4, the hydroquinone in ring B, and the
aminosugar attached to C-7 in ring A) only the hydroquinone proved able to reduce MbIV to MbIII. This
was demonstrated by comparing DOX to its aglycone doxorubicinone and to approved or investigational
analogues which lacked the primary alcohol (daunorubicin), the methoxy residue (4demethoxydaunorubicin), or the hydroquinone (aclarubicin). In reducing MbIV to MbIII, DOX suppressed
peroxidation of arachidonic acid to malondialdehyde much more potently than did ascorbate or GSH or
simple hydroquinone. Interestingly, DOX-dependent MbIV reduction and inhibition of lipid peroxidation
were accompanied by irreversible loss of the optical and fluorescent properties of DOX, which were
indicative of cleavage and degradation of the tetracyclic ring. Similar degradation occurred when DOX
reacted with H2O2-activated heme in place of MbIV. HPLC/UV/Electron Spray Ionization-Mass
Spectrometry analyses offered unprecedented evidence for degradation of DOX into several low mass
compounds, with preliminary evidence for the release of one-ring membered oxidation products. These
results show that the prooxidant activity of DOX toward cell constituents, mediated by quinone-derived
H2O2 and oxidation of myoglobin to MbIV, is balanced by antioxidant properties mediated by
hydroquinone-dependent reduction of MbIV and inhibition of lipid peroxidation. The “antioxidant” mode of
action of DOX, making MbIV unable to attack the cell, casts doubts on the oxidative nature of
cardiotoxicity and explains why antioxidants would be of limited value in protecting patients against
cardiotoxicity. On the other hand, suicide degradation of DOX with MbIV or H2O2-activated heme
unravels that biologic sources of heme iron might influence cellular pharmacokinetics of anthracyclines,
expanding our knowledge of the complex interactions between iron and the untoward effects of these
drugs.
1
12
11
10
9
2
3
8
4
5
6
7
13
14
POSTER 136
IRON AND ATHEROSCLEROSIS: ALTERATION OF MONOCYTE ADHESION TO ENDOTHELIAL
CELL MONOLAYER
A.E.R. Kartikasari, N.A. Georgiou, H. van Kats-Renaud, F.L.J. Visseren, B.S. van Asbeck, J.J.M Marx
Department of Internal Medicine and Eijkman-Winkler Institute for Microbiology, Infectious Diseases and
Inflammation, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands
Background: Atherosclerosis has been associated with several important environment and genetic risk
factors. Progression may involve interaction of the endothelium with leukocytes and platelets as well as
the influences of cytokines, vasoactive molecules and procoagulant factors. This defensive response is
characterized by inflammatory changes and may lead to plaque formation and, furthermore, to plaque
rupture, thrombosis, and myocardial or cerebral infarction. Several studies have currently linked elevated
iron stores and high plasma iron concentration to an increased risk of atherosclerosis. Using an in vitro
cytoadherence assay, we assessed the effect of iron-rich and iron-withholding conditions on human
monocyte (MN) adherence to human umbilical vein endothelial cells (HUVEC). The expression of the
adhesion molecules: ICAM-1, VCAM-1 and ELAM-1 on HUVEC, as well as LFA-1 on MN was also
studied.
Materials and methods: HUVEC were isolated and grown as monolayers for one or two passages before
seeding on 12- or 96-well plates. Peripheral blood mononuclear cells were obtained from blood by Ficoll
gradient separation, and MN of >90% purity was isolated by counter current centrifugal elutriation. After
treatment with 10-50 µM Ferrous Ammonium Sulfate (FAS) or 10-50 µM Ferric Citrate (FC), with or
without TNF-α stimulation, the degree of MN adherence to HUVEC was determined by a BCECFfluorescence-based assay. The same experiments were performed in the presence of iron chelators:
desferrioxamine, deferiprone, salicylaldehyde isonicotinoyl hydrazone and diethylenetriaminepenta acetic
acid. Viability was ascertained using MTT or trypan blue exclusion tests. ICAM-1, VCAM-1 and ELAM-1
expression on HUVEC, as well as LFA-1 on MN, were measured by FACS analysis. Statistical analysis
was done using the Students t-test, and p-values of <0.05 were considered significant.
Results: Both FAS and FC caused a two-fold increase of MN adhesion to HUVEC, both in the presence
and absence of the proinflammatory cytokine TNF-α. A concordant increase in ICAM-1, VCAM-1 and
ELAM-1 expression on HUVEC as well as LFA-1 on MN, was observed. The inclusion of iron chelators
counteracted the iron-induced MN adherence to HUVEC.
Conclusions: Iron upregulates the adherence of MN on HUVEC and is associated with increased
expression of adhesion molecules. Moreover, both cell membrane permeable and impermeable iron
chelators were able to prevent these effects. These results suggest a role of iron in MN adherence to
vascular endothelium, which is one of the earliest events in atherosclerosis.
POSTER 137
CHANGED IRON REGULATION IN SCRAPIE-INFECTED NEUROBLASTOMA CELLS
S. Fernaeus, K. Bedecs and T. Land
Dept. Neurochemistry and Neurotoxicology. Stockholm University, S-106 91 Stockholm, Sweden
Prion diseases are fatal neurodegenerative disorders that are characterized by accumulation of the
scrapie prion protein (PrpSc), the pathogenic form of the endogenously expressed cellular prion protein
(PrPC). PrPC is a glycoprotein expressed on the surface in many cell types. Its function is still unknown,
but several studies have suggested that PrPC might play a role in the regulation of copper homeostasis
and affect the activity of Cu/Zn-superoxide dismutase (SOD). Brains of scrapie infected mice have shown
increased iron content and iron-induced oxidative stress [1], and primary neurons from PrP knock out
mice display reduced Cu/Zn-SOD activity accompanying with increased neuronal sensitivity to oxidative
stress [2], indicating a role for PrPC in cellular oxidative homeostasis.
We have analyzed the cytosolic labile iron pool (LIP) using a calcein based fluorescence method [3],
studied the protein levels of transferrin receptor 1 (TfR), iron regulatory proteins 1 and 2 (IRP1 and IRP2,
respectively) by Western blot analysis and the activities of IRP1 and IRP2 by band-shift assay in
chronically scrapie-infected mouse neuroblastoma N2a cells (ScN2a) as compared to the uninfected cells
(N2a).
We have observed that ScN2a cells have significantly lower cytosolic LIP, lower activities of IRP1 and
IRP2, and lower protein levels of TfR, IRP1 and IRP2. The IRPs respond normally to iron and chelator
treatment, however, iron chelator treatment does not result in the activation of IRPs to wild-type levels.
These results do not show any dysfunction of the iron regulatory system in ScN2a cells though indicate
the possibility of a lowered need for iron in scrapie-infected cells. We are analysing mitochondrial iron
status, which might give us valuable information concerning the demand for iron in the ScN2a cells.
References:
1. Kim, N-H., Park, S-J., Jin, J-K., Kwon, M-S., Choi, E-K., Carp, RI., and Kim, Y-S. (2000) Brain
Res. 884, 98-103.
2. Brown, DR., Nicholas, RJ., and Canevari, L. (2002) J. Neurosci. Res. 67, 211-224.
3. Epsztejn, S., Kakhlon, O., Glickstein, H., Breuer, W., and Cabantchik, ZI. (1997) Anal. Biochem.
248, 31-40.
POSTER 138
NEGATIVELY CHARGED RESIDUES FROM α-HELIX ARE IMPORTANT FOR SELF-ASSEMBLY OF
YEAST FRATAXIN
D.L. McClain, O. Gakh, G. Isaya, Departments of Pediatric & Adolescent Medicine and Biochemistry &
Molecular Biology, Mayo Clinic and Foundation, Rochester MN
Friedreich Ataxia (FRDA), an autosomal recessive neuro- and cardio-degenerative disease, is
characterized by a deficiency of frataxin, a mitochondrial protein encoded for in the nucleus and
synthesized in the cytosol. Yeast frataxin knockouts are characterized by defects in iron-sulfur cluster
biogenesis, loss of respiratory competence, and accumulation of iron in mitochondria. Similar results
were observed in cell lines derived from FRDA patients. Previously, it was demonstrated that in the
presence of ferrous iron, recombinant yeast frataxin (mYfh1p) assembles into a stable complex of 840
kDa and is able to store over 2000 Fe(III) atoms in a mineral core (Adamec et al., 2000; Gakh et al.,
2002; Nichol et al., submitted). A ferroxidation reaction catalyzed by mYfh1p induces the first step of
assembly, α  α3, and size exclusion chromatography has shown that the oligomeric α3 species is used
as a building block for the assembly of mYfh1p into α48 (Park et al., 2002). In this study, site directed
mutagenesis in conserved regions of the mature protein was used to identify surface residues involved in
iron interactions and the process of self-assembly. Mutations were introduced by PCR and overexpressed in E. coli. Protein extracts were separated using size exclusion chromatography and collected
fractions analyzed by SDS PAGE. Results obtained thus far indicate that residues in the negatively
charged patch of the first alpha helix are important for iron binding and self-assembly. This work is
supported by grant AG15709 from the NIH/NIA. D.L.Mc. is supported by grant R25 GM 55252 from the
NIH.
References:
1. Adamec, Jiri, et al., Iron-Dependent Self-Assembly of Recombinant Yeast Frataxin: Implications for
Friedreich Ataxia. Am J Hum Genet, 2000. 67: p. 549-562.
2. Gakh, Oleksandr, et al., Physical Evidence that Yeast Frataxin is an Iron Storage Protein.
Biochemistry, 2002. 41(21): p. 6798-6804.
3. Nichol H, Gakh, O., Pickering, I.J., Isaya, G., Graham, N.G. The structure of yeast frataxin iron cores:
An X-ray absorption spectroscopic study. Submitted.
4. Park, Sungjo, et al., The Ferroxidase Activity of Yeast Frataxin. J Biol Chem, 2002. 277(41): p.
38589-38595.
POSTER 139
FRATAXIN IN AMITOCHONDRIAL PROTIST TRICHOMONAS VAGINALIS
P. Doležal1, M. Embley2, J. Tachezy1
1
Charles University, Czech Republic; 2The Natural History Museum, UK
The anaerobic protist Trichomonas vaginalis is a human sexually transmitted pathogen. Its cellular
structure substantially differs from the general eukaryotic scheme. Lacking mitochondria, it is unable to
carry out oxidative phosphorylation. Instead, Trichomonas vaginalis possesses hydrogenosomes, which
are double membrane bound organelles in which pyruvate is oxidized to acetate, CO2 and H2 with
concomitant synthesis of ATP. The key reactions are mediated by FeS proteins: pyruvate:ferredoxin
oxidoreductase, ferredoxin and hydrogenase. It has been shown that the gene expression as well as the
activity of these proteins is fully dependent on the availability of iron. Although hydrogenosomes possess
a rather high amount of iron, there is no information about iron homeostasis in these organelles as well as
about the mechanisms of FeS cluster formation.
Frataxin is a small protein with a puzzling role in mitochondria of eukaryotes. In human, a deficiency in
frataxin causes Friedreich's ataxia which is associated with an increased level of mitochondrial iron. It has
been suggested that frataxin is involved in various functions such as FeS cluster formation or iron
storage. Searching an EST database of T. vaginalis, we found a fragment of a frataxin homologue in the
genome of this parasite. The screening of a T. vaginalis genomic DNA library resulted in the identification
of a 366 bp ORF, coding for a protein of 121 amino acids. Southern blot analysis showed the presence of
at least three copies of the frataxin genes in the genome of T. vaginalis. Surprizingly, the 5ÚTR region of
frataxin does not contain the transcription initiator element that was shown to be indispensable for gene
transcription in T. vaginalis. Thus, the expression of the frataxin gene was verified by a nuclear run-on
assay, which confirmed that the frataxin gene is transcribed. We further demonstrated that initiation of
frataxin gene transcription is regulated by iron availability. The frataxin transcription was about 2-fold
increased when the trichomonads were maintained under iron-restricted conditions. The N-terminal part
of the deduced frataxin protein sequence contained an 8 amino acid extension similar to presequences
targeting the proteins to hydrogenosomes. It contained an SRS/IM motif with arginine at position –2
relative to the putative cleavage site, which further suggested translocation of frataxin to the organelles.
To study the frataxin subcellular localization, we constructed a vector for stable transfection of T.
vaginalis. The frataxin gene was fused in frame with a doubled hemaglutinin tag (HA), and transfected
cells were selected for neomycin resistance. The 5´ and 3´ untranslated regions of the gene coding for
hydrogenosomal malic enzyme, were used to ensure efficient frataxin transcription.
The phylogenetic analysis showed affinity of T. vaginalis frataxin to other eukaryotic homologues,
although it represents the most divergent type. Together with the recent study of a cysteine desulfurase
(IscS) in T. vaginalis, these analyses indicate a common mechanism for FeS cluster assembly in
mitochondriate and amitochondriate eukaryotes.
POSTER 140
QUANTITATIVE GENETIC ANALYSIS OF BRAIN AND PERIPHERAL IRON IN MICE
B. C. Jones, J. Wiesenger, J. Bouwen, K. McCarthy, J. L. Beard. Departments of Biobehavioral Health,
Nutrition, and Neuroscience and Anatomy, The Pennsylvania State University.
Iron management in the brain is crucial for normal development and functioning. Too little iron in infancy
and the brain fails to develop properly. In aged individuals, too little iron contributes to diseases such as
restless legs syndrome and neuroleptic malignant syndrome. Too much iron has been implicated both to
alter the course of neurobehavioral development and in neuro-degenerative disease. Because of earlier
research that showed genetic-based differences in brain and peripheral iron content, we sought to
examine these genetic differences further using a panel of recombinant inbred (RI) mice. Male and
female (n=5-7) mice from 15 of the BXD/Ty RI panel were assayed for regional brain iron content and
liver iron content. The brain regions assayed were those shown previously to be sensitive to iron
deprivation and included the medial prefrontal cortex (MPF), nucleus accumbens (NA), caudate-putamen
(CP) and ventral midbrain (VMB). Because of its importance in mesolimbic and nigrostriatal dopamine
systems, our focal tissue was the VMB, containing the ventral tegmentum and substantia nigra. This area
contains the perikarya of the dopamine neurons that project to NA and VMB. We first performed some
genetic correlations, using strain means as “raw” data in order to determine which organ iron contents
were related. We observed correlation coefficients of -0.29 and -0.47 for males and females,
respectively, between VMB and liver iron content. While these correlations were suggestive, they were
not statistically significant. This suggests to us that peripheral and central iron regulatory systems are
weakly linked, if at all. We also observed robust correlations of 0.74 and 0.61 (p<0.05 each) between
VMB and CP iron contents for males and females and 0.53 and 0.62 (p<0.05 each) respectively between
VMB and NA iron contents. Correlations between VMB and MPFC for iron content were 0.13 and 0.25
respectively and nonsignificant. Heritability estimates for iron content in the VMB were 0.48 for both
sexes and in the liver, 0.9 and 0.75, for males and females respectively. VMB iron contents were then
subjected to quantitative trait loci (QTL) analysis to identify associated chromosomal polymorphic
locations. QTL analysis revealed several suggestive loci in both sexes. Most notably, QTL analysis
identified a marker in common for males and females on chromosome 7 (D7Mit371) and one unique to
males on chromosome 14 (D14Mit129). These data must be considered as preliminary because only 15
of the RI strains were available for study. Nevertheless the quantitative genetic approach is shown here
as heuristic for describing and then understanding the genetic basis for individual differences in brain and
peripheral iron management.
POSTER 141
THE MITOCHONDRIAL PROTEIN FRATAXIN CHAPERONES AND STORES IRON BY COUPLING
STEPWISE ASSEMBLY WITH A TWO-PHASE IRON OXIDATION REACTION
O. Gakh1, S. Park1, A. Mangravita2, G. C. Ferreira2 and G. Isaya1, 1Departments of Pediatric & Adolescent
Medicine and Biochemistry & Molecular Biology, Mayo Clinic and Foundation, Rochester, MN 55905;
2
Department of Biochemistry & Molecular Biology, College of Medicine and H. Lee Moffitt Cancer Center
and Research Institute, University of South Florida, Tampa, FL 33612
We have investigated the mechanism of action of frataxin, a conserved mitochondrial protein involved in
iron metabolism and neurodegenerative disease. Previous studies revealed that the yeast frataxin
homologue (mYfh1p) is activated by Fe(II) in the presence of O2 and assembles stepwise into a 48subunit multimer (α48) that sequesters >2000 atoms of iron in a ferrihydrite mineral core (1-4). Here we
show that mYfh1p assembly is driven by two sequential iron oxidation reactions: A fast ferroxidase
reaction catalyzed by mYfh1p induces the first assembly step (α →α3), followed by a slower autoxidation
reaction that promotes the assembly of higher order oligomers yielding α48. Depending on the ionic
environment, stepwise assembly is associated with accumulation of ≤50-75 Fe(II)/subunit. Initially, this
Fe(II) is loosely bound to mYfh1p and can be readily mobilized by chelators or made available to the
mitochondrial enzyme ferrochelatase to synthesize heme. However, as iron oxidation and mineralization
proceed, Fe(III) becomes progressively inaccessible and a stable iron-protein complex is produced. We
conclude that by coupling iron oxidation with stepwise assembly, frataxin can successively function as an
iron chaperon or an iron store.
Supported by grants RPG-96-051-04-TBE (to G.C.F.) from the American Cancer Society, and AG15709
(to G.I.) from the National Institute on Aging
1.
2.
3.
4.
Adamec J, Rusnak F, Owen WG, Naylor S, Benson LM, et al. 2000. Iron-Dependent SelfAssembly of Recombinant Yeast Frataxin: Implications for Friedreich Ataxia. Am J Hum Genet
67: 549-62
Gakh O, Adamec J, Gacy MA, Twesten RD, Owen WG, Isaya G. 2002. Physical evidence that
yeast frataxin is an iron storage protein. Biochemistry 41: 6798-804
Park S, Gakh O, Mooney SM, Isaya G. 2002. The ferroxidase activity of yeast frataxin. J Biol
Chem 30: 38589-38595
Nichol H, Gakh, O., Pickering, I.J., Isaya, G., Graham, N.G. Submitted. The structure of yeast
frataxin iron cores: An X-ray absorption spectroscopic study.
POSTER 142
IRON CHAPERON AND STORAGE PROPERTIES OF HUMAN FRATAXIN, THE PROTEIN DEFICIENT
IN FRIEDREICH ATAXIA
H. A. O’Neill1,2, S. Park1,2, O. Gakh1,2, A. Mangravita3, G. C. Ferreira3, M. Ramirez-Alvarado2, and G.
Isaya1,2, Departments of 1Pediatric & Adolescent Medicine and 2Biochemistry & Molecular Biology, Mayo
Clinic and Foundation, Rochester, MN 55905; 3Department of Biochemistry & Molecular Biology, College
of Medicine and H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa,
FL 33612
Friedreich ataxia (FRDA) is an autosomal recessive degenerative disease caused by a deficiency of
frataxin, a conserved mitochondrial protein involved in iron metabolism. When expressed in E. coli, the
mature form of human frataxin assembles into a stable homopolymer that binds ~10 atoms of iron per
subunit. The iron-loaded homopolymer is detected on non-denaturing gels by either protein or iron
staining demonstrating a stable association between frataxin and iron. As analyzed by gel filtration and
electron microscopy, the homopolymer consists of globular particles of ~1 MDa and ordered rod-shaped
polymers of these particles that accumulate small electron-dense cores (1) the structure of which
resembles 6-line ferrihydrite and not other minerals (2). Near and far UV circular dichroism (CD)
spectroscopy of purified monomer and polymer revealed structural differences between these two forms.
The polymer has an increase in the beta-sheet content as compared to the monomer suggesting that the
monomer undergoes a conformational change upon assembly. This change is reversible; when the
polymer is disassembled with increasing concentrations of SDS, the far UV CD spectrum of the polymer
converts to the spectrum characteristic of the monomer. During iron loading of the polymer, a ferroxidase
reaction with a stoichiometry of 2 Fe(II)/O2 is detected at Fe(II)/subunit ratios <0.5. When the polymer is
incubated with an excess of ferrous iron, ferroxidation is rapidly overcome by a slower autoxidation
reaction with a final stoichiometry of ~4Fe(II)/O2. The ferrous iron accumulated inside the polymer is
available to the chelator α,α’-bipyridine as well as purified ferrochelatase allowing production of heme
under aerobic conditions in the presence of protoporphyrin IX. Iron becomes progressively less available
as it is oxidized within the polymer. Similar to yeast frataxin(3-5), human frataxin has the ability to
chaperon and store iron.
Supported by grants RPG-96-051-04-TBE from the American Cancer Society (to G.C.F.), grant AG15709
from the NIH/NIA (to G.I.), and a grant from the Muscular Dystrophy Association (to G.I.). H.O. is
supported by NRSA 44748 from the NIH/NINDS.
1.
2.
3.
4.
5.
Cavadini P, O'Neill HA, Benada O, Isaya G. 2002. Assembly and iron binding properties of
human frataxin, the protein deficient in Friedreich ataxia. Hum Mol Genet 33: 217-27
Nichol H, Gakh, O., Pickering, I.J., Isaya, G., Graham, N.G. The structure of yeast frataxin iron
cores: An X-ray absorption spectroscopic study. Submitted.
Adamec J, Rusnak F, Owen WG, Naylor S, Benson LM, et al. 2000. Iron-Dependent SelfAssembly of Recombinant Yeast Frataxin: Implications for Friedreich Ataxia. Am J Hum Genet
67: 549-62
Gakh O, Adamec J, Gacy MA, Twesten RD, Owen WG, Isaya G. 2002. Physical evidence that
yeast frataxin is an iron storage protein. Biochemistry 41: 6798-804
Park S, Gakh O, Mooney SM, Isaya G. 2002. The ferroxidase activity of yeast frataxin. J Biol
Chem 30: 38589-95
POSTER 143
DEGRADATION OF FRATAXIN MONOMER BY A THERMOLYSIN-LIKE MITOCHONDRIAL
PEPTIDASE
N.E. Babady, H.A. O’Neill, R. Punyashthiti and G. Isaya, Department of Pediatric & Adolescent Medicine
and Biochemistry & Molecular Biology, Mayo Clinic and Foundation, Rochester, MN.
Friedreich Ataxia (FRDA) is a severe neurodegenerative disease with onset in early childhood.
This autosomal recessive disease is caused by expansion of an intronic GAA repeat in gene X25, which
encodes the protein frataxin. The expansion causes a reduction rather than a loss of function of frataxin
(1). Our laboratory has proposed that frataxin is a mitochondrial iron-storage protein. Reduced levels of
frataxin result in an inability to handle mitochondrial iron properly, which leads to impaired oxidative
phosphorylation and increased oxidative stress. Currently, there is no cure for FRDA.
In previous studies, Cavadini et al. (2) characterized the two-step processing of human frataxin by
mitochondrial processing peptidase (MPP). Frataxin was shown to undergo a sequence of cleavages
from precursor (p-fxn) to intermediate (I-fxn) to mature (m-fxn) form, carried out by MPP. Two additional
products, d1-fxn and d2-fxn, were observed and shown to result from N-terminal cleavage of m-fxn. These
degradation products were hypothesized to result from the activity of an unknown protease. Here, we
present the initial characterization of this protein, designated Frataxin Degrading Peptidase (FDP).
Total cell extracts from mouse heart, human heart and different systems expressing human
frataxin (E.coli, S.cerevisiae) were analyzed by SDS-PAGE and western blotting with polyclonal anti
human frataxin antibody. The degradation products, d1-fxn and d2-fxn, were observed in all these
systems. Mutant FRDA mice expressing 30-40% residual frataxin show low steady-state levels of d2-fxn
indicating that the FDP is constitutively expressed regardless of frataxin levels. The two cleavage sites
were then identified through purification of d1-fxn and d2-fxn from E.coli cells expressing m-fxn, and
determination of their molecular mass by electrospray ionization mass spectrometry (ESI-MS). The
products were shown to corresponds to residues 75-210 (d1-fxn, MW: 15,025) and residues 78-210 (d2fxn, MW: 14,666). The cleavage sites were found to be similar to the consensus sequence of the
prokaryotic protease thermolysin. In the presence of thermolysin cleavage to d2-fxn was observed and
was inhibited by metal chelators. In addition, a recombinant m-fxn with a single amino acid substitution
introducing an acidic residue (D) at the P1 position of the d1-fxn cleavage site inhibited degradation of mfxn.
In conclusion, a conserved pattern of degradation, m-fxn to d1-fxn to d2-fxn, is observed during
import of the human frataxin precursor into isolated rat liver mitochondria, during expression of the human
frataxin precursor in S.cerevisiae or under native conditions in the mouse heart. The peptidase
responsible for this reaction in E.coli has a cleavage specificity similar to that of thermolysin and is
inhibited by metal chelators. These results support the hypothesis that a thermolysin-like
metallopeptidase of prokaryotic origin is responsible for degradation/inactivation of human frataxin in the
mitochondrial matrix. This could be a unique mechanism for regulating the level of the iron storage protein
frataxin, which lacks a traditional iron responsive element. Given that FRDA is caused by a reduction in
the level of frataxin, the characterization of FDP could lead to the development of specific FDP inhibitors
to slow the rate of frataxin turnover and increase the levels of functional protein.
This work was supported by a grant from the Muscular Dystrophy Association
1. Campuzano,V., et al., 1997. Frataxin is reduced in Friedreich ataxia patients and is associated with
mitochondrial membranes. Hum Mol Genet 6:1771-80.
2. Cavadini, P., et al., 2000a. Two-step processing of human frataxin by mitochondrial processing
peptidase: precursor and intermediate forms are cleaved at different rates. J Biol Chem 275: 4146941475.
POSTER 144
DETERMINANTS OF SERUM FERRITIN IN THE METABOLIC SYNDROME
C. Bozzini, O. Olivieri, G. Villa, N. Martinelli, A. Bassi, F. Pizzolo, S. Friso, V. Lotto, R. Corrocher, D.
Girelli.
Department of Clinical and Experimental Medicine, and Institute of Clinical Chemistry, University of
Verona
Background: Serum ferritin is frequently increased in subjects with one or more of the features of the
Metabolic Syndrome (MS). The mechanisms underlying the relationship between ferritin and MS are
poorly understood. Recently, it has been highlighted an important association between MS and C-reactive
protein, a sensitive marker of inflammation. The aim of this study was to determine which component of
the MS best correlates with serum ferritin.
Methods: we studied 216 patients of both sexes with MS, defined as the presence of at least three of the
2
following features: obesity (BMI >27 Kg/m ), fasting glucose ≥110 mg/dl or established diabetes mellitus,
high triglycerides (≥150 mg/dl), low HDL-cholesterol (<40 mg/dl in males; <50 mg/dl in females),
hypertension. 485 subjects without MS were used as controls. Besides serum ferritin levels and the
above mentioned parameters, in all subjects we assessed fasting insulin (from which Insulin Resistance
was calculated following the Homeostasis Model Assessment), high sensitivity C-reactive protein
(HSCRP), and the presence of the C282Y mutation in the HFE gene.
Results: serum ferritin was higher in MS subjects than in controls (P<0.001). Similarly, the prevalence of
hyperferritinemia (defined as ferritin levels above the sex-specific upper quintiles of the control
distribution) was higher in MS subjects than in controls (25.5% vs 18.6%, P<0.05 by χ2). Serum ferritin
increased linearly with the increasing number of MS features. In a series of logistic regression models
(using hyperferritinemia as dependent variable) or linear regression model (using log-transformed ferritin
values), HSCRP in the upper quintile (OR 2.37, 95% CI 1.13 to 4.95) and fasting glucose ≥110 mg/dl (OR
2.28, 95% CI 1.36 to 3.83) emerged as the only significant and independent predictors of ferritin values.
Main conclusions: among the classical features of MS, high glucose levels appear as the best predictor of
serum ferritin. Data on HSCRP suggest that subclinical inflammation independently contribute to further
increase serum ferritin in MS.
POSTER 145
EXAMINATION OF THE ACTIVE SITE OF HUMAN FERROCHELATASE
H.A. Dailey, C.-K. Wu, P. Horanyi, and W. Missaoui, Biomedical & Health Sciences Institute, University of
Georgia, Athens GA 30602
Ferrochelatase catalyzes the terminal step in heme biosynthesis, the insertion of ferrous iron into
protoporphyrin to form protoheme IX. In eukaryotes this enzyme is nuclear encoded, synthesized in the
cytoplasm in a precursor form and translocated into the mitochondrion (and chloroplasts in plants). The
mature enzyme is bound to the matrix side of the inner mitochondrial membrane. In animal cells
ferrochelatase is a homodimer with monomer molecular weight of approximately 43,000. It possesses a
[2Fe-2S] cluster that is coordinated by four cysteine residues.
The crystal structure of human ferrochelatase has previously been determined at 2.0 Ǻ and clearly
demonstrated the presence of the active site pocket that is present on a hydrophobic face of the molecule
that is proposed to face the mitochondrial membrane. The active site pocket contains several highly
conserved amino acid residues including R164, Y165, H263, F337, D340, H341, and Q343.
Currently two models for catalysis of iron insertion exist. One, based upon the crystal structure of the
monomeric, water soluble Bacillus subtilis ferrochelatase, proposes that deprotonation of the porphyrin
macrocycle and iron insertion are catalyzed by the conserved active site histidine (H263 in human). In
this model both deprotonation and metal insertion occur from a single side. The second model is based
upon the crystal structure of human ferrochelatase, a number of kinetic studies on specific mutants, and
nonenzymatic, solution model studies. This second model proposes that deprotonation occurs via the
histidine, but that metallation occurs from the opposite side of the active site pocket, possibly involving
R164 and Y165. Both models suggest that the metallation reaction involves distortion of the porphyrin
macrocycle.
In the present work we have examined the crystal structures of three mutants of human ferrochelatase :
H263C, H341C, and F337A. The mutant H263C has no enzyme activity while the other two mutants
have significantly decreased activity. Mutation of H263 results in the reorientation of R164, H341, Q343
and F337 in the active site. Likewise the mutant H341C possesses the same reorientation of R164, Q343
and F337. No other residue side chain in the molecule appears to change orientation. However, in the
mutant F337A the residues of H341, Q343 and R164 retain their wild-type positions.
These data are explained by the fact that a hydrogen bond network exists among residues H263, H341
and Q343 which is disrupted by mutation of any of these residues. Normally F337 is restrained from
movement by the presence of the side chain of Q343. However, disruption of the hydrogen bond network
and the reorientation of Q343 allows F337 to swing into the active site pocket. Since F337A has no effect
on the bond network, there is no movement of side chains in this mutation. We propose that the
observed reorientation of this select set of residue side chains mimics what occurs during the catalytic
cycle of the enzyme. We propose a model where deprotonation of the pyrrole proton of the porphyrin
macrocycle via transfer to H263 causes a disruption of the hydrogen bond network with the resultant
movement of F337. The phenyl ring of F337 then participates in the distortion of the macrocycle thereby
facilitating iron insertion.
POSTER 146
GENE EXPRESSION PROFILING OF b2MICROGLOBULIN KNOCK-OUT MICE USING THE "IRON
CHIP"
Martina Muckenthaler 1, Pedro Rodrigues 2,3, Maria Macedo 3, Belen Minana1, Elsa Cardoso3, Matthias
W. Hentze 1 and Maria de Sousa2,3
EMBL (1) Instituto de Ciências Biomédicas Abel Salazar (2) and IBMC (3)
Heidelberg, Oporto University
There is growing evidence that the immunological system has a role in the regulation of iron overload.
Recent work indicates that MHC class I itself may be involved (1). The mechanism or mechanisms at play
are currently unknown. b2microglobulin (b2m-/-) knock-out mice are a previously established model of
spontaneous iron overload resembling genetic hemochromatosis (2). Here we present the analysis of
genes involved in iron metabolism in b2m-/- mice using a specialized cDNA-based microarray platform
('IronChip' Version 2.0) (3). Male b2m-/- and wild-type mice on a B6 genetic background were maintained
on a standard diet and sacrificed at
3.5 months of age. Liver and duodenum were removed and snap frozen in liquid nitrogen. Liver nonheme iron was measured by the bathophenanthroline method. Total RNA was extracted from different
tissues and b2m-/- mice were analyzed on the 'IronChip' compared to wild-type B6 mice. The differential
expression of selected genes was confirmed by Northern analysis. Non-heme iron. As reported earlier (3)
the hepatic iron content of the ko mice was significantly higher (1087±301 mg/g dry wt) than that in B6
mice (293±59 mg/gdry wt).
Iron gene profiles. Distinct iron gene profiles were observed in the duodenum and liver of b2m-/- mice
compared to B6 controls. Duodenum: The iron transporters DMT1 and ferroportin1 show increased
mRNA expression in the b2m-/- mice. Liver: The mRNA level of TfR-1 is decreased, and expression of Lferritin and ferroportin 1 is increased in b2m-/- mice, while DMT1 is equally expressed in b2m-/- mice and
control mice. These results may help explain the spontaneous iron overload in b2m-/- mice. They also
point to tissue differences in the development of iron overload. The increase of the duodenal iron
transporter expression may explain the documented increase in iron absorption in b2m-/- mice (4). The
increase in L-ferritin mRNA and the decrease in TfR1 mRNA in the liver indicate that this organ is
responding "normally" to the iron accumulation resulting from increased mucosal transfer at the intestinal
level (4). It is interesting to note that mice with defects in the hereditary hemochromatosis gene HFE (B6
and 129S6/SvEvTac genetic background, 8 weeks old) do not respond with increased duodenal iron
transporter mRNA levels
(see also abstract by Muckenthaler et al.). The expression increase in DMT-1 and ferroportin1 mRNA
presented here highlights differences between Hfe-/- and b2m-/- mice and may contribute to the
understanding of why compound mutant mice lacking both Hfe and its interacting protein b2m deposit
more tissue iron than mice lacking Hfe only (Levy et al., 2000).
1. Cardoso, EMP et al. Blood, 100: 4239-4241, 2002
2. De Sousa et al., Immunol Letters 39:105-111, 1994
3. Muckenthaler et al., Blood (in press).
4. Santos, M et al.,J Exp Med. 184: 1975-1985, 1996
5. De Sousa, M. Soc.Exp.Biol.Symp.32:393-410, 1978
6. Levy, J.E. etal., J Clin Invest 105:1209-16, 2000
POSTER 147
IRON CHELATION INHIBITS CYTOMEGALOVIRUS-INDUCED RECOVERY OF HOST CELL
MITOCHONDRIAL TRANSMEMBRANE POTENTIAL
W. Crowe*, L. Maglova*, A. Reeves#, P. Ponka&, J. Russell*. *Syracuse University, #Cornell University,
&
McGill University
Shortly following infection of cultures MRC-5 fibroblasts by human cytomegalovirus (HCMV), host cells
shrink, labile metal levels increase, and mitochondrial dysfunctions are induced. We have observed that
the mitochondrial transmembrane potential (∆ΨM) depolarizes, and that initially interconnected
mitochondrial filaments, fragment and cluster in the perinuclear region. These observed phenomena are
consistent with an apoptotic-like response.
Subsequent events that may be necessary for host cell survival may include dispersion of
numerous punctuate mitochondria and repolarization of ∆ΨM, reductions in labile metal levels and host
cell enlargement (cytomegaly). Iron chelation (salicylaldehyde isonicotinoyl hydrazone, SIH) will however
prevent many of these apparent HCMV antiapoptotic processes and will reduce HCMV yields by more
than 3 log units.
To evaluate how the functional activity of mitochondria is affected by HCMV infection,
deconvolution microscopy was used to measure changes in fluorescence of mitochondria loaded with the
mitochondrial voltage-sensitive dye, JC-1. Image stacks of both green fluorescence (528 nm)
representing low ∆ΨM and red fluorescence (617 nm) represents high ∆ΨM were collected from living cells
at different stages of infection. The ratio (617/528) of deconvoluted fluorescence at the medial plane was
used to obtain an index of ∆ΨM and the results are reported in the table below. In addition, we are using
the VisionX software system (Cornell University) to automatically trace each visible mitochondrial filament
and thereby measure its length and characterize its form.
mock
HCMV
6 h.p.i.
HCMV
24 h.p.i.
HCMV
48 h.p.i.
HCMV
72 h.p.i.
∆ΨM control
(n=3)
1.00 ± 0.18
0.31 ± 0.03
0.94 ± 0.10
1.73 ± 0.24
1.69 ± 0.17
∆ΨM SIH
(n=3)
-
0.59 ± 0.13
-
0.47 ± 0.07
-
Data were normalized to mean mock levels.
As can be seen from the data presented in the table there was ~70% decrease in ∆ΨM of HCMV-infected
cells at 6 hours post infection (h.p.i.) compared to mock-infected cells (cells treated with cell lysate without
virus). However, by 24 h.p.i. the ∆ΨM had recovered to mock levels and by 48 h.p.i. had increased to
~170%. Furthermore, when the iron chelator SIH (100 µM) was included in the cell culture media
following infection, there was an apparent suppression of the initial depolarization at 6 h.p.i. and the
hyperpolarization at 48 h.p.i. was prevented.
