Messenger Ribonucleic Acid Levels of Pregnancy-Associated Plasma Protein-A and

BIOLOGY OF REPRODUCTION 61, 1083–1089 (1999)
Messenger Ribonucleic Acid Levels of Pregnancy-Associated Plasma Protein-A and
the Proform of Eosinophil Major Basic Protein: Expression in Human Reproductive
and Nonreproductive Tissues 1
Michael T. Overgaard,4 Claus Oxvig,4 Michael Christiansen,5 James B. Lawrence,6 Cheryl A. Conover,6
Gerald J. Gleich,7 Lars Sottrup-Jensen,2,4 and Jesper Haaning3,4
Department of Molecular and Structural Biology,4 University of Aarhus, 8000 Aarhus C, Denmark
Department of Clinical Biochemistry,5 Statens Serum Institut, 2300 Copenhagen S, Denmark
Endocrine Research Unit,6 Mayo Clinic, Rochester, Minnesota 55905
Department of Immunology and Medicine,7 Mayo Clinic, Rochester, Minnesota 55905
ABSTRACT
PAPP-A/proMBP, the complex of pregnancy-associated plasma protein-A (PAPP-A) and the proform of eosinophil major basic protein (proMBP), circulates at increasing levels during pregnancy. The major site of synthesis is the placenta, in which PAPPA mRNA has been localized to the syncytiotrophoblast and the
placental X cells, whereas proMBP mRNA has been localized to
the placental X cells only. The function of PAPP-A/proMBP and
its components has remained speculative for years. Recently,
however, it has been shown that PAPP-A specifically cleaves insulin-like growth factor (IGF) binding protein-4 in an IGF-dependent manner. Female reproductive and nonreproductive tissues have previously been reported to contain PAPP-A immunoreactivity, based on studies using preparations of anti(PAPPA/proMBP), now known to recognize both PAPP-A and proMBP,
and other irrelevant antigens. To analyze for the presence of
PAPP-A and proMBP mRNA, a sensitive semiquantitative reverse
transcription (RT) polymerase chain reaction (PCR) method was
developed. Reverse-transcribed poly(A)1 RNA was used as a
template in a competitive PCR. PAPP-A and proMBP mRNA levels were normalized against the level of b-actin mRNA. Both
mRNA species were significantly more abundant in term placenta than in other tissues analyzed. All analyzed tissues, including endometrium, myometrium, colon, and kidney, contained both PAPP-A and proMBP mRNA.
INTRODUCTION
Pregnancy-associated plasma protein-A (PAPP-A) has
been described as a large placental glycoprotein, present in
the serum of pregnant women in increasing concentrations
throughout pregnancy [1]. PAPP-A in pregnancy serum is
disulfide-linked to the proform of eosinophil major basic
protein (proMBP), forming an approximately 500-kDa 2:2
complex, denoted PAPP-A/proMBP [2, 3]. The cDNA sequence of PAPP-A shows that the serum form is derived
from a preproprotein with a putative 22-residue signal peptide, a propart of 58 residues, and the 1547-residue circulating mature polypeptide [4, 5]. The sequence shows no
global similarity to any known protein, but it contains the
two sequence motifs common to the metzincins, a superAccepted May 25, 1999.
Received March 11, 1999.
1
This work was supported by the Danish Science Research Council,
by the Novo Nordic Foundation, and by National Institutes of Health grant
AI 09728.
2
Correspondence: Lars Sottrup-Jensen, Department of Molecular and
Structural Biology, University of Aarhus, Gustav Wieds Vej 10C, 8000
Aarhus C, Denmark. FAX: 45 8612 3178; e-mail: lsj@mbio.aau.dk
3
Current address: M&E Biotech A/S, Kogle Allé 6, 2970 Hørsholm,
Denmark.
family of metalloproteases [5], three Lin-12/Notch repeats
known from the Notch protein superfamily, and five short
consensus repeats known from components of the complement system [4].
ProMBP is composed of a strongly acidic 90-residue
propart [6] and a highly basic mature portion of 117 residues [7]. It is an extensively and very heterogeneously glycosylated proteoglycan, migrating in SDS-PAGE as a smear
corresponding to 50–90 kDa, not visible using Coomassie
Brilliant Blue staining [2, 8]. ProMBP has been isolated
from the blood of pregnant women in complex only with
either PAPP-A, angiotensinogen, or complement C3dg [2,
9]. However, proMBP has recently been studied in cultures
of interleukin-5-stimulated umbilical cord stem cells, and it
is processed in the maturing eosinophil granule to 14-kDa
MBP and localized to the granule core [10]. The mature
MBP is a cytotoxic protein, constituting more than 50% of
the protein content of the granules in eosinophil leukocytes
[11, 12]. It is released from the eosinophil leukocyte by
degranulation, and plays multiple roles in the effector functions of these cells [13].
PAPP-A not complexed with proMBP cannot be isolated
from pregnancy serum [3], but it has recently been detected
in conditioned media from human fibroblasts. Further, it
was established that PAPP-A cleaves insulin-like growth
factor (IGF) binding protein-4 in an IGF-dependent manner
[14]. The function of proMBP in pregnancy is unknown. It
has been reported that the PAPP-A/proMBP complex is absent from maternal serum in pregnancies in which the
mother is carrying a fetus with Cornelia de Lange syndrome [15].
