Σήμα μετάδοσης γλυκοκορτικοειδών: κλινικές προεκτάσεις

Σήμα μετάδοσης γλυκοκορτικοειδών: κλινικές προεκτάσεις

Clinical and Molecular Aspects of
Glucocorticoid Resistance and
Hypersensitivity:
Expanding Clinical Implications to
Complex Human Disorders
The Impact of New Biology
George P. Chrousos, MD,
University of Athens
The Nobel Prize in Physiology or Medicine 1950
Edward C. Kendall, Tadeus Reichstein, Philip S. Hench
Philip Showalter Hench ( 1896 – 1965)
Glucocorticoid Signaling
•
New Biology
•
Molecular Actions of Glucocorticoids
•
Pervasiveness of Glucocorticoid Actions
•
Expanding Clinical Implications
Glucocorticoid Signaling
•
New Biology
•
Molecular Actions of Glucocorticoids
•
Pervasiveness of Glucocorticoid Actions
•
Expanding Clinical Implications
NEW BIOLOGY- POSTGENOMIC ERA
Human genome:
About 20 thousand protein-coding genes
100-140 thousand transcripts
(mRNA, ncRNA)
200-260 thousand proteins
Single nucleotide polymorphisms (snps or snvs),
microsatellites or copy number variants (cnvs):
About 3 million snp’s
About 20 million microsatellites
>700 cnv’s (100kb -many million bases)
Over 10 k disease-related mutations
Human epigenome:
Imprinted genes, miRNAs, GpC islands
September 2012
Complex Systems
•
•
•
•
•
Multiple interactants
Self-organizing
Adapting through feedback loops
Resilient to perturbations
Emergent properties
NEW BIOLOGY- COMPLEX SYSTEMS
Units: Genes, molecules, cells
Modules: Functionally inter-linked
collections of units such as genes,
molecules, neurons, or other cells
Networks: Functionally inter-linked
collections of modules
Systems: Functionally inter-linked
collections of networks
Programs: Functional temporal
connections between systems
Complex Systems
• Systems nested within systems
• Networks and programs nested
within systems
Glucocorticoid Signaling
•
New Biology
•
Molecular Actions of Glucocorticoids
•
Pervasiveness of Glucocorticoid Actions
•
Expanding Clinical Implications
STRESS SYSTEM
Amygdala CRH
HIPPOCAMPUS
LC/NE
CRH/AVP
ACTH
F
NE/E
iCRH
GH/IGF-1
LH/T
TSH/T3

Immune Response
Inflammation
IL-6
Glucocorticoid Receptor
Signaling
Ligand
Cell Membrane
Ligand Binding
GR
Import
HSPs
Nucleus
Transactivation
/Transrepression
Assembly
TF
Export
Membrane-nongenotropic
GREs
mRNA1
TFE
mRNA2
Chromatin Modulators
SWI/SNF
Complex
Co-activator Complex
p300/CBP
p160
DRIP/TRAP
Complex
p/CAF
Transcription Initiation
Complex
GR
RNA
polymerase II
GREs
Ubiquitination/
Proteasomal
Degradation
Translated RNA
Running on DNA
Human Glucocorticoid Receptor
TGA, a
ATG
Exon
1
2
3
4
5
6
7
8
cDNA GRa
1
2
3 4 5 678
cDNA GRß
1
2
34 5 6 7 8
GRa
Immunogenic Domain
TGA, ß
9ß
9a
9a
9ß
LBD
DBD
1
777
A/B
C
D
E
Functional Domains of Human
Glucocorticoid Receptor a
1
420
Immunogenic Domain
480
777
DBD
LBD
568
HSP Binding
456
Dimerization
653
777
Nuclear Translocation
NL1
479 506
NL2
526
777
Transactivation
AF1
AF2
77
262
526
556
TGA, a
ATG
Human GR Gene
1
2
3
4
5
6
7
TGA, 
8
9ß
9a
Alternative Use of Exon 9a or 
cDNA GRa
1
2
3 4
77
6 7 8
262
420
cDNA GR
9a
DBD HR
N-terminal domain
1
5
LBD
480
777
GRa-A
1
2
3 4
77
6 7 8
9
DBD HR
N-terminal domain
1
5
262
420
480
LBD
742
GR-A
