The Journal of Clinical Endocrinology & Metabolism Vol. 86, No. 2 847-854
Copyright © 2001 by The Endocrine Society
Pregnancy-Associated Plasma Protein-A Accounts for the Insulin-Like Growth Factor (IGF)-Binding Protein-4 (IGFBP-4) Proteolytic Activity in Human Pregnancy Serum and Enhances the Mitogenic Activity of IGF by Degrading IGFBP-4 in Vitro1
Dongwon Byun,
Subburaman Mohan,
Myunghi Yoo,
Christopher Sexton,
David J. Baylink and
Xuezhong Qin
Musculoskeletal Disease Center, J. L. Pettis Memorial Veterans
Affairs Medical Center, and Loma Linda University (D.B., S.M., C.S.,
D.J.B., X.Q.), Loma Linda, California 92357; and Department of
Endocrinology, SoonChunHyang University Hospital (M.Y.), Seoul, 140-743
Korea
Address all correspondence and requests for reprints to: Dr. Xuezhong Qin, Musculoskeletal Disease Center, J. L. Pettis Veterans Affairs Medical Center (151), 11201 Benton Street, Loma Linda, California 92357. E-mail: xuezhong.qin{at}med.va.gov
 |
Abstract
|
|---|
Pregnancy-associated plasma protein-A (PAPP-A) has been identified as
the insulin-like growth factor (IGF)-dependent IGF-binding protein-4
(IGFBP-4) protease produced by human fibroblasts. Recently, we found
that serum proteases induced during human pregnancy cleaved IGFBP-4 in
both an IGF-II-dependent and an IGF-II-independent fashion. This study
sought to determine whether PAPP-A is the predominant IGFBP-4 protease
in human pregnancy serum (PS) and to assess the in vitro
role of serum PAPP-A. Immunoprecipitation with PAPP-A antibody
effectively depleted PAPP-A from the PS and completely abolished both
IGF-II-dependent and IGF-II-independent IGFBP-4 proteolytic activity in
PS. Direct addition of PAPP-A antibody to PS completely blocked IGFBP-4
proteolysis and partially blocked IGFBP-5 proteolysis, but had no
effect on IGFBP-3 proteolysis. To evaluate the role of serum PAPP-A, we
tested whether PAPP-A in PS modulated the inhibitory activity of
IGFBP-4 on IGF-II-induced cell proliferation in human osteosarcoma MG63
cells. The wild-type IGFBP-4 (WTBP-4; 200 ng/mL) failed to inhibit
proliferation of the cells treated with PS (0.1% or 0.3%) alone or in
combination with IGF-II (40 ng/mL), whereas the inhibitory effect of
WTBP-4 was observed in the cells treated with nonpregnancy serum alone
or in combination with IGF-II (P < 0.05). In
contrast to WTBP-4, a protease-resistant IGFBP-4 was able to inhibit
proliferation of the cells treated with PS alone or in combination with
IGF-II (P < 0.05). In the presence of PAPP-A
neutralizing antibody, the inhibitory effect of WTBP-4 on proliferation
of the cells treated with IGF-II and PS was restored. In summary, these
data demonstrate 1) that PAPP-A represents the predominant IGFBP-4
protease in PS; 2) that PAPP-A may in part contribute to IGFBP-5, but
not IGFBP-3, proteolytic activity in PS; and 3) that PAPP-A enhances
the bioactivity of IGFs in vitro by degrading IGFBP-4.
 |
Introduction
|
|---|
THE INSULIN-LIKE GROWTH factors (IGFs) are
mitogenic polypeptides that regulate growth by stimulating cell
proliferation and differentiation in addition to exhibiting important
metabolic effects (1, 2, 3, 4). The biological activity and
availability of IGFs have now been known to be modulated by the six
high affinity IGF-binding proteins (IGFBPs) in body fluids
(1, 2, 3, 4). During pregnancy, there is a rapid and progressive
increase in both maternal and fetal tissue growth, which obviously
increases the demand for growth-stimulating factors. In this regard,
IGFs and IGFBPs have been suggested to be important regulators of fetal
growth (5, 6, 7, 8). In normal, nonpregnancy serum (NPS), IGFs
are predominantly sequestered into a ternary 150-kDa complex comprising
IGF, IGFBP-3, and the acid-labile subunit (9, 10).
Although formation of this large complex may prolong the half-life of
IGFs, it precludes IGF from effectively crossing the capillary
endothelium to act on target tissues. In extracellular compartments,
IGFs are also likely to be complexed with IGFBPs, which could reduce
the local free IGF concentrations. Therefore, the release of IGFs from
the IGFBP/IGF complex in both the circulation and the local tissue
environment becomes critical for IGFs to act on both maternal and fetal
tissues during pregnancy.
Proteolysis of IGFBPs by specific IGFBP proteases leads to the
generation of IGFBP fragments that exhibit reduced affinity with IGFs
compared with their intact counterparts (11, 12, 13) and thus
may play an important role in the release of IGFs from the IGFBP/IGF
complexes. Since the early reports on the increased IGFBP proteolysis
in serum during pregnancy in 1990 (14, 15), a number of
studies have been performed to further characterize the
pregnancy-induced proteolysis of IGFBPs in serum (16, 17, 18).
It is generally accepted that an increase in IGFBP proteolysis
contributes to the increase in free serum IGF concentrations observed
during pregnancy (12, 19).
Recent studies on identification of the IGF-dependent IGFBP-4
protease produced by human fibroblasts in vitro has led to a
breakthrough in which the long sought after IGF-dependent IGFBP-4
protease was determined to be the pregnancy-associated plasma protein-A
(PAPP-A) (20). Recent studies demonstrate that PAPP-A is
also the major IGFBP-4 protease present in human ovarian follicular
fluid (21). In circulation, PAPP-A exists as a
PAPP-A/pro-MBP complex that consists of two 200-kDa PAPP-A subunits
that are disulfide bound to each of two mutually disulfide-bridged 50-
to 90-kDa pro forms of eosinophil major basic protein (pro-MBP)
subunits (22). Therefore, serum PAPP-A/pro-MBP complex
migrates as a more than 400-kDa band. Under reducing conditions, the
PAPP-A monomer migrates as a 200-kDa protein band. PAPP-A/pro-MBP is
detectable in pregnancy serum 46 weeks after conception,
progressively increases to a concentration of approximately 50 µg/mL
in late pregnancy serum (PS), and then rapidly declines postpartum
(23, 24). Although a low serum level of PAPP-A has been
used as an indicator of certain genetic fetal developmental disorders
such as Downs syndrome (25) and Cornelia de Lange
syndrome (26, 27), the role of PAPP-A, except for acting
as an IGFBP-4 protease, remains unknown. Recent studies from our
laboratory demonstrate that addition of IGF-II to human PS dramatically
increased IGFBP-4 proteolysis, but did not alter cleavage site
(Met135-Lys136) in human
IGFBP-4 (28). Although these studies suggest that PAPP-A
is likely to contribute to the pregnancy-induced IGFBP-4 proteolysis,
it remains to be determined whether PAPP-A is the major IGFBP-4
protease in PS that is responsible for cleavage of IGFBP-4 in both the
presence and absence of IGF-II.