Recently, PAPP-A and proMBP in conjunction with SP1
have been shown to be effective markers for detecting fetuses affected with Down syndrome in Weeks 7–12 of gestation [16–19]. In addition, proMBP has been suggested as
a serum and histologic marker for the malignant potential
in trophoblastic neoplasia [20, 21].
In term placenta, PAPP-A mRNA is synthesized by the
syncytiotrophoblast and by the trophoblast-derived septal X
cells, as determined by in situ hybridization [22]. In the
same study, PAPP-A was colocalized, using proMBP-adsorbed polyclonal anti(PAPP-A/proMBP), to the septal X
cells and the syncytial lining. Both proMBP and proMBP
mRNA have been localized to the septal X cells by immunofluorescence and in situ hybridization, respectively
[23, 24]. In nonpregnant individuals, synthesis of PAPP-A
has been reported in a number of tissues, e.g., the corpus
luteum [25], endometrium [26–28], prostate [29], testis
[30], liver, pancreas, myocardium, spleen, bone marrow
[31], trophoblastic tumors [32, 33], and breast carcinoma
1083
1084
OVERGAARD ET AL.
Extraction of mRNA and cDNA Synthesis
FIG. 1. Comparison of amplification products. The lengths of the PCR
products with genomic DNA (product 1), cDNA (product 2), or internal
standard (product 3) as template are indicated for b-actin (see Table 1 for
other product sizes). As there is a substantial difference in the product
sizes between PCR products 1 and 2, contamination with genomic DNA
in the mRNA preparation would have been easily detected. A and B represents the 59- and 39-competitive PCR primers, respectively. Boxes represent exons in genomic DNA or cDNA. Shaded boxes represent the part
of the cDNA that is deleted to generate the IS, either by excision of a
restriction fragment or by primer-mediated deletion (see Materials and
Methods section).
[34, 35]. These investigations were based on techniques using polyclonal antibodies, which are now known to recognize several other proteins, including eosinophil MBP,
recombinant proMBP, SP1, and haptoglobin [2, 36–39].
We measured the levels of PAPP-A and proMBP mRNA
in a number of reproductive and nonreproductive tissues
using a sensitive semiquantitative reverse transcription (RT)
polymerase chain reaction (PCR) assay. The method is
based on coamplification of the cDNA and a deletion variant thereof that is used as internal standard (IS). The
amounts of PAPP-A and proMBP mRNA are normalized
against the total amount of mRNA in the sample, determined as the amount of b-actin mRNA.
MATERIALS AND METHODS
Tissue Samples
Term placental tissue (outer maternal side) from cesarean
sections was provided by the Department of Gynecology
and Obstetrics, Aarhus University Hospital, Aarhus, Denmark. First-trimester trophoblast tissue was from the Danish
Cancer Society, Aarhus. Prostate tissue from hyperplasias
and adenocarcinomas was provided by the Department of
Experimental Clinical Oncology, Aarhus University Hospital. Mononuclear cells from bone marrow, prepared as
described [40], were obtained from the Department of Hematology, Aarhus County Hospital, Denmark. Normal
breast tissue, and samples from lobular and ductal breast
carcinomas were provided by the Department of Pathology,
Aarhus County Hospital. Samples from ascending colon
and kidney cortex were from Mayo Clinic, Rochester, MN.
Samples from ovary, endometrium, myometrium, and tuba
uterina, provided by the Department of Gynecology and
Obstetrics, Aarhus University Hospital, were from hysterectomies from normal postmenopausal women (age , 50
yr). A blood sample was drawn from a pregnant woman
(first trimester). All tissue samples were stored in liquid
nitrogen.
Frozen tissue samples were pulverized using a mortar
embedded in dry ice. Approximately 20 mg tissue powder
or 106 cells were then lysed in 1 ml lysis/binding buffer
(0.5 M LiCl, 10 mM EDTA, 5 mM dithiothreitol, 1% SDS,
100 mM Tris-HCl, pH 8.0) using a glass homogenizer
(Wheaton, Millville, NJ). Poly(A)1 RNA was isolated using
the Dynabeads mRNA DIRECT kit (Dynal A/S, Oslo, Norway), according to the manufacturer’s instructions.
Poly(A)1 RNA was eluted from the oligo(dT) Dynabeads
by incubation in 20 ml 2 mM EDTA for 2 min at 658C.
First-strand cDNA was synthesized immediately thereafter
by incubating 50% of the eluted poly(A)1-RNA for 60 min
at 428C with 4 units avian myeloblastosis virus reverse transcriptase, 10 pmol oligo(dT)24, 1 pmol 59-AAACCCATTTTATTGCAGGGAGG-39 (MBP-specific primer [nt 840–
818 in the proMBP cDNA sequence]), 1 pmol 5 9 CTGTGGTTGTGTGACAAATGGC-39 (PAPP-A-specific
primer [nt 4936–4915 in the PAPP-A cDNA sequence]), 40
units RNasin (Promega, Leiden, Holland), 1 mM dNTP, and
5 mM Mg21, in 20 ml of the supplied buffer. All reagents,
except primers, were from Promega (Madison, WI). The
remaining poly(A)1 RNA was processed in parallel without
addition of reverse transcriptase. The resulting cDNA was
diluted (1:4n, n 5 1 to 10) in dideoxy H2O and used directly
as template for competitive PCR, or stored at 2208C until
use.