AF-1
113 141 203 211 226
Serine phosphorylation sites
Alternative Use of Translation Initiation Sites
27
27
GRa-B
GR-B
86
86
GRa-C1
GR-C1
90
90
GRa-C2
GR-C2
98
98
GRa-C3
GR-C3
316
316
GRa-D1
GR-D1
331
331
GRa-D2
GR-D2
336
336
GRa-D3
GR-D3
456
Dimerization
Nuclear localization
NL1
NL2
G-protein -interacting domain
(GR N-terminal Isoforms)
479 506
526
485
14-3-3-binding domain
Transactivation 77
AF1
AF2
777
777
777
262
526 556
262
419
753 768
Chrousos & Kino Science STKE 2005
Plasma Membrane
Glucocorticoid
Export Pump
Membrane GR
Ligand Availability
Cortisone:Inactive
GRa N-terminal Isoforms
11-HSD1/2
GR N-terminal Isoforms
GRa
Cortisol: Active
Import
HSPs
Export
Dimerization
p38 MAPK, JNK,
14-3-3, HSPs
GRa N-terminal
Isoforms
Heterodimerization/
Competition
Proteasomal
Degradation
Proteasomal
Degradation
Transcription Factors
GR N-terminal
Isoforms
Coactivators/Corepressors
Histone Deacetylases
DRIP/TRAP, SWI/SNF Complexes
Transactivation
/Transrepression
GREs
Nucleus
Chrousos & Kino Science STKE 2005
«Μηδέν άγαν»
‘Everything in moderation’
Inscription at the Oracle of Delphi
Homeostatic Effect
Cacostasis
Eustasis
Cacostasis
Allostasis
Deficiency
Allostasis
Optimum
Excess
Homeostatic System Activity
Chrousos Nature Rev 2009
The HPA Axis at Different Generalized
Glucocorticoid Sensitivities
Glucocorticoid
Hypersensitivity
Glucocorticoid
Resistance
Normal
(--)
(---)
(-)
Hypothalamus
CRH/AVP
Pituitary
Gland
(---)
CRH/AVP
CRH/AVP
ACTH
ACTH
(-)
ACTH
Adrenal
Glands
Androgens
Androgens
DOC, B
DOC, B
Androgens
DOC, B
cortisol
cortisol
Cortisol
Glucocorticoid Resistance: Clinical
Manifestations
Apparently normal glucocorticoid function in most cases
Asymptomatic
Chronic fatigue (glucocorticoid deficiency?), hypoglycemia
Mineralocorticoid excess
Hypertension
Hypokalemic alkalosis
Androgen excess
Children: Ambiguous genitalia at birth, clitoromegaly, premature adrenarche,
gonadotropin-independent precocious puberty
Females: Acne, hirsutism, male-pattern hair loss, menstrual irregularities,
oligo-anovulation, hypofertility
Males: Acne, hirsutism, oligospermia, adrenal rests in the testes, hypofertility
Increased HPA axis activity (CRH/AVP and ACTH hypersecretion)
Anxiety
Adrenal rests (oligospermia)
Pituitary corticotropinoma
Glucocorticoid Resistance:
Diagnostic Evaluation
Absence of clinical features of Cushing syndrome
Normal or elevated ACTH
Elevated cortisol concentrations
Increased 24-hour urinary free cortisol excretion
Normal circadian and stress-induced pattern of
cortisol and ACTH
Resistance of the HPA axis to dexamethasone
Thymidine incorporation : Increased resistance to
dexamethasone-induced suppression
Dexamethasone-binding assays: Decreased concentration or
affinity of the GR
Molecular studies: Mutations/deletions of the GR Gene
receptor; functional studies of mutant receptors
Mutations that Cause Familial/Sporadic
Glucocorticoid Resistance Syndrome
1268T>C 1430G>A 1676T>A
R477H
I559N
V423A
1
2
3
1712T>C
V571A
4
5
delA
Splice
6
1777A>T
M593L
7
8
Immunogenic DBD
Domain
GRa
1430G>A
R477H
2035G>A 2185G>A
G679S
V729I
1676T>A
I559N
2209T>C
F737L
9a
2241T>G
I747M
2318T>C
L773P
9