The purpose of this study was 3-fold: 1) to determine whether PAPP-A is
the major protease in PS, 2) to determine whether the IGFBP-3 and
IGFBP-5 proteases induced during human pregnancy are different from
PAPP-A, and 3) to determine whether PAPP-A in PS regulates IGFBP-4
availability and thus the activity of IGFs in vitro.
 |
Materials and Methods
|
|---|
Reagents
Blood samples from pregnant women were collected in
SoonChunHyang Hospital, South Korea, for clinical purposes according to
approved protocols. The samples were shipped on dry ice to the United
States and stored at -80 C before use. The 6xHis-tagged recombinant
human IGFBP-4 was prepared as previously described (29, 30). Recombinant IGFBP-3 and IGFBP-5 are gifts from Dr. A.
Sommer (Celtax Corp., Palo Alto, CA) and Dr. K. Lang (Roche Molecular Biochemicals, Penzberg, Germany). Purified
polyclonal antihuman PAPP-A IgG (catalogue no. A0230) and normal
IgG (catalogue no. X0936) produced in rabbits were purchased from
DAKO Corp. (Carpinteria, CA). Recombinant human IGF-II was
obtained from Bachem (Torrance, CA).
[125I]NaI was purchased from NEN Life Science Products (Wilmington, DE). Reagents for SDS-PAGE were
obtained from Bio-Rad Laboratories, Inc. (Hercules, CA).
All other chemicals and reagents were of reagent grade and were
obtained from Sigma (St. Louis, MO).
Western [125I]IGF-II ligand blot
analysis
[125I]IGF-II Western ligand blot
analysis was performed as previously described (31).
Briefly, proteins were separated on SDS-PAGE gels under nonreducing
conditions and electrically transferred to Transblot nitrocellulose
membranes (catalogue no. 162-0097, Bio-Rad Laboratories, Inc. Hercules, CA). The membrane was first washed with 100 mL
buffer A (150 mmol/L NaCl and 20 mmol/L Tris, pH 7.4) containing 0.1%
Triton X-100 for 1530 min and then blocked with 100 mL buffer A
containing 0.1% BSA for 1 h. Each BSA-treated membrane was
incubated with 10 mL buffer A containing 0.1% BSA, 0.1% Tween-20, and
1,000,000 cpm [125I]IGF-II tracers (200300
µCi/µg protein) for 2 h. All incubations were undertaken at
room temperature with gentle shaking. The membranes were then washed
with buffer A containing 100 mL 0.1% Tween-20, five times each
for 2030 min each time. The membranes were exposed to x-ray
film for 310 h.
Immunoprecipitation of PAPP-A from serum
One hundred microliters of NPS or PS were incubated with 100
µL protein A-agarose for 30 min with frequent manual mixing. After
centrifugation, the supernatant was collected. This step was repeated
three times. Then the supernatant was divided into three aliquots and
incubated with vehicle (PBS), normal IgG (40 µg), and PAPP-A
polyclonal antibody (40 µg), respectively, overnight with gentle
shaking. The samples were then mixed with 100 µL protein A agarose
with frequent manual mixing for 1 h. The supernatant was
collected. This step was repeated one more time. All of the incubations
were performed on ice or at 4 C. The supernatant was then collected and
used to perform PAPP-A immunoblot analysis and IGFBP protease
assays.
PAPP-A Western immunoblot analysis
Ten microliters of treated serum sample (equivalent to 2 µL
undiluted serum) were resolved on a 6% SDS-PAGE gel under both
reducing and nonreducing conditions. The proteins were transferred to a
Transblot nitrocellulose membrane and subjected to immunoblot analysis
using rabbit anti-PAPP-A IgG according to the manufacturers
(DAKO Corp., Carpinteria, CA) instructions. Briefly, membranes were
washed in double distilled water for 15 min and blocked with 10 mL
washing buffer (20 mmol/L Tris, 138 mmol/L NaCl, and 0.1% Tween-20, pH
7.4) containing 0.5% dry skim milk (blocking buffer) for 1 h. The
blocked membranes were then incubated with 10 mL blocking buffer
containing 1 µg/mL PAPP-A antibody for 1 h. After washing the
membranes with the washing buffer three times, the membranes were
incubated for 1 h with 10 mL of 1:20,000 diluted secondary
antibody (ImmunoPure Goat Anti-Rabbit IgG, peroxidase conjugated,
Pierce Chemical Co., Rockford, IL) in blocking buffer and
then washed five times with 20 mL washing buffer for 15 min each time.
All incubations were performed at room temperature with gentle shaking.
Finally, each membrane was incubated with 15 mL SuperSignal West Pico
chemiluminescent substrate (Pierce Chemical Co.) for 5 min
and exposed to x-ray film (Fuji Photo Film Co., Ltd.,
Tokyo, Japan) for 15 min.
IGFBP protease assay
IGFBP-4 protease assays were performed by incubating the
recombinant IGFBPs with serum as previously described (28)
with minor modifications, as described in the figure legends.
Cell proliferation assays
Cell proliferation assays were performed as previously described
with minor modifications, as described in the figure legends (29, 30).
Statistical analysis
Statistical analysis of the data was performed by ANOVA followed
by multiple comparison. The data were expressed as the mean ±
SEM.
 |
Results
|
|---|
Effect of PAPP-A immunodepletion from or addition of PAPP-A
neutralization antibody to PS on subsequent proteolysis of IGFBPs
To determine whether PAPP-A represents the predominant protease
responsible for the serum IGFBP-4 proteolytic activity induced during
pregnancy, we determined whether PS depleted of PAPP-A exhibited
IGFBP-4 proteolytic activity. Consistent with a previous report
(22), immunoreactive pro-MBP/PAPP-A in the PS migrated as
a more than 400-kDa band under nonreducing conditions (band A in lanes
4 and 5, Fig. 1A
). Under reducing
conditions, PAPP-A dissociated from the pro-MBP/PAPP-A complex and
migrated as an approximately 200-kDa band (band a in lanes 4 and 5,
Fig. 1B
). It has been reported that the highly glycosylated pro-MBP
migrated as a smear of 50- to 90-kDa protein (22).