Preparation of IS Templates
The IS is a deletion variant of the respective cDNA PCR
product (Fig. 1) that can be amplified with the same primers
as the cDNA. The PAPP-A IS was constructed by primermediated deletion as previously described [41]. Briefly, the
59-CAGTCAGCTGCTCAACGGAAGGACTCACATTGG39 (nt 4712–4731 and 4789–4805 in the PAPP-A cDNA
sequence) was used with 5 9 -GGAGGCTCTGGGACTGCAC-39 (nt 4904–4886) as primers in a PCR, using firststrand cDNA from placenta as template, to make a 62basepair (bp) deletion variant of the PAPP-A cDNA PCR
product with the same primer binding sequences as the
PAPP-A cDNA. An MBP IS was constructed by excision
of a HinP1I-MspI fragment (nt 447–522 in the proMBP
cDNA sequence), resulting in a 76-bp deletion variant of
the cDNA PCR product. The b-actin IS was constructed by
excision of a HinP1I-MspI fragment (nt 1045–1136 in the
b-actin cDNA sequence), resulting in a 92-bp deletion variant. For construction of a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) IS, the primer 59-AACGGGAAGCTCACTGGCATGATGACATCAAGAAGGTGGTG39 (nt 674–694 and 765–787 in the GAPDH cDNA sequence)
was used with 59-CCACCACCCTGATGTCGTAGC-39 (nt
977–957) in a PCR using first-strand cDNA as template to
make a 73-bp deletion variant of the GAPDH PCR product
with the same primer binding sequences as the cDNA PCR
product. The IS’s were purified from agarose gels and verified by sequence analysis. The fixed amount of IS added
to each PCR was taken from the same batch stored in
ready-to-use aliquots at 2208C.
Primers for Competitive PCR
Primers for competitive PCR were as follows: b-Actin:
5 9 -CACCCAGCACAATGAAGATCAAG-3 9 (nt 1003–
1025) and 59-GTCAAGAAAGGGTGTAACGCAAC-39 (nt
1207–1185); PAPP-A: 59-CAGTCAGCTGCTCAACGGAA-
1085
PAPP-A AND proMBP mRNA LEVELS
TABLE 1. Size of PCR products of the indicated templates in base pairs.
Template
Genomic DNA
cDNA
Internal
standard
b-actin
GAPDH
PAPP-A
MBP
317
1529
nda
408b
205
304
189
298
113
231
127
222
a
b
The intron/exon structure is not determined for PAPP-A.
The 59 primer used spans an intron.
39 (nt 4712–4731) and 59-GGAGGCTCTGGGACTGCAC39 (nt 4904–4886); MBP: 59-TTAGTCAAGCTTGGTTTACTTGC-39 (nt 423–445) and 59-GGAAGTCTTCTGAGGCAGTGG-39 (nt 720–700); and GAPDH: 59-AACGGGAAGCTCACTGGCATG-3 9 (nt 674–694) and
5 9 -CCACCACCCTGTTGCTGTAGC-3 9 (nt 977–957).
Numbers in parentheses refer to the positions in the corresponding cDNA sequences. Gene and cDNA sequences
were obtained from GenBank (accession numbers:
GAPDH, J02642 and J04038; b -actin, X00351 and
M10277; MBP, X14088 and M34462; PAPP-A, X68280).
All primers were from DNA Technology (Aarhus, Denmark).
Competitive PCR
All PCRs were performed in a total volume of 50 ml
with 1.5-unit SuperTaq (HT Biotechnology, Cambridge,
UK), 0.25 nM dNTP (Pharmacia, Upsala, Sweden), 80
pmol of each primer, SuperTaq buffer, and 1 ml internal
standard template (except blank control) in glass tubes using an Abacus thermal cycler (Denzyme, Aarhus, Denmark)
with a ramp rate of 48C/sec. Diluted aliquots of all reagents
(stored at 2208C) were used to prepare a reaction mixture
of which 49 ml was pipetted to each tube in a series of PCR
experiments. A series included reactions with a dilution series of first-strand cDNA from one tissue, a dilution series
from another tissue, one control with IS as the only template, and one blank control (which was taken from the
master PCR mixture before addition of the IS). After addition of 1 ml diluted cDNA template, 37 cycles of PCR
were performed using the following parameters: 948C for
30 sec (90 sec in the first cycle), annealing for 30 sec (see
below), and 728C for 40 sec (400 sec in the last cycle).
Annealing temperatures were 62 8 C for b -actin and
GAPDH, 608C for PAPP-A, and 588C for MBP. The amount
of IS added to each PCR was determined empirically so
that the dilution used for each IS template was in a linear
region of the double logarithmic plot of the PCR product
as a function of the dilution factor (not shown). This ensured that the amplifications were in the exponential phase
throughout the 37 cycles. The PCR primers in each primer
pair were positioned on different exons, enabling an easy
detection of possible genomic DNA contamination (Fig. 1
and Table 1). No genomic DNA contamination of the
cDNA preparations were observed in any of the tissues
examined.
FIG. 2. Agarose gel (2.5%)-electrophoresis of PCR products using PAPPA competitive primers. Lanes 1–5: cDNA dilution (1:4 1 to 1:45) of cDNA
from kidney as template; lanes 6 and 13: control with only internal standard as template; lane 7: control with no template added; lanes 8–12:
cDNA dilution (1:46 to 1:410) of cDNA from placenta as template. A and
B are PAPP-A cDNA (189 bp) and PAPP-A internal standard (127 bp)
amplification products, respectively. The remaining samples from lanes 4
and 5, and lanes 9 and 10 were used for further quantification.
min linear gradient from 0.3 to 0.7 M NaCl in TE buffer,
pH 7.5 (10 mM Tris-HCL, 5mM EDTA) at 608C (Fig. 3).