The HCMV-induced hyperpolarization of ∆ΨM, redistribution and development of punctate mitochondria
are consistent with HCMV restoring mitochondrial function. Thus this ability of HCMV to reverse an
apparent apoptotic-like response, that accompanies infection, may be part of the HCMV antiapoptotic
response. Iron chelation (SIH) was able to inhibit the recovery of host cell mitochondrial function
suggesting that the anti-HCMV action of SIH may be linked to its ability to inhibit HCMV induced recovery
and stimulation of mitochondrial functional activity.
POSTER 148
MITOCHONDRIAL CARRIER PROTEINS INVOLVED IN IRON HOMEOSTASIS
E. Lesuisse (1), E. R. Lyver (2), S. A. B. Knight (2), A. Dancis (2). (1) Laboratoire d’Ingénierie des
Protéines et Contrôle Métabolique, Institut Jacques Monod, Université Paris, France (2) Dept. Medicine,
Div Hematology/Oncology, University of Pennsylvania, Philadelphia, USA
Mitochondrial carrier proteins (MCPs) are a large family of proteins, consisting of 35 members in
Saccharomyces cerevisiae. These proteins are present in metazoans but not in bacteria, and they
transport varied substrates between cytoplasm and mitochondria. We wondered if one or more of these
transporters plays a role in iron metabolism. Haploid yeast strains each deleted for a single MCP were
analyzed for regulated cell surface ferric reductase and high-affinity ferrous transport activities. Two
mutants were identified with altered activities: yhm1 and mrs3. The yhm1 deletion had non-repressing
iron uptake activities and also exhibited loss or damage to mitochondrial DNA (mtDNA). The mrs3
deletion had abnormal levels when compared with the wild-type but the effect was not as dramatic as
yhm1. Mrs3 is highly similar to Mrs4, with 70% amino acid identity. In order to explore the functional
redundancy between these two genes, a mrs3/4 double mutant was generated. The level of
misregulation of iron uptake activities in the mrs3/4 mutant was similar to that observed for the yhm1
mutant, although only the yhm1 mutant underwent mtDNA loss. Iron uptake in the mutants from different
iron sources was increased including from ferric citrate, ferrioxamine B, ferrichrome and TAF. Iron uptake
with Fe-55 radionuclide was measured over 1 h. Both the yhm1 and mrs3/4 mutants had increased and
similar cellular iron levels compared with the control strains, but the distribution within the cells showed
striking differences. The labeled cells were fractionated into highly purified mitochondria and cytoplasmic
fractions. The iron distribution within yhm1 cells showed very high mitochondrial iron and low cytoplasm
iron levels, while in the mrs3/4 cells there was high cytoplasmic iron levels and lower mitochondrial iron
levels. Future work will be directed towards determining how these mitochondrial carrier proteins alter
iron distribution within cells.
POSTER 149
TRANSLATIONAL SILENCING OF CERULOPLASMIN EXPRESSION IN MONOCYTIC U937 CELLS IS
DIRECTED BY A NOVEL STRUCTURAL ELEMENT IN THE CERULOPLASMIN mRNA 3’UNTRANSLATED REGION
P.L. Fox, P. Sampath, B. Mazumder, and V. Seshadri, Department of Cell Biology, Lerner Research
Institute, Cleveland Clinic Foundation, Cleveland, OH
Ceruloplasmin (Cp) is a multi-functional, copper protein made by the liver and secreted into the blood. Cp
has important roles in iron homeostasis and in inflammation. Its role in iron metabolism was originally
proposed because of its ferroxidase activity, and because of its ability to stimulate iron loading into apotransferrin and iron efflux from tissues. This role has been confirmed by the finding of iron overload in
patients with hereditary Cp deficiency and in mice with targeted Cp gene disruption. A role for Cp in
inflammation is suggested by its increased plasma abundance during the acute phase reaction, by
increased hepatocyte expression by inflammatory mediators, and by its synthesis by activated
monocyte/macrophages. Cp’s function in macrophages is not clear but it may regulate iron release or it
may participate in host-defense against pathogenic microbes.
Transcript-selective translational control of eukaryotic gene expression is often directed by a structural
element in the 3’-untranslated region (UTR) of the mRNA. In the case of Cp, we have previously shown
that synthesis of the protein by U937 monocytic cells is induced by interferon (IFN)-γ but is halted by a
delayed translational silencing mechanism requiring the binding of a cytosolic inhibitor to the Cp 3’-UTR.
Silencing requires the essential elements of mRNA circularization, i.e., eIF4G, poly(A)-binding protein,
and poly(A) tail. By progressive deletions from both termini, we have now determined the minimal silencing element in the Cp 3’-UTR. A minimal, 29-nt element was shown by RNA electrophoretic mobility shift
assay to be sufficient for binding of the IFN-Gamma-Activated Inhibitor of Translation (GAIT), an as-yet
unidentified protein or complex. The interaction of GAIT with the Cp 3’-UTR GAIT element was shown to
be functional by an in vitro translation assay using a rabbit reticulocyte lysate; a synthetic GAIT element
used as a decoy completely overcame translational silencing by inhibitory extracts from IFN-γ-treated
U937 cells. Mutation analysis showed that the GAIT element contained a 5-nt terminal loop, a 3-bp helix,
an asymmetric internal bulge, and a proximal 6-bp helical stem. Two invariant loop residues essential for
binding activity were identified. A unique feature of the structure is the distal, 3-bp helix that requires weak
base-pair interactions for activity. Ligation of the GAIT element immediately downstream of a luciferase
reporter conferred the translational silencing response to the heterologous transcript in an in vitro
translation assay and in vivo in transfected U937 cells. The silencing of Cp translation in IFN-γ-activated
monocytic cells may prevent excess Cp accumulation in inflammatory sites, and may minimize adverse
consequences of Cp-mediated oxidative damage or alterations to iron homeostasis during inflammation.
It will be of interest to determine whether this translational control mechanism is common to other
functionally-related transcripts expressed by monocyte/macrophages.
POSTER 150
TARGETED DISRUPTION OF THE MURINE DMT1 GENE
H. Gunshin, C. DiRenzo, Y. Fujiwara, N.C. Andrews, Children's Hospital Boston, Harvard Medical School,
Howard Hughes Medical Institute
Divalent metal transporter 1 (DMT1) is a mammalian iron transporter that has been shown to play an
important role in iron uptake by duodenal enterocytes and erythroid precursors. Its role in liver iron
uptake has not yet been studied directly. We injected iron dextran into homozygous mk mutant mice,
which carry a severe loss-of-function mutation (G185R) in the gene encoding DMT1. This resulted in
marked Kupffer cell and hepatocyte iron accumulation, suggesting that full DMT1 function was not
necessary for liver iron uptake. To address the possibility that the G185R form of DMT1 retains residual
iron transport activity, we developed mice homozygous for a null mutation in the DMT1 gene (universal
DMT1-/-) by homologous recombination in mouse embryonic stem cells, production of chimeric mice, and
passage of the null DMT1 allele through the mouse germline.
Universal DMT1-/- mice are viable but very pale compared to their wild type and heterozygous (DMT1+/+
and +/-) littermates. Universal DMT1-/- mice showed severe postnatal growth-retardation, and they
survived no more than 7 days. Their body weights at postnatal day (P) 4 were approximately half those
of DMT1+/+ and +/- littermates, though they had been only slightly runted at birth.
Peripheral blood smears from universal DMT1-/- mice showed hypochromic, microcytic cells with marked
anisocytosis and poikilocytosis. The animals were severely anemic at P4, though non-heme Fe
concentrations in liver, spleen and kidney were elevated as compared to unaffected controls. When
corrected for organ and body weights, the amounts of iron in spleen and kidney were similar to controls,
but liver iron remained elevated. Iron dextran injection led to further iron accumulation in DMT1-/- Kupffer
cells and hepatocytes, again suggesting that an alternative, non-DMT1 iron transport pathway was
operative.
DMT1+/- mice exhibited lower liver iron concentrations than DMT1+/+ mice, though there was no
significant difference in their hematological parameters. Some DMT1+/- mice appeared smaller than their
wild type littermates at P10, but their growth caught up by the time they were weaned. This suggests that
even a mild deficit in iron may influence early growth.
Observations from this study lead to the following conclusions: 1. Universal DMT1-/- mice are viable and
have high liver iron stores, indicating that materno-fetal iron transfer does not require fetal DMT1. 2.
Universal DMT1-/- mice display severe anemia but do produce some hemoglobin, suggesting that DMT1
is of primary importance in erythroid iron acquisition, but not absolutely required. 3. Universal DMT1-/mice generally die within several days after birth. The precise cause of death has not yet been
determined. Further investigation will be necessary to determine whether the phenotype of universal
DMT1-/- mice is more severe than that of homozygous mk mice carrying the G185R point mutation in
DMT1.
POSTER 151
IN VIVO ANALYSIS OF HUMAN FERROCHELATASE MUTANTS
A. E. Medlock, T. C. Ross, W. Missaoui, & H. A. Dailey, Biomedical and Health Sciences Institute,
University of Georgia, Athens, GA 30602
Ferrochelatase catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme IX.
The enzyme from many sources including human, Bacillus subtilis, and Saccharomyces cerevisiae has
been well characterized in terms of structure and in vitro assays, while little is known of the origin of the
ferrous iron or the entry or exit of substrates and products from the active site in vivo. An in vivo assay for
B. subtilis has been described by Olsson et al. ((2002) J. Bact., 184, p 4018-4024). This prokaryotic
system has provided some interesting results that contrast observations from the in vitro assays leading
the authors to question the unnatural environment provided by the in vitro assays.
To asses our in vitro assay and to better understand the acquisition of ferrous iron and porphyrin
and the release of heme in eukaryotic organisms, we are constructing an in vivo assay system to look at
the effect of mutations both within the active site and also distant from the active site. We have
constructed a yeast vector based on the pRS425 plasmid (Christianson, T. W., Silorski, R. S., Dante, M.,
Shero, J. H., & Hieter, P. (1992) Gene, 110, p. 119-122) that contains the S. cerevisiae ferrochelatase
promoter and targeting sequence upstream of NcoI and HindIII restriction site. We have cloned wild-type
human ferrochelatase and approximately a dozen human ferrochelatase mutants into our vector and have
studied their activity in terms of supporting the growth of the ferrochelatase-deficient (Hem15) strain of S.
cerevisiae GG231-4A (Labbe-Bois (1990) JBC, 265, p. 7278-7283). We have compared the growth rates
GG231-4A expressing these mutant forms of ferrochelatase to that of the wild-type enzyme to asses their
activity. Growth of these mutants is similar to what we see in our in vitro assays most notably mutations
that reside in or near the active site. We have looked at several mutations that reside at the surface of
the enzyme away from the active site, such as S130A and D383A, and shown that growth rates of
GG231-4A expressing these mutants are slower. We propose that this in vivo assay along with the in
vitro assay may provide us with a more complete picture of enzyme activity and provide insight into entry
of substrates and release of product.
POSTER 152
CHARACTERIZATION OF IRON METABOLISM IN MICE WITH A TARGETED DELETION OF IRON
REGULATORY PROTEIN 1
Esther Meyron-Holtz1, Timothy LaVaute2, Manik Ghosh1, Tracey A. Rouault1
1
National Institute of Child Health and Human Development, Bethesda, MD 20892,2 Department of
Psychiatry, University of Wisconsin-Madison Madison, WI
To understand the mechanism of systemic iron regulation we generated mice with targeted deletions of
iron regulatory proteins, IRP1 and IRP2. The IRP2-/- mice develop a progressive neurodegenerative
disease and acquire iron and ferritin (Ft) deposition in specific cell types in the intestinal mucosa and the
central nervous system. The IRP1-/- mice in contrast have no apparent pathology. Gel retardation
assays of many different tissues reveal that at steady state IRP1 is predominantly in its non-IRE binding
state and presumably functions mainly as an active cytosolic aconitase. Nevertheless the loss of IRP1
has some effect on the regulation of iron metabolism.
IRP1 and IRP2 distribution are distinct throughout the body. In situ hybridization, northern and western
blot indicate that kidney, liver and brown fat are tissues high in both IRP1 mRNA and protein, while
forebrain and cerebellum are rich in IRP2.
Ft levels in tissues from IRP1 and IRP2 knock out mice reflect this pattern. In normally IRP1 rich tissues
of the IRP1-/- mice, Ft levels are elevated compared to wild type mice. This suggests that those tissues
that depend mostly on IRP1 show some impairment in their ability to repress Ft synthesis when IRP1 is
absent. In contrast, tissues with predominant IRP2 expression show no misregulation of Ft in the
absence of IRP1 but severe Ft elevation when IRP2 is missing. We conclude that the loss of the
predominant IRP in a tissue will lead to misregulation of targets. However this misregulation leads to
severe pathology in the central nervous system only.
POSTER 153
SEVERE ANEMIA IN TRANSGENIC K19LT MICE, AN ANIMAL MODEL OF TUMORIGENESIS
R.Soares1,2, W. Jiang1, H. Makui1, F. Veillette1, A-M Mes-Masson1, M.Santos1,2
1
CHUM, Notre Dame hospital, Montreal, Canada; 2UnIGENe-IBMC, Univ. of Porto, Portugal;
Anemia is frequently encountered in cancer, due either to the malignant disease itself or to its
treatment. The importance of the management of anemia in cancer patients is highlighted by the fact that
anemia can produce both poor tumor oxygenation, which compromises therapy, and incapacitating
fatigue, which has an adverse effect in the quality of life. In fact, recent studies have shown that treatment
of anemia with recombinant erythropoietin (rEPO) may increase survival in cancer patients and reduce
the need for blood transfusions. However, only about 50% of patients respond adequately to rEPO.
The establishment of an experimental animal model of cancer-related anemia (CRA) can greatly help
the identification of new therapeutic targets and combination therapies for the management of anemia.
In this report, we characterize iron metabolism in Keratin 19 large T-antigen (K19LT) transgenic mice.
In this model, the expression of the polyomavirus large T-antigen, an immortalizing oncogene, is targeted
to epithelial cells by the keratin 19 promoter. These mice develop lung neoplasia and hyperplasias in
other tissues.
Erythroid parameters were determined in transgenic K19LT and control FBV mice. RBC counts, Hb
and HCT values were significantly decreased when compared to control mice, while MCV values were
increased. Transgenic K19LT mice had lower serum iron concentrations and transferrin saturation than
FBV control mice. Body iron stores were directly assessed by measuring iron concentrations in the liver
and the spleen. Transgenic K19LT mice had considerably lower hepatic and spleen iron concentrations
than controls, FBV mice. Finally, we studied the expression of several genes involved in iron metabolism,
such as Nramp2, ferroportin1, hepcidin 1 and 2, and transferrin receptor by Western blot, RT-PCR, and
flow cytometry. The expression pattern of these genes was altered in transgenic K19LT mice compared
to control mice.
Taken together, these results show that in transgenic K19LT mice besides cancer-related anemia
functional iron deficiency occurs. This mouse offers a sensitive model to test if the combination of rEPO
and iron is a reliable tool to correct CRA and whether or not such a combination has a negative or
positive effect towards tumor growth.
POSTER 154
HFE DEFICIENCY INCREASES SUSCEPTIBILITY TO CARDIOTOXICITY AND EXACERBATES
CHANGES IN IRON METABOLISM INDUCED BY DOXORUBICIN
Carlos J. Miranda1, Ricardo Soares1,3, Hortence Makui1, Marc Bilodeau2, Jeannie Mui4, Hojatollam Vali4,
Richard Bertrand1, Nancy Andrews5, Manuela M. Santos1, 3
Hôpital Notre-Dame and 2Hôpital Saint-Luc, Centre hospitalier de l'Université de Montréal, Montréal,
Québec, Canada
3
UnIGENe, Instituto de Biologia Molecular e Celular, Porto, Portugal
4
Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada
5
Howard Hughes Medical Institute, Department of Pediatrics, Harvard Medical School, Children's
Hospital, Boston, MA, USA
1
Anthracyclines such as doxorubicin (DOX) are considered to be among the most effective drugs for
the treatment of solid tumors. However, therapy with these agents is limited by the onset of acute and
chronic cardiotoxicity. Because the myocardial damage caused by anthracyclines is largely irreversible,
the identification of high-risk patients and prevention by the concurrent use of cardioprotective agents
would be as important as the efficacy of treatment.
The possible involvement of iron in DOX-induced cardiotoxicity became evident from studies in which
iron chelators were shown to be cardioprotective. Ultimately, excess body iron stores, especially in the
heart, may influence susceptibility to cardiotoxicity.
Iron overload is found in hereditary hemochromatosis, a genetic disorder prevalent in individuals of
European descent, which may lead to iron accumulation in the liver and heart, among other organs. The
defective gene in hemochromatosis, Hfe, encodes a MHC class I-like protein. We hypothesized that Hfe
deficiency may increase susceptibility to iron-related toxicity induced by DOX.
-/Acute cardiotoxicity and iron changes were studied after treatment with DOX in Hfe knockout (Hfe )
-/mice and wild type mice. DOX-induced iron metabolism changes were intensified in Hfe mice, which
accumulated significantly more iron in the heart, liver and pancreas, but less in the spleen compared to
wild type mice. In addition, Hfe-deficient mice exhibited significantly greater sensitivity to DOX-induced
elevations in serum creatine kinase and aspartate aminotransferase. Increased mortality after chronic
DOX treatment was observed in Hfe-/- mice (62%) and Hfe+/- mice (43%) compared to wild type mice
(12%). DOX-treated Hfe-/- mice had a higher degree of mitochondrial damage and iron deposits in the
heart than wild type mice.
These data demonstrate that Hfe deficiency in mice increases susceptibility to DOX-induced
cardiotoxicity and suggest that genetic mutations related to defects in iron metabolism may contribute to
its cardiotoxicity in humans.
POSTER 155
IDENTIFICATION OF MULTIPLE QUANTITATIVE TRAIT LOCI (QTL) THAT AFFECT SERUM, LIVER,
AND CEREBELLUM IRON LEVELS IN C57BL/6J AND DBA/2J MICE
G.S. Gerhard, M.A. Grundy, J. Abraham, T.J. Stout, D. Blizard, D. Vandenbergh, Z.K. Shihabi, J. Spicher,
G. Vogler, J. Lakoski, N. Sharkey, L. Larsson, J. Strauss, R. Mitchell, S. Hofer, G.M. McClearn, N.
Gorman, P.R. Sinclair, and M.J.Chorney.
Department of Pathology, Dartmouth Medical School, Hanover, NH; Penn State University Center for
Developmental and Health Genetics, University Park, PA; Department of Pathology, Wake Forest
University School of Medicine, Winston-Salem, NC; Departments of Biochemistry, and Pharmacology,
Dartmouth Medical School, Hanover, NH; Penn State College of Medicine, Hershey, PA.
Introduction: Despite the common use of inbred mouse strains for studies on iron metabolism, relatively
little data is available on the naturally occurring genetic differences that may impact such studies.
Several parameters of iron metabolism, including serum iron levels and hepatic iron stores, can differ by
several-fold among commonly used inbred mouse strains. We hypothesized that the genetic differences
among mouse strains contribute to the phenotypic variability in iron metabolism. To test this hypothesis,
we have performed quantitative trait locus (QTL) analysis on serum, liver, and cerebellum iron levels
measured on a population of 378 F2 mice derived from the C57BL/6J (B) and DBA/2J (D) inbred mouse
strains.
Methods: Serum iron levels were determined spectrophotometrically. Non-heme iron determination in
liver and cerebellum was performed by the method of Torrence and Bothwell adapted to a 96 well plate
format. DNA was harvested from each F2 mouse and subjected to genotyping at 94 polymorphic
markers spaced throughout the genome. QTL analysis was performed with the R/QTL software package.
Results: A significant gender difference was present in liver iron, with the average level in females over
twice that found in males. The mean serum iron level in males was about 10% higher than in females,
while no gender difference was found in cerebellum iron levels. No correlation in iron levels was found
among the three tissues. QTL analysis of serum iron data revealed three chromosomal loci with LOD
scores greater than 3.0. Two other loci with LOD scores of 2.5 were identified within 5 cM of the
chromosomal locations of HFE and beta-2-microglobulin, two genes known to be involved in the
regulation of serum iron. Sequence analysis identified amino acid substitutions in both genes in B and D
mice. For liver iron, a sex-specific QTL with a LOD score greater than 3 was found on the X
chromosome, with several autosomal QTL identified when gender was used as a covariate. In the
cerebellum, several QTL with LOD scores between 2 and 3 were found.
Discussion: Few studies have sought to analyze iron metabolism in multiple tissues. With no correlation
found between levels of iron in serum, liver, and blood, our results suggest that the regulation of iron
levels is tissue specific in these mice. Multiple genes also appear to influence iron levels in each
compartment. No potential candidate genes were identified at several QTL suggesting that novel genes
may be identified that are involved in iron metabolism. The approach used for these studies provides the
basis for subsequent efforts to identify these genes.
Conclusions: We conclude that iron levels in serum, liver, and cerebellum are influenced by multiple QTL
in BXD F2 mice and appear to be regulated as independent compartments.
POSTER 156
REVERSIBLE INHIBITION OF HEME CRYSTALLIZATION BY ANTIMALARIALS AND OTHER
COMPOUNDS: IMPLICATIONS FOR DRUG DISCOVERY
C. Chong and D. Sullivan
Dept. of Pharmacology, School of Medicine and W. Harry Feinstone Dept. of Molecular Microbiology and
During intraerythrocytic infection, Plasmodium falciparum parasites crystallize toxic heme
released during hemoglobin catabolism into insoluble crystals called hemozoin. The almost 0.4 M reactive
heme is prevented from production of oxygen radicals by Fenton chemistry. Quinolines like chloroquine
inhibit heme crystallization to kill the malaria parasite. The detailed kinetics of crystal inhibition by the
antimalarial quinolines have not been previously studied. Using a new high-throughput assay based on
the differential solubility of free versus crystalline heme in basic solutions, we examine the kinetics of
heme crystallization. Chloroquine (IC50 = 4.3 µM) and quinidine (IC50 = 1.5 µM) show reversible inhibition
of heme crystallization. This inhibition decreases by increasing amounts of heme crystal seed, but not by
greater amounts of heme substrate. An increase in pH from pH 4 to 4.8 decreases extension product.
The decrease reverses at a local maxima of pH 5.3, before the rapid decrease to zero at pH 6.0. The new
crystallization assay enabled a library screen of existing drugs for inhibition like that of the quinoline
antimalarials. Nitrogen heterocycle cytochrome P450 inhibitors also reversibly block heme crystallization,
including the azole antifungal drugs clotrimazole (IC50 = 12.9 µM), econazole (IC50 = 19.7 µM),
ketoconazole (IC50 = 6.5 µM), and miconazole (IC50 = 21.4 µM). Fluconazole does not inhibit. Subcellular
fractionation of parasites treated with subinhibitory concentrations of ketoconazole demonstrate
copurification of hemozoin and ketoconazole. Likewise, ketoconazole binds to heme crystals during in
vitro hemozoin extension assays. The abundance of FDA-approved drugs that inhibit cytochrome P450
by binding heme opens the possibility that currently approved medicines may be of interest in developing
new antimalarial treatments. Such antimalarials may hinder the development of resistance by attacking
the parasite in multiple ways.
POSTER 157
ELEVATED ZINC PROTOPORPHYRIN IX IN ANEMIC ERYTHROCYTES PROTECTS FROM MALARIA
DISEASE BY INHIBITION OF HEME CRYSTALLIZATION
D Sullivan, J. Iyer*, L. Shi and A. Shankar*
*Helen Keller International, Jalan Bungur Dalam 23A, Kemang, Jakarta, Indonesia
The intraerythrocytic Plasmodium falciparum parasite converts most of host hemoglobin heme
into a nontoxic heme crystal. Erythrocyte zinc protoporphyrin IX, normally present at 0.5 µM, which is a
ratio of 1:40,000 hemes, can elevate tenfold in some of the anemias associated with malaria disease
protection. In this work zinc protoporphyrin IX does not form crystals alone nor does it extend on
preformed heme crystals. Zinc protoporphyrin IX, like chloroquine, inhibits both seed crystal formation
and crystal extension with an IC50 of 5 µM. Field emission inlens scanning electron microscopy depicts
the transition and inhibition of heme monomer aggregates to heme crystals with and without seeding of
preformed hemozoin templates. The in vitro crystal product has a different morphology than purified P.
falciparum hemozoin heme crystals. Zinc protoporphyrin IX, like the quinolines, binds to heme crystals in
a saturable, specific, pH and time dependent manner. The ratio at saturation is approximately one zinc
protoporphyrin IX per 250 hemes of the crystal. Unlike the quinolines, zinc protoporphyrin IX binds
measurably in the absence of heme. Isolated ring and trophozoite stage parasites have an elevated zinc
protoporphyrin IX to heme ratio six to ten times that in the erythrocyte cytosol. The ratio of zinc
protoporphyrin IX to heme is also up to tenfold higher than normal within heme crystals purified from
Plasmodium parasites.
These data implicate a process where elevated zinc protoporphyrin IX in anemic erythrocytes
protects from malaria by inhibition of heme crystallization.
POSTER 158
A CHANGE IN STEADY STATE EXPRESSION OF THE DUODENAL IRON TRANSPORTERS IS NOT
REQUIRED FOR IRON OVERLOAD IN ADULT HFE DEFICIENT MICE
T. Herrmann1, M. Muckenthaler2, F. van der Hoeven3, K. Brennan2, S.G. Gehrke1, N. Hubert2, C. Sergi4,
H.-J. Groene3, I. Kaiser1, I. Gosch1, M. Volkmann1, H.D. Riedel1, M. Hentze2, A.F. Stewart5, W. Stremmel1
1
Department of Internal Medicine IV, University of Heidelberg, 69115 Heidelberg, Germany
2
European Molecular Biology Laboratory, 69117 Heidelberg, Germany
3
German Cancer Research Center, 69120 Heidelberg, Germany
4
Department of Paediatric Pathology, St. Michael’s University Hospital, Bristol, U. K.
5
Biotec, Technical University of Dresden, 01307 Dresden, Germany
Introduction:
Hereditary hemochromatosis (HH) patients show progressive iron overload as a result of increased
duodenal iron absorption. It was hypothesized that mutations in the HH gene HFE cause
misprogramming of the duodenal enterocytes as if they were iron deficient, resulting in increased iron
transporter expression. Previous reports concerning steady state expression levels of the duodenal iron
transporters DMT-1 and ferroportin in HH patients and animal models are controversial. In the present
study we generated an Hfe deficient mouse line. We quantified the expression of Dmt-1 (IRE, non-IRE
and 1A variant), ferroportin, Dcytb, hephaestin, and transferrin receptor 1 in Hfe -/- mice and control
wildtype littermates and analyzed the expression levels in regard to a possible correlation with liver iron
content.
Methods:
A mouse line with a conditional Hfe allele containing two loxP sites in intron 2 and intron 5, respectively,
was generated. By crossing this line with a general Cre deleter line we generated a mouse line carrying
an Hfe null allele lacking exons 3 to 5 which code for the alpha-2, the alpha-3 and the transmembrane
domain of the Hfe protein. To verify the phenotype, liver iron content was quantified by histochemistry and
atomic absorption spectroscopy, and serum transferrin saturation was calculated from total iron binding
capacity and serum iron level. The mRNA expression of the duodenal iron transporters Dmt-1 and
ferroportin in ten weeks old mice was quantified by Northern analysis and quantitative RT-PCR and
protein expression levels were analyzed by Western blotting. In addition, the mRNA expression of Dcytb,
hephaestin and transferrin receptor 1 in the proximal duodenum were analyzed by quantitative RT-PCR.
Results:
The generated Hfe deficient mice displayed the expected elevations of liver iron content and serum
transferrin saturation. The iron accumulated mainly in the parenchymal cells of the liver and was excluded
from the Kupffer macrophages. No difference in the expression of Dmt-1 and ferroportin was observed in
Hfe -/- mice in comparison to wildtype control littermates. Furthermore, hephaestin and transferrin
receptor 1 mRNA expression remained unaltered, while the brush border ferrireductase Dcytb was
significantly increased in Hfe -/- mice. No correlation between liver iron content and expression of any of
the analyzed genes could be detected.
Discussion:
Both mRNA and protein levels of Dmt-1 and ferroportin remain unaffected by the Hfe deletion. A lack of
correlation between Dmt-1 and ferroportin protein expression with the amount of liver iron suggests that
elevated iron transporter expression is not required for high liver iron overload. Upregulation of Dcytb
expression might increase the availability of ferrous iron for the apical iron transporter Dmt-1 and thus
increase the Dmt-1-mediated iron absorption without change in Dmt-1 expression level.
Conclusions:
These data show that the generated Hfe -/- mice do not display features of iron deficiency in the
duodenum, hallmarked by an increase in mRNA and protein levels of Dmt-1 and ferroportin. As the
protein levels of both iron transporters are unaffected by the Hfe mutation, the increase in iron absorption
observed in Hfe -/- mice is most likely mediated by an increase in Dmt-1 and/or ferroportin activity. The
increase in duodenal ferrireductase Dcytb mRNA levels may indicate a role of this protein in this process.
POSTER 159
ON LINE MONITORING OF IRON REDISTRIBUTION BETWEEN CYTOSOL AND MITOCHONDRIA IN
P19 DIFFERENTITATED NEURONAL CELLS
Silvina Epsztejn1, Ursula Rauen2, Reiner Sustmann3 and Z. Ioav Cabantchik1
1
Department of Biological Chemistry, Hebrew University, Jerusalem 91904, Israel , 2 Institut für
Physiologische Chemie Universitat Essen, D-45122 Essen and 3 Institut für Organische Chemie,
Universitat Essen, D-45117, Essen Germany
Mitochondria play an important role in cell iron metabolism due to their direct involvement in the synthesis
of heme and S-clusters and in cell iron homeostasis. Several diseases have been associated with gene
products that are expressed in mitochondria and affect those functions and others whose precise role in
iron metabolism has yet to be identified. Neurodegenerative and other diseases involve cell death
phenomena that have been linked to mitochondrial damage caused by reactive oxygen species. The
latter are assumed to be generated by pro-oxidants formed in mitochondria (M) and catalyzed by labile
iron, based on the protective effect afforded by iron chelators. However the relationship between
mitochondrial labile iron pool (mLIP) and cytosolic labile iron pool (cLIP) has not been characterized in
neuronal cells of either normal or pathological character.
In order to obtain information about the relationship between cLIP and mLIP we have used regular and
confocal fluorescence microscopy imaging and fluorescence plate reading of attached cells (reviewed in
Esposito et al 2002 and Petrat et al 2002a). As an experimental model of neurons we used cells derived
from P19 murine embryonic carcinoma cells that were differentiated with retinoic acid and were devoid of
dividing cells. The neuronal cells were found to be equipped with the major functional components of cell
iron metabolism such as TF receptors, DMT1, ferritin and the cell iron regulatory pathways that respond
to iron loads and iron deprivation. Fluorescent metalosensors such as calcein CAL (loaded as CAL-AM)
(Epsztejn et al. 1997) and fluoresceinated-phenanthroline PG 488:520 nm) distributed homogeneously in
the cells whereas rhodamine B-[(1,10-phenanthrolin-5-yl) aminocarbonyl] benzylester (RPA) (564:610
nm) (Petrat et al. 2002b) concentrated predominantly in the mitochondria. Pairs of such probes (RPA and
CAL or RPA and PG) were used for simultaneous tracing of changes in cLIP and mLIP in response to
iron loads (10 µM of Fe-hydroxyquinoline FHQ or ferrous ammonium sulfate FAS) or to chelation
treatment (100 µM DFO, phenanthroline or salicylaldehyde hydrazone). Exposure of cells to the fast
permeating FHQ led to a concomitant quenching of CAL in C and M and and of RPA in M, reflecting the
fast access of iron metal into those compartments. Similar exposure to FAS led to a gradual change in
cLIP mediated by DMT1, reaching µM plateau levels in ~45’, while the mLIP raised swiftly (< 3’) to µM
levels and remained stable. Treatment with DFO which enters into cells but apparently spares M caused
a reduction in cLIP as well as in mLIP, indicating that M Fe might also fall back into the C. Our results
indicate that M of P19 derived neuronal cells have high affinity transfer systems for transporting iron
between C and M so that effects on cLIP are swiftly reflected in mLIP and vice versa. The changes in
mLIP per se cause no apparent damage to M as reflected in the M membrane potential following iron
loads or deprivation. It is the co-participation of pro-oxidant species that might trigger labile iron to engage
in ROS formation and ensuing M damage.
This work was supported by the C. Smith Inst. of Psychobiology, Jerusalem, Israel.
Espósito, B.P., et al. (2002) A review of fluorescence methods for assessing labile iron in cells and
biological fluids. Anal. Bichem. 304:1-18
Epsztejn, S. et al. (1997). Fluorescence analysis of the labile iron pool of mammalian cells. Anal.
Biochem. 248, 31 – 40.
Petrat, F. et al. (2002a). Selective determination of mitochondrial chelatable iron in viable cells with a new
fluorescent sensor.Biochem. J. 362, 137 – 147.
Petrat, F et al. (2002b) The Chelatable Iron Pool in Living Cells: A Methodically Defined Quantity, Biol.
Chem., 383: 489 – 502,
POSTER 160
DIETARY HEME IRON AND ISCHEMIC HEART DISEASE
D.L. van der A¹, P.H.M. Peeters¹, D.E. Grobbee¹, J.J.M. Marx², Y.T. van der Schouw¹
¹ Julius Center for Health Sciences and Primary Care, University Medical Center, Utrecht, The
Netherlands
² Eijkman-Winkler Institute for Microbiology, Infectious Diseases and Inflammation, University Medical
Center, Utrecht, The Netherlands
A detrimental role of iron in cardiovascular disease has been supported by in vitro as well as in vivo
studies. Two biological mechanisms have been proposed, both based on the catalyst activity of iron in
free radical production. On the one hand, iron may promote oxidation of low density lipoproteins, which is
involved in the early steps of atherosclerosis; on the other iron may increase myocardial injury after blood
flow restoration, following an ischemic event. It is proposed that the difference in incidence rates of heart
disease between men and women could be explained by differences in iron stores. In women there is a
marked increase in incidence rates of heart disease after menopause, which may be the result of lacking
the biological preventative of monthly iron loss through menses. So far, the focus has been on the role of
body iron stores in relation to cardiovascular events, whereas the role of dietary iron, particularly heme
iron, has been shed insufficient light on.
We conducted a prospective cohort study among 15,768 women without prevalent heart disease, aged
49-70 at enrolment between 1993 and 1997. Follow-up was complete until January 1, 2000. Linkage with
the Dutch national hospital discharge diagnosis database provided us with information on incident
ischemic heart disease (codes I20-I25). Through the national municipal registry we were informed on the
date of death of cohort members and causes of death were inquired through the women’s general
practitioners. During a mean follow-up of 4.3 years, 223 cases of ischemic heart disease were
documented. Cox proportional hazards analysis was used to compute hazard ratios of ischemic heart
disease for higher quartiles of heme iron intake versus the lowest. All analyses were adjusted for classical
cardiovascular risk factors and nutrition variables.
Dietary heme iron intake was associated with a 1.66-fold (95% Confidence Interval (CI): 1.07-2.58)
increased risk of ischemic heart disease. This effect was not modified by smoking, hypertension,
hypercholesterolemia or diabetes. Furthermore, we investigated the effect of time since menopause
combined with heme iron intake. Women who are premenopausal or experienced menopause less than
10 years ago and who consume low levels of heme iron are taken as reference group. Compared with
these women, women who reached menopausal age more than 10 years ago and who consume
relatively high levels of heme iron are at increased risk (Hazard Ratio (HR) = 1.64; 95% CI: 1.00-2.71). It
seems that time since menopause has no effect on ischemic heart disease risk in women who consume
low levels of heme iron (HR = 0.90; 95% CI: 0.58-1.40) and that premenopausal women and women who
experienced menopause less than 10 years ago who consume high levels of heme iron have a 1.33-fold
increased risk of ischemic heart disease (95% CI: 0.78-2.27).
Our results indicate that women in the highest quartile of heme iron intake have an increased risk of
ischemic heart disease compared with women in the lowest quartiles. Especially, the effect seems to be
important for women who reached menopausal age more than 10 years ago.
POSTER 161
LOW IRON DIET IS A USEFUL MAINTENANCE THERAPY AFTER IRON REDUCTION WITH
PHLEBOTOMY AND LOW IRON DIET IN PATIENTS WITH CHRONIC HEPATITIS C
Fumiaki Kimura, Tamano-Municipal Hospital
Introduction Phlebotomy has been shown to improve serum alanine aminotransferase (ALT) in patients
with chronic hepatitis C (CHC). However, rebound phenomenon after stopping or during phlebotomy has
been reported. This study was carried out to investigate the effect of low iron diet (LID) on patients (Pts)
with CHC after iron reduction using phlebotomy and LID. Methods Phlebotomy was repeated biweekly on
12 Pts until serum ferritin levels reached 10 ng/ml. Before and after starting this therapy, instruction for
LID, that is restriction of dietary iron intake to 8 mg/day, was given to each Pt every month. 3-day
weighed diet records were kept by Pts regularly, utilized for instruction of LID and calculation of nutrient
intakes with the Standard Tables of Food Composition in Japan(4th revision). Results ALT levels were
significantly improved from 88.9±38.4 to 44.1±8.0 IU/L (p<0.01, paired t-test) with phlebotomy and LID.