However, under nonreducing conditions, no proteins of 5090 kDa
reacted with this polyclonal antibody, which was raised against
purified pro-MBP/PAPP-A complex. A minor band of approximately 180 kDa
was detected in both PS and NPS under nonreducing conditions (band C in
lanes 16, Fig. 1A
). Detection of this protein band was not affected
by PAPP-A depletion. Under reducing conditions, a sharp band of
approximately 60 kDa (band d in lanes 16, Fig. 1B
) was recognized. It
is unlikely that this band represented the pro-MBP disassociated from
the pro-MBP/PAPP-A complex, as the intensity of this band was not
increased in PS compared with the NPS and was not affected by
immunodepletion with PAPP-A antibody. A very faint band of
approximately 80 kDa (band c in lanes 16, Fig. 1B
) was also observed,
whose identity is unclear. After immunodepletion with PAPP-A antibody,
PAPP-A was not detectable under either reducing or nonreducing
conditions (Fig. 1
). In addition to the bands of expected molecular
masses, an extra band of a smaller size (band B in lanes 4 and 5, Fig. 1A
; band b in lanes 4 and 5, Fig. 1B
), which was not present in the
NPS, was immunodepleted. This band may represent the proteolytic
fragment of PAPP-A, as its intensity was increased after long-term
storage of PS at -20 C. Band A (lanes 2, 3, 5, and 6, Fig. 1A
) and
band e (lanes 2, 3, 5, and 6, Fig. 1B
) were only present in the serum
sample treated with normal IgG or anti-PAPP-A IgG. These extra bands
were probably due to nonspecific interaction of proteins in the serum
or IgG preparations with the secondary antibody or with the
chemiluminescent substrate used in the immunoblotting assays.

View larger version (45K):
[in this window]
[in a new window]
|
Figure 1. Immunoblot analysis of PAPP-A in human NPS
and PS after immunoprecipitation with normal IgG or anti-PAPP-A IgG.
Both NPS and PS were immunoprecipitated with PAPP-A antibody, normal
IgG, or vehicle (phosphate-buffered saline) as described in
Materials and Methods. The proteins in the treated serum
(equivalent to 3 µL original serum) were separated on a 6% SDS-PAGE
gel under nonreducing (A) or reducing (B) conditions, and subjected to
immunoblot analysis using polyclonal anti-hPAPP-A IgG (Materials
and Methods). The data shown here are representative of three
independent experiments.
|
|
Next, we determined whether PAPP-A depletion from PS blocked IGFBP-4
proteolysis. Consistent with our previous observations
(28), proteolysis of IGFBP-4 by PS was observed in the
absence of IGF-II, when IGFBP-4 was incubated with a larger dose of PS
for a longer period of time (Fig. 2
).
When the incubation time and the dose of PS were reduced, IGFBP-4
was only cleaved in the presence of exogenous IGF-II (Fig. 2
). After
immunodepletion of PAPP-A with the PAPP-A antibody, both the
IGF-II-dependent and the IGF-II-independent IGFBP-4 proteolytic
activities in PS were completely abolished (Fig. 2
). In contrast,
immunoprecipitation with normal control IgG (Fig. 2
) or anti-cSrc IgG
(data not shown) had no effect on IGFBP-4 proteolysis. To further
confirm that PAPP-A is the predominant IGFBP-4 protease in PS, we
determined whether direct addition of PAPP-A antibody in the protease
assays could affect proteolysis of IGFBP-4. Preincubation of 0.3 µL
PS with 0.011 µg anti-PAPP-A IgG dose dependently inhibited IGFBP-4
proteolysis (Fig. 3
). At a dose of 1
µg, anti-PAPP-A IgG completely blocked the IGFBP-4 proteolysis
induced by 0.3 µL PS. These data provide strong evidence that PAPP-A
is the predominant IGFBP-4 protease in PS, which is responsible for
both IGF-II-dependent and IGF-II-independent IGFBP-4 proteolytic
activities in human serum induced during pregnancy.

View larger version (56K):
[in this window]
[in a new window]
|
Figure 2. Effect of PAPP-A depletion on IGFBP-4
proteolytic activity in PS. One hundred and fifty nanograms of IGFBP-4
were incubated with serum immunoprecipitated with PAPP-A antibody,
normal IgG, or vehicle in the presence of 50 ng IGF-II or vehicle. The
final volume of the reaction mixture (18 µL) contained 8 µL DMEM
with 1 mmol/L CaCl2. A, IGFBP-4 was incubated with 0.6 µL
equivalent PS for 14 h at 37 C. B, IGFBP-4 was incubated with 1.2
µL equivalent PS for 20 h at 37 C. The reaction mixtures were
then subjected to IGF-II ligand blot analysis.
|
|

View larger version (33K):
[in this window]
[in a new window]
|
Figure 3. Effect of direct addition of PAPP-A antibody
on IGFBP-4 proteolytic activity in PS. Ten microliters of 3-fold
diluted PS were incubated with normal IgG or anti-PAPP-A IgG at the
indicated amount in the presence of 5 µL DMEM/1 mmol/L
CaCl2. After a 3-h incubation at room temperature, 150 ng
IGFBP-4 and 100 ng IGF-II were added to the reaction mixture. After an
additional 17 h of incubation at 37 C, the digested samples were
subjected to IGF-II ligand blot analysis.
|
|
As it has been previously reported that IGFBP-3 and IGFBP-5
proteolytic activities in serum were also induced during pregnancy
(14, 15, 17), we determined whether addition of
anti-PAPP-A IgG affected the proteolytic activity of IGFBP-3 and
IGFBP-5 in NPS and PS. To avoid underestimation of the proteolytic
activity of PAPP-A toward degradation of these IGFBPs, IGFBP-3 or
IGFBP-5 was incubated with serum for various periods of time in the
presence of control IgG or anti-PAPP-A IgG. After a 5-h incubation at
37 C, the rate of IGFBP-5 proteolysis was similar in the presence of
control IgG and anti-PAPP-A IgG (Fig. 4A
). As the incubation time was reduced
to 2 h or 40 min, IGFBP-5 proteolysis was progressively reduced,
although not completely blocked, by anti-PAPP-A IgG. In contrast,
proteolysis of IGFBP-3 was not affected by anti-PAPP-A IgG under
the conditions that allow kinetic analysis of IGFBP-3 proteolytic
activity (Fig. 4B
). These data suggest that PAPP-A in part contributes
to IGFBP-5, but not IGFBP-3, proteolytic activity in PS.