The dilution, DIeq, that would have resulted in equimolar
amounts of cDNA and IS PCR products, was calculated
from equation 1,
DI eq(x) 5 D 3
CP
IP
(1)
where CP is the amount of cDNA PCR product, determined
as the total A260 absorption; IP is the amount of IS PCR
product, determined as the total A260 absorption corrected
for the difference in size from the cDNA product; D is the
actual dilution of the cDNA preparation; and DIeq(x) is the
dilution that would result in equal molar amounts of IS and
cDNA PCR product (x is either PAPP-A, proMBP, b-actin,
or GAPDH).
A DIeq value was determined for each of the gene products as the mean value obtained from PCR of two independent dilution series and from two cDNA dilutions in
each series. Finally, the specific abundance of PAPP-A or
proMBP mRNA, A(x), was determined (equation 2):
A (x) 5
DI eq(x)
DI eq(b2actin)
(2)
where A(x) is the specific abundance of individual mRNA
species. Thus, the specific abundance is a measure of the
mRNA level of the gene of interest, normalized against a
measure of the total mRNA in the sample. Given a constant
Quantification
For each dilution series, the two samples, with about
equal amounts of cDNA and IS PCR products, as judged
by gel-electrophoresis in a 2.5% agarose gel (Fig. 2), were
separated on a Hewlett-Packard (Palo Alto, CA) 1084
HPLC instrument equipped with a Waters (Milford, MA)
Gen-Pak FAX nonporous ion-exchange column using a 20-
FIG. 3. A260 elution profile from ion exchange chromatography separation of PCR products with PAPP-A-specific primers. Buffer components,
dNTP, and primers eluted first. The PCR products of cDNA (A) and internal
standard (B) templates were well separated and could easily be quantified.
The vertical arrow denotes change of scale (8-fold increase in sensitivity).
1086
OVERGAARD ET AL.
FIG. 4. Specific abundances of PAPP-A
and proMBP mRNA in the tissues tested,
normalized against the average term placenta specific abundance. Standard deviations are shown as error bars. The number
of samples for each tissue is indicated
above the columns.
amount of b-actin mRNA molecules per cell, which is a
reasonable assumption, the specific abundance, A, is independent of the amount of tissue used.
RNA Dot Blot Analysis
A 32P-labeled PAPP-A cDNA fragment pPA-1 [4] and a
32P-labeled MBP PCR product (see above) were hybridized
to a human RNA master blot (Clontech, Palo Alto, CA) according to the manufacturer’s instructions. After two washes
with 0.15 M NaCl, 15 mM sodium citrate, 0.1% SDS, pH
7.0, at 658C for 30 min, autoradiography was performed for
24 h using a phosphorimager (Molecular Dynamics). The human RNA master blot contains samples from 50 different
tissues spotted on the membrane (see Fig. 5 legend). RNA
amounts from all tissues were normalized against eight different housekeeping gene transcripts on the master blot.
RESULTS
Quantification Procedure
Messenger RNA was extracted from frozen, homogenized tissue samples using oligo(dT)-coupled magnetic
beads. This is a fast and easy protocol ensuring minimal
degradation. To increase the sensitivity, proMBP-, PAPPA-, and oligo(dT)-specific primers were used in the firststrand cDNA synthesis. Serial dilutions were made with the
pool of cDNA obtained from the RT reaction. These cDNA
dilutions were used as templates in competitive PCRs, with
fixed amounts of gene-specific IS template added. From
measurements of the b-actin mRNA levels, the specific
abundance was calculated for each tissue as detailed in the
Materials and Methods section. We also measured the levels of GAPDH mRNA. As expected, the specific abundance
of GAPDH mRNA showed minimal variation (128 6 58
[SD]). This validates normalization against b-actin mRNA.
PAPP-A and ProMBP mRNA Levels
We measured the specific abundance of PAPP-A and
proMBP mRNA in a total of 43 samples from 13 different
tissues, using the semiquantitative RT-PCR method described above. The results are summarized in Figure 4, in
which the mean specific mRNA abundance for each tissue
is shown relative to the specific abundance in term placenta,
which contained the highest level measured for both PAPPA and proMBP. The specific abundance of PAPP-A and
proMBP mRNA was dramatically lower in first-trimester
placenta than in term placenta (75- and 17-fold, respectively). It is also evident that all tissues examined contained
measurable amounts of both mRNA species (Fig. 4). In
endometrium from postmenopausal women, the PAPP-A
mRNA level was 250-fold lower than in term placenta.
Most other tissues examined had a specific PAPP-A mRNA
abundance 500- to 3000-fold lower than in term placenta.
In bone marrow cells, in which proMBP mRNA is expected
at a relatively high level, the specific proMBP mRNA abundance was 230-fold lower than in term placenta. In breast
tissue it was 800-fold lower than in term placenta, whereas
the proMBP mRNA abundance was more than 1300-fold
lower than in term placenta in all other tissues tested.
Analysis of the mRNA in 1.5 ml whole blood drawn
from a pregnant woman showed a very low b-actin mRNA
level and no detectable PAPP-A or proMBP mRNA. Thus,
blood present in tissue samples cannot interfere with the
measurements of the specific abundances of mRNA species.