TGA, ß
TGA, a
ATG
1268T>C
V423A
1922A>T
D641V
LBD
1712T>C
V571A
Immunogenic Domain
1777A>T
M593L
1922A>T
D641V
DBD
2035G>A
G679S
2185G>A
V729I
LBD
2209T>C
F737L
2241T>G
I747M
2318T>C
L773P
Characterization of GR Mutants
• GRE-binding Defective:
GRaR477H
• Ligand-binding Defective:
GRaV571A, D641V, G679I, V729I
• AF-1 Defective:
GRa(77-262)
• AF-2 Defective:
GRaI747M, L773P
• Entire LBD Deletion:
GR514stop
Luciferase Activity (RLU)
4
2
0
0
0
-6
Dex (log M)
V729I
6
4
2
0
0
-6
Dex (log M)
0
-6
Dex (log M)
8
8
I747M
6
4
2
0
0
-6
Dex (log M)
6
6
4
4
2
2
0
L773P
6
4
2
0
0
-6
Dex (log M)
Luciferase Activity (RLU)
R477H
V571A
0
-6
Dex (log M)
0
-6
Dex (log M)
0
-6
Dex (log M)
0
-6
Dex (log M)
8
8
8
8
GR
6
4
2
0
0
-6
Dex (log M)
Luciferase Activity (RLU)
0
D641V
6
4
2
77-262
6
4
2
0
0
-6
Dex (log M)
Luciferase Activity (RLU)
8
Luciferase Activity (RLU)
2
8
Luciferase Activity (RLU)
6
Luciferase Activity (RLU)
8
Luciferase Activity (RLU)
4
GRa
Luciferase Activity (RLU)
6
Luciferase Activity (RLU)
Control
Luciferase Activity (RLU)
Luciferase Activity (RLU)
8
8
8
0
G679S
6
4
2
0
514stop
6
4
2
0
0
-6
Dex (log M)
Familial/Sporadic Glucocorticoid
Resistance Syndrome:
Fluorescence recovery after photobleaching
(FRAP) analyses reveal deficient interaction
of the activated GR with chromatin
Kino T
SPE, RBMB, NICHD
Glucocorticoid Hypersensitivity: Clinical
Manifestations/ Diagnosis
Increased glucocorticoid function:
Cushing syndrome manifestations
Obesity
Metabolic syndrome
etc.
Decreased or normal HPA axis activity
1A
Glucocorticoid Hypersensitivity
ER22/23EK
TthIIII
N363S D401H
T504S
S651F
2314insA
BclI
1
2
3
4
5
6
7
9a
TGA, a
ATG
NTD
8
DBD
9
TGA, 
LBD
Charmandari, Chrousos .. JCEM 2009
A
B
CV-1
MMTV
0.5
Relative Luciferase Activity (RLU)
2
hGRaWT
hGRaD401H
0.4
0.3
0.2
0.1
0
0
-12 -11 -10
-9
-8
-7
-6
Dexamethasone Concentration (Log M)
-5
hGRaWT
1
1
1
1
1
hGRaD401H 0
1
3
6
10
(Ratio)
Charmandari, Chrousos .. JCEM 2009
Clinical Manifestations in Tissue-specific Hypersensitivity or
Resistance to Glucocorticoids
Glucocorticoid Excess
Glucocorticoid Deficiency
AFFECTED
AREA
Central
Nervous
System
GLUCOCORTICOID
HYPERSENSITIVITY
GLUCOCORTICOID
RESISTANCE
Liver
Increased gluconeogenesis*
and liposynthesis, insulin resistance Hypoglycemia, increased
insulin sensitivity
Fat
Accumulation of visceral*
fat
Muscles
Insulin resistance*
Blood Vessels
Hypertension*
Bone
Stunted growth,
osteoporosis
Inflammation/
Immunity
Immune suppression,
suppressed inflammation
Insomnia, anxiety,
depression, defective cognition
*dysmetabolic syndrome
Fatigue, somnolence,
Malaise, defective cognition
Loss of weight
Increased insulin sensitivity
Hypotension
Increased inflammation/
autoimmunity
Differing HPA Axis and
Other Tissue Glucocorticoid Sensitivities
Glucocorticoid Effects in
CNS/Liver/Fat/Blood Vessels
Glucocorticoid Sensitivity
CNS/Liver/Fat/Blood Vessels
HPA Axis
High
Free Cortisol
High
Normal
Low
Normal
Low
Low
Normal
Normal
High
High
Normal
Normal
Low
Low
High
Low
High
Very Low
Very High
Normal
High
Low
Normal
Primary Pathologic Changes
Influence on Various
GR Functions
Genetic/Epi Background
Exogenous Factors etc.