View larger version (72K):
[in this window]
[in a new window]
|
Figure 4. Effect of direct addition of PAPP-A antibody
on IGFBP-5 and IGFBP-3 proteolytic activity in PS. Two microliters of
undiluted PS were preincubated with 10 µg normal IgG, anti-PAPP-A
IgG, or vehicle for 3 h at room temperature. Then approximately
100 ng IGFBP-3 or IGFBP-5 were added, and the incubation was
carried out at 37 C. A, IGFBP-5 was incubated with serum for 40 min,
2 h, and 5 h, respectively. B, IGFBP-3 was incubated with
serum for 10 and 22 h, respectively. The digested samples were
subjected to IGF-II ligand blot analysis.
|
|
Effect of PAPP-A in modulating IGFBP-4 availability and the
mitogenic activity of IGF-II
We previously demonstrated that IGFBP-4 protease in PS cleaved
IGFBP-4 between Met135 and
Lys136, and that the IGFBP-4 analog missing
residues 121142 exhibited similar IGF-binding activity
(30), but was resistant to IGFBP-4 protease in PS, as
determined by cell-free in vitro protease assays
(28). To evaluate the role of PAPP-A, we compared the
effect of wild-type IGFBP-4 (WTBP-4) and the protease-resistant IGFBP-4
analog (PRBP-4) on cell proliferation of MG63 cells treated with IGF-II
and human serum. MG63 cells were chosen because they do not produce
IGFBP-4 protease/PAPP-A (20, 30). As shown in Fig. 5A
, treatment with WTBP-4 (200 ng/mL) did
not reduce cell proliferation in the cultures treated with IGF-II (40
ng/mL) and PS (0.3%). Under identical conditions, the PRBP-4 analog
reduced cell proliferation by 30% (P < 0.01). In
contrast, treatment with the WTBP-4 and PRBP-4 inhibited cell
proliferation to a similar extent when cells were treated with IGF-II
and NPS (P > 0.05).

View larger version (34K):
[in this window]
[in a new window]
|
Figure 5. Effects of IGFBP-4 (WTBP-4) and PRBP-4 on
cell proliferation in MG63 cells treated with IGF-II and serum. A, MG63
cells were seeded in 96-well plates in DMEM/0.1% BSA at 1000
cells/well. After an overnight incubation, the indicated effectors were
added at the following concentrations: IGF-II, 40 ng/mL; WTBP-4 or
PRBP-4, 200 ng/mL; and PS or NPS, 0.3%. After an additional 72 h
of incubation, the nucleic acid contents in cells were determined.
Values (mean ± SEM; n = 8) labeled with
different letters are significantly different from each other
(P < 0.05). Similar results were obtained from
independent experiments in which 0.1% serum or 100 ng/mL IGFBP-4 was
used. B, CM from each treatment group was collected and pooled before
quantitation of the nucleic acid contents in the cells. Sixty
microliters of the pooled CM were subjected to IGF-II ligand blot
analysis.
|
|
IGF-II ligand blot analysis revealed approximately 50% degradation of
the PRBP-4 in the conditioned medium (CM) collected from cell cultures
treated with IGF-II and PS (Fig. 5B
). However, no substantial loss of
the PRBP-4 was evident in the CM collected from the cell cultures
treated with IGF-II or from the cell cultures treated with IGF-II and
NPS. Therefore, the partial degradation of PRBP-4 was contributable to
the proteases present in PS. In our previous studies no apparent
degradation of PRBP-4 was observed when 150 ng PRBP-4 was incubated
with 0.3 µL PS for 17 h at 37 C (28). In this study
PS was apparently overdosed relative to the amount of PRBP-4 in the CM
(0.3 µL PS:20 ng PRBP-4) and the incubation period was much longer
(72 h at 37 C). It is possible that PRBP-4 was cleaved at an
alternative site(s) in the presence of excess serum after a prolonged
incubation. Consistent with this speculation, results from cell-free
protease assays revealed that WTBP-4 (80 ng) was completely degraded
after incubation with 0.1 µL PS for 10 h at 37C, whereas very
little PRBP-4 was cleaved even after a 22-h incubation (Fig. 6
). However, when PRBP-4 was incubated
with a larger dose of PS (2 µL) for 22 h, a partial degradation
of PRBP-4 was observed regardless of the addition of PAPP-A antibody to
the assays. Although the 24-kDa N-terminal WTBP-4 proteolytic fragment
could be detected by IGF-II ligand blotting, no PRBP-4 proteolytic
fragments capable of binding to IGF-II were detectable. These data
suggest that the activity of the nonspecific proteases in PS capable of
cleaving PRBP-4 is extremely low compared with the activity of PAPP-A
on WTBP-4.

View larger version (43K):
[in this window]
[in a new window]
|
Figure 6. Effect of PAPP-A antibody on proteolysis of
PRBP-4. Eighty nanograms of either WTBP-4 or PRBP-4 were incubated with
indicated amount of PS and 50 ng IGF-II at 37 C for the indicated
period of time. The digested samples were then separated by a 12%
SDS-PAGE gel under nonreducing conditions and subjected to IGF-II
ligand blot analysis. The arrow shows the N-terminal
proteolytic fragment of WTBP-4.
|
|
As PAPP-A in PS is able to cleave IGFBP-4 in the absence of exogenously
added IGF-II (28) (Fig. 2
), we also compared the potencies
of WTBP-4 vs. PRBP-4 in inhibiting PS- or NPS-induced cell
proliferation. As PAPP-A activity is reduced in the absence of IGF-II,
WTBP-4 or PRBP-4 was preincubated with serum and then added to cell
culture medium. As shown in Fig. 7
, addition of IGF-II (40 ng/mL) significantly increased cell
proliferation for cells treated with NPS (group 3 vs. 5),
but not PS (group 4 vs. 6). Consistent with the data
presented in Fig. 5A
, cell proliferation was significantly inhibited by
PRBP-4, but not WTBP-4, when cells were preincubated with PS and IGF-II
(group 9 vs. 10). On the other hand, WTBP-4 and PRBP-4
treatments after preincubation with IGF-II and NPS were equally
inhibitory (group 7 vs. 8). In the absence of exogenous
IGF-II, PRBP-4, but not WTBP-4, significantly inhibited cell
proliferation induced by PS (group 13 vs. 14), whereas
PRBP-4 and WTBP-4 were equally potent in reducing NPS-induced cell
proliferation (group 11 vs. 12). These data demonstrate that
PRBP-4 is more potent than WTBP-4 in reducing cell proliferation
induced by PS alone or in combination with IGF-II.

View larger version (24K):
[in this window]
[in a new window]
|
Figure 7. Effects of WTBP-4 and PRBP-4 on cell
proliferation in MG63 cells treated with serum alone or in combination
with IGF-II. Trypsinized MG63 cells were washed with DMEM/0.1% BSA and
seeded in 96-well plates at 1000 cells/well. WTBP-4 or PRBP-4 peptide
(400 ng) was incubated with 2 µL serum and 80 ng IGF-II at 37 C for
15 h. The reaction mixture (24 µL) contained 0.1% BSA and 5
µL DMEM/1 mmol/L CaCl2. All effectors used in this
experiment were incubated at 37 C for 15 h. The preincubated
effectors were diluted with DMEM/0.1% BSA and added to cell cultures,
such that the final concentrations of IGF-II and IGFBP-4 peptide in the
cell culture medium were 40 and 200 ng/mL equivalent, respectively.