Dot Blot Experiments
In addition to the tissue samples analyzed by semiquantitative RT-PCR, a rapid screen for tissues producing high
amounts of PAPP-A or proMBP mRNA was carried out.
This was done by hybridizing a specific 32P-labeled PAPPA or proMBP cDNA probe to a membrane containing RNA
from 50 different human tissues. As expected, placenta
showed a very high signal for both mRNA species. The
only other tissue with a PAPP-A signal above background
was kidney (Fig. 5A). With this method, proMBP mRNA
was detected in placenta and bone marrow, and at very low
levels in kidney (Fig. 5B).
DISCUSSION
Here we report that PAPP-A and proMBP mRNA is synthesized by female reproductive tissues, i.e., ovary, tuba
PAPP-A AND proMBP mRNA LEVELS
FIG. 5. A human RNA master blot hybridized with A) a 32P-labeled
PAPP-A cDNA clone (pPA-1) [4] and B) a 32P-labeled MBP PCR product.
The blot contains mRNA from 50 different human tissues, in addition to
control DNA samples: A1, whole brain; A2, amygdala; A3, caudate nucleus; A4, cerebellum; A5, cerebral cortex; A6, frontal lobe; A7, hippocampus; A8, medulla oblongata; B1, occipital pole; B2, putamen; B3,
substantia nigra; B4, temporal lobe; B5, thalamus; B6, subthalamic nucleus; B7, spinal cord; C1, heart; C2, aorta; C3, skeletal muscle; C4,
colon; C5, bladder; C6, uterus; C7, prostate; C8, stomach; D1, testis; D2,
ovary; D3, pancreas; D4, pituitary gland; D5, adrenal gland; D6, thyroid
gland; D7, salivary gland; D8, mammary gland; E1, kidney; E2, liver; E3,
small intestine; E4, spleen; E5, thymus; E6, peripheral leukocyte; E7,
lymph node; E8, bone marrow; F1, appendix; F2, lung; F3, trachea; F4,
placenta; G1, fetal brain; G2, fetal heart; G3, fetal kidney; G4, fetal liver;
G5, fetal spleen; G6, fetal thymus; G7, fetal lung; H1, yeast total RNA;
H2, yeast tRNA; H3, E. coli rRNA; H4, E. coli DNA; H5, Poly r(A); H6,
human genomic repeat DNA; H7, human DNA; H8, human DNA.
uterina, endometrium, and myometrium from postmenopausal women in addition to placenta. Synthesis of both
species also occurs in nonreproductive tissues, i.e., kidney,
colon, prostate, prostate carcinoma, bone marrow cells,
breast, and breast carcinoma. The specific abundance of
PAPP-A and proMBP mRNA differs greatly between tissues; term placenta has more than 200-fold higher levels
1087
than any nonplacental tissue tested. This finding indicates
that the main site of both PAPP-A and proMBP synthesis
during pregnancy is the placenta. The low mRNA levels of
nonplacental tissues are reflected in the very low serum
concentrations of PAPP-A and proMBP antigen in nonpregnant individuals [17].
Both PAPP-A and proMBP are among the most highly
expressed genes in placenta, representing 1% (PAPP-A)
and 5% (proMBP) of the total number of clones in two
placental cDNA libraries (UniGene at http://inhouse.
ncbi.nlm.nih.gov/UniGene/Hs.Home.html, library 398 and
399, respectively). In these libraries, PAPP-A and proMBP
clones were among the five most abundant. Therefore, the
mRNA specific abundances calculated here for a number
of tissues are low compared to the levels in placenta (Fig.
4). In placenta, both PAPP-A and proMBP mRNA are readily detected by in situ hybridization [23–25]. In bone marrow, proMBP mRNA could not be detected by this technique (unpublished results), even though the mature MBP
constitutes more than 50% of the total granule protein of
the eosinophil leukocyte [12]. This highlights the relevance
of an RT-PCR-based assay for the determination of PAPPA and proMBP mRNA levels.
We found that the ratio between the specific abundance
of proMBP and PAPP-A mRNA in placenta is not constant
during pregnancy: levels of both mRNA species are lower
in first-trimester placenta than in term placenta, but the level of PAPP-A mRNA increases relatively more than the
level of proMBP mRNA. This is in good agreement with
the change in the molar ratio of proMBP and PAPP-A serum levels, which goes from a 10-fold excess of proMBP
in the first trimester to a 4-fold excess in the third trimester
[9].
The finding that PAPP-A mRNA is synthesized in all the
examined tissues, reproductive as well as nonreproductive,
is surprising, and indicates that PAPP-A functions outside
pregnancy. The recent demonstration that PAPP-A in conditioned media from human fibroblasts specifically cleaves
IGF binding protein (BP)-4 [14], which is an inhibitor of
IGF action, makes it likely that PAPP-A plays a localized
role in the IGF/IGFBP-4 system. Because none of the tissues analyzed transcribe only one of the two mRNA species, it is tempting to hypothesize that proMBP plays a role
in regulation of PAPP-A activity. Specifically, we speculate
that proMBP is an inhibitor of PAPP-A proteolytic activity.