Effector Molecules
Ligand-binding Activity
Nuclear Translocation
Transactivation
Alteration of Glucocorticoid Receptor
(GR) Activity
Secondary Pathologic Changes
due to Alteration of Glucocorticoid Action
in Specific Tissues
Politically Correct 1980’s
• Hypothesis-driven Research
ANATHEMA
• “Shotgun Research”
• “Fishing expedition”
Politically Correct 2000’s
• Discovery-driven Research
NO LONGER ANATHEMA
• “Shotgun Research”
• “Fishing Expedition”
Finding Molecules
that Potentially Alter GR Action
Yeast Two-hybrid Screening
Using GR Fragments as Baits
Molecules Identified
GRa
1
777
NTD
DBD HR
LBD
NL-1
Nuclear translocation
Transactivation
AF-1
272
Smad6
G protein 
GRIP1
1
NL-2
AF-2
419
480
COUP-TFII
CDK5/p35
Brx
Vpr
777
594
1462
774
NRB
AD-1
NRs
CBP
p300
694 743
FLASH
AD-2
Yeast Two-hybrid Screening
Using GR LBD as Bait
Human GR
Immunogenic Domain
DBD
LBD
AF1
1
77
262
420
480
777
Bait Fragment
LexA System/Human Jurkat Cell cDNA Library
CLOCK transcription factor
Circadian Rhythm Transcription Factor CLOCK/BMAL1
Regulates the Transcriptional Activity
of the Glucocorticoid Receptor
through Acetylation
Nancy Nader1 George P. Chrousos2 and Tomoshige Kino1
1: Program in Reproductive and Adult Endocrinology, Eunice Kennedy
Shriver National Institute of Child Health and Human Development,
National Institutes of Health, Bethesda, MD 20892, USA
2: First Department of Pediatrics, Athens University Medical School,
Athens 11527, Greece
FASEB J 2009
Circadian Fluctuation of Plasma Cortisol
24 hr Plasma Cortisol Sampling
HPA Axis
Paraventricular
Nucleus (PVN)
(-)
CRH/AVP
Pituitary Gland
(-)
ACTH
Adrenal Gland
Cortisol
Plasma Cortisol
Surachiasmatic
Nucleus (SCN)
How does glucocorticoid action fluctuate
in target tissues?
8 am
8 pm
Dark
8 am
• 3-10 % of the mammalian
transcriptome oscillates with a 24 h
rhythm.
• CLOCK System-controlled genes and
downstream output genes are involved.
CLOCK/BMAL1:
Circadian Rhythm Transcription Factors
• Master regulators of the circadian rhythms both in
the central nervous system and peripheral
tissues/organs.
• Basic helix-loop-helix (bHLH)-PER-ARNT-SIM
(PAS) superfamily of transcription factors.
• CLOCK is a histone acetyltransferase (HAT) with
homology to the p160s.
CLOCK/BMAL1 Represses GR-induced
Transcriptional Activity through Acetylation
In the Absence of
Acetylation by CLOCK
In the Presence of
Acetylation by CLOCK
BMAL1
GR
CLOCK
Acetylation
A
A
GREs
NTD
GRE Binding
A
GREs
GR-induced
Transcriptional Activity
GR-induced
Transcriptional Activity
1
A
DBD HR
LBD
420 480 520
777
GR
K480 K492 K494 K495
Acetylation Sites
Interaction with CLOCK
Influence of Circadian Clock
on HPA-Axis
Circadian Center
Other Inputs
Stress
SCN
-
PVN
AVP
ACTH
A circadian rhythm indirectly
regulates function of all
organs/tissues through
modulation of glucocorticoid
actions.
-
F/B
TARGET
TISSUES
Target Tissue
Circulating F/B
Effect
CRH
Circadian fluctuation
Time
Peripheral CLOCK Regulates Targettissue Glucocorticoid Receptor
Transcriptional Activity in a Circadian
Fashion in Man
Charmandari E, Chrousos GP, Lambrou
GI, Pavlaki A, Koide H, Ng SSM and Kino
PLoS ONE 6(9): e25612, 2011
OVERNIGHT
DEXAMETHASONE TEST
24 H SAMPLING
PLASMA CORTISOL
24 H CORTISOL
A.