After a 72-h incubation, the cellular nucleic acid contents were
measured. The data shown here are representative of two independent
experiments. Values (mean ± SEM; n = 8) labeled
with different letters are significantly different from each other
(P < 0.05).
|
|
To further confirm that the lack of inhibition of WTBP-4 on cell
proliferation in the presence of PS was due to degradation of IGFBP-4
by PAPP-A, we preincubated WTBP-4 with PS in the presence of
anti-PAPP-A IgG or normal IgG and determined the consequence of
blockage of IGFBP-4 proteolysis on IGF-II-induced cell proliferation.
IGF-II ligand blot analysis confirmed that WTBP-4 was essentially
undetectable in the preincubated mixture of WTBP-4/IGF-II/PS/control
IgG (or vehicle), whereas no apparent loss of WTBP-4 was observed in
the preincubated mixture of WTBP-4/IGF-II/PS/anti-PAPP-A or
WTBP-4/IGF-II/NPS/anti-PAPP-A (Fig. 8A
).
The rate of cell proliferation was determined in cells treated with
these preincubated effectors (Fig. 8B
). In the presence of IGF-II (40
ng/mL) and 0.1% PS, WTBP-4 at a concentration of 200 ng/mL failed to
inhibit cell proliferation in the presence of normal control IgG,
whereas the inhibitory effect was restored in the presence of
anti-PAPP-A IgG. In contrast, addition of PAPP-A antibody had no effect
on the proliferation of the cells treated with WTBP-4, IGF-II, and NPS.
These data demonstrate that blocking PAPP-A-mediated IGFBP-4
proteolysis with anti-PAPP-A IgG can restore the inhibitory effect of
IGFBP-4 on cell proliferation, and that PAPP-A acts to regulate IGF
action through degrading IGFBP-4.

View larger version (41K):
[in this window]
[in a new window]
|
Figure 8. Effect of PAPP-A antibody on the inhibitory
effect of IGFBP-4 on cell proliferation. MG63 cells were seeded in
96-well plates in DMEM/1% CS at 1000 cells/well. After a 6-h
incubation, the medium was replaced with DMEM/0.1% BSA. Effectors were
added 24 h later. IGFBP-4 peptide (400 ng) was incubated with 2
µL serum and 80 ng IGF-II in the presence of 10 µg PAPP-A antibody
or normal IgG at 37 C for 15 h. The reaction mixture (24 µL)
buffer contained 0.1% BSA and 5 µL DMEM/1 mM
CaCl2. All effectors used in this experiment were incubated
at 37 C for 15 h to avoid potential artifacts. A, Two microliters
of preincubated mixture containing 33 ng added IGFBP-4 were subjected
to IGF-II ligand blot analysis. B, The preincubated effectors were
diluted with DMEM/0.1% BSA and added to cell cultures such that the
final concentrations of IGF-II and IGFBP-4 peptide in the cell culture
medium were 40 and 200 ng/mL equivalent, respectively. After a 72-h
incubation, the cellular nucleic acid contents were measured. The
experiment was performed in duplicate, and the data were pooled. Values
(mean ± SEM; n = 12) labeled with different
letters are significantly different from each other
(P < 0.05).
|
|
 |
Discussion
|
|---|
PAPP-A has recently been identified to be the IGF-dependent
IGFBP-4 protease produced by human fibroblasts (20). In
this study we clearly demonstrate that PAPP-A represents the
predominant, if not the sole, IGFBP-4 protease in PS and is distinct
from the pregnancy-induced IGFBP-3 proteases. Moreover, we provided
evidence for the first time that PAPP-A in part contributes to IGFBP-5
proteolytic activity induced during pregnancy and that PAPP-A in PS
plays an important role in regulating IGFBP-4 availability and, thus,
IGF activity in vitro.
In our recent studies we clearly demonstrated that the IGFBP-4
proteolytic activity in human PS was largely dependent on the presence
of IGF-II (28). However, significant proteolysis of
exogenously added IGFBP-4 was observed after prolonged incubation with
PS in the absence of exogenous IGF-II (28). As the amount
of total endogenous IGFs contained in the PS included in the assays is
far below the concentration of IGF-II required for protease activation,
the proteolysis of IGFBP-4 by PS in the absence of exogenous IGF-II
could not be explained by the possible activation of IGF-II-dependent
IGFBP-4 protease by the endogenous IGFs in the serum. It was therefore
speculated that a significant amount of IGF-II-independent IGFBP-4
protease might also be present in PS (28). However, this
speculation was not supported by the findings from this study. First,
depletion of PAPP-A from the PS with PAPP-A antibody abolished the
IGFBP-4 proteolysis observed in both absence and presence of added
IGF-II, even after an extensive digestion. Second, addition of PAPP-A
antibody directly to the PS dose dependently inhibited and, eventually,
abolished IGFBP-4 proteolysis. However, we observed that PRBP-4, which
lacks the previously identified cleavage site
(Met135-Lys136), was
partially cleaved when it was incubated with a much larger dose of PS
after a prolonged incubation in either cell cultures or cell-free
protease assays. This limited degradation was not due to the action of
PAPP-A, as the PAPP-A neutralization antibody did not affect
proteolysis of the PRBP-4. As the rate of the degradation of the PRBP-4
by nonspecific IGFBP-4 proteases in PS is extremely low, we conclude
that PAPP-A is the predominant IGFBP-4 protease induced during
pregnancy, which accounts for both the IGF-II-dependent and the
IGF-II-independent IGFBP-4 proteolytic activities in PS.
IGFBP-3 and IGFBP-5 proteolytic activities in human serum also increase
during pregnancy (14, 15, 17, 28). Although the identities
of these pregnancy-induced proteases have remained elusive, previous
studies in rats suggest that matrix metalloproteases contribute to
pregnancy-induced IGFBP-3 proteolysis (32, 33). In human
PS, proteases with molecular masses of more than 150 and 7090 kDa
were able to cleave IGFBP-3 (34). The 70- to 90-kDa
protease was determined to be plasminogen, whereas the identity of the
more than 150-kDa protease was not known. More recently, a 50-kDa
protease partially purified from human PS exhibited properties similar
to those of distintegrin metalloprotease and was able to cleave IGFBP-3
and IGFBP-5 (18). It was also reported that human placenta
trophoblasts secret a disintegrin metalloprotease similar to the
IGFBP-3 protease in human PS (35). More recently, Shi
et al. (36) showed that ADAM 12, a disintegrin
metalloprotease, exhibited IGFBP-3 proteolytic activity and was present
in PS. We recently reported that IGFBP-3 proteolytic activity in PS
was not enhanced by the addition of IGF-II; rather, high doses of
IGF-II inhibited proteolysis of IGFBP-3 (28). These
previous studies together with our finding that the addition of PAPP-A
antibody to PS had no effect on pregnancy-induced IGFBP-3 proteolysis
after both short-term and long-term incubations suggest that PAPP-A
does not contribute to IGFBP-3 proteolytic activity in PS.