The inhibitory effect of proMBP may not be complete,
since the PAPP-A/proMBP complex isolated from pregnancy serum did show proteolytic activity [14]. In the majority
of tissues, the mRNA abundance relative to term placenta
is higher for PAPP-A than proMBP. However, the molar
concentration of PAPP-A in the tissue may not necessarily
exceed that of proMBP. All mRNA levels are expressed
relative to the level in term placenta (Fig. 4), where the
proMBP mRNA abundance is higher than that of PAPP-A
[22]. Interestingly, in the tissues in which proMBP or MBP
are known to be present in excess of PAPP-A, i.e., bone
marrow cells (eosinophil leukocytes) and placenta, the specific abundance of proMBP mRNA is higher than that of
PAPP-A relative to term placenta.
Earlier reports on localization of PAPP-A in tissues have
resulted in contradicting results, and the question of nonplacental PAPP-A synthesis has been a subject of controversy (see [42, 43] for recent reviews). All previous investigations have been based on polyclonal antisera, and a
number of reports have appeared describing the polyspecificity and heterogeneity of different preparations of these
1088
OVERGAARD ET AL.
antisera [2, 36–38]. Some investigators have further purified the antisera preparations to minimize the polyspecificity, but only one [22] has taken into account that polyclonal antisera raised against PAPP-A, now known to be
PAPP-A/proMBP, invariably will recognize the proMBP
part of the PAPP-A/proMBP complex, as well as the mature
eosinophil MBP [2]. To address the question of PAPP-A
synthesis, and to detect and discriminate between PAPP-A
and proMBP antigens in tissues, mRNA assays and monoclonal antibodies, respectively, must be used.
Testing different tissues for the presence of specific
mRNAs is routinely done by RNA blotting techniques such
as Northern or dot blotting. But Northern blotting of large
mRNA species such as the PAPP-A mRNA, is technically
difficult, and the sensitivity is relatively low. We attempted
to detect PAPP-A and proMBP mRNA in a range of tissues
by screening a commercial RNA dot blot containing normalized amounts of RNA from 50 different human tissues.
A positive response above background was seen for placenta, kidney (very low), and bone marrow (only proMBP).
Hence neither PAPP-A nor proMBP is synthesized in nonplacental tissues in quantities comparable to those in the
placenta. We thus developed the semiquantitative RT-PCR
assay described above. RT-PCR has been shown to be
1000- to 10 000-fold more sensitive than traditional RNA
blotting techniques [44, 45], and we were able to detect and
quantitate both PAPP-A and proMBP mRNA in all the tissues tested. In a number of these, such as colon, prostate,
and uterus (endometrium and myometrium), neither PAPPA nor proMBP mRNA was detectable when the commercial RNA dot blot was screened with PAPP-A or proMBP
specific probes (Fig. 5), clearly demonstrating the higher
sensitivity and the usefulness of the RT-PCR assay. The
actual quantification of the products from the competitive
PCR is done by ion exchange chromatography on an HPLC
system, an accurate method that involves a minimum of
post-PCR handling [46, 47].
An alternative way of detecting specific mRNA synthesis is by in situ hybridization. This technique has the advantage that it locates the cells that synthesize the mRNA
but the disadvantage of being less sensitive, as mentioned
above. The fact that the mRNA levels detected in this study
in several of the tissues tested are relatively low reflects
that the synthesis of PAPP-A and proMBP mRNA is limited to a few specific cells in the tissue. Immunohistochemical investigations with monoclonal antibodies are in progress. These studies confirm localization of the antigens in
and around a very limited number of cells within each tissue (unpublished results).
ACKNOWLEDGMENTS
We thank Dr. Peter Hockland, Dr. Vibeke Jensen, Dr. Axel Formann,
Dr. Peter Ebbesen, Dr. Michael Borre, and Dr. Niels Jørgen Secher for
generously providing tissue samples.
REFERENCES
1. Lin TM, Halbert SP, Kiefer D, Spellacy WN, Gall S. Characterization
of four human pregnancy-associated plasma proteins. Am J Obstet
Gynecol 1974; 118:223–236.
2. Oxvig C, Sand O, Kristensen T, Gleich GJ, Sottrup-Jensen L. Circulating human pregnancy-associated plasma protein-A is disulfidebridged to the proform of eosinophil major basic protein. J Biol Chem
1993; 268:12243–12246.
3. Oxvig C, Sand O, Kristensen T, Kristensen L, Sottrup-Jensen L. Isolation and characterization of circulating complex between human
pregnancy-associated plasma protein-A and proform of eosinophil major basic protein. Biochim Biophys Acta 1994; 1201:415–423.
4. Kristensen T, Oxvig C, Sand O, Moller NP, Sottrup-Jensen L. Amino
acid sequence of human pregnancy-associated plasma protein-A derived from cloned cDNA. Biochemistry 1994; 33:1592–1598.
5. Haaning J, Oxvig C, Overgaard MT, Ebbesen P, Kristensen T, SottrupJensen L. Complete cDNA sequence of the preproform of human
pregnancy-associated plasma protein-A. Evidence for expression in
the brain and induction by cAMP. Eur J Biochem 1996; 237:159–163.
6. Barker RL, Gleich GJ, Pease LR. Acidic precursor revealed in human
eosinophil granule major basic protein cDNA [published erratum appears in J Exp Med 1989; 170:1057]. J Exp Med 1988; 168:1493–
1498.
7. Wasmoen TL, Bell MP, Loegering DA, Gleich GJ, Prendergast FG,
McKean DJ. Biochemical and amino acid sequence analysis of human
eosinophil granule major basic protein. J Biol Chem 1988; 263:
12559–12563.
