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Original Studies |
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
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 4–6 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 Down’s 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 15–30 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 (200–300 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 20–30 min each time. The membranes were exposed to x-ray film for 3–10 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 manufacturer’s (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 1–5 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 50–90 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 1–6, 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 1–6, 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 1–6, 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.
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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 0.01–1 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.
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). 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.
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We previously demonstrated that IGFBP-4 protease in PS cleaved
IGFBP-4 between Met135 and
Lys136, and that the IGFBP-4 analog missing
residues 121–142 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).
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). 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.
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), 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.
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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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 70–90 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.
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Acknowledgments |
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Footnotes |
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Received April 13, 2000.
Revised August 31, 2000.
Revised October 4, 2000.
Accepted October 13, 2000.
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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]
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