It was recently reported that the addition of PAPP-A antibody had no
effect on IGFBP-5 proteolytic activity in follicular fluids, which
suggests that PAPP-A is not an IGFBP-5 protease (21). In
contrast, our data suggest that PAPP-A in PS may in part degrade
IGFBP-5, as the addition of PAPP-A antibody to PS substantially reduced
the proteolysis of IGFBP-5. In the previous study (21)
IGFBP-5 was apparently overdigested with the proteases in follicular
fluids, as very little intact IGFBP-5 remained. Under this condition,
determination of the relative contribution of IGFBP-5 proteases was not
allowed. This argument was supported by the findings that very little
effect of PAPP-A antibody on IGFBP-5 proteolysis was observed after 5-h
incubation with PS, whereas partial blockage was observed after the
incubation time was reduced to 40 min. These findings are consistent
with our preliminary findings that partially purified IGF-II-dependent
IGFBP-4 protease from human osteoblasts CM cleaved IGFBP-5, but not
IGFBP-3 (Qin, X., et al., unpublished data). However, unlike
the IGFBP-4 proteolytic activity in PS, the IGFBP-5 proteolytic
activity was only reduced, but was not completely blocked, by PAPP-A
antibody (Fig. 4A
). Therefore, although PAPP-A is the predominant
IGFBP-4 protease in PS, it may represent only one of the major serum
IGFBP-5 proteases induced by human pregnancy.
Next, we used two different approaches to test the hypothesis that
PAPP-A in PS plays a significant role in regulating IGFBP-4
availability and, consequently, IGF-II activity in vitro. In
the first approach, we compared the effect of PRBP-4 vs.
WTBP-4 in inhibiting the proliferation of cells treated with PS or NPS
alone or in combination with IGF-II. Consistent with our hypothesis, we
found that PRBP-4, but not WTBP-4, inhibited the proliferation of cells
incubated with PS and IGF-II. This difference was not observed in cells
treated with NPS and IGF-II under identical conditions. In addition,
our data suggest that PRBP-4 was more potent than WTBP-4 in inhibiting
the proliferation of cells treated with PS alone. Approximately 50% of
the 0.1% PS-induced cell proliferation was blocked by 200 ng/mL
PRBP-4. These results were consistent with previous findings that 100
ng/mL IGFBP-3 inhibited 0.2% PS-induced proliferation of chick
embryo fibroblasts by 75% (IGFBP-3 is more resistant to proteolysis
than IGFBP-4) and that IGFBP-3 was more inhibitory to the proliferation
of cells treated with NPS compared with PS (37). In the
second approach, we used PAPP-A blocking antibody to inactivate PAPP-A
in PS and analyzed the consequence of this inactivation. We found that
IGFBP-4 was able to inhibit IGF-II-induced cell proliferation when
PAPP-A was added along with PS. Consistent with this idea, we recently
reported that the endogenously produced PAPP-A by human osteoblasts may
also act to regulate IGF actions based on the findings that 1) PRBP-4
was much more potent than the WTBP-4 in inhibiting IGF-II-induced cell
proliferation in these cells (30); and 2) PAPP-A is the
predominant IGFBP-4 protease produced by these cells (38).
Taken together, these findings suggest that PAPP-A endogenously
produced by cells or PAPP-A circulating in PS can enhance the mitogenic
activity of IGFs by degrading the inhibitory IGFBP-4. In this regard,
it is tempting to speculate that PAPP-A produced by tissues such as
placenta may serve as both a local and a systemic cogrowth factor
through degrading IGFBP-4. As our data suggest that PAPP-A also
contributes to pregnancy-induced IGFBP-5 proteolytic activity in human
serum, future studies need to be carried out to determine the role of
PAPP-A in modulating the bioavailability as well as the biological
activity of IGFBP-5.
 |
Acknowledgments
|
|---|
We thank Drs. Chulhee Kim, Kyoil Suh, and Haehyeog Lee
(SoonChunHyang University Hospital, Seoul, Korea) for providing us with
human serum, and the Media Development Department at the J. L.
Pettis V.A. Medical Center for illustrations. We also thank Dr. John
Farley for valuable discussion, and Ms. Carol Farrell for assistance
with manuscript editing.
 |
Footnotes
|
|---|
1 This work was supported by NIH Grants R01-AR-45210 and
R03-AR-45081-01 (to X.Q.) and R01-AR-31062 (to S.M.), Loma Linda
University seed money grants (to X.Q.), and facilities of the J.
L. Pettis V.A. Medical Center. 
Received April 13, 2000.
Revised August 31, 2000.
Revised October 4, 2000.
Accepted October 13, 2000.
 |
References
|
|---|
-
Canalis E. 1993 Insulin-like growth factors
and the local regulation of bone formation. Bone. 14:273276.[Medline]
-
Rosen CJ, Donahue LA, Hunter SJ. 1994 Insulin-like
growth factors and bone: the osteoporosis connection. Proc Soc Exp Biol
Med. 206:83102.[Abstract]
-
Clemmons DR. 1997 Insulin-like growth factor
binding proteins and their role in controlling IGF actions. Cytokine
Growth Factor Rev. 8:4562.[CrossRef][Medline]
-
Mohan S, Baylink DJ. 1999 IGF system components
and their role in bone metabolism. In: Rosenfeld R, Roberts C, eds. The
IGF system. Totowa: Humana Press; 457496.