8. Oxvig C, Haaning J, Hojrup P, Sottrup-Jensen L. Location and nature
of carbohydrate groups in proform of human major basic protein isolated from pregnancy serum. Biochem Mol Biol Int 1994; 33:329–
336.
9. Oxvig C, Haaning J, Kristensen L, Wagner JM, Rubin I, Stigbrand T,
Gleich GJ, Sottrup-Jensen L. Identification of angiotensinogen and
complement C3dg as novel proteins binding the proform of eosinophil
major basic protein in human pregnancy serum and plasma. J Biol
Chem 1995; 270:13645–13651.
10. Popken-Harris P, Checkel J, Loegering D, Madden B, Springett M,
Kephart G, Gleich GJ. Regulation and processing of a precursor form
of eosinophil granule major basic protein (ProMBP) in differentiating
eosinophils. Blood 1998; 92:623–631.
11. Gleich GJ, Loegering DA, Maldonado JE. Identification of a major
basic protein in guinea pig eosinophil granules. J Exp Med 1973; 137:
1459–1471.
12. Peters MS, Rodriguez M, Gleich GJ. Localization of human eosinophil granule major basic protein, eosinophil cationic protein, and eosinophil-derived neurotoxin by immunoelectron microscopy. Lab Invest 1986; 54:656–662.
13. Gleich GJ, Adolphson CR, Leiferman KM. The biology of the eosinophilic leukocyte. Annu Rev Med 1993; 44:85–101.
14. Lawrence JB, Oxvig C, Overgaard MT, Sottrup-Jensen L, Gleich GJ,
Hays LG, Yates III JR, Conover CA. The insulin-like growth factor
(IGF)-dependent IGF binding protein-4 protease secreted by human
fibroblasts is pregnancy associated plasma protein-A. Proc Natl Acad
Sci USA 1999; 96:3149–3153.
15. Westergaard JG, Chemnitz J, Teisner B, Poulsen HK, Ipsen L, Beck
B, Grudzinskas JG. Pregnancy-associated plasma protein A: a possible
marker in the classification and prenatal diagnosis of Cornelia de
Lange syndrome. Prenatal Diagn 1983; 3:225–232.
16. Brambati B, Macintosh MC, Teisner B, Maguiness S, Shrimanker K,
Lanzani A, Bonacchi I, Tului L, Chard T, Grudzinskas JG. Low maternal serum levels of pregnancy associated plasma protein A (PAPPA) in the first trimester in association with abnormal fetal karyotype.
Br J Obstet Gynaecol 1993; 100:324–326.
17. Qin QP, Christiansen M, Oxvig C, Petterson K, Sottrup-Jensen L,
Koch C, Noergaard-Pedersen B. Development, comparison and performance of four double monoclonal immunofluorometric assays for
PAPP-A·proMBP complex in first trimester maternal serum screening
for Down syndrome. Clin Chem 1997; 43:2323–2332.
18. Christiansen M, Oxvig C, Wagner JM, Qin QP, Nguyen TH, Overgaard MT, Larsen SO, Sottrup-Jensen L, Gleich GJ, Noergaard-Pedersen B. The proform of eosinophil major basic protein: a new maternal serum marker for Down syndrome. Prenatal Diag 1999; (in
press).
19. Haddow JE, Palomaki GE, Knight GJ, Williams J, Miller WA, Johnson A. Screening of maternal serum for fetal Down’s syndrome in the
first trimester. N Engl J Med 1998; 338:955–961.
20. Gurian KV, Podratz KC, Elg SA, Twiggs LB, Lurain JR, Wagner JM,
Gleich GJ. Major basic protein as a marker of malignant potential in
trophoblastic neoplasia. Am J Obstet Gynecol 1996; 175:632–637.
21. Rhoton-Vlasak A, Wagner JM, Rutgers JL, Baergen RN, Young RH,
Roche PC, Plummer TB, Gleich GJ. Placental site trophoblastic tumor: human placental lactogen and pregnancy-associated major basic
protein as immunohistologic markers. Hum Pathol 1998; 29:280–288.
22. Bonno M, Oxvig C, Kephart GM, Wagner JM, Kristensen T, SottrupJensen L, Gleich GJ. Localization of pregnancy-associated plasma
protein-A and colocalization of pregnancy-associated plasma proteinA messenger ribonucleic acid and eosinophil granule major basic protein messenger ribonucleic acid in placenta. Lab Invest 1994; 71:560–
566.
PAPP-A AND proMBP mRNA LEVELS
23. Wagner JM, Hustin J, Bonno M, Kephart GM, Gurian KV, Gleich GJ.
Pregnancy-associated major basic protein: deposition of protein and
expression of mRNA at the maternal-fetal junction in early and late
gestation. Placenta 1994; 15:625–640.
24. Bonno M, Kephart GM, Carlson CM, Loegering DA, Vernof KK,
Gleich GJ. Expression of eosinophil-granule major basic protein messenger ribonucleic acid in placental X cells. Lab Invest 1994; 70:234–
241.
25. Chegini N, Lei ZM, Rao CV, Bischof P. The presence of pregnancyassociated plasma protein-A in human corpora lutea: cellular and subcellular distribution and dependence on reproductive state. Biol Reprod 1991; 44:201–206.