During maintenance periods ( 24.5±4.5 months) ALT levels was 42.3±8.2 IU/L and no rebound
phenomenon was detected with LID alone. However, there was a small increase in ferritin levels during
maintenance periods from 11.1±2.5 to 30.5±13.1 ng/ml (p<0.005). According to 3-day weighed diet
records, mean dietary iron intakes before and after diet instruction were 17.1±5.2 mg/day and 7.9±3.7
mg/day, respectively. Discussion LID alone has also been reported to improve ALT levels in Pts with
CHC. But the efficacy is not so strong. After induction of storage iron to low level in this study, ALT levels
were controlled in good levels with LID alone. Conclusions These findings suggest that in the low iron
storage state, dietary iron through portal vein would be critical to control inflammation in Pts with CHC.
POSTER 162
POTENTIAL DISCRIMINATION OF CIRHHOTIC LIVER FROM DISEASE-FREE LIVER BY PROTON
TRANSVERSE RELAXATION RATE MAGNETIC RESONANCE IMAGING (R2-MRI)
T.G. St. Pierre1, P. Clark1, W. Chua-anusorn1, G. Jeffrey2, J. Olynyk2, B. de Boer3
1
School of Physics and 2Dept. of Medicine, The University of Western Australia, AUSTRALIA
3
PathCentre, QE II Medical Centre, Nedlands, WA 6009, AUSTRALIA
Proton transverse relaxation rate (R2) images of the liver enable visualisation of the spatial variation of
tissue iron concentration. The distribution of R2 throughout the liver typically presents as a normal
distribution. However, the presence of tumour or cirrhosis can distort R2 histograms from a normal
distribution [1,2]. Generally the local tissue R2 is an average from separate populations of hydrogen
protons owing to chemical and physical compartmentalisation at the microscopic scale. A two-component
model of transverse relaxation [3] distinguishes one population of hydrogen protons with a fast rate of
decay in transverse magnetisation (R2f) from a second population with a slow rate of decay in transverse
magnetisation (R2s).
The aim of this study was to define statistical parameters on the R2, R2f, and R2s distributions in liver
tissue with the potential to discriminate liver cirrhosis from disease-free liver. The parameters selected
included Kuiper’s statistic (KP-V) for normalcy of the distribution, and the fourth order moment (kurtosis)
of the distribution (a measure of the extent of the tails on the distribution). The volunteers in the study
were divided into 3 groups: (i) healthy volunteers (n = 8); (ii) patients known to have cirrhosis from liver
biopsy diagnosis (Knodell system staging of 5-6) as well as patients activated for liver transplantation with
HIC in the normal range (0.2 – 1.8 mg Fe/ g dry tissue (n = 8); (iii) patients known to have cirrhosis from
liver biopsy with HIC above the normal range (n = 14, HIC range from 2 – 34.7 with mean± SD of 16.2 ±
10.3 mg Fe/ g dry tissue). The etiologies of cirrhosis included hepatitis C, ethanol abuse, and iron
overload.
Table 1 shows the means and standard deviations of the R2 distribution parameters for the three groups
of volunteers. In each case the distributions of parameters for the volunteers with cirrhosis are
significantly different from those for the healthy volunteers. It is noteworthy that the distributions of the
ratio of R2s KP-V to R2f kurtosis for the cirrhotic volunteers showed no overlap with that measured for the
healthy volunteers suggesting that this parameter may have potential as a non-invasive probe of liver
cirrhosis.
Table 1: Mean ±SD of selected R2 parameters from 3 groups of volunteers.
Parameter
(i) Healthy
Cirrhosis
p-value*
n=8
(ii) Normal HIC (iii) Increased
i - ii
i - iii
n=8
HIC, n=14
R2 KP-V
0.19 ± .05
0.13 ± 0.06
0.15 ± 0.04
0.04
0.03
R2 kurtosis
4.1 ± 0.8
3.2 ± 0.9
3.0 ± 0.9
0.04
0.02
R2f kurtosis
4.1 ± 0.8
3.3 ± 0.9
2.9 ± 0.7
0.05
0.01
R2s KP-V
0.087 ± 0.014
0.130 ± 0.033
0.168 ± 0.033
0.01
<0.001
R2s KP-V : R2f
0.022 ± 0.006
0.040 ± 0.008
0.063 ± 0.022
0.001 <0.001
kurtosis
* significant difference between groups when P < 0.05 (Mann-Whitney U test)
[1] Clark PR & St. Pierre TG. Magn. Reson. Imaging, 18 (2000): 431-438.
[2] Clark PR, Chua-anusorn W & St. Pierre TG. Magn. Reson. Imaging, (2003) in press.
[3] Clark PR, Chua-anusorn W & St. Pierre TG. Magn. Reson. Med., (2003) in press.
ii - iii
0.39
0.66
0.26
0.02
0.01
POSTER 163
MILD ANEMIA AND A DIMINISHED STRESS-INDUCED ERYTHROPOIETIC RESPONSE IN
ACERULOPLASMINEMIC MICE
S. Cherukuri1,2 and P.L. Fox1, 1Department of Cell Biology, The Lerner Research Institute, Cleveland
Clinic Foundation, Cleveland, OH, 2Department of Biology, Cleveland State University, Cleveland, OH
Erythropoiesis is regulated by cytokines, growth factors, and by the availability of iron to the erythropoietic
system. Ceruloplasmin (Cp) is a multi-copper ferroxidase that drives the incorporation of iron into
transferrin by converting ferrous iron to ferric iron and hence has a critical role in iron transport.
Aceruloplasminemic patients and mice with targeted Cp gene disruption have iron deposits in liver,
pancreas, basal ganglia, and other organs. In patients, the absence of Cp leads to elevated serum ferritin,
low serum iron, and mild hypochromic and microcytic anemia, suggesting a possible role for Cp in
erythropoiesis. Previous findings from our laboratory, i.e., the transcriptional regulation of Cp expression
by iron deficiency and the presence of hypoxia-responsive elements in the 5’-flanking region of the Cp
gene, also suggest a role for Cp in erythropoiesis. To test whether Cp-deficient mice have a decreased
erythropoietic response, we studied the kinetics of the response of Cp-null mice to stresses that induce
+/+
+/–
–/–
erythopoiesis. Cp , Cp , and Cp littermates were subjected to phenylhydrazine-induced hemolytic
anemia, and complete blood counts and reticulocyte indices were measured during the stress and the
recovery phases. Before application of the stress, Cp–/– mice were mildly anemic with lower hemoglobin
concentration (11.3 ± 0.2 g/dl in Cp–/– mice vs. 12.4 ± 0.5 g/dl in Cp+/+ mice), mean cell hemoglobin (11.6
± 0.2 pg vs. 13.8 ± 0.4 pg), mean cell volume (45.1 ± 0.2 fl vs. 48.6 ± 0.2 fl), and reticulocyte-hemoglobin
content (13.8 ± 0.5 pg vs. 15.7 ± 0.2 pg). Cp–/– mice and Cp+/+ mice became severely anemic upon
phenylhydrazine-induced hemolytic anemia, but Cp–/– mice showed a markedly diminished erythropoietic
response to the stress compared to wild-type mice. Infusion of purified human Cp into phenylhydrazinetreated Cp–/– mice during the recovery phase caused a larger increase in the reticulocyte-mean cell
volume and reticulocyte-hemoglobin content compared to Cp–/– mice not given Cp injections. The
decreased erythropoietic response of Cp–/– mice was confirmed using two other erythropoiesis-inducing
stresses. Cp–/– mice subjected to either acute bleeding or hypoxia exhibited a 2- to 3-day lag in the
recovery of blood hemoglobin, and throughout the recovery phase had significantly lower hematocrit and
hemoglobin values compared to wild-type mice. The mild anemia in Cp–/– mice and their diminished
response to erythropoiesis-inducing stress may be due to the inefficient recycling of iron between the
reticuloendothelial system and the erythropoietic system. Our findings reveal a role for Cp in basal
erythropoiesis, and that this role becomes more significant after stress.
POSTER 164
ANTICANCER ANTHRACYCLINES PROTECT CARDIAC CELLS FROM IRON LOADING: A
PARADOX DUE TO FORMATION OF REACTIVE OXYGEN SPECIES AND INDUCTION OF FERRITIN
SYNTHESIS
G. Corna1, P. Santambrogio2, G. Minotti3 and G. Cairo1. 1Inst. General Pathology, Univ. Milan, 2DIBIT
H.S.Raffaele, Milan, 3Dept. Drug Sci. Univ. Chieti, ITALY
Doxorubicin (DOX) is an anticancer anthracycline whose therapeutic efficacy is limited by cardiotoxicity.
Both iron and reactive oxygen species (ROS), possibly formed during DOX metabolism, have been
implicated as molecular determinants of cardiotoxicity. The mechanisms through which iron and ROS
interact while inducing cardiotoxicity are nonetheless controversial (Minotti et al. 1999 FASEB J). Iron
could act by converting ROS like superoxide and hydrogen peroxide into more potent species like
hydroxyl radicals. In addition, or alternatively, ROS and other DOX metabolites might target proteins of
iron metabolism and facilitate an amplification of the free iron pool which catalyses oxidative injury. In
fact, we have recently shown that DOX can inactivate iron regulatory proteins (IRP-1 and IRP-2) through
a sequential action of DOXol, an alcohol metabolite of DOX, and ROS on IRP-1 and an effect of ROS
alone on IRP-2 (Minotti et al. 1998 FASEB J; Minotti et al. 2001 Cancer Res). In the present study, to
better understand the role of iron in anthracycline cardiotoxicity, the rat cardiomyocytes cell line H9c2 has
been exposed to concentrations of DOX similar to those found in the plasma of patients undergoing
chemotherapy. Given our previous demonstration that DOX modulates IRP activity in cardiomyocytes
(Minotti et al. 2001 Cancer Res), we examined the expression of proteins of iron metabolism controlled by
IRPs. In agreement with an elevation of IRP-1 activity induced by DOX, we found an increased amount of
transferrin receptor (TfR) in H9c2 cells exposed to 5-10 µM DOX; however, also ferritin content was
increased, with preferential stimulation of the H subunit. Higher ferritin expression could not be attributed
to IRP-1 activation but was accounted for by the ability of DOX to enhance ferritin mRNAs levels, in
particular H subunit mRNA; whether concomitant down-regulation of IRP-2 caused by DOX contributed to
such an effect cannot be excluded at this time. The expression of the stress responsive genes catalase
and aldose reductase was also induced in DOX-treated H9c2 cells, suggesting that ROS production was
increased. Since a cytoprotective effect of ferritin has been documented (Torti and Torti, 2002 Blood), we
investigated whether pre-exposure to DOX could protect H9c2 cells against a damage caused by an iron
load. MTT assays showed that pre-treatment with DOX significantly improved the resistance to ironinduced cell death. Therefore, these results suggested that a ROS-mediated transcriptional increase of
ferritin expression resulted in a cytoprotective effect, in spite of increased uptake of Tf-bound iron.
Because secondary alcohol metabolites would not be involved in reactions like these, we performed
experiments with either 5-iminodaunorubicin (5-iminoDNR, a DOX analogue that forms its secondary
alcohol metabolite but much less ROS due to the replacement of the quinone group with an imino moiety)
or naphthazarin (NZ, a model compound forming ROS but not secondary alcohol metabolites). Measuring
oxidation of DCFH as an index of ROS production confirmed that 5-iminoDNR was less effective than
DOX and NZ at producing ROS, while ELISA assays showed that it was much less potent than DOX and
NZ at stimulating the accumulation of ferritin, mainly the H subunit. Consistently, 5-iminoDNR was unable
to protect cardiomyocytes against iron-induced cytotoxicity, while NZ showed a protective effect similar to
that of DOX. Having demonstrated an increase of TfR, we also examined the fate of iron taken up by
55
55
H9c2 cells exposed to Fe-transferrin. Analysis of Fe-labeled proteins by non-denaturing PAGE
showed a strong increase in the amount of ferritin-associated radioactivity in DOX-treated cells, indicating
that the increased amount of iron entering cardiomyocytes after exposure to DOX is promptly
sequestered into ferritin shells. In conclusion, in the experimental model of H9c2 cardiomyocytes,
oxidative stress does not play any role in amplifying the iron pool and hence exacerbating DOX toxicity
but, through induction of ferritin synthesis, makes iron unavailable to initiate free radical reactions, thus
preventing a subsequent iron-mediated damage. Therefore, the present results lend support to the
concept that the role of iron and ROS in inducing cardiotoxicity might not be confined to “simple” ironcatalyzed oxidative events and probably extends to more severe perturbations of iron
compartmentalization and related metabolic events.
POSTER 165
BOVINE HAEMOCHROMATOSIS DOES NOT RESULT FROM THE C282Y MUTATION OF HFE
N.M. Joshi, M. Heritage, S.R. Wang, D. O'Toole*, A.P. Walker, Centre for Hepatology, Royal Free and
University College Medical School, University College London, UK and *Wyoming State Veterinary
Laboratory, University of Wyoming, USA
Bovine haemochromatosis has been reported in line-bred Salers cattle. The disease has inheritance
consistent with an autosomal recessive trait. It is symptomatic, showing fibrosis, cirrhosis,
hypogonadotrophic hypogonadism and joint involvement. Venesection treatment was reported to be
effective. In the liver of affected animals, iron is deposited predominantly in hepatocytes and the liver iron
concentration can exceed the threshold required for induction of fibrosis and cirrhosis (~20,000 µg/g dry
weight). The phenotype of bovine haemochromatosis is similar to that of human HFE-related
haemochromatosis. The majority of human patients with this disease are homozygous for the founder
C282Y mutation. This study was undertaken to identify the bovine HFE orthologue and investigate it as a
candidate gene for bovine haemochromatosis, based upon phenotypic parallels with the human HFErelated disease.
Exons of the putative bovine HFE orthologue were identified by several approaches including crossspecies PCR, RT-PCR, cDNA analysis and hybridization of bovine genomic libraries with human HFE
probes.
Sequence analysis of the locus in control bovine DNA indicated that it likely encodes the bovine HFE
orthologue rather than an antigen-presenting MHC-type protein. Like human HFE, only two of the four
key tyrosine residues, which are important for peptide binding in classical MHC class I proteins, are
conserved. A proline, predicted to kink the α2 domain of HFE and narrow the peptide binding-like cleft,
is conserved in the bovine locus. Four conserved cysteines, involved in disulphide bonds in the α2 and
α3 domains of HFE and MHC class I proteins, are also conserved in the bovine locus. However,
sequence analysis of the putative bovine HFE orthologue in three obligate carriers of bovine
haemochromatosis showed that the bovine equivalent of the C282Y mutation was not present.
In conclusion, a putative bovine HFE orthologue has been identified with sequence hallmarks
characteristic of a non-classical MHC-like molecule. These studies indicate that bovine
haemochromatosis does not result from C282Y mutation of the bovine HFE orthologue. Physical
mapping and full analysis of the bovine locus in additional samples will be completed before considering
non HFE-related candidate genes. A genetically defined, symptomatic animal model of
haemochromatosis will be important for investigation of new therapeutic strategies.
POSTER 166
IRON-CITRATE TOXICITY AGAINST ASEXUAL-STAGE PLASMODIUM FALCIPARUM PARASITES
M. Hodges, M Loyevsky, V.R. Gordeuk, Howard University
In the malaria parasite Plasmodium falciparum, the need for non-heme iron has been demonstrated
by the inhibitory effect of iron chelators such as desferrioxamine (DFO) on parasite growth. Furthermore,
the presence of the labile iron pools in the malaria-infected erythrocytes has been shown directly using an
iron-sensing probe, calcein (Loyevsky et al., 1999). These facts imply that P. falciparum possesses
mechanisms to regulate iron homeostasis. As a first candidate for the protein regulating iron homeostasis,
we have recently began characterizing an iron-response element binding protein-like protein from P.
falciparum (PfIRPa), and showed that recombinant PfIRPa displays aconitase activity under conditions
favoring the assembly of iron-sulfur clusters. The major function of the tricarboxylic acid cycle enzyme,
aconitase, is to convert citrate into isocitrate.
A recent report suggested that citrate may promote iron-mediated cell damage (Chen et al., 2002).
Hence, a plasmodial cytosolic aconitase could conceivably lessen iron-citrate toxicity by reducing citrate
concentration in cells that also have cytosolic iron available to bind to citrate. To determine if iron in
conjunction with citrate could be deleterious for asexual parasites grown in erythrocyte culture, we
examined the effect of ferric ammonium citrate (FAC), ferric citrate, sodium citrate, ferric chloride (FeCl3)
and ammonium chloride on parasite growth. FAC inhibited parasite growth by 50% and ferric citrate by
34% at 0.5 mM concentration, whereas sodium citrate, FeCl3, and ammonium chloride did not.
Interestingly, DFO, which at 10 µM slightly inhibited parasite growth, partially reversed the inhibitory effect
of FAC when used in tandem with FAC (p<0.001), indicating that the toxicity was associated with iron
when citrate was also present. The lack of an inhibitory effect by sodium citrate, ferric chloride and
ammonium chloride used as single agents suggests that only the combination of iron and citrate results in
toxicity.
A role for citrate in intracellular iron metabolism has been proposed because IRP1 in the presence of
high labile iron is converted into a cytosolic aconitase (Rouault et al., 1992), which in turn converts citrate
into isocitrate. In almost all organisms, citrate occupies a pivotal position in several important biochemical
pathways, such as synthesis of cytoplasmic acetyl CoA to serve as a starting material for the synthesis of
fatty acids and cholesterol. In man, among the various citric acid cycle intermediates, citrate is present in
the circulation at highest levels, ~135 µM (Inoue et al., 2002). Also, citrate has iron-chelating activity and
can function physiologically as an iron carrier (Trinder and Morgan, 1997). When mammalian transferrin
is highly saturated, some plasma iron may be found bound to citrate. Despite its metabolic importance for
organisms, citrate is potentially toxic when it complexes with iron. In a variety of in vitro systems, citrate3+
Fe complexes lead to increased oxidant damage (Minotti and Aust , 1987).
Our results suggest that iron-citrate complexes are toxic for the asexual malaria parasites in culture.
It seems possible that asexual malaria parasites may use PfIRPa/aconitase to regulate both the
intracellular labile iron pool by incorporating iron into the iron-sulfur cluster and citrate concentration by
converting citrate into isocitrate.
References.
M. Loyevsky et al. (1999). Chelation of iron within the erythrocytic Plasmodium falciparum parasite by iron
chelators. Mol Biochem Parasitol 101: .43-59.
O.S. Chen et al. (2002) Genetic analysis of iron citrate toxicity in yeast: Implications for mammalian iron
homeostasis. Proc Natl Acad Sci U S A 99:16922-16927.
T.A. Rouault et al. (1992) An iron-sulfur cluster plays a novel regulatory role in the iron-responsive
element binding protein. Biometals 5:131-140.
K. Inoue et al. (2002) Structure, function, and expression pattern of a novel sodium-coupled citrate
transporter (NaCT) cloned from mammalian brain. J Biol Chem. 277:39469-39476.
D. Trinder, E. Morgan (1997) Inhibition of uptake of transferrin-bound iron by human hepatoma cells by
nontransferrin-bound iron. Hepatology 26:691-698.
G. Minotti, S.D. Aust (1987) An investigation into the mechanism of citrate-Fe2+-dependent lipid
peroxidation. Free Radic Biol Med 3:379-387.
POSTER 167
THE ACTIONS OF SMALL MOLECULAR WEIGHT BIOIRON--IRON CITRATE COMPLEX--IN THE
BRAIN
C. C. Chiueh and P. Rauhala, LCS, NIMH, NIH, Bethesda, MD 20892-1264 USA and Institute of
Biomedicine, University of Helsinki, Helsinki, FIN-00014 Finland
Citrate and isocitrate are parts of the tricarboxylic cycle of mitochondria. They readily chelate with iron to
form bidentate and tridentate complexes of iron citrate and iron isocitrate, respectively. In the presence of
oxygen and background irradiation (hv), the redox cycling of ferrous and ferric citrate complexes leads to
the generation of reactive oxygen species such as cytotoxic hydroxyl radicals (•OH) that can be trapped
by salicylate and assayed as 2,3- and 2,5-dihydroxysalicylate. The generation of •OH depends on the
incubation or body temperature. UV irradiation increases further the generation of •OH. In contrast to the
classic Fenton reaction (oxygen, EDTA-Iron complex and ascorbate), iron citrate-induced •OH generation
can be blocked by not only iron chelator (i.e., EDTA and deferroxamine) but also antioxidants including
ascorbate, GSH, •NO, and S-nitrosoglutathione (GSNO).
In the biological system iron citrate complex modulates mitochondrial aconitase activity and plays a
critical role in the homeostasis of intracellular levels of bioiron. In the brain bioiron maintains normal
functions of neurotransmission. Bioiron serves as a cofactor for tyrosine hydroxylase, the rate limiting
enzyme in the biosynthesis of catecholamines including brain dopamine. Selective inhibition of the
nigrostriatal dopaminergic neurotransmission is known to cause Parkinson’s disease. Iron complexes are
age-dependently accumulated in the brain especially Parkinson's patients. Tissue levels of bioiron in
several brain regions of the basal ganglia can reach as high as those in the hepatic tissue. It is known
that high levels of bioiron, oxygen, and dopamine lead to the generation neuromelanin pigments in the A9
nigral dopaminergic neurons of the midbrain substantia nigra compacta area. It has been proposed that
heavily pigmented neurons die first during senescence and following the administration of the
parkinsonian syndrome causing neurotoxin, MPTP, which selectively destroys A9 and A8 nigral neurons
but spares other A10, A12 and A16 brain dopamine neurons. Little iron complexes are accumulated in
the rat midbrain thereby rendering rat A9 nigral neurons less vulnerable to MPTP. However, intranigral
infusion of ferrous citrate (0 to 16.4 nmol) into rat midbrain caused oxidative stress, lipid peroxidation and
delayed nigral death reflected by axonal degeneration and severe dopamine depletion in the caudate
nucleus. This dose-dependent nigrostriatal degeneration due to iron citrate begins 3 to 5 days following
+
+
the treatment with iron citrate complex that is as potent as the toxic metabolite of MPTP, MPP . MPP is
known to chelate iron for the promotion of intracerebral transportation. Iron chelators protect cells and
neurons against MPP+-induced oxidative stress and apoptosis. Therefore, bioiron-induced oxidative
stress may play a pivotal role in the neurodegenerative mechanism underlying MPTP-induced
parkinsonism and perhaps the idiopathic Parkinson's disease.
In addition to Parkinson's disease, excess levels of small molecular weight bio-iron may also induce
oxidative stress, brain lipid peroxidation, axonal degeneration, and neuronal death in other degenerative
brain disorders such as Hallervorden-Spatz syndrome (excess biorion accumulation), hemorrhagic stroke
(hemoglobin), Alzheimer's dementia (toxic β-amyloid precursor fragments), and neuroAIDS (toxic viral
protease). Both in vitro and in vivo studies suggest that GSNO-mediated •NO/cGMP-dependent and independent neuroprotection may be useful to counteract small molecular weight iron complex-induced
oxidative stress, neurodegeneration, and brain atrophy. Therefore, it is necessary to keep brain bioiron
levels in check for not only maintaining normal brain functioning and also preventing oxidative stress.
POSTER 168
IMPROVEMENT OF HAEMOGLOBIN CONCENTRATION AND IRON STATUS IN CHILDREN WITH
CHRONIC ADENO-TONSILLAR INFECTION AFTER SURGICAL TREATMENT
R.J.Ulvik, BioMetal Research Group, Institute of Medicine, University of Bergen, 5021, H.H.Elverland,
Department of Otorhinolaryngology, University of Tromsø, 9038, Norway
Children with chronic or frequently recurrent infections are disposed to anaemia due to iron deficient
erythropoiesis caused by the inflammatory process, manifest iron deficiency or both. This may hinder
normal cognitive and motor development. A normal iron status in childhood is therefore important. The
prevalence of anaemia depends on age, the number, duration and intensity of the infectious episodes,
and the cut-off value of haemoglobin concentration used to define anaemia. In children with chronic
adeno-tonsillar infections selected for surgical treatment, prevalence figures of 17 – 23 % and 4 % have
been reported with cut-off limits for haemoglobin of 12.0 –12.1 g/dL and 11.1 g/dL respectively at the
time of surgery. Studies on the longitudinal effect of surgery on the iron status and haemoglobin
concentration, are largely absent.
The present study was done to find the prevalence of abnormal iron status and anaemia in children
undergoing surgical tonsillectomy, adenoidectomy or both, and to see if surgery had any persistent
favourable effect on the iron status six months later.
Of 119 children with a median age of 5.8 years ( range 1.8-14.5 years) who were consecutively
considered for surgery, preoperative analysis of the haemoglobin-, serum-ferritin- and erythrocyteprotoporphyrin IX - (EPP-IX) concentration was performed in 114 patients.The analysis was repeated at
the six month postoperative control in 99 of the patients. Cut-off limits for the haemoglobin concentration
were 11.1, 11.5 and 11.9 g/dL for children < five, five to eight and > eight years of age respectively.
Serum – ferritin < 15 ug/L indicated iron deficiency with depleted iron stores and EPP-IX > 1.9 umol/L
indicated iron deficient erythropoiesis.
64.9 % of the children were anaemic preoperatively. This figure was reduced to 10.1% at the six months
control. The median haemoglobin concentration increased from 11.3 to 12.7 g/dL (p<0.001), while the
median EPP-IX fell from 1.8 to 1.5 umol/L ( p<0.001). The median serum-ferritin value changed
insignificantly from 17 to 16 ug/L. 17.5 % of the patients with preoperative anaemia, also had pathological
serum-ferritin and EPP-IX. Serum-ferritin, but not EPP-IX correlated positively with increasing age.
The prevalence of preoperative anaemia was unexpectedly high in our material. The considerable
increase in the median haemoglobin concentration of more than 1 g/dL in six months, was evidence of a
substantial iron deficient erythropoiesis at the time of surgery and probably also several children had
manifest iron deficiency. The reduced EPP-IX proved that adequate iron supply was reestablished for
erythropoiesis. However, only extra iron supplementation would have told if the patients indeed had got a
sufficient amount of iron to reach their optimal haemoglobin concentration at the control. Since this was
not done we conclude that the improved haemoglobin- and iron status was primarily explained by the
relief from the inflammatory condition. Also better nutrition after treament could have contributed to the
profitable result.
The low diagnostic usefulness of serum-ferritin in patients with inflammatory disease, is illustrated by the
finding that 31.1 % of the anaemic patients and 50 % of the non-anaemic patients had preoperative
serum-ferritin > 15 ug/L. How many of these patients had their serum-ferritin falsely increased as a
response to the inflammatory process, we do not know. Most probably several of the anaemic patients
had a real iron deficiency, and several of the non-anaemic patients probably had a haemoglobin
concentration below their optimal level. A falsely high preoperative serum-ferritin, would be reduced after
treatment. On the other hand, patients with a real preoperative iron deficiency would have an increased
serum-ferritin after treatment due to increased iron absorption. These two processes influencing the
serum-ferritin in opposite directions, may explain the stable median value of serum-ferritin.
To summarize, we found that iron deficient erythropoiesis and anaemia is common among children
suffering from chronic upper respiratory infections, and that surgial treatment significantly improves the
iron status of these patients. Furthermore, many of these patients will benefit from iron supplementation
after treatment.
POSTER 169
Withdrawn
POSTER 170
HOW DISRUPTION OF IRON HOMEOSTASIS IN HOST EPTHELIAL CELLS BY NEISSERIA
MENINGITIDIS MAY AFFECT PATHOGENESIS: A NOVEL MODEL
R.A. Bonnah1, M. Muckenthaler2, H. Carlson1, J. Larson1, C. Enns1, M. Hentze2 and M. So1
1
Oregon Health & Science Univesity, Portland OR, USA and 2European Molecular Biology Laboratory,
Heidelberg, Germany.
Delineation of the inter-play between pathogen and host may provide us with new and innovative antimicrobial agents and vaccination targets. One promising area involves understanding the molecular basis
of iron withholding by the mammalian host, and the mechanisms by which microbial pathogens
circumvent these processes. We have previously shown that Neisseria meningitidis (meningococcus or
MC) and the closely related pathogen Neisseria gonorrhoeae (gonococcus or GC) alter cellular iron
homeostasis of target epithelial cells at multiple levels. First, and perhaps most importantly, infection by
these pathogens results in downregulation of host Tf Receptor 1 (TfR-1) mRNA in epithelial cells. This
correlates with infected cells having decreased surface and cycling TfR over time. There is also a
redistribution of TfR-1 from intracellular compartments to the epithelial cell surface at early infection time
points. The result of TfR downregulation and slower endocytosis is that infected cells internalize much
less Tf (~50%). Neisseria likely alters host cell iron homeostasis to enhance the supply of iron to the
bacterial populations that reside both within and outside of host cells.
To better understand how MC infection affects the host iron regulatory network, we analyzed the
expression profile of genes involved in iron metabolism in MC infected human epithelial cells. As a tool
we used a specialized cDNA microarray platform ('IronChip' Version 3.0). We find that MC infected A431
cells exhibit a gene expression profile similar to the one observed in uninfected, iron-deficient A431 cells
(treated with Desferrioxamine). We also made use of the MC mutant strains lpxA and pilE, which lack
LPS or cannot enter into cells, respectively. These analyses reveal that the majority of gene responses
persist independent of bacterial LPS or MC being able to attach to or invade the host cell (pilE). This
suggests that the cellular iron deficiency response is generated as a result of extracellular bacterial
growth and is independent of LPS. Interestingly, the reduction of TfR-1 mRNA protein levels is only
observed in the wild-type and LPS-deficient MC strain but not in the pilE mutant, suggesting that these
effects require attachment and possibly intracellular infection. We are currently investigating whether a
change in the iron regulatory protein (IRP-1) activity and/or TNFα induction, and/or intracellular MC
infection can explain the effects observed on TfR-1 and H-ferritin expression.
Our study demonstrates how MC and other pathogens may manipulate host cells rendering them unable
to respond appropriately to iron depletion. Our new paradigm of host-pathogen interplay hypothesizes
how disruption of the host iron homeostasis network could effectively deliver iron to both extracellular and
intracellular bacterial populations.
POSTER 171
L-FERRITIN OVEREXPRESSION RESULTS IN NUCLEAR AND CYTOPLASMIC INCLUSIONS AS
WELL AS CRYSTALLINE CATARACT: LESSONS FROM A MURINE MODEL OF HEREDITARY
HYPERFERRITINEMIA CATARACT SYNDROME
DG Brooks1, T Baradet2, I Devaux3, C Beaumont3 and D Stambolian2. 1) Division of Medical Genetics; 2)
Departments of Ophthalmology & Genetics, University of Pennsylvania, Philadelphia, PA; 3) INSERM
U409, Faculte X. Bichat, Paris France.
INTRODUCTION: Loss of protein solubility is an important pathogenic mechanism in chronic diseases.
The extreme of protein insolubility is crystallization, a rare fate for native proteins in vivo. Striking crystals
of L-ferritin are found in the lens in human Hereditary Hyperferritinemia Cataract Syndrome (HHCS).
HHCS is defined by autosomal dominant inheritance of cataracts, hyperferritinemia and specific
mutations of the L-ferritin gene. These mutations disrupt function of the iron responsive element in the 5'
untranslated region of the L-ferritin mRNA, thereby dis-inhibiting L-ferritin translation. L-ferritin is over
expressed in HHCS independently of iron and accumulates as ferritin 24-mers that crystallize to form
cataracts in human lens.
METHODS: To understand the natural history of this rare example of in vivo protein crystallization, a
transgenic mouse model was developed. A typical HHCS mutation (A40G) was engineered into the
normal mouse L-ferritin gene and three lines of mice were established with this mutated transgene. Iron
staining was perfomed with Perl's stain. Monospecific rabbit anti-L-ferritin antibodies (a generous gift of
Drs. P. Santambrogio and P. Arosio) were used for immunostaining and for ferritin quantitaion by ELISA.
RESULTS: The transgenic mice are generally healthy and over express mouse L-ferritin between 5 and
20 fold in serum. In the line with highest ferritin expression, large ferritin inclusions are observed in the
cytoplasm and nucleus of many tissues; in liver, pancreas and heart these inclusions contain stainable
iron. Chemical measurement of the non-heme iron in these transgenic mice shows a 1.5-2 fold increase
in liver and spleen as compared to control littermates. By immuno-histochemistry ferritin levels in lens
increase from undetectable in nontransgenic mice to readily detectable levels in transgenic animals. This
ferritin is diffusely cytoplasmic in most lens epithelial cells but is occasionally found as large cystoplasmic
inclusions. Cortical lens fibers have ferritin-rich aggregates that range from unordered inclusions through
paracrystalline arrays to frank crystals of ferritin.
CONCLUSIONS: This transgenic mouse model recapitulates the human HHCS cataract phenotype as
well as highlighting a sequence of events in protein deposition: protein over-expression, aggregation into
inclusions, organization into paracrystalline arrays and crystallization. Some of the steps in ferritin
deposition may be unique to the lens microenvironment where crystals are particularly problematic
because they diffract light causing glare, which interferes with human vision. A unique feature of this
mouse model is robust nuclear ferritin, this suggests that ferritin can enter the nucleus when markedly
over expressed. The failure of these mice to exhibit a detectable movement disorder suggests that wild
type ferritin is well tolerated in contrast to mutant L-ferritin in neuroferritinopathy.
POSTER 172
DEFICIENT INSULIN SECRETION IN A MOUSE MODEL OF HEMOCHROMATOSIS
R.S. Ajioka, J.P. Kushner, D.A. McClain, Dept. Internal Medicine, University of Utah School of Medicine,
Salt Lake City, UT.
Introduction: Hemochromatosis is one of the most common inherited disorders among Caucasians of
northern European descent. The disease is most often due to homozygosity for a G to A transition at
nucleotide 845 in the hemochromatosis gene (HFE). C282Y homozygotes hyperabsorb dietary iron and
have a clinical phenotype that ranges from an elevated transferrin saturation to iron overload severe
enough to damage the liver, heart, joints and endocrine organs. Diabetes is a late complication of
hemochromatosis, generally becoming manifest only when the insulin resistance associated with ironinduced cirrhosis is established. We hypothesized that an insulin secretory defect occurs as iron
overload develops and precedes the second “hit” of insulin resistance.
Methods: To test the hypothesis, we utilized a murine model of iron overload. Effects of iron overload on
glucose response were analyzed in control mice and in mice with targeted deletions of the Hfe gene (Hfe
-/-) or equivalent to the C282Y mutation (Hfe Y/Y) and mice fed a diet supplemented with 2% carbonyl
iron. Standard methods of measurement were used to quantify insulin and glucose. Islet cell responses
were measured in isolated perifused islets.
Results: Eleven week-old mice homozygous for the Hfe deletion (Hfe -/-) had a mean liver iron content of
1091 µg/g compared to 230 µg/g in age-matched controls (Hfe +/+). Acute insulin responsiveness was
measured by intraperitoneal administration of glucose (3 mg/g) and measuring the serum insulin
concentration 5 m later. Fasting glucose levels did not differ between groups but fasting insulin levels (µ
IU/ml) were 0.19±0.03 (n=8) in control mice versus 0.11±0.04 (n=3) in Hfe -/- mice (P < 0.05). The
insulin/glucose ratio following the glucose load in control mice was 1.6±0.2 (n=8) versus 0.9±0.2 (n=8) in
Hfe -/- mice (P < 0.01). To determine the mechanism of the decreased insulin secretory capacity we
measured total pancreatic insulin content and islet insulin content in acid-ethanol extracts. Islets were
prepared by collagenase digestion and separation on a discontinuous Ficoll gradient. Total pancreatic
insulin content (µg/pancreas) in control mice was 8.5±0.2 (n=6) versus 5.0±0.2 (n=9) in Hfe -/- mice (P <
0.05). Islet insulin content (mg/islet) was 0.156±0.034 (n=8) in control mice versus 0.12±0.032 (n=10) in
Hfe -/- mice (P < 0.038).
Discussion: The basis for the sensitivity of the beta cell to iron is not completely understood. Excess iron
in the pancreas may lead to oxidative damage but high iron levels might also compete with zinc for
transport via the divalent metal transporter DMT1. Zinc is required for packaging of insulin in secretory
granules. Despite decreased insulin secretory capacity, the Hfe -/- mouse does not develop diabetes.
This may indicate that a second “hit” of insulin resistance is required to produce diabetes.
Conclusions: These data suggest a) pancreatic insulin content is decreased in Hfe -/- mice; b) insulin
deficiency is directly related to iron loading; c) Insulin secretory capacity by viable islet cells is not
impaired in Hfe mutant mice; d) impaired insulin production is not sufficient to produce diabetes in this
model; e) insulin resistance is not found in Hfe -/- mice; f) decreased pancreatic insulin content and
secretion is most likely due to iron-induced beta cell destruction.