-
Baker J, Liu J, Robertson EJ, Efstratiadis A. 1993 Role of insulin-like growth factors in embryonic and postnatal
development. Cell. 75:7382.[CrossRef][Medline]
-
Nonoshita LD, Wathen NC, Dsupin BA, Chard T, Giudice
LC. 1994 Insulin-like growth factors (IGFs), IGF-binding proteins
(IGFBPs), and proteolyzed IGFBP-3 in embryonic cavities in early human
pregnancy: their potential relevance to maternal-embryonic and fetal
interactions. J Clin Endocrinol Metab. 79:12491255.[Abstract]
-
Giudice LC, Zegher FD, Gargosky SE, et al. 1995 Insulin-like growth factors and their binding proteins in the term and
preterm human fetus and neonate with normal and extremes of
intrauterine growth. J Clin Endocrinol Metab. 80:15481555.[Abstract/Free Full Text]
-
Sakai K, Iwashita M, Takeda Y. 1997 Profiles of
insulin-like growth factor binding proteins and protease activity in
the maternal circulation and its local regulation between placenta and
decidua. Endocr J. 44:409417.[Medline]
-
Baxter RC, Martin JL. 1989 Structure of the Mr
140,000 growth hormone-dependent insulin-like growth factor binding
protein complex: determination by redistribution and affinity labeling. Proc Natl Acad Sci USA. 86:68986902.[Abstract/Free Full Text]
-
Lee CY, Rechler MM. 1995 A major portion of the
150-kilodalton insulin-like growth factor binding protein (IGFBP)
complex in adult rat serum contains unoccupied, proteolytically nicked
IGFBP-3 that bind to IGF-II preferentially. Endocrinology. 136:668678.[Abstract]
-
Liu F, Baxter RC, Hintz RL. 1992 Characterization
of the high molecular weight insulin- like growth factor complex in
term pregnancy serum. J Clin Endocrinol Metab. 75:12611267.[Abstract]
-
Lassarre C, Binoux M. 1994 Insulin-like growth
factor binding protein-3 is functionally altered in pregnancy plasma. Endocrinology. 134:12541262.[Abstract]
-
Kanzaki S, Hilliker S, Baylink DJ, Mohan S. 1994 Evidence that human bone cells in culture produce insulin-like growth
factor-binding protein-4 and -5 proteases. Endocrinology. 134:383392.[Abstract]
-
Hossenlopp P, Sergovia B, Lassarre C, Roghani M, Bredon
M, Binoux M. 1990 Evidence of enzymatic degradation of
insulin-like growth factor-binding proteins in the 150K complex during
pregnancy. J Clin Endocrinol Metab. 71:797805.[Abstract]
-
Giudice LC, Farrell EM, Pham H, Lamson G, Rosenfeld
RG. 1990 Insulin-like growth factor binding proteins in maternal
serum throughout gestation and in the puerperium: effects of a
pregnancy-associated serum protease activity. J Clin Endocrinol
Metab. 71:806816.[Abstract]
-
Wang HS, Chard T. 1992 Chromatographic
characterization of insulin-like growth factor- binding proteins in
human pregnancy serum. J Endocrinol. 133:149159.[Abstract]
-
Claussen M, Zapf J, Braulke T. 1994 Proteolysis of
insulin-like growth factor binding protein-5 by pregnancy serum and
amniotic fluid. Endocrinology. 134:19641966.[Abstract]
-
Kubler B, Cowell S, Zapf J, Braulke T. 1998 Proteolysis of insulin-like growth factor binding proteins by a novel
50-kilodalton metalloproteinase in human pregnancy serum. Endocrinology. 139:15561563.[Abstract/Free Full Text]
-
Hasegawa T, Hasegawa Y, Takada M, Tsuchiya Y. 1995 The free form of insulin-like growth factor I increases in circulation
during normal human pregnancy. J Clin Endocrinol Metab. 80:32843286.[Abstract]
-
Lawrence JB, Oxvig C, Overgaard MT, Sottrup-Jensen L,
Gleich GJ, Hays LG, Yates III JR, Conover CA. 1999 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. 96:31493153.[Abstract/Free Full Text]
-
Conover CA, Oxvig C, Overgaard MT, Christiansen M,
Giudice LC. 1999 Evidence that the insulin-like growth factor
binding protein-4 protease in human ovarian follicular fluid is
pregnancy associated plasma protein-A. J Clin Endocrinol Metab. 84:47424745.[Abstract/Free Full Text]
-
Oxvig C, Haaningh J, Kristensen L, et al. 1995 Identification of angiotensinogen and coplement C3dg as novel proteins
binding the proform of eosinophil major basic protein in human
pregnancy serum and plasma. J Biol Chem. 270:1364513651.[Abstract/Free Full Text]
-
Folkersen J, Grudzinskas JG, Hindersson P, Teisner B,
Westergaard J. 1981 Pregnancy-associated plasma protein A:
circulating levels during normal pregnancy. Am J Obstet Gynecol. 139:910924.[Medline]
-
Westergaard J, Teisner B, Grudzinskas JG. 1983 Serum PAPP-A in normal pregnancy: relationship to fetal and maternal
characteristics. Arch Gynecol. 233:211216.[CrossRef][Medline]
-
Casals E, Aibar C, Martinez JM, et al. 1999 First
trimester biochemical markers for Downs syndrome. Prenatal Diagn. 19:811.[CrossRef][Medline]
-
Aitken DA, Ireland M, Berry E, Crossley JA, Macri JN,
Burn J, Connor JM. 1999 Second-trimester pregnancy associated
plasma protein-A levels are reduced in Cornelia de Lange syndrome
pregnancies. Prenatal Diagn. 19:706710.[CrossRef][Medline]
-
Westergaard JG, Chemnitz J, Teisner B, Poulsen HK, Ipsen
L, Beck B, Grudzinskas JG. 1983 Pregnancy-associated plasma
protein-A: a possible maker in the classification and prenatal
diagnosis of Cornelia de Lange syndrome. Prenatal Diagn. 3:225232.[Medline]
-
Byun D, Mohan S, Kim C, et al. 2000 Studies on the
human pregnancy-induced insulin-like growth factor binding protein
(IGFBP)- 4 poetesses in serum: determination of IGF-II dependency and
localization of cleavage site. J Clin Endocrinol Metab. 85:373381.[Abstract/Free Full Text]
-
Qin X, Strong D, Baylink DJ, Mohan S. 1998 Structure-function analysis of the human insulin-like growth factor
(IGF) binding protein (hIGFBP)-4. J Biol Chem. 273:2350923516.[Abstract/Free Full Text]
-
Qin X, Byun D, Strong DD, Baylink DJ, Mohan S. 1999 Studies on the role of human insulin-like growth factor-II (IGF-II)
dependent IGF binding protein (hIGFBP)-4 protease in human osteoblasts
using protease resistant IGFBP-4 analogs. J Bone Miner Res. 14:20792088.[CrossRef][Medline]
-
Scharla SH, Strong DD, Mohan S, Baylink DJ, Linkhart
TA. 1991 1,25-Dihydroxyvitamin D3
differentially regulates the production of insulin-like growth factor I
(IGF-I) and IGF-binding protein-4 in mouse osteoblasts. Endocrinology. 129:31393146.[Abstract]
-
Fowlkes JL, Suzuki K, Nagase H, Thrailkill KM. 1994 Proteolysis of insulin-like growth factor binding protein-3 during rat
pregnancy: a role for matrix metalloproteases. Endocrinology. 135:28102813.[Abstract]
-
Wu HB, Lee CY, Rechler MM. 1999 Proteolysis of
insulin-like growth factor binding protein-3 in serum from pregnant,
non-pregnant and fetal rats by matrix metalloproteases and serine
proteases. Horm Metab Res. 31:186191.[Medline]
-
Bang P, Fielder PJ. 1997 Human pregnancy serum
contains at least two distinct proteolytic activities with the ability
to degrade insulin-like growth factor binding protein-3. Endocrinology. 138:39123917.[Abstract/Free Full Text]
-
Irwin JC, Suen LF, Cheng BH, Martin R, Cannon P, Deal
CL, Giudice LC. 2000 Human placental trophoblasts secrete a
disintegrin metalloproteinase very similar to the insulin-like growth
factor binding protein-3 protease in human pregnancy serum. Endocrinology. 141:666674.[Abstract/Free Full Text]
-
Shi Z. Xu W. Loechel F. Wewer UM. Murphy LJ. 2000 ADAM 12, a disintegrin metalloprotease, interacts with insulin-like
growth factor-binding protein-3. J Biol Chem. 275:1857418580.[Abstract/Free Full Text]
-
Blat C, Villaudy J, Binoux M. 1994 In vivo
proteolysis of serum insulin-like growth factor (IGF) binding protein-3
results in increased availability of IGF to target cells. J Clin
Invest. 93:22862290.