26. Bischof P, Schindler AM, Urner F, Mensi N, Herrmann WL, Sizonenko PC. Pregnancy-associated plasma protein-A (PAPP-A): concentration in uterine fluid and immunohistochemical localization in the endometrium. Br J Obstet Gynaecol 1984; 91:863–869.
27. Sjoberg J, Wahlstrom T, Seppala M. Pregnancy-associated plasma protein A in the human endometrium is dependent on the effect of progesterone. J Clin Endocrinol Metab 1984; 58:359–362.
28. Wahlstrom T, Koskimies AI, Tenhunen A, Rutanen EM, Yki-Jarvinen
H, Julkunen M, Sjoberg J, Seppala M. Pregnancy proteins in the endometrium after follicle aspiration for in vitro fertilization. Ann NY
Acad Sci 1985; 442:402–407.
29. Sjoberg J, Wahlstrom T, Seppala M, Rutanen EM, Koistinen R, Koskimies AI, Sinosich MJ, Teisner B, Grudzinskas JG. Seminal plasma
levels of PAPP-A in normospermic and oligospermic men and tissue
localization of PAPP-A in the male genital tract. Arch Androl 1985;
14:253–261.
30. Schindler AM, Dayer A, Bischof P. Immunohistochemical localization
of pregnancy-associated plasma protein-A in the male genital tract.
Hum Reprod 1986; 1:55–59.
31. Schindler AM, Bischof P. Histochemical localization of pregnancyassociated plasma protein A in fetal, infant, and adult organs and
comparison between antisera. Gynecol Obstet Invest 1984; 18:88–94.
32. Wahlstrom T, Teisner B, Lee JN, Grudzinskas JG, Seppala M, Folkersen J. Placenta-associated plasma protein-A (PAPP-A, SP4) in trophoblastic tumours. Acta Pathol Microbiol Scand A 1981; 89:65–66.
33. Horne CH, Ranking R, Bremner R. Pregnancy specific hormones as
markers for gestational trophoblastic disease. Int J Pathol Gynaecol
1984; 3:27–40.
34. Kuhajda FP, Abeloff MD, Eggleston JC. Pregnancy-associated plasma
protein A: a clinically significant predictor of early recurrence in stage
II breast carcinoma. Hum Pathol 1985; 16:228–235.
35. Kuhajda FP, Eggleston JC. Pregnancy-associated plasma protein A
and extensive necrosis. Clinically significant predictors of early re-
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
1089
currence in stage I estrogen receptor-negative breast carcinoma. Lab
Invest 1985; 53:101–107.
Chemnitz J, Folkersen J, Teisner B, Sinosich MJ, Tornehave D, Westergaard JG, Bolton AE, Grudzinskas JG. Comparison of different
antibody preparations against pregnancy-associated plasma protein-A
(PAPP-A) for use in localization and immunoassay studies. Br J
Obstet Gynaecol 1986; 93:916–923.
Kuhajda FP, Katumuluwa AI, Pasternack GR. Expression of haptoglobin-related protein and its potential role as a tumor antigen. Proc
Natl Acad Sci USA 1989; 86:1188–1192.
Bueler MR, Bersinger NA. Antiserum to pregnancy-associated plasma
protein A (PAPP-A) recognizes human haptoglobin. Br J Obstet Gynaecol 1989; 96:867–869.
Bersinger NA, Zakher A, Huber U, Pescia G, Schneider H. A sensitive
enzyme immunoassay for pregnancy-associated plasma protein A
(PAPP-A): a possible first trimester method of screening for Down
syndrome and other trisomies. Arch Gynecol Obstet 1995; 256:185–
192.
Kristensen JS, Ellegaard J, Hansen KB, Clausen N, Hokland P. Firstline diagnosis based on immunological phenotyping in suspected
acute leukemia: a prospective study. Leuk Res 1988; 12:773–782.
Celi FS, Zenilman ME, Shuldiner AR. A rapid and versatile method
to synthesize internal standards for competitive PCR. Nucleic Acids
Res 1993; 21:1047.
Oxvig C, Haaning J, Wagner JM, Sottrup-Jensen L, Gleich GJ. Placental Proteins. In: Polin R, Fox W (eds.), Fetal and Neonatal Physiology, vol. 1, 2nd ed. Philadelphia: WB Saunders Company; 1998:
103–114.
Bersinger NA, Altermatt HJ, Birkhauser MH, Brizot ML, Kappeler
A, Muller MD, Schneider H. Non-placental production of pregnancyassociated plasma protein A (PAPP-A): old and new evidence. Early
Pregnancy 1997; 3:96–101.
Byrne BC, Li JJ, Sninsky J, Poiesz BJ. Detection of HIV-1 RNA
sequences by in vitro DNA amplification. Nucleic Acids Res 1988;
16:4165.
Wang AM, Doyle MV, Mark DF. Quantitation of mRNA by the polymerase chain reaction [published erratum appears in Proc Natl Acad
Sci USA 1990; 87:2865]. Proc Natl Acad Sci USA 1989; 86:9717–
9721.
Gaus H, Lipford GB, Wagner H, Heeg K. Quantitative analysis of
lymphokine mRNA expression by a nonradioactive method using
PCR and anion exchange chromatography. J Immunol Methods 1993;
158:229–236.
van-Hille B, Lohri A, Reuter J, Herrmann R. Nonradioactive quantification of mdr1 mRNA by polymerase chain reaction amplification
coupled with HPLC. Clin Chem 1995; 41:1087–1093.