POSTER 173
SIMULTANEOUS GENETIC ABLATION OF BOTH IRP1 AND IRP2 GENES LEADS TO EARLY
EMBRYONIC LETHALITY
S. R. Smith and T. A. Rouault
Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National
Institutes of Health
Iron regulatory proteins (IRPs) 1 and 2 are cytoplasmic RNA-binding proteins that regulate cellular iron
metabolism. In iron starved cells, IRP1 and IRP2 bind specific mRNA structures known as iron responsive
elements (IREs) and post-transcriptionally regulate proteins involved in iron uptake, storage, and
utilization. To assess the biological significance of the IRPs, knockout targeting constructs were prepared
through homologous recombination. Mice homozygous for the IRP2 deletion (IRP2 -/-) only and those
mice that are heterozygous for IRP1 (IRP1 +/-) in combination with complete loss of the IRP2 gene
display CNS iron misregulation. We have found that embryos with complete genetic ablation of both IRP1
and IRP2 do not survive to birth; the IRP1 -/- ; IRP2 -/- genotype is embryonic lethal.
Embryo harvesting techniques were used to analyze the stage of embryogenesis at which lethality takes
place. Embryos from E12-E6.5 days were resected and their genotype was analyzed. Blastocysts
between E3.5 -E4.5 days were collected from superovulated female mice. The blastocysts were
genotyped through PCR. Blastocysts were whole mounted and analyzed for levels of iron related
proteins and to characterize morphology.
Between embryonic days E6.5 - E12 no double knockouts were found. The double knockout occurred at
E3.5 -E4.5 days. Initial attempts to culture these blastocysts revealed inner cell mass and trophoblast
giant cells in wild-type embryos with normal development. However, double knockout E3.5-E4.5 embryos
displayed significant defects in growth and development
Results indicate that at least one normal allele of IRP1 or IRP2 is essential for postnatal survival. IRP1
and IRP2 blastocysts display an impaired potential for outgrowth. This embryonic defect reflects a
development defect of the pre-implantation embryo thereby leading to embryonic lethality.
POSTER 174
MITOCHONDRIAL CONTROL OF CELLULAR IRON METABOLISM
E. S. Jacobson, A.J. Troy and K.J. Nyhus. McGuire Veterans Affairs Medical Center and Virginia
Commonwealth University
Because infection represents a contest between host and invader for the host’s iron stores, we
studied the regulation of iron uptake in Cryptococcus neoformans. We identified random mutations
for regulation of the uptake-related plasma membrane ferric reductase by plating a UV-mutagenized
population on high iron agar and overlaying colonies with soft agar containing the ferrous-binding
chromogen, bathyphenanthroline disulfonate. Regulatory mutants were identified by their red
haloes. Using meiotic analysis we were able to assign the mutants to one of four chromosomal loci.
A representative from each locus was subjected to transformation with a library obtained from the wild
type; complementing clones were subjected to transposon-mutagenesis and those knocked out were
sequenced from the point of the transposon insertion. We were able to determine the identity of
three of the four mutated loci, and all were nuclear genes with mitochondrial functions. The first,
FRR1, is homologous to the Saccharomyces cerevisiae (yeast) mitochondrial transport genes, MRS3
and MRS4. The next to be identified, FRR4, is homologous to the human mitochondrial iron exit
protein which is affected in Friedrich’s ataxia, called frataxin, and also is homologous to the yeast
frataxin homologue, termed YFH1. The third locus, FRR3, is homologous to the yeast ISU1 and
ISU2 genes, which are required for synthesis of mitochondrial iron-sulfur clusters. Since random
mutagenesis represents a statistical query of the genome, we conclude that mitochondrial iron
metabolism contains many mutational targets, is very complex and is central to the cellular iron
economy.
POSTER 175
ALTERATIONS IN SULFUR AMINO ACID METABOLISM IN FRIEDREICH'S ATAXIA CELLS
Gino Cortopassi1, Eleonora Napoli1, Franco Taroni2 and Guolin Tan1;1Dept. of Molecular Biosciences,
1311 Haring Hall, University of California, Davis, CA 95616; 2Istituto Neurologico ‘Carlo Besta’,Via
Celoria, Milan, Italy
Deficiencies in the mitochondrial protein frataxin cause the neuro- and cardio-degenerative disease
Friedreich’s ataxia. Five hypotheses for frataxin’s physiological function in human cells have been
proposed, including as a mitochondrial iron transporter, iron-sulfur cluster assembler, iron-storage protein,
antioxidant and stimulator of oxidative phosphorylation. We have carried out microarray analysis of gene
expression in frataxin-deficient and frataxin-transfected human cells in three cell types. The most
consistent cell-autonomous effects of frataxin-deficiency are on genes involved in Sulfur Amino Acid
(SAA) homeostasis, which are downregulated, in concordance with an involvement of frataxin in
biogenesis or maintenance of Iron Sulfur Clusters (ISCs), and probing specific transcripts and proteins
involved in ISC synthesis indicates a specific deficiency in these molecules in FRDA cells. Biochemical
analysis of Amino acid level indicates a specific alteration in SAA concentrations in this disease. The
second largest number of genes altered was in the apoptotic category, predominantly an up-regulation of
genes involved in the Fas/TNF pathway, suggesting an apoptotic mechanism for the cell degeneration in
FRDA. Furthermore an FRDA-specific cellular deficiency is rescued by small molecules related to these
microarray-based hypotheses. Thus these results support only one of the current hypotheses for
frataxin's function in human cells, and suggest that there are multiple steps in the FRDA pathophysiologic
mechanism, which could each be relevant for pharmacotherapy.
POSTER 176
DECREASED LIVER HEPCIDIN EXPRESSION IN THE HFE KNOCKOUT MOUSE
R.E. Fleming1,3, K.A. Ahmad1, J.R. Ahmann1, M.C. Migas1, R.S. Britton2, B.R. Bacon2, A. Waheed3, W.S.
Sly3. 1Pediatrics; 2Internal Medicine and 3Edward A. Doisy Department of Biochemistry & Molecular
Biology, Saint Louis University School of Medicine, St. Louis, MO.
Introduction: Hepcidin is a circulating antimicrobial peptide which has been proposed to regulate the
uptake of dietary iron and its storage in reticuloendothelial macrophages. Transgenic mice lacking
hepcidin expression demonstrate abnormalities of iron homeostasis similar to Hfe knockout mice and to
patients with HFE-associated hereditary hemochromatosis (HH).
Objective: To determine if there exists an association between liver hepcidin expression and the iron
homeostasis abnormalities observed in HH, we compared liver hepcidin mRNA content in wild-type and
Hfe knockout mice.
Methods: Hfe wild-type and knockout mice on an AKR background were sacrificed at 4 weeks and 8
weeks of age. 8 week old mixed-strain Hfe wild-type and knockout mice were placed on a 2% carbonyl
iron diet for 2 weeks. Liver and spleen samples were obtained from each group of mice, and iron
concentrations were measured. Liver hepcidin mRNA was quantified by RNA blot analysis and/or realtime RT-PCR and normalized to ribosomal RNA.
Results: At four weeks of age, Hfe knockout mice had significantly decreased liver hepcidin mRNA
expression compared to wild type mice. The decreased hepcidin expression was associated with hepatic
iron deposition, elevated transferrin saturations, and decreased splenic iron concentrations. At eight
weeks of age, despite marked hepatic iron loading, Hfe knockout mice demonstrated liver hepcidin mRNA
expression similar to that observed in wild-type mice. Placing 8 week-old wild-type and Hfe knockout mice
on a 2% carbonyl iron led to a similar degree of hepatic iron loading in each group. However, while the
wild-type mice demonstrated a mean 5-fold increase in liver hepcidin mRNA, no change was observed in
the Hfe knockout mice. The lack of an increase in liver hepcidin expression in these iron-loaded Hfe
knockout mice was associated with sparing of iron deposition into the spleen.
Conclusion: Liver hepcidin expression is decreased (relative to iron stores) in Hfe knockout mice
compared with wild-type mice. We speculate that decreased hepcidin expression relative to body iron
stores contributes to the iron homeostasis abnormalities characteristic of HH.
POSTER 177
EFFECT OF IRON TREATMENT ON CIRCULATING CYTOKINE LEVELS IN ESRD PATIENTS
RECEIVING RECOMBINANT HUMAN ERYTHROPOIETIN
G. Weiss*, U. Neyer#, G. Mayer+
*) Department of Internal Medicine and +) Division of Nephrology, University Hospital of Innsbruck,
Austria and #) Department of Nephrology and Dialysis, Feldkirch Hospital, Austria
Background: Anemia in patients with ESRD is treated with recombinant human erythropoietin (rhuEPO)
often in combination with iron. However, iron catalyzes the formation of toxic radicals which might
promote vascular damage, is a nutrient for microorganisms and negatively affects immune pathways, thus
increasing the risk for severe infections.
Methods: We investigated 28 patient on chronic hemodialysis who were randomized to receive either
rhuEPO alone (n=15) or rhuEPO in combination with intravenous iron (n=13) for a period of twelve
weeks. We analyzed iron therapy associated changes in cytokine patterns and endogenous radical
formation.
Results: Tumor necrosis factor–alpha (TNF-α le ve ls we re incre a s e d in ES RD pa tie nts a t s tudy e ntry
and then decreased significantly over time in subjects receiving additional iron while they increased with
rhuEPO alone. In contrast we found serum concentrations of the anti-inflammatory cytokine interleukin
(IL)-4 to increase with iron therapy. A significant negative correlation between iron availability, as
determined by transferrin saturation (TfS), and TNF-α levels (p= 0.008) and a positive one between TfS
and IL-4 (p=0.02) pointed to the potential role of iron to induce immunological changes. Interestingly, iron
therapy resulted in a slight decrease in the amounts of endogenous peroxides, which may be referred to
reduced TNF-α concentrations since peroxide concentrations were positively correlated to TNF-α levels
(p=0.046) and negatively to TfS (p=0,02).
Conclusions: Iron supplementation in ESRD patients down-regulates pro-inflammatory immune effector
pathways and stimulates the expression of the anti-inflammatory cytokine IL-4. Such a condition is
detrimental for host response towards invading pathogens. However, tissue damage by radicals such as
endogenous peroxides may be reduced in this condition due to impaired TNF-α formation.
POSTER 178
EXPRESSION OF HUMAN MITOCHONDRIAL FERRITIN IN YFH1 STRAIN PROTECTS
YEASTS FROM FRATAXIN DEFICIENCY AND PARTIALLY RESTORES MITOCHONDRIA
FUNCTIONALITY
A. Campanella (1), G. Isaya (3), H. A. O'Neill (3), B. Corsi (1), P. Santambrogio (1), A. Cozzi (1),
P. Arosio (2), S. Levi (1)
Department of Biological and Technological Research, IRCCS H. San Raffaele, Milano, 20132
Italy; (2) Section of Chemistry, Faculty of Medicine, University of Brescia, 25100 Italy; (3)
Departement of Pediatric and Adolescent Medicine, Mayo Clinic and Fondation, Rochester, USA.
Studies on transfectant HeLa cell models indicated that mitochondrial ferritin (MtF) plays an
important role in regulating mitochondrial iron trafficking and in protecting the organelle form ironinduced damage (1). This hypothesis is supported by the finding of large amounts of MtF in the
erythroblasts from subjects with sideroblastic anemia, peculiar cells that are viable despite
massive mitochondrial iron loading (2). Friedreich’s ataxia (FRDA) is caused by frataxin
deficiency, through a mechanism not completely elucidated, and is associated to mitochondria
iron deregulation, which is thought to be a major cause of pathogenicity (3). We considered that
MtF might protect from the mitochondria iron excess in FRDA. The simplest model of FRDA are
the yeast strains deficient in YFH1 (∆yfh1), the frataxin homologue, which develop mitochondrial
iron loading. To test the hypothesis, we cloned human MtF in the high copy number pG3 vector
for expression in yeast. Analysis of the transformed cells showed that the protein is efficiently
expressed, it is properly processed into the mature protein, and assembles in ferritin shells
localized in the mitochondria. MtF functionality was demonstrated by the in vivo incorporation of
radioactive iron. We concluded that MtF acts as an iron storage protein also in yeast, which lack
of any ferritin type. Yeast strain ∆yfh1 grow slowly in SD minimal medium, does not grow in nonfermentable solid media and is highly sensitive to H2O2. However, when this strain was
transformed with the pG3-HMtF vector (∆Yfh1 [HMtF]), the growth rate in minimal medium
increased, growth capacity in non-fermentable solid media was restored and resistance to H2O2
increased. In addition we observed that the PCR-detectable mitochondrial DNA disappeared after
the growth in minimal medium added of
14 µM iron in the ∆Yfh1 strain but not in the ∆Yfh1 [HMtF] strain. Moreover we analysed aconitase
and succinate dehydrogenase activities as markers of the Fe/S production. In SD minimal
medium∆Yfh1 [HMtF] and ∆Yfh1 strains show similar activities, lower than ∆Yfh1 [YFH1], while
these activities were strongly decreased in the ∆Yfh1 but not in the ∆Yfh1 [HMtF] in presence of
14 µM iron. In conclusion, the findings indicate that MtF partially complement the phenotype
caused by frataxin deficiency by restoring respiratory activity, increasing resistance to oxidative
damage, protecting mitochondrial DNA and the residual activity of Fe/S enzymes. This is
consistent with the MtF capacity to sequester excess iron, and reduce local iron toxicity.
Supported by grant n° GP0075Y01 from Telethon (to S.L.)
(1) Corsi B, Cozzi A, Arosio P, Drysdale J, Santambrogio P, Campanella A, Biasiotto G, Albertini
A, Levi S. Human mitochondrial ferritin expressed in HeLa cells incorporates iron and affects
cellular iron metabolism. J Biol Chem. 2002 Jun 21;277(25):22430-7.
(2) Cazzola M, Invernizzi R, Bergamaschi G, Levi S, Corsi B, Travaglino E, Rolandi V, Biasiotto
G, Drysdale J, Arosio P. Mitochondrial ferritin expression in erythroid cells from patients with
sideroblastic anemia. Blood. 2003,101(5):1996-2000.
(3) Campuzano V, Montermini L, Molto MD, Pianese L, Cossee M, Cavalcanti F, Monros E,
Rodius F, Duclos F, Monticelli A, et al. Friedreich's ataxia: autosomal recessive disease caused
by an intronic GAA triplet repeat expansion. Science. 1996 , 271(5254):1423-7.
POSTER 179
EXPRESSION OF DCYTB IN PHAGOCYTIC CELLS
Y. Sánchez, J. McGregor, A.T. McKie, King’s College London
Previous studies have shown the presence of a ferric reductase activity in leukocytes and HL-60 cells, the
latter of which was found to be NADH-independent A candidate for this activity is Dcytb, originally
identified as a 30kDa ferric reductase located on the apical membrane of intestinal enterocytes.
Preliminary data have confirmed a high level of expression of Dcytb in human peripheral neutrophils,
murine bone marrow-derived macrophages, and HL-60 cells differentiated into neutrophils and
macrophages. The aim of the present study was to investigate the level and site of expression of Dcytb in
the named cells by semi-quantitative RT-PCR and immunohistochemistry. Additionally, cells were
stimulated with immuneregulators or cytokines in order to mimic infection and therefore analyse any
changes in Dcytb expression. High expression of Dcytb in these granulocytes would suggest that Dcytb
may play an important role in host defense by recycling ferrous iron at the plasma and phagosomal
membranes to aid the killing of pathogens via Fenton chemistry.
King’s College London, London, UK
POSTER 180
APPROACHES FOR REPRESSING FRATAXIN IN HELA CELLS USING SMALL INTERFERING RNAS
(siRNAs)
I. Zanella, C. De Cato, G. Biasiotto, G.M. Gerardi, F. Taroni, A. Albertini, P. Arosio.
Brescia, and Division of Biochemistry and Genetics Istituto Nazionale Neurologico "Carlo Besta", Via
Celoria,11 – 20133 Milano, Italy
Frataxin is a mitochondrial protein defective in Friedreich’s ataxia (FRDA). Its involvement in iron
metabolism is indicated by the finding of iron deposits in the heart of subjects with FRDA and by a
massive iron deposition in the mitochondria of yeast cells deficient the in the frataxin homologue, Yfh1p. It
has been indicated that frataxin acts as an iron binding protein, or that it has a role in the assembly of
iron-sulphur clusters, but its actual role in mitochondrial iron handling is unclear. Yeasts have been largely
used as models for the FRDA, although they differ markedly from mammalian cells in the regulation of
iron homeostasis, and lack of the iron storage proteins, ferritins. Thus, simple, frataxin-deficient,
mammalian cell models may be useful to study the role of the protein in the regulation of cytosolic and
mitochondrial iron. In addition, they can be used to verify whether mitochondrial ferritin (MtF) may protect
mitochondria from the iron excess caused by frataxin deficiency, as it seems to occur in the yeast. We
started exploring the new technology of small interfering RNA (siRNA) to repress frataxin expression in
HeLa cells. To this aim we produced by in vitro transcription 5 double-strand siRNAs complementary to
different regions of frataxin mRNA, which were transfected in the cells. Blotting experiments with specific
antibodies showed that all ds-siRNA repressed frataxin expression down to 10-40% of the controls. A
time curse analysis using the most efficient siRNA showed that the highest repression was obtained at
24-48 h post transfection with a residual Frataxin level of about 10%. Preliminary results indicate that the
artificial frataxin down-regulation causes a significant reduction of cell proliferation rate, while does not
seem to affect the level of cytosolic L ferritin. Work is in progress for a detailed characterization of HeLa
cell frataxin-deficient phenotype and to study the effects of MtF expression on it. In conclusion, present
data show that siRNA is an efficient technology for the transient down-regulation of frataxin. It can be
extended to constitutive or inducible down-regulation by using proper vectors, now in construction.
POSTER 181
PRO-INFLAMMATORY CYTOKINES MODULATE THE EXPRESSION OF IRON TRANSPORT
FACTOR GENES IN HUMAN INTESTINAL CELLS
O, Han, S, Bu, A, Penner, V, Chilton, Oklahoma State University, OK 74078, USA
Pro-inflammatory cytokines are thought to play a key role in iron homeostasis in response to the chronic
infection and inflammation. It has been previously reported that pro-inflammatory cytokines down-regulate
intestinal iron transport across the human enterocyte by increasing cellular iron levels (Han et al., 1997)
but its mechanism of action remains unknown. To elucidate how pro-inflammatory cytokines influence
intestinal iron metabolism and transport, fully differentiated human intestinal Caco-2 cells grown on
microporous membrane inserts were treated for up to 48 hours with pro-inflammatory cytokines, human
IL-1β (30 ng/ml), human IL-6 (30 ng/ml) or human TNFα (5 ng/ml) in the basolateral compartment. After,
cytokine treatment, the expression of genes involved in intestinal iron metabolism was measured. Our
observation showed that while expression of divalent metal transporter1 (DMT1) and transferrin receptor
(TfR) protein levels initially increased (up to 110%) and then decreased, the putative ferroxidase
hephaestin and iron exporter, ferroportin1 protein levels remain decreased (by 30-50%) by the treatment
with the individual pro-inflammatory cytokines. These results suggest that pro-inflammatory cytokines may
regulate iron transport in human intestinal cells by modulating the expression of iron transport factor
genes.
POSTER 182
ALFA-SYNUCLEIN TRANSGENIC MICE SHOWS IRON ACCUMULATION IN BRAIN.
FE-SYNUCLEIN INTERACTION: A POSSIBLE MECHANISM FOR NEURODEGENERATION
IN PARKINSON DISEASE
Ruma Raha-Chowdhury1,2, Piers C Emson2, Maria Spillantini1, and Timothy M Cox3.
1Cambridge Centre for Brain Repair, University of Cambridge, 2Neurobiology Programme, The
Babraham Institute, Babraham, 3Department of Medicine, University of Cambridge, UK.
Parkinson’s disease (PD) is a common neurodegenerative disease characterized by loss of dopaminergic
neurones in the substantia nigra pars compacta (SNc), and also Lewy bodies in some surviving
neurones. Accumulation of -synuclein in Lewy bodies and neurites is a pathological hallmark of
Parkinson Disease (PD). Mutations in the -synuclein are only found in familial PD, suggesting
that altered -synuclein function can trigger neurodegeneration. The aetiology of PD is unknown,
however, iron is abundant in brain areas that degenerate in this disorder. Accordingly, we examined iron
deposition in human brain and mouse brain tissues. Transgenic mice expressing human αsynuclein (human wild type, A30P and A53T mutations) were used in this study to investigate the
functional role of α-Synuclein and iron in neurodegeneration. We investigated non-haem iron in
mouse brain and
liver samples. The expression of iron-metabolism genes: ferritin, divalent metal transporter1 (DMT1),
ferroportin 1 (MTP1), hephaestin (HEPH), hepcidin, and transferrin receptor (TFR) were determined by insitu hybridisation and RT PCR in the brain, duodenum and liver of synuclein mice. Several of these
mRNA transcripts were examined in Parkinson’s disease (PD), Alzheimer’s disease (AD)
and control brain samples. The results showed that with age, the accumulation of iron in the brain of
-synuclein/transgenic mice was greater than in age-matched controls (108.01 g/g vs.
207.14 g/g, in control brain vs in transgenic mice brain tissues, p<0.001). There was no
difference in non-heam iron concentrations in the liver, of transgenic or control mice (264 g/g in
controls vs. 294 g/g in transgenic mice). -synuclein was found to colocalise with ferritin
in globus pallidus, substantia nigra, thalamus and several other brain regions. Ferritin, DMT1 and MTP1
have higher expression in α-synuclein mice than control mice. Our findings implicate the activity of
iron transporters in the neurodegenerative process in PD. We propose the iron has a specific role in the
pathogenesis of PD and other neurodegenerative diseases.
POSTER SESSION 3
POSTER 183
IRON STORAGE IN SALMONELLA
A.K. Robinson1, P. Golby1, P.A. Barrow2 and S.C. Andrews1, 1University of Reading and 2Institute of
Animal Research, Compton, UK
Pathogenic bacteria such as Salmonella enterica are of major importance as food-borne pathogens and
iron acquisition plays a vital role in their physiology and pathogenicity. Mutations affecting iron (II) and
(III) uptake, mediated by FeoB and TonB-dependent pathways (respectively), inactivate all known primary
iron-uptake systems of S. enterica and affect the bacteria's ability to occupy certain compartments within
the host (such as the gut). S. enterica is closely related to the model bacterium Escherichia coli. E. coli,
like S. enterica, contains at least four genes that may have roles in iron storage: at 75 min is bfd encoding
the 64-residue [2Fe-2S]-containing ‘bacterioferritin-associated ferredoxin’ (Bfd), and bfr encoding
bacterioferritin (Bfr); at 43 min is ftnA encoding a ferritin (FtnA), and ftnB encoding a ferritin-like protein
(FtnB). E. coli uses FtnA to store iron under iron-rich conditions during post-exponential growth. This
provides a source of iron that partially compensates for iron deficiency during iron-restricted growth.
Given the importance of iron to bacterial colonisation and survival in vivo, we have initiated a study aimed
at investigating the role of the iron-storage systems of S. enterica in gut colonisation and pathogenicity.
Various ‘iron-storage mutations’ were produced in S. enterica by substitution of central segments of the
corresponding structural genes with antibiotic-resistance cassettes, followed by allelic exchange using the
temperature-sensitive plasmid, pDM4. Mutations were moved between strains using P22 transductions
to create double and triple mutants. S. enterica iron-storage mutants were pre-cultured in iron-replete
media to enable the accumulation of iron stores within iron-storage proteins (where present). Subsequent
inoculation into iron-replete media did not reveal any major difference in growth phenotype between the
various strains. However, upon inoculation into iron-restricted minimal medium containing 50 µM DTPA
(an extracellular ferric-iron chelator) there was a substantial decrease in growth rate in some strains. All
of the ftnA mutants were strongly affected and additional knockout of the bfr gene enhanced this effect
somewhat. Also, compared to WT, the ftnB/bfr double mutant had a small reduction in growth yield.
Plasmids harbouring ftnA and bfr expressed from their own promoters were transformed into the ftnA
knockout strain to determine whether the growth phenotypes could be complemented. The plasmid
expressing ftnA rescued the cells but the plasmid expressing bfr did not.
Whole-cell iron assays showed that, as for E. coli, there is an approximate doubling of iron content during
the transition from mid-log to stationary phase. Additionally the ftnA and bfr knockout strains had a lower
iron content than the WT in stationary phase and combination of the ftnB and bfr mutations resulted in a
slight reduction in iron content. The iron deficiency caused by the ftnA and bfr mutations was enhanced
by combining the mutations in a single strain. Currently, the effects of the iron-storage and -transport
mutations on virulence and gut colonisation are being investigated.
The above results suggest that intracellular iron, stored within FtnA, can partially compensate for poor
availability of exogenous iron and is thus consistent with previous work in E. coli. However, the effect of
the bfr mutation on growth and iron content differs from the results obtained for E. coli where the bfr
single mutation had no effect on iron content or growth, even when combined with the ftnA mutation.
These differences may be due to differing expression patterns of ftnA and bfr in S. enterica as compared
to E. coli. As far as we are aware, this is the first time that an iron-storage defect and iron-restricted
growth deficiency have been found to be associated with a bfr mutation for any bacterium.
POSTER 184
DMT1: AN IRON 2+ AND COPPER 1+ TRANSPORTER
M. Arredondo, C. V. Mura, A. Esparza and M. T. Núñez. Laboratorio de Micronutrientes, Instituto de
Nutrición y Tecnología de los Alimentos, Departmento de Biología, Facultad de Ciencias, and Millennium
Institute for Advanced Studies in Cell Biology and Biotechnology, Universidad de Chile.
Introduction. DMT1, also known as Nramp2 and DCT1, is the transporter responsible for iron uptake from
the intestinal lumen into the enterocyte. Electrophysiological evidence suggests that DMT1 can also be a
transporter for other metals. In this work, we examined the potential role of DMT1 as a copper transporter
in intestinal Caco-2 cells.
Methods. Cells: Caco-2 cells grown in bicameral inserts were used as test model. Antisense treatment: in
a strategy to decrease DMT1 expression, cells were transfected with eight different antisense
oligonucleotides. After 3 days of treatment, the cells were assayed for DMT1 protein expression and
DMT1 function. Copper and iron uptake and competition experiments. To measure uptake, cells were
59
incubated at 37°C in saline supplemented with 5 µM Fe-ascorbate (1:50, mol:mol) or the Fe-NTA
64
64
complex (1:2.2, mol:mol), or with 5 µM Cu as the Cu-histidine complex (1:10, mol:mol) in the apical
medium. 59Fe or 64Cu radioactivity was evaluated in cell extracts. Competition studies between Fe2+ and
Fe3+, and Cu2+ and Cu1+ uptake were performed with cells grown in polycarbonate cell culture inserts.
When the effect of copper on iron uptake was tested, the apical chamber contained 5 µM 59Fe as the 5
µM 59Fe-ascorbate (1:50, mol:mol) or the Fe-NTA complex (1:2.2, mol:mol), plus 0.5-250 µM Cu as a Cuhistidine complex (1:10, mol:mol). When the effect of iron on copper uptake was tested, the apical
chamber contained 5 µM 64Cu-histidine with or without 250 µM ascorbate, plus 0.5-250 µM Fe3+ either as
FeCl3-NTA (1:2.2 mol:mol), or as freshly prepared FeSO4.
Results. Treatment of cells with one DMT1 antisense oligonucleotide resulted in 80% and 48% inhibition
of iron and copper uptake, respectively, while seven other antisense oligonucleotides produced smaller or
no inhibition of apical iron uptake. Cells incorporated considerable amounts of copper as Cu1+, while Cu2+
transport was about ten-fold lower. Cu1+ inhibited apical Fe2+ transport. Fe2+, but not Fe3+, effectively
inhibited Cu1+ uptake. The iron content of the cells influenced both copper and iron uptake. Cells with low
iron content transported 4-fold more iron, and 3-fold more copper than cells with high iron content.
Conclusions. The above results demonstrate that DMT1 is a physiologically relevant Cu1+ transporter in
intestinal cells. The findings reported here are germane for the understanding of the basic mechanisms of
intestinal copper absorption and their interplay with intestinal iron absorption. They are also relevant for
the establishment of strategies to prevent excessive copper intestinal absorption in pathologies such as
hereditary hemochromatosis, where DMT1 activity is upregulated.
POSTER 185
FUNCTIONAL ROLE OF DMT1 (IRE+) OF HUMAN HEPATOCYTES AND DMT1-OVEREXPRESSED
HEPATOMA CELLS FOR NON-TRANSFERRIN-BOUND IRON UPTAKE
M. Shindo, K. Ikuta, J. Inamura, K. Ohnishi, H. Shinzaki, Y. Torimoto, Y. Kohgo
Third Department of Internal Medicine, Asahikawa Medical College, Midorigaoka Higashi 2-1, Asahikawa,
Hokkaido 078-8510, Japan
Most of cells requiring iron, such as erythroid precursors, usually take up iron from transferrin (Tf) by
transferrin receptor (TfR)-mediated endocytosis. Because most of serum iron is bound to Tf, the role of
non-transferrin bound iron (NTBI) is considered to be limited. However, under the conditions of iron
overload, Tf is fully saturated with iron, and a substantial amount of serum NTBI is detected. Although it is
known that serum NTBI is rapidly cleared by liver and iron is preferentially deposited in hepatocytes, the
detail of the mechanisms was poorly understood. Recent advances of iron regulatory molecules
demonstrate that dietary iron is transported into intestinal enterocytes by divalent metal transporter 1
(DMT1), and DMT1 transports divalent cations, such as Fe2+, Zn2+, Cu2+ and Ni2+, and is a major duodenal
ferrous iron transporter. DMT1 is also suggested to play an essential role in iron transport from TfR
mediated recycling endosome to intracytoplasm. In the present study, we raised antiserum against human
DMT1 (iron responsive element: IRE+) protein derived from DMT1 mRNA with IRE and investigated the
distribution of DMT1 in human liver and hepatoma cell line HLF. We also established DMT1overexpressed HLF cells and analyzed the functional role of DMT1 in ferrous iron uptake into liver.
Immunohistochemical study revealed that the expression of DMT1 was mainly observed in the membrane
side of duodenal enterocytes and enterocyte cell line Caco2, whereas the DMT1 in normal hepatocytes
and HLF cells was preferentially located in cytoplasm and weakly in cell surface. In addition, the irondepleted HLF by desferrioxamine (DFO) treatment increased the membrane expression of DMT1,
suggesting that the intracellular iron concentration regulates the DMT1 expression in hepatocytes via iron
responsive protein (IRP)/IRE system. Because the viability of the cells treated with DFO was not suitable
for the period of functional study in HLF cells, we overexpressed DMT1 in HLF by transfecting DMT1
cDNA. This transfectant expressed DMT1 protein in both cytoplasm and cell membrane similar with the
HLF cells treated with DFO. Tf-dependent and Tf-independent iron uptake by this transfectant was
59
performed by using Fe. Although DMT1-overexpressed HLF did not change the Tf-dependent iron
uptake, these cells took up significant amounts of non-transferrin bound ferrous iron from outside to inside
of cells. These results indicate that the DMT1 (IRE+) may play an important role in transporting the NTBI
into cells by different pathway from Tf-TfR dependent iron uptake in hepatocytes.
POSTER 186
CHARACTERIZATION OF THE HEME-INDUCED OXIDATION OF IRP2
H. Ishikawa, K. Ishimori, I. Morishima, K. Iwai. Osaka City University, Osaka, Kyoto University, Kyoto,
CREST, Saitama Japan
Iron regulatory proteins (IRPs) are the central regulators of iron metabolism by controlling the expression
of genes involved in iron metabolism via binding to iron responsive elements (IREs) found on the mRNAs
encoding those molecules in iron-depleted condition. In mammalian cells, two IRPs, IRP1 and IRP2, have
been identified to date. While IRP1 and IRP2 share 58% overall amino acid identity, they do seem to
exhibit different biochemical properties and mechanisms of regulation. High iron levels promote the
assembly of an Fe-S cluster in IRP1 that has aconitase activity, with concomitant loss of IRE-binding
activity. In iron-depleted cells, IRP1 loses both the Fe-S cluster and the aconitase activity, but exhibits the
IRE-binding activity. On the other hand, the IRE-binding activity of IRP2 is controlled by the stability of
protein. In iron-replete cells, IRP2 is oxidized and degraded by the ubiquitin-dependent protein
degradation. A specific 73 amino acid domain is thought to be important for the iron-dependent
degradation (IDD) of IRP2, which is called the IDD domain. Recently, we have found that the oxidation of
IRP2 is induced by the addition of hemin and oxygen in vitro. Although the recognition of the specific
oxidative modification of IRP2 by HOIL-1 ubiquitin-protein ligase causes the degradation of IRP2, the
heme-induced oxidation mechanism of IRP2 has not been characterized.
Since the deletion IDD domain from IRP2 resulted in the inhibition of the ubiquitin-dependent degradation
of IRP2, the heme binding to the IDD domain is crucial for the oxidative modification which provokes
ubiquitination of IRP2. To characterize the oxidation mechanism for IRP2, we examined the heme binding
to the IDD domain and the axial ligands for heme iron in IRP2. In the presence of ferric heme, IRP2 gave
the g-values at 2.41, 2.27, and 1.91 on their EPR spectra, which are typical of cysteine-ligated low-spin
hemoproteins. The EPR spectrum of the isolated IDD domain also exhibited similar g-values to those of
IRP2, indicating that cysteine in the IDD domain is one of the axial ligands of the heme iron in IRP2. The
absorption and resonance Raman spectra for the heme-bound IDD, however, clearly showed that
reduction of the heme iron replaces the axial cysteine with histidine. Since the molecular oxygen could
bind only to the ferrous heme iron, the axial histidine facilitates ligation of molecular oxygen to the heme
iron to generate reactive oxygen species essential for the oxidative modification to the ubiquitination of
IRP2. To further characterize the heme-induced degradation mechanism of IRP2, the identification of the
modification site recognized by HOIL-1 is now in progress.
POSTER 187
A NEW MECHANISM FOR MEMBRANE IRON TRANSPORT IN PSEUDOMONAS AERUGINOSA
I. J. SCHALKa, K. POOLEb, M. VINCENTc, J. GALLAYc, M. A. ABDALLAHa and F. PATTUSa
a
Laboratoire des Récepteurs et Protéines Membranaires, UPR 9050, CNRS, ESBS, Bld Sébastien Brant,
67 400 Illkirch, France.
b
Department of Microbiology and Immunology, Queen's University, Kingston, Ontario, Canada K7N 3N6.
c
Laboratoire pour l’Utilisation de rayonnement Electromagnétique (LURE), CNRS-CEA-MDRI, Université
Paris-Sud, Bâtiment 209D, 91898 Orsay Cedex, France.
Under iron limiting conditions, Pseudomonas aeruginosa secretes a fluorescent siderophore called
pyoverdin (PaA), which, after complexing iron, is transported back into the cells via its outer membrane
receptor FpvA. FpvA is a TonB-dependent outer membrane transport protein. The siderophore PaA is
composed of a fluorescent chromophore (a derivative of the 2,3-diamino-6,7-dihydroxyquinoline) bound to
an 8 amino acids peptide.
In our laboratory, we have shown that the fluorescence spectroscopy properties of iron-free PaA allow
transfer of fluorescence resonance energy (FRET) between tryptophans of the FpvA receptor and ironfree PaA when excited at 290 nm (1-3). Using FRET, we have shown that all FpvA receptors on the
3
bacterial cell surface are loaded with iron-free PaA under iron limiting conditions (1 and 3). [ H]PaA',
55
[ Fe]PaA-Fe and a kinetically stable chromium-PaA complex have been used to demonstrate that iron
loading of the receptor occurs through a siderophore displacement mechanism in vivo, rather than an
exchange of the metal ion between PaA and PaA-Fe (4). In vivo, the kinetics of formation of this FpvAPaA-Fe complex is regulated by the TonB protein (3). Moreover, the fluorescence properties of iron-free
PaA revealed that after PaA-Fe uptake and dissociation, the PaA molecule is recycled into the
extracellular medium (4). This technic of Fluorescence Resonance Energy Transfer (FRET) between the
PaA chromophore and the FpvA tryptophans in vivo was used to monitor the kinetics of PaA
displacement by PaA-Fe at the cell surface, the dissociation of iron from the siderophore and the
recycling of PaA back to the receptor on the outer membrane of the bacteria in real time (4). At last, time
resolved fluorescence spectroscopy showed the existance of at least two different conformations for the
FpvA-PaA complex (5). The dihydroquinoline moiety of PaA in both conformers is fully protonated, or
coordinated by protein charged groups, but the polarity of its environment, its solvent accessibility, and its
3+
3+
rotational dynamics are different. In the presence of Ga or Al ions, the solvent accessibility and
mobility of the dihydroquinoline moiety are intermediate between those observed for the metal-free ones.