-
Qin X, Byun D, Lau K-H. William, Baylink DJ, Mohan
S. 2000 Evidence that the interaction between insulin-like growth
factor (IGF)-II and IGF binding protein (IGFBP)-4 is essential for the
action of the IGF-II dependent IGFBP-4 protease. Arch Biochem Biophys. 379:209216.[CrossRef][Medline]
This article has been cited by other articles:

|
 |

|
 |
 
Y. Ning, A. G. P. Schuller, C. A. Conover, and J. E. Pintar
Insulin-Like Growth Factor (IGF) Binding Protein-4 Is Both a Positive and Negative Regulator of IGF Activity in Vivo
Mol. Endocrinol.,
May 1, 2008;
22(5):
1213 - 1225.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
C. A. Conover
Insulin-like growth factor-binding proteins and bone metabolism
Am J Physiol Endocrinol Metab,
January 1, 2008;
294(1):
E10 - E14.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
M. Rehage, S. Mohan, J. E. Wergedal, B. Bonafede, K. Tran, D. Hou, D. Phang, A. Kumar, and X. Qin
Transgenic Overexpression of Pregnancy-Associated Plasma Protein-A Increases the Somatic Growth and Skeletal Muscle Mass in Mice
Endocrinology,
December 1, 2007;
148(12):
6176 - 6185.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
S. C. Harrington, R. D. Simari, and C. A. Conover
Genetic Deletion of Pregnancy-Associated Plasma Protein-A Is Associated With Resistance to Atherosclerotic Lesion Development in Apolipoprotein E-Deficient Mice Challenged With a High-Fat Diet
Circ. Res.,
June 22, 2007;
100(12):
1696 - 1702.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
B. S Miller, J. T Bronk, T. Nishiyama, H. Yamagiwa, A. Srivastava, M. E Bolander, and C. A Conover
Pregnancy associated plasma protein-A is necessary for expeditious fracture healing in mice
J. Endocrinol.,
March 1, 2007;
192(3):
505 - 513.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
X. Qin, J. E. Wergedal, M. Rehage, K. Tran, J. Newton, P. Lam, D. J. Baylink, and S. Mohan
Pregnancy-Associated Plasma Protein-A Increases Osteoblast Proliferation in Vitro and Bone Formation in Vivo
Endocrinology,
December 1, 2006;
147(12):
5653 - 5661.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
Z. T. Resch, R. D. Simari, and C. A. Conover
Targeted Disruption of the Pregnancy-Associated Plasma Protein-A Gene Is Associated with Diminished Smooth Muscle Cell Response to Insulin-like Growth Factor-I and Resistance to Neointimal Hyperplasia after Vascular Injury
Endocrinology,
December 1, 2006;
147(12):
5634 - 5640.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
F. Prefumo, S. Canini, A. Crovo, D. Pastorino, P. L. Venturini, and P. De Biasio
Correlation between first trimester fetal bone length and maternal serum pregnancy-associated plasma protein-A (PAPP-A)
Hum. Reprod.,
November 1, 2006;
21(11):
3019 - 3021.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
Z. T. Resch, C. Oxvig, L. K. Bale, and C. A. Conover
Stress-Activated Signaling Pathways Mediate the Stimulation of Pregnancy-Associated Plasma Protein-A Expression in Cultured Human Fibroblasts
Endocrinology,
February 1, 2006;
147(2):
885 - 890.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
C. A. Conover, L. K. Bale, S. C. Harrington, Z. T. Resch, M. T. Overgaard, and C. Oxvig
Cytokine stimulation of pregnancy-associated plasma protein A expression in human coronary artery smooth muscle cells: inhibition by resveratrol
Am J Physiol Cell Physiol,
January 1, 2006;
290(1):
C183 - C188.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
A. Kumar, S. Mohan, J. Newton, M. Rehage, K. Tran, D. J. Baylink, and X. Qin
Pregnancy-associated Plasma Protein-A Regulates Myoblast Proliferation and Differentiation through an Insulin-like Growth Factor-dependent Mechanism
J. Biol. Chem.,
November 11, 2005;
280(45):
37782 - 37789.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
L. K Bale and C. A Conover
Disruption of insulin-like growth factor-II imprinting during embryonic development rescues the dwarf phenotype of mice null for pregnancy-associated plasma protein-A
J. Endocrinol.,
August 1, 2005;
186(2):
325 - 331.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
S. Oesterreicher, W. F. Blum, B. Schmidt, T. Braulke, and B. Kubler
Interaction of Insulin-like Growth Factor II (IGF-II) with Multiple Plasma Proteins: HIGH AFFINITY BINDING OF PLASMINOGEN TO IGF-II AND IGF-BINDING PROTEIN-3
J. Biol. Chem.,
March 18, 2005;
280(11):
9994 - 10000.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
M. Matsui, B. Sonntag, S. S. Hwang, T. Byerly, A. Hourvitz, E. Y. Adashi, S. Shimasaki, and G. F. Erickson
Pregnancy-Associated Plasma Protein-A Production in Rat Granulosa Cells: Stimulation by Follicle-Stimulating Hormone and Inhibition by the Oocyte-Derived Bone Morphogenetic Protein-15
Endocrinology,
August 1, 2004;
145(8):
3686 - 3695.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
C. A. Conover, L. K. Bale, M. T. Overgaard, E. W. Johnstone, U. H. Laursen, E.-M. Fuchtbauer, C. Oxvig, and J. van Deursen
Metalloproteinase pregnancy-associated plasma protein A is a critical growth regulatory factor during fetal development
Development,
March 1, 2004;
131(5):
1187 - 1194.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
Z. T. Resch, B.-K. Chen, L. K. Bale, C. Oxvig, M. T. Overgaard, and C. A. Conover
Pregnancy-Associated Plasma Protein A Gene Expression as a Target of Inflammatory Cytokines
Endocrinology,
March 1, 2004;
145(3):
1124 - 1129.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
G. M. Rivera and J. E. Fortune
Selection of the Dominant Follicle and Insulin-Like Growth Factor (IGF)-Binding Proteins: Evidence that Pregnancy-Associated Plasma Protein A Contributes to Proteolysis of IGF-Binding Protein 5 in Bovine Follicular Fluid
Endocrinology,
February 1, 2003;
144(2):
437 - 446.
[Abstract]
[Full Text]
[PDF]
|
 |
|