-D
-D
CS
NS
+D
+D
8 am
DARK
8 pm
8 am
NS
CS
TARGET TISSUE RESPONSE
B.
TARGET TISSUE SENSITIVITY
HS
N
R
THRESHOLD
FOR HARMFULNESS
Chrousos, IJO 1998
CORTISOL CONCENTRATION
Nader, Chrousos, Kino TEM 2010
Glucocorticoids reset the “slave”
CLOCKs but not the “master” CLOCK,
and phase-shift their circadian rhythm by
modulating expression of several clockrelated genes..
Perturbation of either the CLOCK
SYSTEM or the HPA axis leads to similar
metabolic and immune pathologies
related to hypercortisolism:
Obesity and Metabolic Syndrome
Immune Dysfunction, as if due to a
hyperactive stress system
Examples of pathology due to aberrant
coupling of the CLOCK and HPA axis:
 Chronic stress, evening F elevations
 Endogenous/exogenous Cushing s.
 Trans-time-zone travel
 Nightshift work
All above conditions are associated with
a high risk for CVD
Yeast Two-hybrid Screening
Using GR 263-419 as Bait
Human GR
Immunogenic Domain
DBD
LBD
AF1
1
77
262
420
480
777
Bait Fragment
LexA System/Human Jurkat Cell cDNA Library
 Subunit of Trimeric Guanine
Nucleotide-binding Proteins (G)
Trimeric G Protein Complex
(from Clapham DE & Neer EJ Ann Rev. Pharmacol. Toxicol. 1997 37: 167-203)
G as a Negative Regulator of GR
Transactivation
Effect of G2 Overexpression
on GR Transactivation
HCT116
1.5
MMTV
G2 (-)
G2 (+)
Effect of G1/2 siRNAs on
Endogenous Glucocorticoid-responsive
Tyrosine Aminotransferase (TAT) Activity
HTC
Control siRNA
TAT Activity
(x10-3 O.D.331/g total RNA)
Lucifrease Activity (RLU)
G1/2 siRNAs
1.2
0.9
0.6
0.3
20
15
10
5
0
Dex
0
0 -12 -10 -8 -6 -4
Dexamethasone
Concentration (logM)
*
n.s.
-
+
Kino ..Chrousos J Cell Biol 2005
PI
Blot
-
+
anti-GRa Ab
Control IgG
G and G, but not Ga,
are Co-precipitated with GR
-
+ Dex
anti-G Ab
Precipitated G
anti-G Ab
Precipitated G
anti-Gai Ab
Precipitated Gai
Kino ..Chrousos J Cell Biol 2005
G Co-migrates with GR
from the Cytoplasm into the
Nucleus
EGFP-G2 DsRed2-GRa
Merge
Dex (-)
Dex (+)
Kino ..Chrousos J Cell Biol 2005
Cyto-trap Yeast Two-hybrid Screening using the
human GR LBD as bait
Human GR
N-terminal Domain
1
DBD
420
LBD
480
777
Bait Fragment
Cyto-trap System/Human Fetal Brain cDNA Library
Cyclin-dependent Kinase 5 (CDK５)
Activator p35
CDK5/p35 (p25)
1.
Member of the Cyclin-dependent serine/threonine kinase family.
2.
Activated by forming a heterodimer with partner p35.
3.
Functions specifically in the CNS and is essential for the
development of the fetal brain (formation of layer structure of the
cerebral neocortex) and for dendritogenesis (sprouting) and
synaptogenesis at any age.
5.
Aberrant activation of CDK5 by proteolytically produced p25 is
associated with development of neurodegenerative disorders,
such as Alzheimer’s disease and amyotrophic lateral sclerosis.