In conclusion, our study on PaA-mediated iron transport in P. aeruginosa revealed a different iron
transport mechanism as the one usually proposed for ferrichrome in E. coli (6 and 7) or suggested by
Stintzi and collaborators (8) for Aeromonas hydrophila. This new mechanism will be discussed in the ligth
of all this new findings.
References:
1. I. J. Schalk et al. (1999) Biochemistry 38, 9357-9365.
2. N. Folschweiller et al. (2000) Mol Memb Biol 17, 123-133.
3. I. J. Schalk et al. (2001) Mol. Microbiol. 39, 351-360.
4. I. J. Schalk et al. (2002) Biochemistry 41, 1663-1671.
5. N. Folschweiller et al. (2002) Biochemistry 41, 14591-14601
6.V. Braun, 1998, Science 282, 2202-2203.
7. V. Braun, 1998, Met Ions Biol Syst 35, 67-145.
8. A. Stintzi et al., 2001, Proc Natl Acad Sci USA 98, 12837-12842.
POSTER 188
EXPRESSION OF THE NON-HEME FERRITIN OF THE ARCHAEON HALOBACTERIUM SALINARUM
IS NOT POST-TRANSCRIPTIONAL REGULATED
S. Reindel, C.L. Schmidt, B.F. Matzanke, University of Luebeck
e-mail: matzanke@physik.uni-luebeck.de
Members of the ferritin superfamily were found in bacteria and eukarya but none were isolated thus far
from archaea. We isolated and purified an iron-containing protein from the euryarchaeon H. salinarum [1].
This protein shows typical features of a ferritin: it is heat stable, the molecular mass of a subunit is 20
kDa, the subunit exhibits a highly α-helical secondary structure (CD spectroscopy) and assembles to a
hollow spherical shell where the iron atoms can be deposited (ESR spectroscopy). We were not able to
determine heme groups (UV/VIS spectroscopy). Normally, the protein shell of a ferritin is built up by 24
subunits, but our size exclusion experiments show a dodecameric assembly. Sequence comparison of
protein fragments with a genome derived protein library of H. salinarum sp. NRC-1 yields 100% identity
with DpsA (DNA binding protein from starved cells). Further sequence alignments (FASTA) show
identities with the DpsA from Synechococcus sp., with the non-heme ferritin from Listeria innocua and
with the non-heme ferritin from Helicobacter pylori. Both non-heme ferritins belong to the new class of
ferritins which exhibit a dodecameric structure. The molecular modelling (MOLMOL) of the halobacterial
DpsA yields a four-helix-bundle structure with one additional smaller α-helix.
Surprisingly, Dps proteins from E.coli (Dps), Bacillus subtilis (MrgA) and Synechococcus sp. Strain PCC
7942 (DpsA) show the same protein properties as the new class of 12-meric ferritins. Differences exist,
however, on a regulatory and a functional level. Dps proteins exhibit non-specific DNA binding [2-4]. The
expression of the dps/dpsA/mrgA mRNA is low in exponentially growing cells and increases as cells enter
stationary phase. The transcription is also induced under conditions of iron starvation and this induction
can be blocked by adding iron. The expression is also induced in response to oxidative stress [5]. In order
to decide whether halobacterial DpsA is functionally a Dps or a ferritin we examined the expression of the
isolated protein under different growth conditions by Northern and Western blot analysis throughout all
growth phases. A comparison of transciption in early and late log growth phase under conditions of iron
starvation did neither show a decrease nor an increase of halobacterial DpsA. The same was found under
conditions of iron deficiency and oxidative stress (1mM H2O2). In iron rich media with and without
hydrogen peroxide, however, an increase was observed. The analysis of the Western blots under the
same growth conditions shows a constant post-transcriptional signal throughout all growth phases.
Our results from the Northern blot analysis show clearly that the expression of the halobacterial dpsA
does not correspond to a transcription found in the dps/dpsA/mrgA system. Furthermore, we can exclude
a post-transcriptional regulation of our protein on the basis of the Western blot analysis. This fact was of
special interest because regulation in archaea is in some cases like in eukarya, in other cases like in
bacteria. The ferritin expression in eukarya is strictly post-transcriptional regulated. Based on our
observations, we can conclude that the regulation of halobacterial DpsA differs from that of the other Dps
proteins. Rather, regulation seems to be similar to what is known about the bacterial ferritin regulation [6,
7].
We thank A. Hiller for excellent assistance (immunization of the rabbit). This work is supported by DFG
grant Ma 916/14-1.
1. Reindel, S., et al. (2002) Biochimica Biophysica Acta, 1598: 140-146.
2. Azam, T.A. and Ishihama, A. (1999) Journal of Biological Chemistry, 274: 33105-33113.
3. Pena, M.M.O. and Bullerjahn, G.S. (1995) Journal of Biological Chemistry, 270: 22478-22482.
4. Chen, L. and Helmann, J.D. (1995) Molecular Microbiology, 18: 295-300.
5. Sen, A., et al. (2000) Archives in Microbiology, 173: 352-357.
6. Abdul-Tehrani, H., et al. (1999) Journal of Bacteriology, 181: 1415-1428.
7. Dundon, W.G., et al. (2001) FEMS Microbiology Letters, 199: 143-149.
POSTER 189
CHANGES IN FERRITIN SYNTHESIS THROUGH A TRANSIENT INCREASE OF THE LABILE IRON
POOL IN HEPG2 CELLS MEDIATED BY INTRAVENOUS IRON PREPARATIONS
B. Sturm, B. Scheiber-Mojdehkar, H. Goldenberg, Department of Medical Chemistry, University of Vienna,
Austria
Patients with anemia of chronic renal failure are treated with recombinant human erythropoetin in
combination with administration of intravenous iron. However, the question of exact dosage regime for
this therapy is not settled. The iron compounds used could enter cells apart from the reticuloendothelial
system and and release potentially toxic labile iron. Our studies on the human hepatoma cells HepG2
representing a model for the liver parenchyma clearly show that these cells are capable of ingesting
intravenous iron polymers, Therefore the liver might be a possible target for intravenous iron. Before
being incorporated into the storage protein ferritin or being released back to the circulation, this iron must
pass the labile iron pool (LIP) which regulates the intracellular iron metabolism, and which can be
dangerous if it overflows.
Measuring the labile iron pool with the fluorescent probe calcein-AM (method developed by Cabantchik et
al.) we show a significant increase in the level of the LIP in HepG2 cells with apparent differences
between the various iron sources. When cells were challenged with a range of doses of intravenous iron
the level of the LIP increased in a time and dose-dependent manner. Furthermore, an increase in the
amount of ferritin, measured by an ELISA, strongly related to the size of the LIP was observed.
We conclude that the different iron preparations, namely ferric saccharate, ferric dextran, ferric gluconate
and ferric pyrophosphate affect the LIP differently and that changes in the level of LIP are correlated with
the synthesis of ferritin.
This work was supported by: FWF, # P14842-PAT and # P11594-MED and Hochschuljubiläumsstiftung
der Stadt Wien # H83/200
POSTER 190
DISSECTING THE INVOLVEMENT OF ACCESSORY PROTEINS IN IRON SULFUR CLUSTER
METABOLISM
E. Skovran, C.T. Lauhon, D.M. Downs, University of Wisconsin at Madison
Our laboratory uses Salmonella enterica as a model organism for understanding microbial physiology and
metabolism. In this organism, we have identified several proteins required for optimal iron sulfur (Fe-S)
cluster metabolism in addition to those encoded by the isc and suf operons. Proteins with both known and
unknown cellular functions comprise this class including; GshA, required for glutathione synthesis; ApbC
(Mrp), a cytosolic ATPase; ApbE, a periplasmic membrane protein; RseC, a cytoplasmic membrane
protein; and YggX, an iron binding protein. Biochemical characterization and phenotypic analysis of isc,
apbC, apbE, rse, gshA, and yggX mutant strains has revealed that distinct subsets of these gene
products are required for optimal function of different Fe-S proteins. Characterization of these mutant
strains continues to provide insight into the cellular roles for multiple proteins in the synthesis and repair
of Fe-S clusters.
POSTER 191
Withdrawn
POSTER 192
THE ROLE OF TRANSFERRIN IN THE ACTIVATION OF HEPATIC STELLATE CELLS:
IMPLICATIONS FOR IRON-INDUCED HEPATIC FIBROSIS IN HEMOCHROMATOSIS
G.A. Ramm1, P.S. Rutherford1, A.C. Hoskins1, K.R. Bridle1,2. 1The Hepatic Fibrosis Group, The
Queensland Institute of Medical Research and The University of Queensland, and 2The Department of
Gastroenterology and Hepatology, Princess Alexandra Hospital, Brisbane, 4029, AUSTRALIA.
Background: Hepatic stellate cells (HSC) are known to be involved in iron-induced fibrogenesis,
however the precise role of iron and iron-proteins in HSC activation is unclear. We have recently
identified and characterized a specific, high affinity receptor on activated HSC, which is not expressed on
quiescent HSC (Bridle et al, Am. J. Pathol, 2003, in press). In addition, we have shown that rat holotransferrin (RTf) significantly alters HSC activation, however, the pathways associated with this regulation
are unknown.
Aims: This study was designed to evaluate the differential gene expression profile of RTf-treated HSC, to
assist in identifying the metabolic pathways involved in iron-induced hepatic fibrogenesis.
Methods: Rat HSC were isolated by sequential pronase and collagenase perfusion followed by density
gradient centrifugation. Following culture on plastic for 5 days HSC were treated with 0.2 mg/ml RTf. RNA
was extracted at 6 and 24 hr ± RTf treatment for use in the Atlas Rat 1.2 cDNA array (Clontech, USA).
Analysis of arrays was performed using AtlasImage 2.01 software. Real-time RT-PCR was used to verify
selected cDNA array results, and expression was standardized to the expression of the house-keeping
gene for S9 ribosomal protein. Results are presented as a -fold difference between RTf-treated and
untreated HSC.
Results: (i) cDNA Array. At 6 and 24 hr post-treatment cyclin D1, cyclin D2 and proliferating cell nuclear
antigen (PCNA), were down-regulated (2.0 and 10-fold; 1.7 and 3.3-fold; 1.4 and 3.3-fold, respectively).
While expression of several chemokine/cytokine genes was significantly upregulated at 6 hr but reduced
by 24 hr. Notably, the CXC chemokine LIX, macrophage inflammatory protein 1-alpha (MIP1α) and
macrophage inflammatory protein-2 precursor were upregulated 2.4, 2.5 and 6.7-fold respectively at 6 hr.
In addition, transferrin had significant effects on genes involved in fibrinolysis such as plasminogen
activator inhibitor 2A (42-fold increase at 6 hr; 13-fold increase at 24hr), urokinase-type plasminogen
activator (uPA) precursor (10-fold and 3.3-fold decrease at 6 and 24 hr, respectively) and matrilysin
precursor (2.0 and 1.4-fold decrease at 6 and 24 hr, respectively). Significant changes were also seen in
cell signaling genes, with focal adhesion kinase (FAK), mitogen-activated kinase p38 and protein kinase
C-delta all downregulated at 6 hr (4.2, 2.0 and 3.3-fold, respectively). At 6 hr post-treatment inducible
nitric oxide synthase and nuclear factor kappa-B were also upregulated (6.2-fold and 1.4-fold,
respectively). (ii) Real-time RT-PCR. cDNA array results were confirmed by RT-PCR showing significantly
decreased expression of cyclins D1 (1.4-fold) and D2 (2.0-fold), PCNA (1.9-fold), uPA (5.7-fold), FAK
(2.1-fold), p38 (2.8-fold) and MMP-7 (2.9-fold) at 6hrs post-treatment. Significantly increased expression
of LIX (3.1-fold) and MIP1α (5.8-fold) were also confirmed.
Conclusions: Transferrin decreased HSC expression of cyclin D1, D2 and PCNA which may contribute
to the reduced proliferation of HSC seen following RTf treatment (Bridle et al, 2003, Am. J. Pathol., in
press). The changes observed in fibrinolysis genes (PAI2, MMP7 and uPA) may suggest a role for
transferrin in HSC matrix re-modelling. The alterations observed in cell signaling genes may suggest that
iron/RTf affects pathways separate from those normally associated with iron uptake. These studies
demonstrate that transferrin is responsible for altered expression of many key genes involved in cell
cycling and signaling, matrix degradation and inflammatory responses. The role of iron or cell signaling
due to RTf/RTf-receptor interactions in HSC gene regulation remains to be determined. While these
studies provide important information relating to transferrin-induced gene expression, further investigation
is required to confirm the role of these genes and their respective gene products in HSC biology and in
hemochromatosis.
POSTER 193
IRON HOMEOSTASIS IN ALVEOLAR MACROPHAGES
Roberta J Ward 1, Daniel Leroy 1, Louise Toussaint 1, Robert R Crichton 1 Christophe Pierreux 2, Louis
Hue 2
Unite de Biochimie 1, Hormone and Metabolic Research Unit 2, , Universite catholique de Louvain
Belgium
Macrophages are reputed to play an important role in iron homeostasis, since they are able both to
acquire and store iron as well as to release iron to transferrin and ferritin. They have two major iron
uptake systems, via phagocytosis and transferrin-transferrin receptor cycle. The latter system is thought
to be regulated via iron regulatory element/iron regulatory proteins. In these present studies the effect of
in vivo iron loading via phagocytosis and iron depletion on activation of iron regulatory proteins as well as
mRNA expression of genes involved in iron uptake and release, i.e. DCT1, Ireg1 and transferrin receptors
has been investigated in alveolar macrophages.
Alveolar macrophages were isolated from rats who had been administered either iron dextran (total dose
= 125mg) or an iron-depleted diet, (Fe<0.001g Fe/kg) for a period of four weeks. The IRP activity in the
alveolar macrophages was assayed by band shift assay while the mRNA expression of transferrin
receptors, DCT1 and Ireg 1 was by RT-PCR.
The IRP activity in the alveolar macrophages was regulated by the iron status of the animal; increasing
(10%) and decreasing (46%) in the iron deficient and iron loaded rats, respectively, by comparison to
controls. In the presence of β-mercaptoethanol there was little evidence of increased IRP activities in any
of the three groups .
Transferrin receptor mRNA expression increased significantly, P<0.01, in iron deficient macrophages
although its expression in the iron loaded macrophages was comparable to controls. Ireg1 mRNA
expression was similar in each of the groups. The lack of activation of the IRPs by β-mercaptoethanol in
the macrophage samples is an interesting observation for which we have no explanation. This is in
contrast to the results from immortilised cell lines (Recalcati et al., 1998) as well as our previous in vivo
studies of iron loaded and iron depleted livers (Ward et al., 1994).
Recalcati S. et al., Blood 91 1059-1066, 1998
Ward RJ. et al., Eur J Biochem 220 927-931, 1994
POSTER 194
MECHANISMS AND REGULATION OF TRANSFERRIN AND IRON TRANSPORT IN A MODEL
BLOOD-BRAIN BARRIER SYSTEM
J. R. Burdo1, D. A. Antonetti2, E. Wolpert2 and J. R. Connor1, Penn State University College of Medicine,
1
Department of Neuroscience & Anatomy,and 2Department of Cellular and Molecular Physiology.
Hershey PA, USA
For peripheral iron to reach the brain, it must transverse the blood brain barrier. The barrier consists of
several physical and enzymatic impediments. Transferrin receptors are present in the vascular
endothelial cells presumably to facilitate movement of transferrin bound iron into the brain parenchyma.
However, a number of significant voids exist in our knowledge about transcytosis of iron into the brain.
Among these are, the exact mode of transferrin and iron transport through vascular endothelial cells has
not been identified, there are conflicting data regarding whether transcytosis involves iron, transferrin or
the transferrin-iron complex, the conditions at the abluminal membrane neither favor release of transferrin
from its receptor nor iron from transferrin, and none of the existing studies address the iron requirements
of the endothelial cells. These gaps in our knowledge are significant not only because iron is an essential
neurotrophic factor but also because the system for delivery of iron into the brain is being viewed as an
opportunity to circumvent the blood-brain-barrier for delivery of neurotoxins for tumors or trophic factors in
neurodegenerative diseases. In this study, we have used a bovine retinal endothelial cell (BREC) culture
system to determine the mechanism of transferrin-iron transport and to test the hypothesis that the iron
status of the endothelial cells would influence iron transport. The endothelial cells are grown in a tight
monolayer on Transwell culture inserts and have been used previously to study solute flux. Transferriniron flux across the BREC monolayer was measured by applying 1 uM fluorescein-transferrin conjugated
with 59Fe in the apical chamber of a Transwell apparatus. To control for accumulation of transferrin and
iron in the basal chamber by non-specific paracellular flux, 286 nM rhodamine-dextran was also loaded in
the apical chamber simultaneously with the transferrin for each of the experimental conditions. Dextran is
not taken up at an appreciable level into endothelial cells, so any accumulation in the basal chamber is
due to paracellular transport. None of the experimental conditions altered the dextran transport. Our
results indicated that iron is transported across endothelial cells as both transferrin bound and not
transferrin bound. The ratio of non transferrin-bound to transferrin bound iron transport is dependent upon
the iron status of the cells. Blocking acidification of endosomes led to a significant decrease in
transcytosis of non-transferrin bound iron but not transferrin. These data indicate that some iron is
removed from transferrin within endosomes and later is made available for release into the brain.
Blocking pinocytosis had no effect on either transferrin or iron transcytosis, which is consistent with
previous reports that pinocytosis makes minimal contribution to transcytosis of nutrient or toxins at the
BBB. These results indicate that there is both transferrin mediated and non-transferrin mediated
transcytosis of iron and that the process is influenced by the iron status of the cells. These data have
considerable implications for common neurodegenerative diseases that are associated with excess brain
iron accumulation and the numerous neurological complications associated with brain iron deficiency.
This research is supported in part by funds from the Restless Legs Syndrome Foundation.
POSTER 195
DEVELOPMENT OF A NOVEL METHOD FOR THE MEASUREMENT OF CELLULAR IRON LEVELS
AND IRON-REGULATED PROTEIN EXPRESSION
Rebecca J. Henderson, Stephanie M. Patton, and James R. Connor (Penn State University College of
Medicine)
Iron responsive element (IRE) and iron regulatory protein (IRP) interactions are of great interest to cell
biologists because cellular iron imbalance is capable of causing oxidative stress, a feature which is linked
to many diseases including Alzheimer’s disease (AD), Parkinson’s disease (PD), hereditary
hemachromatosis, and cancer. The associations between IREs and IRPs are affected by changes in
cellular iron concentrations and are therefore useful as reporters for measuring both the posttranscriptional control of IRE-driven genes as well as real-time cellular iron levels. To study these
dynamic interactions, we have developed a cyan fluorescent protein (CFP) - driven assay that can reflect
cellular iron status via changes in CFP production. Here, we show that HEK-293 cells, either transiently
or stably transfected with an IRE-CFP construct, are responsive to iron chelation 16 hours after the
addition of 50 and 100 µM concentrations of desferroxamine (DFO) as verified by a decrease in both
fluorescence, measured by photon counts, and CFP expression, measured by western blot analysis. The
construct is also responsive to the addition of iron by ferric ammonium citrate (FAC) at 50 and 100 µg/mL
concentrations as demonstrated by an increase in both photon counts and CFP production 16 hours after
treatment. The ultimate goal of this assay is to use it as a tool by which to investigate real-time IRE, IRP
interactions. The knowledge gained from having this assay system has numerous applications across a
broad range of cellular and molecular biology.
POSTER 196
BIOCHEMICAL AND COMPUTATIONAL IDENTIFICATION OF NOVEL IRON-RESPONSIVE
ELEMENT CONTAINING GENES
M. Sánchez1, M. Muckenthaler1, B. Galy1, T. Dandekar1,2, P. Bengert2,3, M.W. Hentze1
1
European Molecular Biology Laboratory, Gene Expression Programme, Meyerhofstrasse 1, D-69117
Heidelberg, Germany.
2
Biozentrum, dept. of bioinformatics, University of Würzburg, Am Hubland, 97074 Würzburg.
3
Biochemistry Centre, INF 365, EG(SFB), 69120 University of Heidelberg.
The expression of several key proteins in iron metabolism are regulated via iron-responsive RNA
elements (IREs) and the binding of iron regulatory protein (IRP)-1 or IRP2. Binding of IRP to a 5’ UTR IRE
results in translational inhibition, while IRP binding to the 3’ UTR IREs in transferrin receptor mRNA
causes its stabilization. All known IREs have C at position 1 and G at position 5 of the loop (C1G5 type),
although in vitro studies identified a U1A5-type as another possible IRE sequence that can bind IRP.
Selection experiments were performed by exogenous addition of recombinant His6-tagged IRP1 to Hela
cell RNA (Deferoxamine, DFO, and Hemin treatments), or with endogenous protein-RNA complexes from
Hela cells treated with DFO or Hemin. In both cases, IRP1 polyclonal antibody and A sepharose beads
were used to specifically isolate IRE/IRP mRNPs. Immunoprecipitated RNAs were tested on an
established cDNA-based microarray (the ”Iron chip”) and with Northern blots to validate and optimize the
procedure. In a complementary fashion, we employed biocomputational approaches to identify new IREcontaining candidate mRNAs.
We have confirmed the specific selection of known “IRE genes” such as the transferrin receptor and Lferritin using immunoprecipitated RNA. Additional cell lines and mouse tissues are being tested, as well
as more comprehensive human and mouse cDNA-based microarrays.
So far, 16 putative IRE candidate genes have been identified that will be tested in the next updated
version of the “Iron chip”.
POSTER 197
THE REGULATION OF IRON ABSORPTION IN THE SMALL BOWEL OF COELIAC DISEASE
N. Sharma, C Tselepis, *J Butterworth and *T Iqbal.
Division of Medical Sciences, University of Birmingham, Birmingham, UK, and * City Hospital,
Birmingham, UK.
Background: In the West, coeliac disease is an important cause of iron deficiency. Its prevalence in the
white population in the UK is estimated to be 1 in 300. The classical small bowel lesion seen in coeliac
disease is sub-total villous atrophy (loss of villous height and deepening of mucosal crypts), with a
marked lymphocytic cell infiltrate. This leads to a reduction in the available surface area for iron
absorption. However, surprisingly iron deficiency anaemia is only found in 20% of patients presenting with
coeliac disease. Recently we have screened coeliac patients for the two most common mutations in the
HFE gene, C282Y and H63D, and have shown i) coeliac patients carried both mutations more frequently
than the general population, ii) coeliac patients carrying the C282Y mutation had higher serum iron levels
than coeliac patients with wild type HFE. The aim of this study is to further characterise DMT-1
expression in coeliac small bowel. In addition investigate whether cytokines present in the inflammatory
infiltrate within the small bowel of coeliac patients can modulate the expression of DMT-1, and ultimately
Iron absorption.
Methods: Immunohistochemistry Frozen sections of i) normal small bowel, ii) coeliac small bowel, and iii)
treated coeliac small bowel were processed for immunohistochemistry using the streptavidin-biotin
indirect immunoperoxidase method according to published methods.
Real Time PCR; RNA was extracted from Caco-2 cells following between 1-24 hr cytokine stimulation
(TNF-α, IL-2, IL-6, and IFN-γ), using Trizol reagent according to manufacturer’s instructions. RNA was
reverse transcribed and the cDNA used in Real Time PCR reactions using a 5’ FAM-TAMRA 3’ labelled
DMT-1 probe. All reactions were performed on a Taqman real time PCR instrument (PE7700 ABI Prism).
Results: Using Immunohistochemistry, we found the distribution of DMT1 within the immature crypt
enterocytes of normal small bowel was predominantly cytoplasmic becoming membranous with migration
along the crypt villous axis. However, in coeliac small bowel DMT-1 was localised both in crypt
enterocytes and at the villous surface where it was localised at the cell periphery and within the cytoplasm
and nucleus. This membranous staining within the immature crypt enterocytes suggests that DMT-1 is
being prematurely targeted to the cell surface for iron absorption.
To test if the associated inflammatory response in coeliac small bowel may contribute to DMT-1
expression Caco-2 cells were stimulated with the cytokines TNF-α, IL-2, IL-6 and IFN-γ Us ing Re a l
Time PCR we have shown that all cytokines tested showed a significant induction in DMT-1 mRNA at 1.5
hrs, varying from between 6 and 20 fold, and this increase in DMT-1 was further substantiated at the
protein level by Western blotting.
Conclusions. In this study we have shown evidence that DMT- 1 is not only over-expressed, but also
prematurely targeted to the cell surface in coeliac small bowel. We further provide in vitro evidence that
this modulation in DMT-1 may be directed by cytokines prevalent in the inflammatory infiltrate of coeliac
small bowel. This may explain in part why iron deficiency anaemia is only found in 20% of patients
presenting with coeliac disease, a disease characterised by villous atrophy.
POSTER 198
THE STRUCTURE OF THE DI-IRON CENTER IN THE DESULFOVIBRIO BACTERIOFERRITIN, IN
THREE DIFFERENT STATES
C.V. Romão(1), S. Macedo(1,2), P.M. Matias(1), E. Mitchell(2), A.V. Xavier(1), J. LeGall(3), M.
Teixeira(1), P. Lindley(1,2), M.A. Carrondo(1)
A bacterioferritin was isolated from the anaerobic bacterium Desulfovibrio desulfuricans ATCC 27774. It
contains a heme quite distinct from the heme B: iron-coproporphyrin III, the first example of such a
prosthetic group in a biological system. The heme has a reduction potential of +140mV (pH 7.6), a value
unusually high compared to that of other bacterioferritins (ca.-200 mV). The molecular biology studies
showed that bacterioferritin and rubredoxin-2 genes form a dicistronic operon which reflects the direct
interaction between the two proteins. The X-Ray structure of this bacterioferritin has been determined at
1.95, 2.05 and 2.35 Å resolution corresponding to different intermediates of the di-iron ferroxidase site.
This is the first time that the structure of the native di-iron center in any BFR has been determined.
Molecular surface and electrostatic potencial calculations suggest the presence of a “new” possible route
for the iron uptake, distant from the 3-fold axis channel hypothesized as an entry for the iron atoms.
1- Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa
Av. República, EAN, Apartado 127, 2781-901 Oeiras, Portugal.
2- ESRF, BP-220, F-38043 Grenoble Cedex, France.
3-Department of Biochemistry, University of Georgia, Athens, Georgia 30602, USA.
POSTER 199
UPTAKE MECHANISMS OF CITRATE AND ALBUMIN BOUND NTBI SPECIES INTO HUMAN
HEPATOMA CELLS
R Rafique1, B Ramesh2, SKS Srai2, JB Porter1. 1Department of Haematology, University College London
Medical School, London, UK. 2Department of Biochemistry & Molecular Biology, Royal Free and
University College Medical School, London, UK.
The liver is the major clearance organ for uptake of non-transferrin bound iron (NTBI). Although the
chemical nature of NTBI has not been defined, it is likely that several species exist and that their
mechanisms of uptake differ. Citrate forms a wide range of oligomeric iron (III) species, which have been
proposed to be the dominant NTBI form in iron-overloaded plasma. However, ultrafiltration experiments
we have undertaken with thalassaemic plasma or with ferric citrate in the presence of human albumin
show a low iron recovery in the ultrafiltrate, suggesting NTBI is predominantly protein bound. Albumin is
present at high concentrations in the blood and has substantial low-affinity iron-binding capacity, both in
the presence and absence of citrate. We hypothesized that albumin may modify hepatocyte NTBI uptake
from citrate and that the mechanisms of citrate and albumin bound iron species may differ. This study
uses human hepatoma cells (HuH7) to examine the effect of albumin on citrate-iron uptake, the
dependence of iron uptake on reduction through Dcytb and the dependence of divalent uptake on DMT1.
59
Initial uptake experiments used an albumin free system with 1µM [ Fe]-ferric citrate at an iron:citrate ratio
of 1:100. Under these conditions, which are relevant to transfusional iron overload, oligomeric species of
iron-citrate predominate. After an incubation of up to 2hrs at 37°C, internalized iron was separated from
membrane-bound iron by a pronase digest at 4°C.
The rates of intracellular and membrane-associated iron uptake from ferric citrate increased linearly with
time and after 1hr more than 80% was internalized. Total uptake reached saturation at an iron
concentration of ~6µM. Optimal iron uptake was observed at physiological pH (7.4) and at iron:citrate
ratios of 1:1-1:100. Albumin (0-40 mg/ml) inhibited intracellular and membrane-bound iron uptake in a
concentration-dependent manner, suggesting that albumin binding decreases availability of citrate iron
species for transport into the cell. Increasing concentrations of ascorbate (0-1000µM), stimulated cellular
iron uptake in the presence or absence of albumin, suggesting that uptake is dependent on the reduction
3+
2+
of ferric iron (Fe ) to the ferrous form (Fe ). The divalent metal manganese (0-100µM) inhibited cellular
iron uptake in the presence and absence of albumin by up to 70%, suggesting that iron and manganese
are internalized by shared transport mechanism(s). Blocking studies with Dcytb antibody failed to inhibit
iron uptake or reductase activity (measured by thiazolyl blue reduction) in the HuH7 cells, in contrast to
previous studies with HuTu-80 cells using the same antibody (McKie A et al, Science, 2001, 291:175559). This suggests other ferric reductase systems are involved. Similarly, DMT1 is unlikely to be directly
involved in HuH7 cells, as antibody to DMT1 failed to inhibit iron uptake at either low (6.5) or high pH (7.4)
values, again in contrast to the effects previously reported of this antibody on iron transport in CaCo2
cells (Tandy S et al, J Biol Chem, 2000, 275:1023-29). The effects of iron loading and chelation on iron
uptake and reductase activity were investigated by pre-incubating cells with ferric ammonium citrate (0-80
µg Fe/ml) or deferoxamine (0-1000 µM) for 24 hrs. Iron loading stimulated the rate of iron uptake but had
no effect on reductase activity, whilst iron depletion resulted in a dose-dependent decrease in the uptake
rate. These changes in the rate of iron uptake in response to modulations in iron status may be due to
the regulation of other transporters.
We conclude that uptake of citrate-NTBI species into hepatocyte like cells is modified by albumin binding.
Furthermore, NTBI uptake is predominantly but not exclusively as ferrous iron but is unlikely to involve
DMT1 at the hepatocyte membrane surface. Iron reduction prior to uptake also appears to be through a
Dcytb-independent mechanism. Thus NTBI uptake and iron reduction into hepatocyte like cells is likely to
involve alternative pathways to those identified for intestinal epithelium like cells such as CaCo2. These
pathways for iron reduction and divalent iron transport need to be identified for hepatocytes.
POSTER 200
HEMIN UPTAKE AND USE AS AN IRON SOURCE BY CANDIDA ALBICANS: ROLE OF CAHMX1
ENCODED HEME OXYGENASE
R. Santos1, N. Buisson1, S. Knight2, A. Dancis2, J.-M. Camadro1 and E. Lesuisse1, Institut Jacques
Monod, France.
Candida albicans, unlike Saccharomyces cerevisiae, was able to use extracellular hemin as an iron
source. Hemin uptake kinetics by C. albicans cells showed two phases: a rapid phase of hemin binding
(with a Kd of about 0.2 µM) followed by a slower uptake phase. Both phases were strongly induced in
iron-deficient cells compared to iron-rich cells. Hemin uptake did not depend on the previously
characterized reductive iron uptake system and siderophore uptake system. CaHMX1, encoding a
putative heme oxygenase, was shown to be required for iron assimilation from hemin. A double ∆Cahmx1
mutant was constructed. This mutant could no more grow with hemin as the sole iron source, although
hemin uptake was not affected. The three different iron uptake systems (reductive, siderophore, hemin)
were regulated independently and in a complex manner. CaHMX1 expression was induced by iron
deprivation, by hemin and by a shift of temperature from 30°C to 37°C. CaHMX1 expression was strongly
deregulated in a ∆efg1 mutant but not in a ∆tup1 mutant. C. albicans colonies forming on agar plate with
hemin as the sole iron source showed a very unusual morphology. Colonies were made up of tubular
structures organized into a complex network. The effect of hemin on filamentation was increased in the
double ∆Cahmx1 mutant. Our study provides the first experimental evidence that heme oxygenase is
required for iron assimilation from heme by a pathogenic fungus.
4. Laboratoire d’Ingénierie des Protéines et Contrôle Métabolique, Institut Jacques Monod, Université
Paris 6/Paris 7, 2 place Jussieu, 75251 Paris cedex 05, France.
5. University of Pennsylvania, Department of Medicine, Division of Hematology/Oncology, BRBII/III
Room 731, 431 Curie Blvd, Philadelphia PA 19104, USA
POSTER 201
IRON HOMEOSTASIS IN THE YEAST SACCHAROMYCES CEREVISIAE :
STUDIES ON THE RELATIONSHIPS BETWEEN THE TRANSCRIPTION FACTORS AFT1P AND
AFT2P BY IDENTIFICATION OF TARGET GENES
M. Courel, J.-M. Camadro, P.-L. Blaiseau, Institut Jacques Monod, France.
In the yeast Saccharomyces cerevisiae, the regulation of iron homeostasis is assumed to be
mainly transcriptional, mediated by a major transcriptional factor, Aft1p. Under iron starvation conditions,
Aft1p activates high-affinity transport-related genes by specific binding to the DNA consensus motif 5’PyPuCACCC-3’, found in the promoters of these genes.
We have described a second transcriptional activator, named Aft2p because of its homology with
Aft1p. Aft2p seems to have a role in iron homeostasis as well. Phenotypic analyses suggest a
requirement of Aft2p in iron homeostasis when Aft1p is absent. Moreover, molecular analyses
demonstrate that Aft2p activates transcription of the Aft1p target gene FET3 in an iron-dependent
manner. All these data are in favor of overlapping functions in the control of intracellular iron use in yeast
for Aft1p and Aft2p.
In order to further establish the relationships between these two proteins, we are currently
performing their functional characterization using global transcriptome analyses and Chromatin
ImmunoPrecipitation (ChIP) experiments. The latter should yield in vivo indication of Aft1p and Aft2p
respective DNA-binding mechanisms, while the global approach should help identifying other putative
target genes.
Laboratoire d'ingénierie des protéines et contrôle métabolique, Dpt Biologie des génomes, Institut
Jacques Monod, UMR 7592, CNRS-Universités Paris 6 & 7, 2 place Jussieu, 75251 PARIS Cedex 05.
POSTER 202
INVOLVEMENT OF MITOCHONDRIA IN IRON-SULFUR CLUSTER ASSEMBLY AND REPAIR OF
IRON REGULATORY PROTEIN-1 IN MURINE CELLS
C. Bouton∗, M-J. Chauveau∗, H. Puccio#, J-C. Drapier∗, Institut de Chimie des Substances Naturelles,
CNRS∗, Gif-sur-Yvette, France. Institut de Génétique et de Biologie Moléculaire et Cellulaire, Université
Louis Pasteur#, Strasbourg, France.
Iron regulatory protein-1 (IRP-1) is a bifunctional metalloprotein which functions either as an [4Fe-4S]
cluster aconitase or as a trans-regulatory factor responsible for maintaining iron homeostasis. Importantly,
the regulation of IRP-1 activities (aconitase/trans-regulatory factor interconversion) depends on the
integrity of the [4Fe-4S] cluster located in the active site of the protein. Indeed, the [4Fe-4S] cluster, which
is attached to three cysteines in the backbone of the protein, is critical for the aconitase activity (of IRP-1)
while a complete disassembly of this cluster is necessary to allow IRP-1 to bind specifically to iron
responsive element (IRE) motif(s) on mRNAs. Stimulation of macrophages with IFN-γ and LPS, mimicking
inflammatory conditions, leads to the conversion of IRP-1 from an aconitase to an iron regulatory factor.
Nitric oxide (NO) is the physiological molecule responsible for this activation and we showed that it
directly promotes complete Fe-S cluster disassembly (1). Because NO production can last for many hours
at inflammatory sites and therefore induces a sustained activation of IRP-1 that can deregulate iron
metabolism, we focused on the fate of this protein as trans-regulator when NO flux stops. By incubating
cytosols from NO-producing macrophages with iron, sulfide and DTT, we first observed rapid and
complete dissociation of IRP-1 from IRE and concomitant and complete recovery of cytosolic aconitase
activity. This indicates that endogenous NO did not lead to irreversible post-translational modification of
IRP-1 since the protein could easily regain the same behavior as that found in control cells. We then
showed that conversion of IRP-1 from a trans-regulator to an aconitase rapidly occurred in intact cells that
were no longer exposed to NO flux. As this phenomenon did not require de novo protein synthesis, this
strongly suggests that it proceeded through reassembly and reinsertion of an intact [4Fe-4S] cluster into
apo-IRP1. We also observed the same phenomenon as regards related mitochondrial aconitase, which is
also a target of NO action. Several proteins that participate in the biogenesis of Fe-S clusters have been
characterized in prokaryotes and eukaryotes. Interestingly, the mitochondrion has been described as the
primary site for Fe-S cluster biogenesis in yeast. We investigated the role of this compartment by
following the Fe-S cluster repair process of both IRP-1 and mitochondrial aconitase in murine cells. To
solve this issue, we first exposed macrophages to a source of NO in order to promote complete Fe-S
cluster disassembly. We then stopped NO flux by extensive washings, and replaced cells in fresh medium
containing an uncoupling agent that depleted mitochondrial membrane potential. Mitochondrial and
cytosolic fractions were prepared, and time-course measurements of both aconitases were performed.
We showed that recovery of cytosolic aconitase activity was drastically delayed in macrophages whose
mitochondria were de-energized (2). Recovery of mitochondrial aconitase activity was also affected but to
a lesser extent. These data showed that Fe-S cluster repair of IRP-1 depends on mitochondrial
membrane potential, implying a role of the mitochondrial ATP-binding cassette transporter ABC7
suspected to export Fe-S cluster (3). We were also interested in another mitochondrial protein, namely
frataxin, whose function might be involved in Fe-S cluster formation (4). By using a mouse model for
Friedreich ataxia (FRDA) that lacks frataxin, we showed that IRP-1 did not assemble a Fe-S cluster, as
indicated by a very low level of cytosolic aconitase activity and a high level of IRE-binding activity in
FRDA mice compared to wild-type mice. These new data again point to the critical connection between
mitochondria and cytosolic compartments for the synthesis and/or repair of the Fe-S cluster in vivo.
(1) Soum E and Drapier J-C, J. Biol. Inorg. Chem, (2003), 8, 226-32
(2) Bouton C, Chauveau M-J, Lazereg S and Drapier J-C J. Biol. Chem. (2002), 277, 31220-27
(3) Bekri S, Kispal G, Lange H, Fitzsimons E, Tolmie J, Lill R and Bishop D.F, Blood (2000) 96,
3256-43.
(4) Mühlenhoff U, Richhardt N, Ristow M, Kispal G and Lill R, Hum. Mol. Gen. (2002), 11, 2025-36
POSTER 203
UNDERSTANDING THE MECHANISMS OF MOVEMENT OF IRON REGULATORY PROTEINS (IRPS)
S. Patton1, D. Piñero2, J. Connor1,
1
Pennsylvania State University College of Medicine, Department of Neuroscience and Anatomy 2New
York University, Department of Nutrition and Food Studies
Disruption of iron homeostasis is a key element in a number of diseases, including Alzheimer’s Disease
(AD), Parkinson’s Disease (PD), and Hereditary Hemochromatosis (HH). In order to maintain cellular iron
homeostasis, iron regulatory proteins (IRPs) function at a post-transcriptional level by binding to iron
regulatory elements (IREs) to control the synthesis of a number of proteins involved in regulating iron
levels, such as ferritin and transferrin receptor. We demonstrated previously in vitro that IRE/IRP binding
and movement were responsive to changes in iron levels. Using a time-sensitive fluorescent expression
system (pTimer), we determined that synthesis of the IRPs occurs homogeneously throughout the cytosol
as opposed to beginning in the perinuclear region and progressing towards the processes as was
previously hypothesized. A fluorescent fusion protein model system was used to show IRP/ER colocalization in HEK-293 cells. We were unable to visualize a significant change in IRP localization with
changes in iron levels in real time due to changes in cell morphology. However, recent experiments
suggest that this movement can be followed directly by subcellular fractionation followed by fluorimetric
analysis. Our next aim was to examine possible mechanisms involved in the movement of IRPs. Gel shift
assays show that inhibition and activation of protein kinase C cause movement of IRPs between
subcellular compartments. Additionally, calcium chelation increases IRE binding and movement of IRPs
into the membrane fraction but doesn't effect the iron or DFO-induced movements. We also examined
the role of the cytokine, IL-1β, in IRP movement. Treatment of HEK-293 cells with IL-1b for 16 hours
resulted in a decrease in IRP/IRE interaction; however, it did not induce IRP movement from the
membrane to the cytosol. In this study, we found that although IRPs are synthesized homogeneously
throughout the cytosol, IRP movement between subcellular compartments is responsive to a number of
different cellular stimuli.
POSTER 204
SERUM CERULOPLASMIN AND ITS FERROXIDASE ACTIVITY ARE NOT DECREASED IN
ALCOHOLIC CIRRHOSIS WITH SEVERE HEPATIC FAILURE: CLINICAL AND
PATHOPHYSIOLOGICAL IMPLICATIONS
C. LE LAN(1), M. ROPERT (2), F. LAINE(1), M. POUCHARD(3), A. LE TREUT(2), R. MOIRAND(1), O.
LORÉAL(1), P. BRISSOT(1)
(1)
Service des Maladies du Foie and INSERM U-522, University Hospital Pontchaillou, (2) Laboratoire de
Biochimie Générale et Enzymologie, University Hospital Pontchaillou, and (3) Centre d'Examens de Santé
CPAM. Rennes, France.
Background. A decrease in serum ceruloplasmin (Cp), a protein involved in iron metabolism through its
ferroxidase1 activity, is classically claimed to be observed in non wilsonian chronic liver diseases with
severe hepatic failure and therefore to be a confounding factor for the diagnosis of Wilson's disease.
Moreover, a simultaneous decrease in ferroxidase activity could be hypothetized as playing a role in the
development of the hepatic siderosis frequently observed in advanced liver diseases. Aims. To test the
validity of these two statements. Patients and Methods. The present study investigated Cp, determined by
immunonephelemetry, and its ferroxidase1 activity (determined by Erel's method, Clin Chem
1998;44:2313) in 33 male patients with severe alcoholic cirrhosis (29 grade B or C for Child-Pugh
classification). 66 healthy male volunteers, selected on strict criteria, constituted the control group. Each
patient was age-matched with 2 controls. Non parametric tests were used for statistical analysis. Results.
The mean values of Cp were significantly higher in cirrhosis as compared to controls (0.37± 0.09 g/L
versus 0.30 ± 0.04 g/L, p<0.001 ; range: 0.21-0.57 and 0.20-0.40, respectively). A significant elevation of
Cp was also observed in the subgroup of 11 cirrhotic patients who had normal serum C-reactive protein
levels. The mean values of ferroxidase1 activity were similar to those obtained in controls (445±133 IU/L
versus 413±53 IU/L; NS). Summary and conclusions. i) Low serum Cp should not be expected in severe
hepatic cirrhosis of non wilsonian origin ; ii) Hepatic siderosis in advanced chronic liver disease is likely
unrelated to a decrease in ferroxidase activity ; iii) Whether the increase in serum Cp is related to an
estrogen effect and/or a local inflammatory process and/or transcriptional activation through the Hypoxia
Inducible Factor-1 deserves to be explored. (European Grant QLT-2001-00444).
POSTER 205
PRODUCTION OF CHIMERIC RECOMBINANT HUMAN HEPCIDIN IN E. COLI
G.M. Gerardi, C. Belingheri, G. Biasiotto, I. Zanella, A. Albertini, P. Arosio.
Brescia, Italy
Hepcidin is a small protein expressed as an 80 amino acid precursor, which is processed into a 20-25
amino acid peptide with 8 cysteins. The protein was first isolated from urines and it was recognised to
have anti-bacterial activity. Animal models showed that the deletion of hepcidin gene causes iron
overload, while its over-expression causes severe iron deficient anemia. The involvement of hepcidin in
the regulation of iron metabolism is further supported by the identification of a form of juvenile hereditary
hemochromatosis that is associated to homozygous disabling mutations of the gene. Much of the studies
on hepcidin functionality are based on the use of probes for mRNA expression, and showed that the gene
is expressed mainly in the liver and that it is actively modulated by iron availability and by inflammation.
Synthetic hepcidin has been obtained in some laboratories and its structure determined, but no functional
studies have been reported so far. The production of recombinant hepcidin would be useful to elicit
antibodies and recognise its presence in circulation, and to identify possible receptors or partners for its
activity. The expression of wild type hepcidin in E. coli is probably difficult, mainly due to its anti-microbial
activity. Thus we chose to express it in fusion with the well-characterized ferritin molecule. In a first
approach the 20 amino acid sequence of Hepcidin was fused to the C-terminus of mouse ferritin H chain
(MoH-Hep). The chimera purified as an assembled, iron-containing ferritin-type molecule with and
apparent molecular weight of about 500 kDa which had a mobility on non-denaturing PAGE similar to that
of MoH ferritin wild-type. The data suggested that the C-terminal extension carrying hepcidin was not
exposed and probably accommodated inside the large ferritin cavity. This is further supported by the
observation that the chimera and MoH-wt elicited antibodies with similar specificity. In addition, the finding
that the chimeric peptide had a faster mobility in non-reducing than reducing SDS-PAGE suggested that
that some of the disulfide bridges of the hepcidin have been formed and therefore it is partially structured.
In a second construct, hepcidin-20 was fused at the N-terminus of mouse-H chain (Hep-MoH). The
chimera was efficiently expressed in E. coli as soluble high molecular weight complex. The product is
apparently iron-free and not recognised by anti-ferritin antibodies. Work is in progress to co-assemble it
with wild type ferritins and to produce specific antibodies. In conclusion, the production of recombinant,
chimeric hepcidin is feasible, but it poses structural problems.
POSTER 206
DAMAGE AND REPAIR OF IRON-SULFUR CLUSTERS IN ESCHERICHIA COLI IN VIVO
Ouliana Djaman and James A. Imlay, Department of Microbiology, University of Illinois, Urbana, IL, USA
61801.
The [4Fe-4S] clusters of dehydratases are rapidly damaged by univalent oxidants, including
hydrogen peroxide, superoxide, and peroxynitrite. The loss of an electron destabilizes the cluster,
causing it to lose its catalytic iron atom and converting the cluster initially to an inactive [3Fe-4S] form.
Continued exposure to oxidants in vitro leads to further iron release. Experiments have demonstrated
that these clusters are repaired in vivo. We have sought to identify the repair mechanism in E. coli.
Proteins encoded by the isc operon are critical for de novo assembly, but we find that most of these are
unnecessary for cluster repair. The iscS gene, which encodes a cysteine desulfurase, appeared to be an
exception, since mutants repaired damaged clusters more slowly than did wild-type cells. This effect
could arise indirectly from the poor health of this strain, however, rather than from direct participation in
the repair process. Because sulfur mobilization should be required only if clusters degrade beyond the
[3Fe-4S] state, we have used whole-cell EPR to visualize the fate of oxidized enzymes in vivo.
Aconitases A and B and fumarase A were overproduced. Brief exposure of cells to hydrogen peroxide
resulted in the appearance of a characteristic [3Fe-4S] signal. When hydrogen peroxide was then
scavenged, the enzyme activities reappeared within minutes, in concert with the disappearance of the
EPR signal. Our data indicate that damaged clusters do not typically decompose beyond the [3Fe-4S]
state in vivo. The apparent deficiency of iscS mutants in regenerating clusters is likely due to their
compromised metabolism. Mutants lacking Suf proteins, glutathione, or NADPH:ferredoxin
oxidoreductase all repair clusters at normal rates.
POSTER 207
ONE OF THE THREE CATECHOLATE SIDEROPHORE RECEPTORS FEPA, CIR, OR IRON IS
SUFFICIENT FOR EFFICIENT COLONIZATION AND INVASION OF SALMONELLA ENTERICA
W. Rabsch1, U. Methner3, W. Voigt1, H. Tschaepe1, and R. Reissbrodt1 P.H. Williams1,
1
Robert Koch-Institut Wernigerode, Germany,
2
Department of Microbiology & Immunology, University of Leicester
3
Bundesforschungsanstalt für Viruskrankheiten der Tiere Jena, Germany
The role of enterobactin, the principal siderophore of Salmonella enterica and its cognate receptors, the
iron regulated outer membrane proteins (IROMPs) FepA and IroN, in virulence is controversial. To clarify
the matter, we construceted single, double and triple mutants of S. enterica serovar Typhimurium and S.
enterica serovar Enteritidis defective in the expression of the IROMPs proposed to be catecholate
receptors FepA, IroN and Cir. The mutants were characterized by assessing the uptake of a number of
catecholate type siderophore (naturally-occurring and chemically synthesised compounds) in growth
promotion tests and by MIC tests with a siderophore-cephalosporin cunjugate. Unique patterns of uptake
were identified for each IROMP. Specifically, FepA and IroN were confirmed to be required for transport
of enterobactin and Cir was shown to function as a receptor for the enterobactin breakdown products,
trimeric, dimeric and monomeric 2,3-dhiydroxybenzoyl-serine. In vitro assays for adherence to and
invasion of the rat small intestinal epithelial cell line IEC6 gave similar results for the parent S. enterica
serovar Enteritidis strains and its fepA and fepA iroN derivatives. However, adherence and in particular
invasion were markedly reduced in the triple mutant missing FepA, IroN and Cir proteins. Similar results
were seen in vivo in experimental infections of day-old chicks. The results point to contribution of the
enterobactin-dihydroxybenzoylserine complex in the virulence of S. enterica serovar Enteritidis,
particularly the invasion of this pathogen seems to be dependent from the enterobactindihydroxybenzoylserines complex in the virulence of S. enterica serovar Enteritidis, particularly for the
process of host cell invasion.
Keywords: Salmonella, enterobactin, 2,3-dihydroxybenzoyl-serine, virulence
POSTER 208
EXPLORING H-FERRITIN-LIKE DNA SEQUENCES IN HUMAN GENOME FOR PUTATIVE
FUNCTIONAL GENES
P. D’Ursi*, E. Rovida*, P. Arosio, I. Zanella, J. Drysdale**
Brescia, Italy; *Istituto di Bioimmagini e Fisiologia Molecolare, CNR, Via F.lli Cervi 93, 20090 Segrate
(Mi), Italy; **Tufts University, Sch. Med., Boston MA 02111, USA
Southern blot analyses and chromosomal hybridization previously showed that the human genome
contains about 15 sequences that are highly homologous to H-ferritin (FTH) cDNA. All of these are
intronless. Most contain polyA tails and flanking repeats sequences and appear to have arisen as
retrotranscripts of an H-like sequence. Most have non-viable mutations and are regarded as nonfunctional processed pseudogenes. In addition to these sequences, BLAST analyses identified other
intronless H-like sequences. One of these corresponds to the recently identified mitochondrial ferritin.
This finding of a functional intronless sequence raises the possibility that some of the others might also be
functional. To explore this possibility we have begun to analyse some of these sequences. Blast analysis
using full H-ferritin cDNA sequence identified 29 DNA fragments with a score < e-4, used as threshold.
After sequence alignment these sequences could be separated into three groups with 14, 7 and 6
members. The sequences of the first group of 14 members extend the length of the HcDNA and are the
most homologous to the H-cDNA. These sequences correspond to those previously identified by
Southern blot, in situ gene hybridization and more recently genome mapping. They had > 88% identity to
the query. Most have mutations that would appear to preclude expression of a functional protein.
However, two have potentially intact protein sequences of the same size as FTH (8 and 11 substitutions,
respectively), and one has a potential ORF encoding a longer sequence with an N-terminal extension. No
corresponding transcript has been found for these three sequences and it may be that they are also nonfunctional processed pseudogenes. The second group of 7 components includes sequences which
overlap 20-60% of FTH-cDNA with identity between 80 and 85%. All potential ORF carried disabling
mutations and none of them was represented in EST database. We concluded that they represent nonfunctional pseudogenic fragments. The third group consists of 6 sequences that overlap about 50% FTHcDNA with 70-80% identity. It includes the previously described mitochondrial ferritin MtF, on
chromosome 5, and five sequences on chromosome X. These sequences are also intronless but lack
polyA stretches and repeat flanking regions characteristic of processed pseudogenes. One, (FTHL17)
has already been described. It encodes a peptide of 183 amino acids and has been found to be
expressed in spermatogonia (Wang PJ, McCarrey JR, Yang F, Page DC. An abundance of X-linked
genes expressed in spermatogonia. 2001 Nat Genet 27:422-6). The other sequences are located in close
proximity. One encodes a peptide of 158 residues while the others have potential N-terminal extensions
of 30-70 amino acids. Interestingly, none has a conserved ferroxidase centre. All three have potential
counterparts in EST databases and may therefore be expressed. Some of these DNAs have now been
cloned in expression vectors, and work is in progress to study the structure and expression of the
corresponding proteins.
POSTER 209
ABSENCE OF A DIRECT ROLE FOR A PUTATIVE NUCLEAR LOCALIZATION SIGNAL IN A
RECENTLY IDENTIFIED DMT1 EXON IN NUCLEAR TARGETING OF THE TRANSPORTER
H.C. Kuo, J. J. Smith, A. Lis, X. Zhou, J. A. Roth, M. D. Garrick, L. Garrick
Departments of Biochemistry, Pharmacology & Toxicology, Pediatrics and Medicine, SUNY at Buffalo,
Buffalo, NY, USA
DMT1 (Divalent Metal Transporter 1), also known as DCT1, Nramp2 or SLC11A2, is responsible for both
uptake of ferrous iron in enterocytes and iron exit from endosomes during the transferrin cycle. There are
four known isoforms: Two mRNA isoforms differ in the 3’UTR; the +IRE form containing a 3’ IRE (Iron
Responsive Element) and –IRE DMT1 lacking this element. The corresponding proteins differ in their Cterminal peptides. A newly identified exon 1A (Hubert & Hentze 2002 PNAS 99:12345) can be present in
place of exon 1B. When 1B is present, the protein’s N-terminus is encoded in exon 2; but when 1A is
present, an additional peptide constitutes the N-terminus. The two C-terminal isoforms localize differently
in a number of cell types. Our laboratory previously showed that the –IRE species of DMT1 localizes to
the nucleus of neuronal cells and models of neuronal cells, but the +IRE DMT1 is limited to the plasma
membrane and cytosolic vesicles. Based on information obtained from the PSORT II prediction program,
exon 1A might have the potential to target DMT1 into the nucleus due to the presence of a nuclear
localization signal (MGKKQPRAA). To test this role, all four isoforms of DMT1 were prepared - 1A+IRE,
2+IRE, 1A–IRE, 2–IRE, each with and without a FLAG® tag on the N terminus. Each was transiently
transfected into two different cell lines: HEK293T cells (immortalized human embryonic kidney cells where
the endogenous isoforms do not appear in the nucleus) and PC12 cells (an adrenal pheochromocytoma
neuronal model where we detected a portion of the endogenous –IRE form in the nucleus). All four
ectopically expressed isoforms failed to appear in the nucleus in both cell lines despite the presence of
the putative nuclear localization signal on 1A–IRE and 1A+IRE constructs. Antibody specific for exon 1A
was made to learn whether this exon was associated with endogenous –IRE DMT1 expression in the
nuclei of PC12 cells. The specificity of this antibody was verified by its ability to detect transient ectopic
expression of the 1A–IRE and 1A+IRE constructs. The results also showed that DMT1 containing exon
1A remained in the plasma membrane and cytosolic vesicles whether ectopically or endogenously
expressed. Therefore we conclude that exon 1A is neither sufficient nor necessary for DMT1 to appear in
the nucleus, although it may yet facilitate nuclear localization indirectly.
POSTER 210
RESTORATION OF AN FERROXIDASE SITE IN ENGINEERED L FERRITIN
Xiaofeng Liu, William Small, Rene Tipton, Elizabeth C. Theil Hildren’s Hospital Oakland Research
Institute, 5700 Martin Luther King Way, Oakland, CA 94609
Ferritin is a 24 subunit protein in animals, composed of cell-specific ratios of H and L subunits. Of the two
subunits, H subunit has a ferroxidase site, which oxidizes ferrous iron rapidly (milliseconds) forming
diferric mineral species. In L subunits, the ferroxidase site is degenerate and likely evolved from the
ubiquitious H subunit by gene duplication. Iron uptake rates in recombinant H and L ferritin homopolymers
differ by 10, 000-fold due to the ferroxidase activity. During fast iron uptake by H-type ferritin,
stoichiometric amount of hydrogen peroxide are produced during the oxidation of Fe2+, decay of the
diferric peroxo intermediate and formation of diferric oxo mineral precursors. A role for the cell-specific
variations of the H/L ferritin subunit ratio may be matching rates of iron uptake to cell need and ability to
detoxify H2O2.
The ferroxidase site has been defined by phylogenetic sequence conservation, mutagenesis, and crystal
structures with metal substituted apoferritins, to consist of E23, E58, H 61, E 103, and Q137 (alternate
numbering E27, E62, H65, E107 and Q 141). Many functional studies on H type ferritins have defined the
mechanisms of iron uptake and oxidation, based on the inferred active site structure. To directly
determine the contribution of each catalytic site residue, and the minimal requirements for fast iron
uptake, we have reconstructed an active ferroxidase site in L ferritin subunits, analyzing the effect on the
Fe2+ oxidation rate for each added active site residue. Site-directed mutagenesis with the Chameleon Kit
was used with the frog L subunit which has diverged much less from the H and is more likely to produce
stable protein after amino acid substitutions of H residues in the L background. Recombinant proteins
were isolated without low Fe and reaction rates were monitored by stopped-flow spectrometry , after the
addition of 48 Fe(initiation complexes) or 480 Fe (bulk mineral).
When the active site residue E 23 was inserted in an H25Y protein, which had Fe uptakes
indistinguishable from L-wild type protein, the double mutant, L- K23E,H25Y, displayed iron uptake
kinetics that were intermediate between wild type L and H or H’(M) ferritin. The triple mutant L- K23E,
H25Y, S137Q had nearly full ferroxidase activity relative to H/M ferritin, revealed by 350 nm spectrum
characteristic of the product of ferroxidase activity, the oxo-differic mineral precursors. However, the
complete absence of the intermediate, the diferric peroxo species, which absorbs at 650 nm, suggests
that the intermediate is not stabilized by the protein ligands. When the activity of the quadruple mutant, LK23E/H25Y/S137Q/T140D was analyzed, it was similar to H ferritin at 350 nm, and the diferric peroxo
species could be detected at 650 nm
In summary, the reconstruction of an active ferroxidase site in L ferritin directly shows the requirement for
E23, E58, H 61, E 103, the importance of Q137 and the influence of residue 140 in determining the
stability of the diferric peroxo transition state complex of ferroxidation by ferritin.
POSTER 211
INTER-INDIVIDUAL DIFFERENCES IN HFE EXPRESSION REGULATE IRON HOMEOSTASIS IN
MICE
S Ludwiczek*, I Theurl*, M Chorney#, E. Artner*, G Weiss*
*Department of Internal Medicine, University Hospital, A-6020 Innsbruck, AUSTRIA, # Departments of
Microbiology, Immunology, The Pennsylvania State University College of Medicine, Hershey, PA
Background: HFE affects the binding of transferrin bound iron to cell surface transferrin receptors (TfR)
thereby modulating cellular iron uptake. However, limited information is available on individual differences
in HFE expression, its regulation by iron availability and the consequences of this on duodenal iron
absorption and liver iron accumulation.
Methods: We studied C57BL/6 mice receiving either an iron rich or an iron depleted diet and studied the
expression of HFE by means of quantitative real-time PCR and Western blots. In parallel, we assessed
the metabolic consequences of this on the expression of TfR, ferritin, DMT-1, ferroportin and hepcidin.
Monitoring of intracellular iron availability was performed by determination of IRP binding affinity via gel
shift assays.
Results: In mice under an iron balanced diet we found an up to fourteen-fold variation in inter-individual
expression of HFE in the duodenum. Mice with high duodenal HFE expression presented significantly
higher levels of TfR and DMT-1 and an increased IRP-1 binding affinity as compared to mice with
reduced HFE levels in the duodenum. HFE expression in duodenum was significantly associated with
serum iron levels and liver HFE expression. Moreover, dietary iron supplementation decreased HFE
expression in the duodenum but not in the liver. This resulted in reduced DMT-1 and FP-1 expression in
the duodenum while the expression of DMT-1, FP-1 and hepcidin in the liver was increased with dietary
overload.
Conclusion: Inter-individual differences of HFE expression exert pivotal regulatory properties on duodenal
iron absorption by modulating the expression of DMT-1 and FP-1. Duodenal HFE levels are modulated by
dietary iron and are increased when iron availability and stores are reduced.
Since duodenal and hepatic HFE expression are closely associated and exert the same regulatory
consequence on iron transport HFE may act as a gate keeper orchestrating iron absorption and liver iron
storage.
POSTER 212
MULTIPLE C/EBP AND AP-1 SITES IN HEPCIDIN PROMOTER CONFER SPATIAL AND CELL
SPECIFIC POSITIVE/NEGATIVE REGULATION OF GENE EXPRESSION
K. Y. Yeh, M. Yeh, H. Yin, and J. Glass. Departments of Medicine and Molecular and Cellular
Physiology; and Feist-Weiller Cancer Center; LSUHSC, Shreveport, LA 71130-3932
Hepcidin (Hepc), an anti-microbial peptide, has been suggested to play a role in the regulation of iron
homeostasis. Hepc is specifically expressed in the liver. Hepc expression is upregulated by endotoxin
(LPS) and iron and is down regulated by iron deficiency [Pigeon et al., JBC 276:7811, 2001; Nicolas et
al., PNAS 98: 8780, 2001; Park et al., JBC 276: 7806, 2001]. The molecular mechanisms by which iron
and LPS alter Hepc expression are largely undefined. Recently, CAAT/enhancer-binding protein a
(C/EBPα ha s be e n s hown to pla y a n im porta nt role in the a ctiva tion of He pc e xpre s s ion [Cours e la ud e t
al., JBC 277: 41163, 2002]. Hepc expression in response to LPS exhibits a biphasic increase with peaks
of expression at about 6 hours and 36 hours after LPS (cf. accompanying abstract). The complex pattern
of response suggests the involvement of additional factors in the regulation of Hepc expression. To
examine transcriptional regulation of Hepc gene, the inter-gene region of rat USF2 and Hepc was cloned
and the transcriptional activity of defined 5’ deleted regions was analyzed by using the pGL3 luciferase
reporter system. The inter-gene region was amplified by nested PCR using rat DNA as the template. Two
forward primers (5'-TTCTGGATCCCTGCTCCCCTCTG-3' and 5'-CAATAAACT
GGCCAGTGTGGCCC-3') and one reverse primer (5'-AGTGAGTCTTGCCTTCTGTCCTGCAG-3') were
synthesized according to the USF2 mRNA sequence in Genbank database (accession number
AB047556) and Hepc mRNA sequences (accession number AF344185). The amplicon was cloned using
the pCR-Script cloning kit (Stratagene) and sequenced (Genbank accession number AY230877). The
region between USF2 and Hepc consists of 1857bp with a TATA box at -34/-24 from the transcription
start site (=1) of Hepc. A series of 5' deletions were assayed in HepG2, Cos1, and U2OS cells. In HepG2
cells there was an ~70 to100-fold increase in luciferase activity with Hepc -113/+50 (-113) and -229 and
only ~1 to 4-fold increase with Hepc -603, -705 and -1715 regions. In Cos1 and U2OS cells the promoter
activity of -113 was 50% and 90% lower and of -603 was 85% and 95% lower than that in HepG2 cells
respectively. Deletion of 60 bp of the 3' end of -113/+50 resulted in 80% reduction of promoter activity, but
had no effect on that of -229/+50. Sequence analysis of the -603 identified multiple putative C/EBPα and
activator protein 1 (AP-1) binding elements. The activity of these elements was tested in cells cotransfected with Hepc -113 or -603 in pGL3 and pcDNA-C/EBPα or pcDNA-Ref1. In HepG2 cells, over
expression of C/EBPα did not further increase the activity of -113, but doubled the activity of -603; over
expression of redox factor 1 (Ref1) modestly reduced the activity of -113, and reduced 70% of the activity
of -603. In Cos1 cells, C/EBPα or Ref1 over expression did not alter the promoter activity of -113, but
reduced 60% and 50% of the activity of -603 respectively. In U2OS cells, over expression of C/EBPα or
Ref1 altered neither -113 nor -603 promoter activity. To determine whether changes in hepatic C/EBP
and AP-1 binding activity and Hepc expression occurred in rats after LPS administration (ip, 1µg/g),
C/EBP and AP-1 activity were measured by EMSA and hepatic Hepc mRNA levels determined by
Northern blots. LPS increased both C/EBPα and AP-1 binding activities at 1 and 2 h and Hepc mRNA
expression by 3-fold at 2 h with a peak at 8 h; by 12 h Hepc mRNA returned to normal. Western blots
showed that hepatic c/EBPα protein (42 and 30 kDa bands of differential translation initiation sites) in the
nuclear extract was high at 0, and 4, reduced to 30% at 8 and did not recover until 24 h after LPS. The
present data demonstrate that in rats the Hepc -113 and -229 promoter regions contain regulatory
elements conferring high levels of cell specific Hepc expression. In addition, C/EBPα stimulates the
region between -229 and -603 in a cell specific manner while AP-1, presumably activated by Ref1,
suppresses Hepc expression. Thus, in the rat liver there is normally a feedback regulation in which
C/EBPα activates and AP-1 inhibits Hepc expression. These regulatory pathways for Hepc expression
must be considered in elucidating the mechanisms by which Hepc serves as the iron stores regulator.
POSTER 213
A MUTAGENESIS APPROACH TO THE ROLES OF TWO CONSERVED HISTIDINES IN THE
PROTON-DEPENDENT IRON TRANSPORTER DMT1
Jie Jin, Haoxing Xu, David E. Clapham and Nancy C. Andrews
Children’s Hospital, Harvard Medical School, Boston, MA 02115, USA
Divalent metal transporter 1 (DMT1, also known as Nramp2 and DCT1), a membrane protein that
contains twelve putative transmembrane segments (TMS), is a proton-dependent iron transporter that
plays important roles in iron metabolism. Protons provide not only a driving force, but also a likely mode
of regulation of DMT1. As histidines are often found to play critical roles in proton-coupled transporters,
two conserved histidines (His267 and His272) in putative TMS VI of DMT1 were targeted in this study.
Using site-directed mutagenesis, they were individually replaced by other residues, expressed in a
mammalian cell line and analyzed by patch clamp. His267 was more sensitive to mutation than His272.
Five out of eight His267 mutations abolished both the proton leak and the iron-induced current. However,
all eight His272 mutations allowed retention of at least the proton leak current. Several mutations altering
these two histidines were found to be active in mediating both the proton leak and the iron-induced
current, but relative to the wild type, they possessed different electrophysiological properties. One
mutation altering His272 resulted in a much higher ratio of iron-induced current to proton leak current.
Several mutations altering H272 retained the proton leak but abolished the iron-induced current,
suggesting that the proton leak and iron transport pathways can be functionally dissociated. These data
suggest that neither His267 nor His272 is essential for the DMT1 iron translocation machinery, but both
may contribute as regulators. His267 and His272 may be differentially involved in regulation of DMT1mediated functions; particularly, His272 may be involved in iron transport but not the proton leak.
Underlying mechanisms are under investigation by multiple approaches.
POSTER 214
IRON INDUCES THE INTERNALIZATION AND DEGRADATION OF THE HIGH AFFINITY IRON
TRANSPORT SYSTEM IN YEAST
DM Ward1, MR Felice2, L Li1, G Musci2 and J Kaplan1
1
Division of Immunology and Cell Biology, Department of Pathology, School of Medicine, University of
Utah, Salt Lake City, Utah 2Dipartimento di Scienze Microbiologiche Genetiche e Molecolari, Università di
MessinaSalita Sperone, 31I-98166 Villaggio S. Agata Messina, Italia
In yeast the high affinity iron transport system is comprised of two plasma membrane proteins: Fet3p, a
multicopper oxidase and Ftr1p, a transmembrane permease. Transcription of the genes that encode
these proteins is under the control of the iron-sensing transcription factor Aft1p, which is activated under
low iron conditions. We demonstrate that high media iron induces the internalization and degradation of
Fet3p in a time and iron-concentration dependent manner. Iron-induced internalization and degradation
is seen under conditions in which transcription of the gene is prevented (through the use of galactosedependent promoters) or when protein synthesis is inhibited by cycloheximide. The iron-induced
internalization of Fet3p can be visualized in cells lacking the vacuolar protease Pep4p. In ∆pep4 cells,
iron induces the transfer of Fet3p from the cell surface to the vacuole. The rate of Fet3p internalization is
reduced in cells deficient in endocytosis (∆end4). Internalization and degradation of surface Fet3p is
highly specific for iron and is not induced by other transition metals. Previous studies demonstrated that
Fet3p and Ftr1p must be synthesized simultaneously for proper surface targeting of a putative
Fet3p/Ftr1p complex. Iron not only induces the internalization of Fet3p but of Ftr1p as well. Site-specific
mutants of Fet3p that are deficient in oxidase activity are appropriately targeted to the cell surface but can
not transport iron. The internalization and degradation of these mutant proteins is also accelerated by
iron. These results demonstrate that regulation of iron transport in yeast is similar to the regulation of
other transition metal transporters and occurs at both transcriptional and post-transcriptional levels. (This
work was supported by a grant from the National Institute of Health (USA-NIDDK- DK-30534)
POSTER 215
OVEREXPRESSION OF TRANSFERRIN RECEPTOR 2 INHIBITS ERYTHROID DIFFERENTIATION OF
MURINE ERYTHROLEUKEMIA CELLS
H-S. Yoon, N. Lok, S. Kim, P. Ponka, Lady Davis Institute for Medical Research-Jewish General Hospital,
and Physiology and Medicine, McGill University, Montreal, QC, Canada.
The transferrin receptor (TfR) is a cell membrane-associated protein that serves as a gatekeeper
regulating cellular uptake of iron (Fe) from transferrin (Tf) by TfR-mediated endocytosis. TfRs are
expressed ubiquitously, but their highest levels of expression are on immature erythroid cells. Importantly,
a second TfR (TfR2) has recently been cloned in humans. TfR2 is highly homologous with the “classical”
TfR but shows different tissue distribution. In this study, we have cloned the mouse TfR2 (mTFR2) by
RACE (rapid amplification of cDNA ends) and sequenced it. The sequence analysis shows approximately
50% and 82% homology to mouse TfR and human TfR2, respectively. Northern blot analysis revealed
that TfR2 mRNA was predominantly expressed in the liver. To analyze the function of TfR2, TfR and TfR2
59
59
were transfected to TfR-deficient Chinese hamster ovary (CHO) cells and Fe uptake from Fe-Tf was
measured in both parent and transfected cells. After the transfection, the cells had a marked increase in
59
Fe uptake but TfR2, as compared to TfR, was less efficient in promoting iron uptake. Moreover,
experiments examining 125I-Tf binding revealed that, as compared to TfR, the affinity of TfR2 to Tf is
significantly lower. Furthermore, W7 (an inhibitor of endocytosis) and bafilomycin (an inhibitor of
endosomal acidification) inhibited 59Fe uptake from 59Fe-Tf in wild-type as well as TfR2-transfected CHO
cells, suggesting that both receptors share similar mechanisms in cellular iron acquisition. It has been
speculated that TfR2 is expressed in erythroid cells since its message is abundant in K562 cells, a human
cell line that can be induced to synthesize hemoglobin. Thus, we deemed it important to determine
whether and what role TfR2 plays in erythroid cells. As expected, the induction of erythroid differentiation
of murine erythroleukemia (MEL) cells was associated with a dramatic increase in TfR mRNA levels. In
sharp contrast, TfR2 mRNA, which shows a relatively high level of expression in uninduced MEL cells,
significantly decreased following the induction of MEL cells to synthesize hemoglobin. Interestingly, the
overexpression of TfR2 in MEL cells significantly attenuated their capacity to undergo erythroid
differentiation and caused a decrease in iron utilization for heme biosynthesis. These data suggest that
the down-regulation of TfR2 is essential for an efficient delivery of iron for heme synthesis during MEL cell
differentiation.
POSTER 216
NITRIC OXIDE APPEARS TO PLAY A ROLE IN IRON-MEDIATED DEGRADATION OF IRON
REGULATORY PROTEIN 2
S. Kim, P. Ponka, Lady Davis Institute for Medical Research, SMBD – Jewish General Hospital and
Departments of Physiology and Medicine, McGill University, Montreal, Quebec, Canada H3T 1E2
Intracellular iron homeostasis is regulated post-transcriptionally by iron regulatory proteins (IRP1 and 2).
When iron in the labile iron pool (LIP) is scarce, IRPs bind to iron responsive elements (IREs) in the 5’
untranslated region (UTR) of the ferritin mRNA and 3’ UTR of the transferrin receptor (TfR) mRNA. Such
binding inhibits translation of ferritin mRNA and stabilizes the TfR mRNA, whereas the opposite scenario
develops when iron in the LIP is plentiful. It has been reported that IRP2 is degraded by the proteasome
in iron replete cells. This degradation is dependent on a 73-amino acid insertion rich in cysteines that are
+
well-known targets for S-nitrosylation by the oxidized form of nitric oxide (NO), nitrosonium ion (NO ).
+
Recently, we have shown that treatment of RAW 264.7 cells with the NO donor sodium nitroprusside
(SNP) targets IRP2 for S-nitrosylation leading to IRP2 degradation via the ubiquitin-proteasome pathway.