CDK5/p35 (p25)
CDK5
1
292
38
183
p35/p25-binding Site
Myristoylation
p35
1
307
Membrane-bound Form
138
p25
91
291
307
138
291
Proteolytic Digestion
CDK5-binding Site
Cytoplasmic Form
Associated with Development
of Neurodegenerative Disorders
CDK5/p35 Phosphorylates Multiple
Serines of the GR
Human GR
S45
1
NTD
S203
S211
262
77
AF-1
DBD
S226
S395
420
490
490
490
Effector Domain
LBD
777
AF-2
777
777
p35/p25-interaction
Domain
LBD: Binding Domain, NTD: Effector Domain
CDK5 Inhibitor Roscovitine Facilitates Cofactor
Accumulation on the Glucocorticoid-responsive
SGK Promoter in ChIP Analyses
Rat Neuronal Cells
SGK/GREs
Fold Induction (x)
0
2
4
6
p300
+
SNF2
-
Dex (-)
Dex (+)
*
Dex (-)
Dex (+)
+
*
+
GR
Dex (-)
Dex (+)
Fold Induction (x)
0
2
4
6
8
Fold Induction (x)
0
1
2
3
4
n.s.
Fold Induction (x)
0
1
2
3
4
Control
-
+
Dex (-)
Dex (+)
n.s.
CDK5 Suppresses Cofactor Accumulation on the GR
through Phosphorylation Kino ..Chrousos Mol Endo
2007
CDK5 Modulates GR Transcriptional Activity in a
Gene-specific Fashion in Microarray Analyses
Rat Neuronal Cells
Dex-responsive Genes
Further Down-regulated by Roscovitine
++
- +
Up-Dex/Down-Rosco
Dex-responsive Genes
Up-regulated by Roscovitine
++
- +
Down-Dex/Up-Rosco
Up-Dex/Up-Rosco
Roscovitine
-responsive
Genes:1958
1301
Dex-responsive
Genes:3626
657
2969
Regulation of GR Transcriptional
Activity by CDK5
In the Absence of GR Phosphorylation by CDK5
Ligand
SWI/SNF
Complex
Other Cofactors ?
HAT Complex
GREs
Transcriptional
Activation
Regulation of GR Transcriptional
Activity by CDK5
In the Presence of GR Phosphorylation by CDK5
Ligand
p35/p25
CDK5
Inhibition of
Cofactor Attraction
P P
Facilitation of
Cofactor Attraction
Cofactor B?
Cofactor A?
P PP P
P P P P
GREs
GREs
Suppression of Transcription
Enhancement of Transcription
Summary
1.
CNS-specific CDK5 physically interacts with GR at its LBD
through the activator p35/p25.
2.
CDK5 phosphorylates GR at multiple serines (45, 203, 211,
226, 395) located in the N-terminal domain.
3.
CDK5/p35 modulates GR transcriptional activity by
changing attraction of cofactors to a promoter in a
promoter- and, probably, CNS region- specific fashion
through phosphorylation..
Yeast Two-hybrid Screening
Using GR 420-489 as Bait
Human GR
Immunogenic Domain
DBD
LBD
AF1
1
77
262
420
480
777
Bait Fragment
LexA System/Human Jurkat Cell cDNA Library
Growth Arrest-specific 5 (Gas5)
What is Non-coding (nc) RNA?
1.
RNAs that do not code proteins.
2.
tRNAs and miRNAs are examples of ncRNAs.
3.
ncRNAs have distinct biologic activities.
4. ~60% of the mouse and 80% of the human genome is
transcribed, producing ~200,000 independent
transcripts, of which ~half consist of ncRNA.
5.
Interestingly, about ~20% of coding and non-coding
genes express both sense and anti-sense RNAs,
forming double stranded RNAs through hybridization of
the sense and antisense strands.
Growth Arrest-specific 5 (Gas5)
• Non-protein-coding RNA (Human Gas5: 598 bases).
• Originally isolated as a gene specifically expressed in
growth-arrested cells (or in G0 phase of the cell cycle).
• Physiologic functions are not known as yet.