SNP-mediated degradation of IRP2 was associated with a dramatic increase in ferritin synthesis. In this
study, we demonstrated that NO scavengers and NO synthase inhibitors blocked iron-dependent
degradation of IRP2. Moreover, SNP-mediated degradation of IRP2 was prevented by the cell permeable
iron chelator salicylaldehyde isonicotinoyl hydrazone which also inhibited ferritin synthesis in SNP-treated
cells. However, SNP did not increase iron levels in the LIP. Importantly, a conversion of one of the
cysteines in the degradation domain of IRP2 prevented nitrosylation of this protein and its consequent
degradation in response to SNP. In addition, this mutation also blocked iron-mediated degradation of
IRP2. These data strongly suggest that nitric oxide plays an important role in the degradation of IRP2 in
iron-replete cells.
POSTER 217
KISS AND RUN: THE ROUTE OF IRON FROM ENDOSOMES TO MITOCHONDRIA CAN BYPASS
THE CYTOSOL IN HEMOGLOBIN-PRODUCING CELLS
A.D. Sheftel1, A.-S. Zhang1,2, O. Shirihai3, P. Ponka1, 1Dept. of Physiology and Medicine, McGill
University, Montreal, QC, Canada; 2Dept. of Cellular and Developmental Biology, Oregon Health
Sciences University, Portland, OR, USA; 3Biocurrents Research Center, Marine Biological Institute,
Woods Hole, MA, USA
During differentiation, immature erythroid cells acquire vast amounts of iron at a breakneck rate.
Proper coordination of iron delivery and utilization in heme synthesis is essential and disruption of this
process likely underlies iron loading disorders such as sideroblastic anemia and myelodysplastic
syndrome with ringed sideroblasts. Iron is taken up by the cells via receptor mediated endocytosis, a
process whereby diferric transferrin (Tf) binds to its cognate receptor (TfR) on the erythroid cell plasma
membrane, followed by internalization of the Tf-TfR complex. Subsequent to endocytosis, the endosome
is acidified by a H+-ATPase, allowing the release of iron from Tf. Through an unknown mechanism, iron
is targeted to the inner membrane of the mitochondria, where the enzyme that inserts Fe into
protoporphyrin IX, ferrochelatase, resides. Although it has been demonstrated that the divalent metal
transporter, DMT1, is responsible for the egress of reduced Fe from the vesicle, the immediate fate of the
iron atoms after their transport across the vesicular membrane remains unknown. Because reduced iron
is a strong pro-oxidant, contributing to free radical formation through Fenton chemistry, it has been
predicted that an iron binding molecule shuttles Fe from the endosome to mitochondria. However, this
much sought iron binding intermediate, that would constitute the labile iron pool (LIP), has yet to be
identified. Thus, we hypothesize that, in hemoglobin-producing cells, there is a direct relaying of Fe from
the endosomal machinery to that of the mitochondria. We have taken two strategies in examining this
supposition: 1) a biochemical approach by which the cytoplasm of cells was loaded with an impermeant
iron chelator, thus intercepting the delivery of Fe by the putative LIP intermediate, and 2) a morphological
approach employing time-lapse confocal microscopy which permits the tracking of iron-loaded
endosomes and mitochondria with high spatial and temporal resolutions. To examine whether iron
delivered by Tf for heme synthesis can bypass the cytosol, we have loaded reticulocytes with a high59
molecular weight version of desferrioxamine, hDFO, prior to incubation with Fe-Tf. The incorporation of
transferrin iron into heme was unaffected by hDFO when compared to controls. Importantly, iron
delivered to these cells in a form that freely diffuses across the membrane, iron-salicylaldehyde
isonicotinoyl hydrazone (59FeSIH2), was significantly prevented from being used for heme synthesis in
hDFO-laden reticulocytes. Using confocal microscopy, as well as polarized light microscopy, we found
that endosomes are very mobile organelles. Immediately following budding from the plasma membrane,
these organelles continuously traverse the cytosol and touch a number of mitochondria multiple times.
Experiments using various pharmacological agents indicate that these movements are mediated by
components of the cytoskeleton which are essential for proper iron delivery for use in heme synthesis.
Together, these data suggest that iron is directly delivered to mitochondria by endosomes in a “kiss and
run” paradigm. Our current studies will examine the required components and regulation of this
interaction using the same experimental strategies as well as a cell free system consisting of isolated
organelles.
POSTER 218
RECOMBINANT IRON-RESPONSE ELEMENT BINDING PROTEIN-LIKE PROTEIN FROM
PLASMODIUM FALCIPARUM DISPLAYS ACONITASE ACTIVITY
M. Loyevsky, E. Yikilmaz, M. Hodges, J.C. Wootton, V.R. Gordeuk, T.A. Rouault, Howard University,
Washington, DC; NICHD, NCBI, NIH, Bethesda, MD
Iron is an indispensable nutrient for the rapidly proliferating asexual forms of plasmodia, but this
element can also generate damaging free radicals. Hence, as yet unknown mechanisms for regulating
iron transport, storage and utilization must be present in P. falciparum. A candidate regulator of iron
homeostasis in asexual erythrocytic malaria parasites is iron-response element binding protein-like
protein (PfIRPa), which was first cloned by Muckenthaler and co-workers (1998). We overexpressed
PfIRPa in a bacterial expression system and showed that it specifically binds to putative plasmodial ironregulatory elements (IREs), which we identified using an algorithm developed by Stephen Altschul
(Loyevsky et al., 2003).
The molecular structure of PfIRPa predicts the capacity to form an iron-sulfur cluster, which is
necessary for aconitase activity. To determine if recombinant PfIRPa has aconitase activity, we used a
modified procedure of Kaptain and coworkers (1991). rPfIRPa contained in imidazole eluate obtained
after the elution of the protein from a His-bind column was activated with an aconitase-regeneration
buffer, which favors the assembly of an iron-sulfur cluster. Aconitase activity was detected using a
colorimetric NADPH-MTT assay coupled to isocitrate dehydrogenase (Figure). As shown in the Figure,
rPfIRPa has aconitase activity under conditions that favor formation of iron-sulfur cluster. Thus, this
experiment provides evidence for the capability of PfIRPa to switch between IRP and aconitase activity.
Our immunofluorescence data suggest that PfIRPa/aconitase is localized within the parasite cytosol in
the trophozoite-infected erythrocyte and not in the mitochondrion. In T. cruzi a single aconitase functions
both in mitochondria and in the cytosol (Saas et al., 2000). This raises the possibility that
PfIRPa/aconitase can potentially function as a TCA enzyme in the mitochondria in different parasitic
stages of P. falciparum (sporozoite, liver or mosquito).
Our search of plasmodial databases suggests that PfIRPa may be the only aconitase present in P.
falciparum. These considerations have prompted us to hypothesize that PfIRPa/aconitase participates in
regulation of intra-plasmodial iron metabolism perhaps in a fundamental manner.
0.10
1
Absorbance, a.u.
0.08
2
1 - Aco
2 - rPfIRPa+
3 - rPfIRPa4 - ConVect
5 - ConElut
6 - ConWID
0.06
aconitase activity. The absorbance of rPfIRPaeluate in aconitase detection mix was measured
at 560 nm after 30 min. incubation at 220C.
0.04
Curve 1: 0.1 IU/ml aconitase (Sigma); Curve 2:
3
4
0.02
activated rPfIRPa (rPFIRP+); Curve 3: no
5
0.00
Figure. Recombinant PfIRPa (rPfIRPa) displays
activation (rPfIRPa-); Curve 4: transfection
6
450
500
550
Wavelength, nm
600
650
control: eluate from Rosetta E. coli transfected
References
1) M.U. Muckenthaler et al. (1998). An IRP-like cDNA from Plasmodium falciparum, accession number
TM
AJ012289, GenBank . 2) M. Loyevsky et al. (2003). Expression of a recombinant IRP-like Plasmodium
falciparum protein that specifically binds putative plasmodial IREs. Mol Biochem Parasitol, in press. 3) S.
Kaptain et al. (1991) A regulated RNA-binding protein also possesses aconitase activity. Proc Natl Acad
Sci USA 88: 10109-10113. 4) J. Saas et al. (2000). A developmentally regulated aconitase related to ironregulatory protein-1 is localized in the cytoplasm and in the mitochondrion of Trypanosoma brucei. J Biol
Chem 275: 2745-2755.
POSTER 219
IDENTIFICATION OF HUMAN ISCA AND HSCB HOMOLOGS
Xavier Brazzolotto, Tracey A. Rouault
Section on Human Iron Metabolism, Cell Biology and Molecular Metabolism, National Institute of Children
Health and Human Development, National Institutes of Health
Bethesda, MD, 20892, USA
Iron–sulfur cluster containing proteins (FeS proteins) are ubiquitous in all living organisms and
synthesis process of their prosthetic group involves a highly conserved protein machinery, ISC proteins,
encoded by the isc operon in bacteria. This operon encodes IscS, a cysteine desulfurase catalyzing the
release for sulfur from cysteine, different scaffold proteins, IscU, IscA and NFU, able to assemble iron–
sulfur clusters as homodimers, a chaperone and its co-chaperone, HscA and HscB respectively, and a
1
2
3
ferredoxin. In human, ISC homologs characterized so far, IscS , IscU and NFU , have been localized
both in mitochondria and in cytosol unlike in yeast where all homologs have been essentially localized in
mitochondria4. In order to further characterize the iron-sulfur cluster biosynthesis process in human, we
have decided to identify and characterize the remaining ISC homologs : IscA, HscA and HscB.
Bacterial and yeast proteins have been used to screen human protein and genome databases.
Results for the chaperone protein, HscA, do not allow us to determine the exact human homolog, as
multiple candidates are found but screening with bacterial and yeast IscA lead to the identification of two
human IscA homologs sharing 25% identity. IscA1 is localized on chromosome 9 and IscA2 localized on
chromosome 14. Finally, screening with the bacterial HscB, allowed us to identify one candidate for
human HscB, localized on chromosome 22.
Initial 5’ RACE experiments and sorting programs predict that all newly identified proteins are
localized in mitochondria. Nevertheless an AUG start codon is found in frame on HscB mRNA and could
1
be used to translate for a predicted cytosolic protein, as it was found in the case of IscS . To check the
sub-cellular localization of these proteins, we are raising specific antibodies for each protein.
Moreover, to distinguish among human HscA homologs, specific protein-protein interactions
between the HscA homologs and the co-chaperone HscB will be analyzed.
1.
2.
3.
4.
Land T. and Rouault T.A., 1998, Mol. Cell, 2, 807-815
Tong W-H. and Rouault T.A., 2000, EMBO J., 19, 5692-5700
Tong W-H. and Rouault T.A., submitted
Mühlenhoff U. and Lill R., 2000, Biochim. Biophys. Acta, 1459, 370-382
POSTER 220
MOLECULAR AND CELLULAR CHARACTERISATION OF TRANSFERRIN RECEPTOR 2
V.N. Subramaniam1,2, L. Summerville1, D.F. Wallace1. 1Membrane Transport Laboratory, The Queensland
Institute of Medical Research and 2Departments of Biochemistry and Medicine, The University of
Queensland, Brisbane, Queensland, Australia.
Transferrin receptor 2 (TfR2) is a type II integral membrane protein with significant homology to transferrin
receptor 1 (TfR1). The role of TfR2 in iron metabolism has not been fully determined. TfR2 can bind
transferrin but with a much-reduced affinity as compared to TfR1. Unlike TfR1, TfR2 contains no ironresponsive elements in its mRNA and does not appear to be regulated by iron. Mutations in TfR2 have
been implicated in a new form of haemochromatosis (type 3). Homozygosity for these mutations leads to
a form of iron overload with phenotypic characteristics very similar to the HFE-related form (type 1).
The aims of this study were to determine the tissue distribution and intracellular localisation of mouse
TfR2 compared to TfR1. The effect of the Y245X mutation (equivalent to the human Y250X) on the
trafficking and localisation of TfR2 was also studied.
Antibodies were raised against a GST-fusion protein containing the amino-terminal domain of mouse
TfR2. Western blot analysis of mouse and rat tissues was performed. Immunofluorescence experiments
were performed on rat liver sections, isolated rat hepatocytes and a rat liver cell line and analysed by
confocal microscopy. Co-immunoprecipitation experiments using TfR2 and TfR1 antibodies were
performed in isolated rat hepatocytes and the liver cell line. Amino-terminal myc-tagged wild type and
Y245X mutant constructs of mouse TfR2 were made. Constructs were transfected into the rat kidney cell
line, NRK-52E, and stably expressing clones were isolated by selection with G418. The localisation of
wild type and Y245X TfR2 was studied using indirect immunofluorescence and confocal microscopy.
Western blot analysis revealed that the TfR2 antibodies recognised a 105 kDa protein expressed
predominantly in the liver. Indirect immunofluorescence experiments revealed that TfR2 is expressed
predominantly in an endosomal compartment. The localisation differs from that of TfR1, although there is
some overlap. Rat hepatocytes express proportionally more TfR2 than TfR1, however the liver cell line
expresses proportionally more TfR1 than TfR2. Co-immunoprecipitation experiments in liver cells
suggests that there is very little interaction between TfR1 and TfR2. The localisation of myc-tagged wild
type TfR2 in NRK cells was similar to the endogenous expression seen in rat hepatocytes and in the cell
line and also did not colocalise significantly with TfR1. The Y245X mutant form of TfR2 localises to the
endoplasmic reticulum.
Our results show that TfR2 is expressed predominantly in the liver whereas TfR1 has a more ubiquitous
expression. TfR2 localises to an endosomal-like compartment but does not co-localise significantly with
TfR1. There appears to be very little interaction between TfR1 and TfR2. The Y245X mutation causes
retention of the mutant protein in the endoplasmic reticulum, suggesting that the carboxyl-terminal
extracellular portion of the protein is required for the trafficking and correct localisation of TfR2.
The tissue distribution and intracellular localisation of TfR2 differs from TfR1 suggesting that they have
separate functions. It is likely that the main role of TfR2 is in the regulation of iron metabolism and not in
transferrin mediated iron uptake as is the case for TfR1. Mutations in TfR2 could cause this regulation to
be impaired resulting in iron overload.
POSTER 221
MOLECULAR AND CELLULAR ANALYSIS OF HEPCIDIN
V.N. Subramaniam1,2, L. Summerville1, M.D. Jones1, L. Rivas3, L. Sly3, P.A. Pedersen4, and D.F.
Wallace1. 1Membrane Transport Laboratory, The Queensland Institute of Medical Research,
2
Departments of Biochemistry and Medicine, 3Department of Microbiology and Parasitology, The
University of Queensland, Brisbane, Queensland, Australia, 4Department of Clinical Biochemistry,
Hospital of Naestved, Naestved, Denmark.
Hepcidin is a recently identified antimicrobial peptide which is highly expressed in the liver. It plays a
critical role in a variety of iron-associated pathways and is proposed to be an iron-regulatory hormone.
Deletion of the hepcidin gene in mice leads to iron overload and a haemochromatosis-like phenotype.
Iron was shown to accumulate in parenchymals cells especially in the liver and pancreas. Conversely,
transgenic mice overexpressing liver hepcidin died perinatally with anaemia typical of severe iron
deficiency. Two mutations in the hepcidin gene have also been associated with a new form of juvenile
haemochromatosis in humans. These studies are further evidence that hepcidin plays an important role in
regulating iron metabolism.
The aims of this study were to analyse the trafficking and regulation of human hepcidin in an in vitro
system.
Human hepcidin was cloned into the mammalian expression vector, pCDNA3.1Myc-His, and transfected
into a human embryonic kidney cell line, HEK-293. Stably expressing cells were selected for analysis.
The expression of the myc-tagged hepcidin was studied by indirect immunofluorescence and confocal
microscopy. Hepcidin secreted into the cell culture medium was purified using conventional ion-exchange
and metal-affinity chromatography. Anti-microbial assays of the purified hepcidin were performed.
Significant staining of hepcidin was detected in perinuclear as well as in smaller vesicular structures.
Immunofluorescence and confocal microscopy analysis using marker proteins revealed that these
perinuclear structures were the Golgi complex. Hepcidin was purified to homogeneity using a combination
of ion exchange and nickel-affinity chromatography. Amino-terminal sequence analysis of the purified
peptide revealed that it was the mature 25 amino-acid form of hepcidin. Biochemical analysis also
showed that the peptide was processed and secreted from the cell into the medium within 60 minutes.
The purified protein was also shown to be active in anti-microbial assays against Bacillus megaterium and
Micrococcus luteus but inactive against the bacteria, Escherichia coli BL21.
Myc-His-tagged human hepcidin expressed in HEK-293 cells was shown to be expressed, processed and
secreted correctly. Staining of hepcidin was observed in the exocytic pathway in the Golgi complex and in
smaller vesicular structures. The secreted peptide was biologically active in anti-microbial assays and can
now be used as a tool to study its regulatory role in iron metabolism.
POSTER 222
GENOMIC ANALYSIS OF mrs4 YEAST, A MODEL OF MITOCHONDRIAL IRON DEFICIENCY
J. Kim, C. Vulpe, University of California at Berkeley
Mrs4p is proposed to be an iron importer for the mitochondria. The mrs4 mutant accumulates more iron
in non-mitochondrial compartment and shows impairment of iron transport into the mitochondria in
comparison to MRS4 wildtype. In the absence of Mrs4p, we would expect a decrease in iron sulfur cluster
and/or heme synthesis and concomitant increase in iron level in other cellular compartments besides the
mitochondria. Decreased mitochondrial iron levels may lead to compensatory mechanisms in order to
mobilize iron from stores within the vacuole or increase cellular uptake. Three independent cDNA
microarray experiments were compared to better understand iron homeostatic mechanisms in the mrs4
mutant. Firstly, genes encoding previously identified key components in iron uptake system at the cell
surface were highly up-regulated. These include the high affinity iron transport pathway, FRE1, CCC2
and FET3 and the siderophore iron transport genes, ARN's and FIT's. These results confirm that
mitochondrial iron deficiency induces cellular uptake pathways likely mediated via the Aft1/2p pathways.
In addition, multiple genes encoding vacuolar proteins were differentially expressed. The vacuole is
known iron reservoir in yeast yet the details of its role in iron homeostasis are not well characterized.
Some of the differentially expressed genes were also shown to be regulated in other expression studies
of metal overload and general environmental stress. Several genes encoding vacuolar genes have not
been shown to be differentially expressed in previous expression studies and may represent mrs4
specific responses. We noted dramatic change in predicted carbon flow through glycolysis and the TCA
cycle. The combination of these changes as well as dramatic up regulation of non-mitochondrial citrate
synthase suggest that citrate levels may be increased in the mrs4 mutant. Chelation by citrate may play a
role in intracellular sequestration of iron. Finally, multiple predicted solute transporters which may play a
role in iron metabolism were identified in this study. Ongoing phenotype analysis of targeted disruption of
candidate genes identified by this analysis will provide additional insight into the role of the proteins
encoded by the genes differentially expressed in mrs4.
POSTER 223
NON-TRANSFERRIN BOUND IRON UPTAKE AND DIVALENT METAL TRANSPORTER 1
EXPRESSION IS INCREASED BY HUMAN HEPATOMA CELLS DURING PROLIFERATION
D. Trinder1, A.W.M. Lee2 and P.S. Oates2. 1School of Medicine and Pharmacology, University of Western
Australia, Fremantle Hospital, Fremantle and 2Physiology, School of Life and Physical Sciences,
University of Western Australia, Crawley, Western Australia, Australia.
Iron is usually transported in the plasma bound to transferrin. However, in conditions of iron
overload the concentration of plasma non-transferrin bound iron (NTBI) is increased. NTBI is extremely
toxic and may cause generation of oxygen free radials and lipid peroxidation. NTBI is rapidly cleared from
the plasma by the liver and is likely to contribute to iron loading of the liver. Divalent metal transporter-1
(DMT1) is a well-characterized transmembrane iron transporter that has been implicated in the cellular
uptake of NTBI. The aim of this study was to determine the effect of proliferation on the uptake of NTBI
and DMT1 expression by hepatoma cells.
We used a human hepatoma (HuH7) cell line stably transfected with transferrin receptor 1 (TfR1)
antisense RNA expression vector to suppress TfR1 expression. The cells were grown for 3 or 6 days till
they reached the proliferating and stationary phases of growth, respectively. The uptake of NTBI in the
59
o
form of Fe(III):citrate was measured by incubating the cells with Fe-citrate for up to 2 hours at 37 C.
DMT1 mRNA and protein expression was measured by Northern and Western blot analysis, respectively.
The rate of iron uptake (1µM Fe citrate) by the proliferating cells (1.47±0.10pmol Fe/mg
protein/min) was increased significantly by 1.5-fold when compared with the stationary cells
(0.99±0.10pmol Fe/mg protein/min; p<0.05; mean±SEM; n=4). The maximum rate of Fe uptake (Vmax) by
the proliferating cells (4.38±0.13 pmol Fe/mg protein/min) was 1.4-fold greater than by the stationary cells
(3.07±0.12 pmol Fe/mg protein/min; p<0.05; mean±SEM; n=4). DMT1 mRNA and protein expression was
increased 1.7 and 1.3 times, respectively, by the proliferating cells compared with stationary cells.
We conclude that the uptake of NTBI increased when the human hepatoma cells were in the
proliferative stage of growth and this was associated with an increase in the expression of DMT1 mRNA
and protein.
POSTER 224
EFFECT OF PHOSPHATE ON BACTERIOFERRITIN-CATALYSED IRON(II) OXIDATION
N. E. Le Brun, H. Aitken-Rogers, C. Singleton, A. Lewin, G. R. Moore.
Centre for Metalloprotein Spectroscopy and Biology, School of Chemical Sciences and Pharmacy,
University of East Anglia, Norwich NR4 7TJ, UK.
Bacterioferritin (BFR) from Escherichia coli is an iron-storage protein consisting of 24 identical subunits,
each of Mw~18.5 kDa, which pack together to form an approximately spherical molecule with a central
cavity. Large amounts of iron can be deposited as a ferric-oxy-hydroxide-phosphate mineral core within
this cavity. In addition to the iron core, BFR contains up to 12 b-type haem groups, which are situated
between symmetry-related subunit pairs, and a dinuclear metal-binding site within each subunit. Previous
work has shown that the dinuclear centre, also called the ferroxidase centre, is essential for rapid iron(II)oxidation, leading to core formation [1,2]. This process occurs via at least three kinetically distinct
phases: in phase 1 two iron(II) ions bind at each of the dinuclear ferroxidase centres; phase 2
corresponds to the rapid oxidation of the iron(II) ions to iron(III), yielding a likely µ-oxo-bridged iron(III)
dimer at each ferroxidase centre; and, phase 3 corresponds to the subsequent build up of the iron core
and is only observed when > 2 iron(II) ions per BFR subunit are added.
The iron cores of ferritins from different sources exhibit significant compositional and structural differences
and one of the major variables is their phosphate content. Cores of mammalian ferritins contain variable
but low amounts and a number of studies have shown that it is not an integral component of the core
structure, being largely surface-associated. The phosphate content of BFR cores is much higher, and the
phosphate is found to be distributed throughout the core, resulting in amorphous and much less dense
structures compared to mammalian ferritin cores [3,4].
Up to now in vitro mechanistic analyses of E. coli BFR iron core formation have been carried out in the
absence of phosphate, leading to the formation of phosphate-free (mammalian ferritin-like) cores. Since
phosphate significantly affects the structure of the core, an investigation of the effect of phosphate on
BFR-catalysed iron(II) oxidation is essential for a full understanding of the in vivo core formation process.
Using a combination of kinetic and spectroscopic methods we have now undertaken such an
investigation. Phosphate affected several aspects of the iron-uptake behaviour of BFR. The rate of the
phase 3 reaction was enhanced significantly in the presence of phosphate. This effect was linear at low
phosphate:iron ratios but decreased at higher ratios. The stoichiometry of the phase 2 reaction was
unaffected by phosphate but the rate was enhanced (although to a lesser extent than for phase 3).
Phosphate continued to have an effect throughout core formation: surprisingly, it significantly increased
the capacity of the BFR core for iron and also altered the rate profile of core formation. Finally, the
electronic properties of BFR-iron(III) species were also affected by the presence of phosphate, such that
extinction coefficients of iron(III)-derived absorbance bands were significantly decreased. These changes
in electronic properties enabled the observation of two previously undetected phases in the iron-uptake
process, which are providing novel mechanistic insight into BFR core formation.
[1] Le Brun, N. E., Thomson, A. J. and Moore, G. R. (1997) Structure and Bonding 88, 103-138
[2] Yang, X., Le Brun, N. E., Thomson, A. J., Moore, G. R. and Chasteen, N. D. (2000) Biochemistry 39,
4915-4923
[3] Mann, S., Bannister, J. V. and Williams, R. J. P. (1986) J. Mol. Biol. 188, 225-232.
[4] Rohrer, J. S., Islam, Q. T., Watt, G. D., Sayers, D. E. and Thiel, E. C. (1990) Biochemistry 29, 259264.
POSTER 225
MUTATIONS IN A TRNA MODIFICATION GENE, MIAE, AFFECT IRON METABOLISM IN
SALMONELLA TYPHIMURIUM
H. K. Lundgren1, R. Leipuviene2 and G. R. Björk3
Umeå University, Department of Molecular biology, S-90187 Umeå, Sweden
1
Hans.Lundgren@molbiol.umu.se, 2Ramune.Leipuviene@molbiol.umu.se,
3
Glenn.Bjork@molbiol.umu.se
Keywords: tRNA, modified nucleosides, metabolism, iron
The miaE gene in Salmonella typhimurium encodes for the modification enzyme (MiaE), which is required
for the hydroxylation of the modified nucleoside 2-methylthio-N6- isopentyladenosine (ms2i6A37) to form
2-methylthio-N-6-(cis-hydroxy) isopentenyladenosine (ms2io6A37) present at position 37 of most tRNA:s
reading UNN codons[1]. This hydroxylation reaction requires molecular oxygen and iron [2]. A
miaE2507::MudJ mutant of S. typhimurium is unable to grow on minimal medium containing succinate,
malate or fumarate as the primary carbon source [3]. Such a mutant also experiences a longer lagphase
than a wildtype Salmonella LT2 when shifted to minimal acetate medium[3]. Anaerobically the miaE
mutant grows as the wildtype Salmonella LT2 at all conditions tested [3]. Detection of carbonylated
proteins was performed using the chemical and immunological reagents of the OxyBlotTM Oxidized
Protein Detection Kit (Oncor). This showed that a miaE mutant has a roughly 30-40% increased
carbonylation level of proteins, which indicates an increased level of oxidative stress. The mutant also
lacks siderophores outside the cell as shown on CAS agar plates [4] and addition of dihydroxybenzoic
acid, which is one of the precursors to the major siderophore, enterochelin, restores growth on succinate.
Addition of FeCl3 as well as FeSO4, magnesium or ferrichrome to the growth medium does not alleviate
the growth or the siderophore defect of a miaE mutant. The specific uptake machinery of enterochelin is
present as shown by outer membrane profiles, which is consistent with the supplementation experiments.
Addition of only serine but no other amino acid enables the miaE mutant to grow on succinate. Serine is
part of the enterochelin pathway and the miaE phenotype is similar to ent mutants, which are defective in
the synthesis of enterochelin. The transcription of the entCEB operon but not the entF operon is
decreased by ~33% as measured using lacZ fusions on the chromosome. Expression of a chromosomal
fur::lacZ translational fusion was decreased by 33% and a iroA::MudJ fusion previously described [5]
showed a similar reduction of expression. A miaE mutant seems to be stalled in stationary phase unable
to resume growth aerobically without the addition of metabolites in the enterochelin biosynthetic pathway
when grown on succinate, malate or fumarate. Interestingly additions of a wide variety of metabolites in
the citric acid cycle (CAC) also enables a miaE mutant to grow [3] which suggests a deficiency in one or
several of the enzymes in the CAC. We suggest that a tRNA modification mediated effect on the
enterochelin biosynthetic pathway possibly through Fur may explain the siderophore deficiency and the
inability to grow on dicarboxylic acids by poor or faulty translation of one of the steps involved.
[1]
[2]
[3]
[4]
[5]
Persson B.C and G. R. Björk. J. Bacteriol. 175, (1993) p7776.
Buck M, Ames B. N. Cell 36, (1984) p523.
Persson B.C et al. J. Bacteriol. 180, (1998) p3144.
Schwyn B and Neilands J.B. Anal Biochem 160, (1987) p47.
Hall H.K and Foster J.W. J. Bacteriol 178, (1996) p5683.
POSTER 226
GENETIC VARIABILITY IN IRON METABOLISM: A QTL APPROACH
Carole Beaumont
Introduction: The genetic basis of mammalian iron metabolism has been extensively studied. Most of the
genes controlling this metabolism have been found either through genetic studies of hereditary iron
disorders or animal models, or through identification of modifier genes in disorders where iron acts as an
aggravating factor (neurological disorders, inflammatory or infectious diseases). To analyze the genetic
control of iron metabolism in physiological conditions, we used the quantitative trait loci (QTL) mapping in
genetic recombinant congenic strains (RCS), a powerful tool specially developed for multigenic trait
analysis. In this approach, the RCS are produced by inbreeding mice of the second backcross generation
of two inbred strains. Each RCS contains 12.5% of genes from the parental donor strain and 87.5% of
genes from the parental background strain. The genes of the donor strain involved in the genetic control
of a multigenic trait become separated into the different RCS and can be studied individually.
Methods: Here we used the C3H/DiSnA – C57BL/10ScSnA (HcB/Dem) RCS where the C3H/DiSnA is the
background strain and the C57BL/10ScSnA the donor strain. To assess the iron status of these animals,
we measured plasma transferrin and non-heme iron content in liver, spleen and plasma from five 3month-old male mice from the two parental strains and from each of the 27 HcB/Dem recombinant
strains. We also measured hepcidin mRNA levels by quantitative RT-PCR in livers of all these mice.
Analysis of variance (ANOVA and Tukey’s tests) with strains as fixed factor and individual experiments as
random factors was used to evaluate the differences between individual strains.
Results: The two parental strains were found to differ widely, the C3H/DiSnA strain having a high iron
load in spleen and liver as compared to the C57BL/10ScSnA donor strain. Analysis of spleen and liver
iron content lead to the identification of a major group (24) of C3H/DiSnA-like strains and a minor group
(3) of C57BL/10ScSnA-like strains. This indicates that a limited number of genes with a strong effect is
responsible for the low iron level in liver and spleen of the C57BL/10ScSnA donor strain. Moreover, the
stain pattern distribution (SPD) of liver and spleen iron parameters is different, indicating that they are
under the control of different genes. A similar situation is observed for transferrin saturation that is greater
in the C57BL/10ScSnA strain than in the C3H/DiSnA and there again, a small set of RCS show s a similar
phenotype as that observed in the C57BL/10ScSnA. There is a partial overlap in SDP between hepatic
iron content and transferrin saturation, suggesting the presence of common genetic determinants.
We then analyzed by linear regression the relationships between tissue iron contents, transferrin
saturation and hepatic hepcidin mRNA. There is a weak but significant correlation (p<0.05) between
spleen iron and hepatic hepcidin mRNA levels. A strong correlation is observed between either the liver
or the spleen iron stores and transferrin saturation (p<0.0001).
Conclusions: This study highlights the genetic variability of the normal iron status and shows that
correlation parameters are depending upon RCS. Thus, the RCS are suitable for mapping the genes
controlling these correlations and the appropriate crosses are currently being generated
POSTER 227
STRAIN, GENDER AND HFE GENE MODULATE HEPATIC HEPCIDIN mRNAs EXPRESSION IN MICE
Brice Courselaud1, Marie-Bérengère Troadec1, Mounia Bensaïd2, Gennady Ilyin1, Seiamak Bahram3,
Pierre Brissot1, Marie Paule Roth2 and Olivier Loréal1.
1
INSERM U522, CHRU Pontchaillou, Rennes, France ; 2Unité de Physiopathologie Cellulaire et
Moléculaire, CNRS UPR 2163, CHU Purpan, Toulouse, France and 3INSERM-CreS, Centre de
Recherche d’Immunologie et d’Hématologie, Strasbourg, France
Hepcidin, a gene mainly expressed in the liver, plays a major role in the control of iron metabolism.
Indeed, in mice, invalidation of this gene leads to the development of an iron overload phenotype which
mimics human genetic hemochromatosis related to HFE gene C282Y mutation. Moreover, it has been
recently reported that, in humans, hepcidin mutations are associated to a juvenile hemochromatosis
phenotype. In addition, it has been reported that males are more prone to the development of hepatic iron
overload during genetic hemochromatosis. These data suggests that an abnormal regulation of hepcidin
related to sex and/or HFE mutations may participate to the phenotypic expression of the disease. The aim
of this work was to analyse in mice the effects of strain, gender and HFE phenotype on hepatic hepcidin
mRNAs steady-state levels. Animals and methods. For this purpose wild-type and HFE KO mice of two
different strains, ie C57BL/6 and DBA2, and of the two genders were included in this study. Liver and
splenic iron load were evaluated in the liver and in the spleen by biochemical assay. HEPC mouse liver
mRNAs contents were evaluated by northern-blot. In addition, specific HEPC1 and HEPC2 liver mRNA
contents were quantified using Taqman real-time RT-PCR technology. Results. 1) Iron load. In wild-type
DBA2 mice of either gender, the hepatic iron concentration (HIC) was slightly higher than in the
corresponding C57BL/6 mice. In addition, in both strains, females had a higher HIC than males. For
splenic iron concentration (SIC), in wild-type, males of the two strains had similar values which were
significantly lower than their corresponding females. As expected, in C57BL/6 and DBA2 HFE KO mice of
the two genders, HIC values were increased compared to those found in their respective wild-type mice.
In addition, they were slightly higher in DBA2 compared to C57BL/6 mice. HFE KO significantly
decreased SIC in DBA2 female mice compared to the wild-type group but did not influence its value in
DBA2 males as well as in C57BL/6 mice of the two sexes. 2) Hepcidin expression. In two wild-type strains
DBA2 and C57BL/6, despite some heterogeneity, we found that females overexpressed hepcidin mRNA
compared to the corresponding males. HFE KO was associated with a significant increase of hepcidin
mRNAs in males of the two strains as well as in C57BL/6 females. However, DBA2 HFE KO females had
a slight decrease of hepcidin mRNAs level. Furthermore, we found that overexpression of hepcidin
mRNAs observed in other HFE KO mice, corresponded in fact to an increase of HEPC1 expression in
male or female C57BL/6 and of both HEPC1 and HEPC2 mRNAs in DBA2 males.
Summary and conclusions. Hepcidin expression i) was higher in both wild-type females strains, which
have a higher splenic iron concentration than males, ii) was increased in three out of four HFE KO
groups, iii) was slightly decreased in females HFE KO DBA2 mice, the only group exhibiting a reduction
of splenic iron concentration compared to their corresponding wild-type animals. Collectively, these data
demonstrate that genetic background related to strain, gender, as well as HFE gene expression may
independently modulates hepcidin 1 and 2 mRNAs expression. Furthermore, whatever the molecular role
of hepcidin, our results suggest that hepcidin may remains inducible in case of iron overload related to
HFE gene invalidation. These results strongly reinforce the hypothesis that besides HFE, other genes
such as hepcidin could be involved in the phenotypic expression of genetic hemochromatosis. In addition,
they support a relationship between the splenic iron content and the hepcidin expression.
POSTER 228
SULFUR DELIVERY FROM CYSTEINE FOR FE-S CLUSTER FORMATION IN YEAST
H.L. Lumppio1,2, E. R. Lyver2, S. A. B. Knight2, D. Pain3, A. Dancis2*
1
Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853
2
Department of Medicine, Division of Hematology-Oncology, University of Pennsylvania, Philadelphia, PA
19104
3
Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey,
Newark NJ 07103
Formation of Fe-S clusters in proteins such as ferredoxins is mediated by a conserved group of proteins.
In eukaryotes, these proteins are present in mitochondria. Here, we developed an assay for tracking
cysteine sulfur to Yah1p, the yeast mitochondrial ferredoxin, identifying key features of this process. Fe-S
cluster formation on apo-Yah1p required the presence of yeast mitochondrial matrix fraction and occurred
linearly for 1 hr. We discovered that the ferredoxin substrate must be converted to its apo form under
anaerobic conditions, and that exposure to air rendered the substrate incapable of being loaded to holoYah1p. Cluster formation also required the addition of ferrous iron, suggesting that stored iron was either
not present or unavailable in the mitochondrial matrix. Yeast mitochondrial matrix fractions lacking Nfs1p
were unable to support cluster formation on Yah1p. By contrast, fractions lacking the yeast frataxin
homolog, Yfh1p, were active. Fe-S cluster proteins, Yah1p and aconitase, were deficient in mitochondria
depleted of Nfs1p or Yfh1p. These results suggest that Nfs1p is required for sulfur delivery for Fe-S
cluster formation while Yfh1p is involved in another step in holoferredoxin formation. Finally, the yeast
mitochondrial matrix was able to support cluster formation on yeast ferredoxin but not on spinach
ferredoxin, indicating a preference of the yeast Fe-S cluster synthesis apparatus for its endogenous
substrate.