2-D Structure of 3’-Terminal Gas5
MRE/GRE(AGUGCA)
GRE (AGUGGU)
Gas5 RNA Competes with DNA GRE
for Binding to GR DBD in vitro
In vitro Binding Assay
Competitors
Gas5 RNA
DNA GRE
Gas5 WT
Gas5 GRE1 Mut
% Binding Inhibition (%)
120
100
DNA GRE WT
“Acceptor”
“Binder”
FAM-DNA GRE WT
FAM
GR DBD
GST
GST Beads
80
60
40
20
0
IC50
DNA GRE: 1.679 M
Gas5 WT: 0.106 M
0
Kino ..Chrousos Science Sig 2010
10-2 10-1
1
10
Concentration (M)
102
Interaction of GR DBD and Gas5
through its RNA “GRE”
GR DBD-DNA GRE
Interaction
GR DBD-RNA “GRE”
Interaction
Gas5 is a Growth-related Inhibitor of GR
Transcriptional Activity
Resting Cells
Replicating Cells
High Gas5 Level
Low Gas5 Level
:Gas5
GR: High Transcriptional Activity
GR: Low Transcriptional Activity
Gas5 Silences GR Transcriptional Activity
in Growth-arrested Cells
Speculation
Gas5 is a growth arrest-related, RNA
“GRE”-harboring co-repressor of the GR
in resting cells, restricting the expression
of glucocorticoid-responsive genes.
This might be an adaptive response to
starvation, saving energy resources..
Gas5 ncRNA Levels in PMCs Correlate with
BMI in Peripheral Mononuclear Cells
y=-0.05307x+5.005 (p=0.0103)
Ct Values (CtGas5-CtRPLP0)
5
4
3
2
0
0
10
20
BMI
30
40
2.5
30
1.5
r=0.533
p=0.036
10
1.0
1.5
1.0
GILZ
20
AMPKa2
AMPKa1
2.0
r=0.031
p>0.05
r=0.030
p=0.031
0.5
0.5
0
0
0
0
10
20
30
0
40
10
20
30
40
BMI
BMI
1.00
GILZ
GILZ
1.0
r=0.025
p=0.040
0.5
0.75
r=0.307
p>0.05
0.50
0.25
0
0
0
10
20
AMPKa1
30
40
0
0.5
1.0
1.5
AMPKa2
10
20
BMI
1.25
1.5
0
2.0
2.5
30
40
Glucocorticoid Signaling
•
New Biology
•
Molecular Actions of Glucocorticoids
•
Pervasiveness of Glucocorticoid Actions
•
Expanding Clinical Implications
Regulated genes
Metabolism
DNA/RNA/Protein synthesis
Cell development
ESTs
Miscellaneous
Clusters
Immune Response
1000
500
Number of Genes Down-regulated
500
1000
Number of Genes Up-regulated
Galon ..Chrousos… FASEB 2002
Subcategories
Subclusters
Complement system
Cytokines
Enzymatic mediators
Inflammation
Innate
Immunity
Chemokines
Innate
Recognition
Scavenger System
Integrins
Apoptosis
Humoral and Cellular
Antigen Presentation
100%
Adaptive
Recognition
Adaptive
Immunity
100%
Galon ..Chrousos… FASEB 2002
Glucocorticoid Signaling
•
New Biology
•
Molecular Actions of Glucocorticoids
•
Pervasiveness of Glucocorticoid Actions
•
Expanding Clinical Implications
Change of Tissue Sensitivity to
Glucocorticoids in Pathologic Conditions
Resistance
• Familial/Sporadic Glucocorticoid Resistance Syndrome
• Bronchial Asthma, Rheumatoid Arthritis, Systemic Lupus
Erythematosus, Ulcerative Colitis
• Acute Respiratory Distress Syndrome/Sepsis
• Depression
Hypersensitivity
• Generalized Glucocorticoid Hypersensitivity Syndrome
• Transient Sporadic Glucocorticoid Hypersensitivity Syndrome
• Visceral-type Obesity-related Dyslipidemia,
Insulin Resistance, Carbohydrate Intolerance and Hypertension
• AIDS-related Insulin Resistance and Lipodystrophy Syndrome
• Depression
Alteration of Tissue Glucocorticoid Sensitivity in
Pathologic States:
Glucocorticoid Receptor Polymorphisms