POSTER 229
ISCS-DEPENDENT IRON-SULFUR CLUSTER ASSEMBLY IN TRICHOMONAD HYDROGENOSOMES
J. Tachezy1, R. Šuťák1, P. Doležal1, M. G. Delgadillo-Correa2, H. Lumppio3, A. Dancis3, L. Sánchez4, M.
Müller4, P. J. Johnson2. 1Charles University, Czech Republic; 2University of California Los Angeles;
3
University of Pennsylvania, 4The Rockefeller University
The formation of iron-sulfur clusters is mediated by a highly conserved group of proteins of which PLPdependent cysteine desulfurase (IscS) is the best characterized member. These proteins were
discovered in bacteria and later identified in the mitochondria of eukaryotes. Assembling of iron-sulfur
clusters is particullarly important for amitochondrial anaerobic eukaryotes, which possess a specific set of
FeS proteins that mediate key steps in their energy metabolism. In Trichomonas vaginalis, a sexually
transmitted human parasite, FeS proteins including pyruvate:ferredoxin oxidoreductase, 2Fe-2S
ferredoxin and hydrogenase, are present in unusual ATP generating organelles, the hydrogenosomes.
Virtually no information is available about FeS cluster assembly in trichomonads as well as in other
amitochondrial organisms. Here we identified and sequenced two IscS homologs in T. vaginalis (TviscS1 and TviscS-2). Both gene products possessed the typical conserved regions implicated in cysteine
desulphurase activity, and N-terminal amino acid extensions, which resembled the presequences that
target proteins to hydrogenosomes. Subcellular localization of IscS with an antibody raised against
recombinant TviscS-2 showed the presence of IscS in the hydrogenosomal cell fraction. Stable
transfection of T. vaginalis with TviscS-1 and TviscS-2 fused with a hemagglutinin tag confirmed that both
gene products are targetted to these organelles. The function of TviscS-1 and TviscS-2 is probably not
identical: TviscS-2, contains a C-terminal sequence signature involved in iscS-iscU interaction that is
absent in TviscS-1 and run-on assays showed that transcription of TviscS-2 was up-regulated under ironrestricted conditions, while transcription of TviscS-1 was not observed under any experimental conditions.
To study the mechanism of FeS cluster formation, an in vitro assay was established based on FeS cluster
35
59
reconstitution of recombinant apo-ferredoxin using S-cysteine or Fe. In vitro formation of holoferredoxin was dependent on the concentration of the hydrogenosomal extract and iron, it was timedependent, and required anaerobic conditions. The reaction was inhibited by iron chelators and thiolalkylating reagents. Phylogenetic analysis of IscS showed a close relationship among all eukaryotic IscS,
indicating a common mechanism for FeS cluster formation in mitochondriates, T. vaginalis, as well as
other amitochondriate eukaryotes.
POSTER 230
IRON-DEPENDENT REGULATION OF FERRITIN AND TRANSFERRIN RECEPTOR IS ABROGATED
DURING TERMINAL DIFFERENTIATION OF NORMAL MOUSE ERYTHROBLASTS
Matthias Schranzhofer1, Javier A. Cabrera1, Hartmut Beug2 and Ernst W. Müllner1
1
Inst. of Medical Biochemistry, Division of Molecular Biology, 2Inst. of Molecular Pathology, Vienna
Biocenter, Dr. Bohr-Gasse 7-9, A-1030 Vienna, Austria
Iron-dependent co-regulation of ferritin (Fer) and transferrin receptor (TfR) is an example par excellence
for specific regulation on the post-transcriptional level in animal cells. The system consists of two iron
responsive proteins (IRP 1 and 2), which bind to specific RNA stem loop structures, called iron responsive
elements (IREs) under low concentrations of biologically available iron in the cell. Such IREs are found in
the 5' untranslated region (UTR) of the iron storage protein ferritin and in the 3'UTR of TfR, which is
responsible for iron uptake into the cell. Binding of IRP to these IREs results in stabilization of TfR mRNA,
whereas translation of Fer mRNA is blocked, leading to an increase of accessible iron in the cell. In
addition to Fer and TfR there is a third mRNA, specific for erythroid cells, which contains an coding for the
enzyme delta- aminolevulinic acid synthase (ALAS-E), which catalyses the rate-limiting step in the
biosynthesis of heme. Due to this constellation, the question arises, how differentiating erythroid cells
would cope with the paradoxical situation that low IRP activity, allowing efficient ALAS-E translation, also
should favor ferritin production and down-regulation of TfR at times of massive iron demand for heme
biosynthesis.
Recently, we established conditions allowing mass cultivation of primary erythroid progenitors derived
from mouse fetal liver. These cells can either be kept as immature erythroblasts or be induced to highly
synchronous differentiation by physiological stimuli. Hence, this system appeared ideally suited to study
the regulation of iron utilization and storage under in vivo-like conditions. Polysome gradients, as well as
Western and Northern blot analysis on proliferating and/or differentiating mouse erythroblasts treated with
the iron chelator desferrioxamine (Des) or high levels of transferrin-bound iron (Tf) demonstrated that
during terminal differentiation expression of Fer and TfR is not modulated by iron anymore, whereas
translation of ALAS-E is still iron-dependent.
These findings corroborate data for ferritin expression in chicken erythroblasts (Mikulits, Blood
94:4321, 1999) and support the model of a direct transfer of iron from the late endosome into
mitochondria via protein:protein interaction, where it is inserted into protoporphyrin IX, recently
designated as "Kiss and Run Hypothesis" (Ponka, Blood 89:1, 1997; Lobmayr, Blood 100:289, 2002)
This work was supported by grants from the "Herzfelder Family Foundation" and the Austrian National
Basic Science Foundation "FWF".
POSTER 231
IRON TRAFFICKING IN THE FET3P, FTR1P IRON TRANSPORT COMPLEX IN THE YEAST PLASMA
MEMBRANE
S. Severance, T.-P. Wang, S. Chakraborty, A. Singh, C. Stoj, A. Romeo, D. Kosman, The University at
Buffalo
High affinity uptake of non-siderophore (‘free’) iron in fungi is due to a pair of plasma membrane
proteins, at least. In Saccharomyces cerevisiae these two proteins are Fet3p and Ftr1p. Fet3p is a
multicopper oxidase (MCO), that like ceruloplasmin and hephaestin, is kinetically specific for Fe2+ as
substrate. Thus, Fet3p is a ferroxidase, catalyzing the oxidation of 4Fe2+ with every O2 consumed. Fet3p
is a type 1 transmembrane protein with a single, C-terminal transmembrane (TM) domain. The MCO
domain, residues 22-555, is extracellular; the C-terminus is intracellular. Ftr1p has 7 TMs, N-terminal out,
C-terminal in. The most mechanistically significant fact about iron transport through this complex is that
uptake is absolutely coupled to ferroxidation; uptake occurs only if ferroxidation is on-going. This
behavior suggests two possible models of iron trafficking through the Fet3p, Ftr1p complex. First, the
Fe3+ produced by Fet3p is channeled to Ftr1p; it does not equilibrate with bulk solvent. Second,
ferroxidation and permeation are energetically coupled in some fashion. These two models are not
mutually exclusive. This presentation includes the data that: 1) establish the orientation and topology of
the Fet3p, Ftr1p complex in the yeast plasma membrane; and 2) are consistent with an iron channeling
model of iron uptake.
Two epitope-tagged versions of Ftr1p were used in indirect immunofluorescence studies to
demonstrate this protein’s orientation and topology. A C-terminal myc tag was accessible only in
permeabilized, but not unpermeabilized yeast spheroplasts, consistent with a cytoplasmic C-terminal
orientation. (HA)2 insertions into four putative loop regions were similarly probed. Whereas 40(HA)241 and
124
were accessible only in permeabilized cells, 174(HA)2175 and 255(HA)2256 were seen in both
123(HA)2
preparations. In addition, C-terminal fluorescent protein fusions to both Fet3p and Ftr1p were fully wild
type, also indicating a cytoplasmic orientation. Lastly, truncation of the putative TM and C-terminus of
Fet3p resulted in the production of a soluble form of Fet3p that was processed normally, but was secreted
into the growth medium.
Mutagenesis in both proteins, and, for Fet3p, in both the soluble form for kinetic studies of
ferroxidation in vitro, and in the membrane-bound form for analysis in iron uptake in vivo, demonstrated
roles for several motifs in both proteins required for iron uptake. This functional mapping implicated
Fet3pE185 and three motifs on Ftr1p in an iron channeling pathway. Thus, 185E→D substitution in Fet3p
increased KM for Fe2+ in vitro 2-fold; in contrast, the increase in vivo was 250-fold. The 185E→A
substitution was more severe causing an 8-fold KM increase in ferroxidation, while completely inactivating
Fet3p in support of iron uptake in vivo. The stronger in vivo phenotype is consistent with a channeling
model in which E185 ‘hands’ iron ‘off’ to Ftr1p. A possible Ftr1p ‘partner’ in this channeling was also
identified. Mutagenesis in a conserved DaxE motif in extracellular loop 6 in Ftr1p demonstrated that both
acidic residues were required for iron permeation. Also, insertions in this loop induced a citrate sensitivity
indicative of ‘leakiness’ in this putative channeling process.
Membrane permeation is the next step in this iron trafficking process. Our studies demonstrate
that a 16RECLE20 motif in TM1 is essential, as is an 157REGLE161 one in TM4 identified by Stearman and
his co-workers. An E→A substitution at any one of the four glutamates inactivates Ftr1p. In contrast,
single E→D substitutions have a graded effect, the strongest being at E20 and E161. The pattern of
effect in double mutants parallels this: the E20D/E161D mutant exhibited <5% wild type activity. Analysis
of these results suggests that E20 and E161 are part of a iron coordination complex that is interTM
domain in nature. Lastly, this putative complex is stabilized by positive ‘caps’: whereas R→K
substitutions are conservative, R→A, Q, or E ones inactivate Ftr1p.
POSTER 232
Withdrawn
POSTER 233
INTERRELATIONSHIP BETWEEN RIBOFLAVIN BIOSYNTHESIS AND IRON TRANSPORT IN THE
YEAST PICHIA GUILLIERMONDII
Y.R.Boretsky, K.E. Kapustiak, M.M. Stenchuk, O.V. Stasyk, V.I.Kutsyaba, A.A. Sybirny. Institute of Cell
Biology National Academy of Sciences of Ukraine, Drahomanov St., 14/16, 79005, Lviv, Ukraine
It is known for decades that yeast P. guilliermondii overproduces riboflavin (vitamin B2, RF) in response
to iron limitation. P. guilliermondii RF overproducing mutants rib80, rib81 and hit1 are hypersensitive to
oxidative stress and possess defects in regulation of iron acquisition. Molecular mechanisms of such
regulation remain unknown.
To study this phenomenon in more details, we cloned and sequenced P. guilliermondii genes coding for
GTP cyclohydrolase (RIB1) and riboflavin synthase (RIB7) – the first and the last enzymes of RF
biosynthetic pathway. Several potential binding sites for AFT1p (major iron sensing transcriptional factor
in S.cerevisiae) were found in their promoter regions suggesting that iron can be directly involved in
transcriptional regulation of RF biosynthetic genes in P. guilliermondii. In addition, the potential binding
sites for YAP1p and Rox1p were also found in both promoters supporting a notion that these
transcriptional factors can be involved in control of RF biosynthesis and iron acquisition in P.
guilliermondii.
Using P. guilliermondii rib1-86 RF deficient mutant carrying leaky mutation in the RIB1 gene, we selected
+
a collection of spontaneous rib revertants. Genetic analysis revealed that mutations in the six novel
genes RED1-RED6 (reduction), as well as in the previously identified genes RIB81, RIB80 and HIT1,
rescue RF auxotrophy of rib1-86 mutant. Newly identified secondary mutations lead to the enhancement
of the RIB1 expression. These mutations are not linked with the RIB1 locus.
Relative to the wild-type strain, all red mutants possessed: (i) increased activity of GTP cyclohydrolase,
riboflavin synthase and elevated levels of RF biosynthesis; (ii) enhanced ferric/cupric reductase activity
and higher non-hemin iron content; (iii) increased sensitivity to transitional metals, as well as to hydrogen
peroxide. Therefore, all selected P.guilliermondii RF overproducing mutants also possessed defects in
regulation of iron acquisition. Noteworthy, previously selected P. guilliermondii mutant strain rib83 (unable
to overproduce riboflavin) has dramatically reduced iron transport and is unable to grow on iron deficient
media. Obtained results demonstrate that iron acquisition and RF biosynthesis are regulated coordinately
in P.guilliermondii.
POSTER 234
DIFFERENCE BETWEEN IN-VIVO AND IN-VITRO DEGRADATION OF IRON REGULATORY
PROTEIN 2 (IRP2)
M. C. Ghosh, E. Bourdon, S. K. Drake, J. Wey and T. A. Rouault, Cell Biology and Metabolism Branch,
NICHD, National Institutes of Health, 9000 Rockville Pike, Bethesda, Maryland 20892
Iron metabolism in mammals is regulated by two iron-regulatory proteins, IRP1 and IRP2 through their
binding to RNA stem-loop motifs known as iron-responsive elements (IREs) [T. A. Rouault and R.
Klausner (1997) Curr. Top. Cell Regul. 35, 1-19]. IRP1 and IRP2 have similar sequences except that
IRP2 contains an extra 73 amino acid domain that is absent in IRP1. IRP2, but not IRP1 undergoes iron
dependent degradation. The 73 amino acid domain is responsible for the degradation of IRP2 [K. Iwai et.
al. (1998) Proc. Natl. Acad. Sci. U.S.A., 95, 4924-4928]. Results of in vitro experiments with this 73 amino
acid degradation domain have shown that the iron-catalyzed oxidation of one of the five cysteines (cys137, cys-168, cys-174, cys-178, cys-201) facilitates iron-dependent degradation of IRP2 [D-K Kang et. al.,
unpublished results].
In an effort to verify whether the same mechanism for the degradation of IRP2 is operative in in-vivo, we
over-expressed the IRP2 mutants with mutations of degradation domain cysteines. Our gel-shift assay
and western blotting results indicate that IRP2 can still be iron dependently degraded when these
cysteines are mutagenized. These observations indicate that mechanism of in-vivo degradation of IRP2 is
different from that of its in-vitro degradation.
POSTER 235
HIGH CAPACITY RNA AFFINITY COLUMN FOR PURIFICATION OF HUMAN IRPS
E. Yikilmaz, CR. Allerson, JJ. Gilligan, AL. Yergey, and TA. Rouault. National Institutes of Health
Iron is essential for life because of its involvement in fundamental cellular processes such as respiration,
photosynthesis, and protection against oxidative stress. Due to its high reactivity, regulatory molecules
tightly control the availability and reactivity of iron in the cell. In humans, two slightly different forms of iron
regulatory proteins (IRP1, IRP2), structural homologs of mitochondrial aconitase, are located in the
cytoplasm. In iron replete cells, IRP1 has functional homology to aconitase and it contains an [Fe4-S4]
cluster in its active site whereas IRP2 doesn’t show any aconitase activity. IRPs exert their
posttranscriptional regulatory effects by binding to RNA stem loops (iron-responsive elements, IREs)
formed in the 5’ or 3’ untranslated regions of ferritin, and transferrin receptor transcripts in iron depletion.
Here we report that, we can overexpress the human IRPs in Pichia pastoris and purify them efficiently in
mg quantities by an IRE affinity column, which is constructed by using the IRP1 binding domain of ferritin
IRE. Besides purification, the IRE-column will be used to investigate the kinetics of IRP-IRE complex
formation and the results will be compared to the values obtained from surface plasmon resonance.
These experiments will allow us to answer some of the questions related to iron regulatory functions of
IRPs at a molecular level.
POSTER 236
IRON-REGULATORY PROTEIN 2 AS IRON SENSOR: IRON-DEPENDENT OXIDATIVE
MODIFICATION OF CYSTEINE
J. Jeong, D.-K. Kang, S.K. Drake, N. Wehr, T.A. Rouault, R. L. Levine, Laboratory of Biochemistry,
National Heart, Lung & Blood Institute and Cell Biology and Metabolism Branch, National Institute of Child
Health and Human Development, National Institutes of Health.
Iron-regulatory protein 2 (IRP2) coordinately regulates the synthesis of many proteins involved in iron
metabolism. Earlier studies established that IRP2 is regulated through control of its degradation, in an
iron-dependent fashion. When cellular levels of iron are low, IRP2 is stable, but when iron stores become
sufficient, IRP2 is rapidly degraded by the proteosome. We previously demonstrated that the degradation
pathway is initiated by an iron dependent oxidative modification of IRP2.
This modification requires a degradation domain of about 7 kD, including 5 cysteine residues. In
vivo experiments with mutant IRP2 established that 2 of these cysteine residues are dispensable. We
therefore produced a degradation domain of 63 residues with 3 cysteines. Oxidative modification of fulllength IRP2 or recombinant peptide was performed by exposure to a model metal-catalyzed oxidation
system consisting of iron, dithiothreitol (DTT), and oxygen from air at 37° C in 50mM Hepes, pH 7.2, and
monitored by amino acid analysis, HPLC-mass spectral analysis, and tandem mass spectrometry.
Oxidized or native peptide was alkylated by iodoacetamide (IAA) before analysis. If the protein or peptide
was not alkylated, reduction of thiols was accomplished by incubation with Tris(2-carboxyethyl)phosphine
(TCEP).
Before exposing the peptide to the oxidizing system, its mass spectrum was homogeneous with
the mass that of the native peptide. After exposure, the mass spectrum became heterogeneous,
containing both residual native peptide and products. The most prominent product, representing ~5% of
the starting material, was 59 amu less than the native peptide when alkylated (native=6,694 amu;
modified=6,635 amu), and 1 to 2 amu less when not alkylated. These results indicate that oxidative
modification converts a cysteine to a product which is 2 amu less than cysteine and which can no longer
be alkylated by iodoacetamide.
Although the mass spectrum became heterogenous, the native form was always readily
deconvoluted so that we could determine the fraction of the peptide which was still native. After a 2 hr
exposure to the oxidizing system, virtually all of the peptide was modified. Amino acid analysis of this and
the native peptide confirmed the loss of one cysteine; no other modifications were detected. Loss of the
cysteine residue required only micromolar concentrations of iron. Examination of the time course of the
oxidation revealed that loss of the native mass coincided with loss of the cysteine residue. Neither loss of
cysteine nor change in mass occurred if the incubation was performed anaerobically nor if iron was
chelated. Thus, the IRP2 degradation domain is susceptible to iron and oxygen-dependent oxidative
modification of a cysteine residue. To determine the structural requirements for the oxidative
modification, we made site-specific mutants including 3 single cysteine to alanine mutants, 3
combinations of double mutants and a triple mutant with the three cysteines changed to alanines. Mass
spectrometric analyses established that the 3 cysteine residues are located at the site of free-radical
generation, and thus likely participate in the binding of iron.
IRP2 regulates iron metabolism, and IRP2 in turn is regulated by iron. We have demonstrated
that the degradation domain forms an iron-binding site which readily undergoes iron-catalyzed oxidative
modification. The oxidative modification proceeds efficiently with a non-enzymatic system consisting of
oxygen, iron, and a reducing agent. However, iron metabolism is exquisitely regulated in vivo so it would
not be surprising to find that other factors, presumably proteins, modulate the reaction.
While the details of the oxidation mechanism of IRP2 remain to be experimentally elucidated, it is
clear that the degradation domain has evolved to become an efficient iron sensor. It contains an ironbinding site which mediates reaction with oxygen to cause covalent modification of the site. This
modification causes a structural change that may be recognized by the proteasome system which then
degrades the oxidatively modified IRP2.
POSTER 237
THE P. FALCIPARUM IRON PATHWAY CHARACTERIZED BY THE DIVALENT METAL
TRANSPORTER HOMOLOGUE
M. Rivarola, L. Shi, J. Gauthier, C. Ng, S. Krishna*, D. Sullivan
Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD USA
*Infectious Diseases, St George's Hospital Med. School, London, SW17 ORE, UK
The intraerythrocytic Plasmodium encounters high milliMolar concentrations of heme iron, yet is
susceptible to microMolar concentrations of iron chelators. The metabolism of iron or heme iron is the
target of both the quinoline and artemisinin class of antimalarials. The critical bioavailable source of iron
has yet to be definitively determined whether from erythrocyte cytosol or hemoglobin. Extraerythrocytic
iron has been experimentally ruled out as a source of iron for the parasite.
The family of Divalent Metal Transporters (DMT) is highly conserved amongst bacteria, yeast,
plants, insects and mammals and functions to transport iron and other metals across membranes by a
proton driven force. PfDMT1 is a single copy gene by Southern blot anlaysis and genome searching. Both
P. yoelii and P. vivax have homologous DMT1 sequences that share 70% identity with PfDMT1 in the
twelve transmembrane region. P. yoelii and P. falciparum have an extra large 25 kDa N-terminal
nontransmembrane tail with no amino acid similarity to other DMT homologues in this region. Northern
blot and tandem competitive-RTPCR analysis of transcription demonstrate message predominately at ring
stage that increases two-fold with iron chelation. Western blot and Immunofluorescence analysis indicate
that PfDMT1is not in the digestive vacuole membrane, a predicted iron source, but instead localizes to
the plasma membrane and a cytostolic microsomal compartment. ImmunoEM also confirms the plasma
membrane location. The native gene with a C-terminal HA epitope tag is unable to be expressed in
bacteria, yeast, mammalian cell lines or Xenopus oocytes. Interestingly however, the identical constructs
fused to 70 amino acids of the nontransmembrane N-terminal ratDCT1 demonstrate expression in
Xenopus oocytes. Unfortunately the expressed fusion protein does not localize to the plasma membrane
of Xenopus oocytes, obviating functional verification.
This work concurs with other investigations that implicate a bioavailable iron source in the
erythrocyte cytosol rather than the digestive vacuole where hemoglobin is degraded.
POSTER 238
ROLE OF HEPHAESTIN IN IRON TRANSPORT IN CACO-2 INTESTINAL EPITHELIAL CELLS
V. Seshadri and P.L. Fox, Department of Cell Biology, Lerner Research Institute, Cleveland Clinic
Foundation, Cleveland, OH
Dietary iron is absorbed and transported across the intestinal epithelium for processing and delivery to
tissues requiring iron or to iron storage sites. In humans, there are no regulated, physiological processes
that excrete iron, and thus iron availability is regulated principally at the level of absorption and transport.
Hephaestin, a homologue of ceruloplasmin, was discovered as the gene defective in the sex-linked
anemic (sla) mouse. Iron absorption by the gut is normal in these mice, but the serosal transport of iron
across the intestinal epithelium is defective leading to mucosal iron accumulation and consequent
hypochromic anemia. Like ceruloplasmin hephaestin is a copper containing protein with ferroxidase
activity. However, unlike ceruloplasmin, hephaestin contains a putative one-pass, trans-membrane
domain at the C-terminal end of the protein and is not secreted. Based on the yeast model of iron uptake
where a membrane bound ferroxidase in conjunction with a permease is responsible for high affinity iron
uptake, it has been proposed that hephaestin in conjunction with ferroportin-1 transports iron across the
basolateral membrane of the intestinal epithelium. For analysis of expression and function of hephaestin,
we have cloned the human hephaestin gene by RT-PCR and 5’-RACE. We have identified two clones
corresponding to transcripts containing different first exons but identical open reading frames. Using
radiolabeled hephaestin cDNA as a probe we have analyzed its expression in a multi-tissue expression
array. Hephaestin expression is high in intestine and colon, moderate in lung, heart and aorta, and low in
other tissues. We have also analyzed the expression of ferroportin-1 and find that, in general, ferroportin1 expression is complementary to hephaestin expression, i.e., in tissues in which hephaestin expression
is high, ferroportin-1 expression is low, and vice versa. An immunohistochemical analysis at the cellular
level shows a similar distinction between these proteins; hephaestin is localized primarily in the
perinuclear region, whereas ferroportin-1 has been localized to the plasma membrane. Together, these
results suggest that hephaestin is unlikely to be physically associated with ferroportin-1, and may function
independently. We have analyzed the regulation of hephaestin expression by iron or by oxygen levels, in
human intestinal epithelial Caco-2 cells. Iron chelators or hypoxia induce the expression of hephaestin by
about 2-fold at both mRNA and protein levels. To investigate the function of hephaestin in gut iron
transport, Caco-2 cells have been stably transfected with a plasmid containing the full-length hephaestin
open reading frame under the control of the cytomegalovirus promoter, and clones that over-express
hephaestin have been isolated. We have measured iron transport from the apical to basolateral aspects
of cells grown to confluence in a transwell chamber. Iron transport is increased by about 2- to 4-fold in
Caco-2 cells that express about twice the normal amount of hephaestin. Together these experiments
indicate an important role of hephaestin in iron transport across intestinal epithelial cells.
POSTER 239
FLUORESCENCE ANALYSIS OF SICKLE ERYTHROCYTE LABILE IRON POOL DISTINCT FROM
NON-HEME MEMBRANE IRON AND FERRITIN IRON
J. Kurantsin-Mills,1, 2 V.R. Gordeuk,1, 3 M. Loyevsky,1, 4 V. Apprey,5 D. Darbari,1, 6 J.A. Kark,1, 3 S. Rana,1, 6
and O. Castro.1, 3Center for Sickle Cells Disease,1 Department of Biophysics and Physiology,2
Department of Medicine,3 Department of, Microbiology,4 Statistical Genetics and Bioinformatics Unit,
National Human Genome Center5 and Department of Pediatrics,6 Howard University College of Medicine
and Howard University Hospital, Washington, DC 20060.
Cellular labile iron is a metabolically active pool of low molecular weight cytosolic iron complexes
that are accessible to strong membrane-permeable chelators (1). We have previously demonstrated the
labile iron levels in intact human erythrocytes can be estimated using the iron-sensing fluorescent probe,
calcein (2, 3).
Sickle erythrocytes have increased ferritin and increased molecular iron on the inner membrane
leaflet, and we postulated that cytosolic labile iron is also elevated. We used the fluorescent
metallosensor, calcein, and a permeant Fe2+ chelator to estimate labile cytosolic Fe2+, and calcein plus an
Fe3+ chelator to estimate total cytosolic labile iron (Fe2+ + Fe3+). We measured membrane non-heme iron
by its reactivity with ferrozine.
As estimated by calcein and Fe2+ chelator, the mean ± SD labile Fe2+ concentration was
significantly lower in hemoglobin SS (n=29) than hemoglobin AA (n=17) erythrocytes (0.56±0.35 µM
versus 1.25±0.65µM; P<0.001). In contrast, as estimated by calcein and Fe3+ chelator, total erythrocyte
labile iron was similar in hemoglobin SS (n=12) and hemoglobin AA (n=10) participants (1.75±0.41µM
versus 2.14±0.93µM; P=0.2). Mean membrane non-heme iron levels were higher in hemoglobin SS cells
than hemoglobin AA cells (0.0016x10-4 versus 0.0004x10-4 fmoles/cell; p=0.01), but much lower than the
mean amounts of total labile iron (1.6-1.8x10-4 fmoles/cell) or hemoglobin iron (18,000-19,000x10-4
fmoles/cell). Both membrane iron and total labile iron were much less than the mean amount of iron
potentially present in erythrocyte ferritin as calculated from results of other investigators (15x10-4 versus
34x10-4 fmoles/cell in HbAA versus HbSS erythrocytes).
We conclude that cytosolic labile iron is not elevated in hemoglobin SS erythrocytes and is
distinct from ferritin iron and elemental membrane iron which is present in only trace amounts.
Reference:
1.
Espósito BP, Epsztejn S, Breuer W and Cabantchik ZI. A review of fluorescence methods for
assessing labile iron in cells and biological fluids. Analytical Biochemistry, 304: 1- 18, 2002.
2.
Loyevsky M, John C, Dickens B, Hu V, Miller JH, Gordeuk VR. Chelation of iron within the
erythrocytic Plasmodium falciparum parasite by iron chelators. Molecular Biochemistry and
Parasitology 101: 43-59, 1999.
3.
Darbaris, D, Loyevsky, M, Gordeuk, VR, Kark, JA, Castro, O, Rana, S, Apprey, V, and
Kurantsin-Mills, J. Fluorescent measurements of the labile iron pool of sickle erythrocytes. Blood,
2003 (in press).
POSTER 240
CHARACTERIZATION OF HUMAN NFU, AN IRON-SULFUR CLUSTER SCAFFOLD PROTEIN
W.-H. Tong, G. N. L. Jameson, B. H. Huynh T. A. Rouault, National Institute of Child Health and Human
Development, Cell Biology and Metabolism Branch, Bethesda, MD 20892 and Department of Physics,
Emory University Atlanta, GA 30322
Iron-sulfur (Fe-S) clusters serve as cofactors in many proteins that have important redox, catalytic and
regulatory functions. In bacteria, biogenesis of Fe-S clusters is mediated by multiple gene products
encoded in the isc and nif operons. In particular, genetic and biochemical studies suggested that scaffold
proteins IscU, Nfu and IscA function as a complementary system for assembly and delivery of
rudimentary Fe-S clusters to apoproteins. Here we report the characterization of human Nfu. Analyses of
genomic DNA, transcripts, and translation products indicate that alternative splicing of a common premRNA results in synthesis of two Nfu isoforms with distinct subcellular localizations. Isoform I is localized
to the mitochondria, whereas isoform II is present in the cytosol and the nucleus. These results are
consistent with previous reports of human IscS and IscU in mitochondria, cytosol and nucleus, and
suggest that the Fe-S cluster assembly mechineries are compartmentalized in higher eukaryotes. We
show that human Nfu contains a labile [4Fe-4S] cluster. The nature and properties of the cluster were
assessed by biochemical analysis, absorption and Mössbauer spectroscopies. The presence of a labile
Fe-S cluster in Nfu supports the proposal that Nfu is an alternative scaffold protein for assembly of
clusters that are subsequently used for maturation of Fe-S proteins.
POSTER 241
FERRIC NITRILOTRIACETATE-INDUCED RENAL TUMOR PROMOTION AND OXIDATIVE DAMAGE:
ATTENUATION BY PROBUCOL
S. Okada, M. Iqbal, Y. Okazaki, Okayama University Graduate School of Medicine and Dentistry
In this study we have investigated the protective effect of probucol, a cholesterol-lowering drug,
with pronounced antioxidant properties on ferric nitrilotriacetate (Fe-NTA)-induced renal tumor promotion,
oxidative damage and histopathological changes in ddY mice. Ferric nitrilotriacetate (Fe-NTA) is a known
complete renal carcinogen as well as renal and hepatic tumor promoter. Fe-NTA treatment enhances
oxidative stress in the renal tissues, and renal ornithine decarboxylase activity (ODC) increases several
folds as compared to saline treated control in 12 hours. We then assessed the effects of probucol fed diet
pretreatment on Fe-NTA induced renal ODC activity, since many antioxidants inhibits proliferative
response and ornithine decarboxylase (ODC) is a well-known biochemical marker of proliferative
response.
Four weeks of probucol fed diet pretreatment resulted a dose dependent inhibition of Fe-NTA
induced renal ODC activity. In oxidative damage studies, Fe-NTA treatment showed a significant increase
in lipid peroxidation (measured as TBARS and 4-hydroxy-2-nonenal modified proteins), and 8-hydroxy-2’deoxyguanosine (8-OHdG) which are accompanied by decrease in GSH level, activities of GSH
metabolizing enzymes and antioxidant enzymes concomitant with histopathological changes in kidney.
These decreases in level of GSH, activities of GSH metabolizing enzymes and antioxidant enzymes were
significantly reversed by probucol fed diet pretreatment in a dose dependent manner. Probucol fed diet
pretreatment also resulted in a dose dependent inhibition of Fe-NTA induced lipid peroxidation (measured
as TBARS and 4-hydroxy-2-nonenal modified proteins), 8-OHdG in kidney and protected the tissue
against the observed histological alterations.
These results indicate that low-molecular-weight iron- induced tumor promotion and oxidative
damage is protected by food that provides antioxidant properties. This study also suggests that probucol
may be useful as a chemopreventive agent, in addition to being a cholesterol lowering and antiatherogenic drug with low toxicity.
POSTER 242
A ROLE FOR THE ISO-IRE FLANKING REGION IN IRE STRUCTURE AND FUNCTION
J. C. Long and E. C. Theil, CHORI (Children’s Hospital Oakland Research Institute), Oakland, California
Ferritin, the iron-storage protein, is critical for ensuring that intracellular iron levels are sufficient, while
maintaining iron at a safe level to prevent production of reactive oxygen species. Ferritin is a large 24
subunit protein, with a tissue-specific composition of H and L subunits. The importance of appropriate
ferritin concentration is reflected by the numerous signals regulating ferritin H and L subunit synthesis and
final H/L ratio. Translation of ferritin H and L subunit mRNA is modulated by binding of Iron Regulatory
Protein (IRP) to a single stem-loop structure, the Iron Regulatory Element (IRE) in the 5’ untranslated
region (UTR). Binding of IRP inhibits cap-dependent ribosome binding and translation initiation. The
ferritin IRE is a 3D structure comprised of a terminal loop and a stem of 10 base pairs interrupted by a
loop/bulge. In addition, the ferritin IRE is flanked by a base-paired region (FL) of variable length which
positions the IRE near the cap. In the H ferritin subunit FL, a conserved triplet of base pairs (GAG-CUC)
has been detected which modulates IRE/IRP function. Mutations within this conserved triplet altered
(1)
ferritin translation . IRP1 binding also altered structure at this site based on RNA probing with radical
nucleases or protein nucleases (2). Further evidence for the importance of IRE flanking regions on IRE
structure/function is the observation that the stem structure of a single Transferrin Receptor (TfR) IRE
was different compared to the same IRE sequence within native flanking sequences, and this structural
change coincided with enhanced IRP2 binding (3). The context-dependent structure change in the Tfr-IRE
was detected with the radical nuclease Cu-1,10-phenanthroline (Cu-phen), a probe for 3D structures
which had been particularly useful for analyzing the IRE stem structures associated with selective IRE
interactions between iso-IREs and IRP2 both in vitro and in HeLa cells (4,5). To assess the role of IRP2 in
combinatorial regulation of iso-IRE regulation, we investigated the impact of IRP2 binding on the
conserved triplet of base pairs in H ferritin subunit FL region compared to IRP1. Full-length frog H ferritin
in vitro transcripts were prepared. In vitro transcripts were incubated with either recombinant IRP1 or
IRP2. RNA/protein complexes were probed with Cu-phen in solution. Cleavage sites were identified by
primer extension of cleavage products followed by electrophoresis on a calibrated gel. We found that
IRP2 did not differ from IRP1 in footprinting at the IRE. However, the increased reactivity at the
conserved triplet previously observed upon IRP1 binding was absent for IRP2 binding. The differential
effect of IRPs at the H ferritin subunit FL region provides further evidence for a role of the FL in
combinatorial iso-IREs structure/function. Interestingly, a corresponding triplet of base pairs in the Lsubunit IRE-FL is also conserved, but is GGG-CCC. Do mRNA-specific FL sequences lead to differential
effects on translational efficiency? If so, selective translation of L ferritin during iron overload may
explain the change in the H/L ratio of liver ferritin protein associated with transfusional iron overload in
disease, where the H/L ferritin mRNA ratio was constant (see poster by Hagar, Jenkins, Vichinsky and
Theil). Further experiments will be carried out to compare structure/function of H and L ferritin IRE and
flanking regions to directly address this issue.
REFERENCES:
1- Dix, D.J., Lin, P.N., McKenzie, A.R., Walden, W.E., and Theil, E.C. (1993) J. Mol. Biol. 231:230.
2- Harrell, C.M., McKenzie, A.R., Patino, M.M., Walden, W.E., and Theil, E.C. (1991). Proc. Nat’l.
Acad. Sci. USA 88:416.
3- Erlitzki, R., Long, J. C., and Theil, E. C. (2002). J. Biol. Chem. 277:4257.
4- Ke, Y., Sierzputowska-Gracz, H., Gdaniec, Z. and Theil, E. C. (2000). Biochemistry 39: 6235.
5- Ke, Y., and Theil, E. C. (2002) J. Biol. Chem. 277: 2373
POSTER 243
EXPLORING THE ROLE OF THE C-SITE IN E. COLI BACTERIAL FERRITIN (EcFtnA)*
F. Bou-Abdallah‡, M. R. Woodhall¶, S. C. Andrews¶, and N. D. Chasteen‡
‡
Department of Chemistry, University of New Hampshire, Durham, NH 03824, USA, and ¶Division of
Microbiology, School of Animal & Microbial Sciences, University of Reading, Whiteknights, PO Box 228,
Reading RG6 6AJ, UK.
At least two ferritins are found in Escherichia coli, the heme containing bacterioferritin (EcBFR) and the
non heme ferritin (EcFtnA). EcFtnA is similar to human H-chain ferritin (HuHF) in that each of the 24
subunits of the two proteins contains a dinuclear ferroxidase consisting of A & B binding sites where the
rapid oxidation of 2 Fe(II) by O2 occurs. However, EcFtnA has a third iron binding site (C-site), in close
proximity of the ferroxidase cen

Scientific Program - International BioIron Society

Transcription

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