1
2
3
4
5
6
7
8
TGA, ß
TGA, a
ATG
Immunogenic
Domain
DBD
9ß
9a
GR Beta SNP
 RA
LBD
Exon 2
1
ER22/23 EK
“Glucocorticoid Resistant”
Better Body Composition
Healthier Metabolic Profile
Better Survival
Lower Risk of Dementia
Depression
N363S
Bcl1
“Glucocorticoid Hypersensitive”
More Body Fat, Less Lean Body
Mass
Insulin Resistance
Metabolic s. manifestations
Depression
Interactions of the Stress and
Immune Systems
Cell Membrane
Kinases
p65
p
p
p50
I-kB
I-kB
NF-kB
GRa
GRa
-
I-kB
+
+
Nucleus
TATA
+
p65
Glucocorticoid
Responsive Elements
IkB gene
p50
TATA
kB binding site
Inflammatory cytokine genes
ICAM gene
I-NOS gene
COX2 gene
PLA2 gene
mRNA
Prostaglandins
Nitric Oxide
Oxidants/Oxidized, denatured
substances
Bacterial
endotoxins
Arachidonic acid
Glucocorticoid
ICAM
TNFa, IL-1, IL-6
Inflammatory
Cytokines
Ischemia/Reperfusion
Intracellular proteins
Inflammation
MODS
SIRS
Gram- Infection
Basal
State
Gram+ Infection
CARS
MODS
SIRS = Systemic Inflammatory Response Syndrome
MODS = Multiple Organ Dysfunction Syndrome
CARS = Counter-regulatory Response Syndrome
100
p<
0.0001
150
p<
0.0001
200
p<
0.0001
300
250
p = 0.03
350
p = 0.03
Plasma TNF-a (pg/ml)
400
50
6
10
5
9
5
6
+3
3
7
9
4
Randomized
10
5
+3
p < 0.0001
+7
9
5
p < 0.0001
p < 0.0001
n=
+10
2
+5
p < 0.0001
200
180
160
140
120
100
80
60
40
20
0
2
+7
6
+5
+10
3
7
p < 0.0001
4
7
p = 0.0006
9
Randomized
3
+5
p = 0.0007
9 5
+3
p = 0.03
n=
Plasma IL-6 (pg/ml)
10 5
5000
4500
4000
3500
3000
2500
2000
1500
1000
500
0
Days
Days
4
p < 0.0001
Plasma IL-1 (pg/ml)
9
Randomized
p < 0.0001
n=
Days
p < 0.0001
0
2
+7
+10
Meduri ..Chrousos NIM 2005
p<
0.0001
9
4
10 5
9
Randomized
3000
6
+3
3
7
p<
0.0001
2
+5
+7
+10
p = 0.002
p < 0.05
3500
5
p<
0.0001
p<
0.0001
n=
Days
Nuclear p50 density
p = 0.02
p = 0.008
0
p < 0.05
Nuclear NF-kB
density
9000
8000
7000
6000
5000
4000
3000
2000
1000
2500
1000
p<
0.0001
1500
p<
0.0001
p<
0.0001
2000
500
0
3000
10
5
+3
9
5
6
3
6
3
+5
7
2
+7
+10
2500
1500
p<
0.0001
p<
0.0001
2000
1000
p<
0.0001
4
p < 0.001
p = 0.007
3500
Nuclear p65
(ug/ml)
9
Randomized
p < 0.01
n=
Days
500
0
n=
Days
9
4
Randomized
10
5
9
+3
5
+5
7
2
+7
+10
Meduri ..Chrousos NIM 2005
Vpr and p300
Vpr
TFIIB
1
p300/CBP
70
84
96
Two different surfaces
GR
p300/CBP
Transactivation Domain
Histone Acetyltransferase
2045 2191 2414
1
Nuclear
Receptors
Vpr
CREB
p/CAF
TFIIB
Tat
p160
E1A
Vpr is an Adaptor Facilitating
Interaction of GR with CBP/p300
LQQLL
Vpr
p300/CBP
RNA Polymerase II
TFIID
TATA
Transcription
Kino... Chrousos J Exp Med 1999, J Virol 2002
HPA Basics
Behavior
Gold
Susman
Nottelmann
Dorn
Development,
Aging
Gold
Susman
Nottelmann
Dorn
Johnson
Cizza
Blackman
Growth
HPA
Axis
HPA
Axis
Metabolism
LC/NE System
Reproductive
System
Mastorakos
Latronico
Charmandari
Zoumakis
Dorn
Magiakou
Charmandari
Tsigos
Peeke
Kino
Immune
System
Sleep
Vgontzas
Friedman
Zoumakis
Schulte
Gold
Oldfield
Doppman
Schuermeyer
Nieman
Cutler
Brandon
Kino
Charmandari
Zachman
Karalis
Sternberg
Wilder
Papanicolaou
Elenkov
Mastorakos
Kino
Neoplasms
Reincke
Latronico
Stratakis
Bornstein