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Studies on Human Pregnancy-Induced Insulin-Like Growth Factor (IGF)-Binding Protein-4 Proteases in Serum: Determination of IGF-II Dependency and Localization of Cleavage Site¹

J. L. Pettis Veterans Administration Medical Center (D.B., S.M., D.J.B., X.Q.), Loma Linda, California 92357; and the Departments of Endocrinology (C.K., K.S., M.Y.) and Gynecology and Obstetrics (H.L.), Soon Chun Hyang University Hospital, Seoul, Korea

Address all correspondence and requests for reprints to: Dr. Xuezhong Qin, Musculoskeletal Disease Center, J. Pettis Veterans Administration Medical Center (151), 11201 Benton Street, Loma Linda, California 92357. E-mail: Xuezhong.Qin{at}med.va.gov

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Insulin-like growth factor (IGF)-binding protein-4 (IGFBP-4), a consistent inhibitor of IGF action, is subject to proteolytic cleavage by the IGF-II-dependent IGFBP-4 protease. However, regulation of the IGF-II-dependent IGFBP-4 protease in vivo is not known. As IGFBP proteases are known to be triggered during pregnancy, we systematically evaluated the changes in IGFBP-4 proteolysis by serum collected throughout human pregnancy. Results from in vitro protease assays using recombinant IGFBP-4 revealed that IGFBP-4 proteolysis determined in both the presence and absence of exogenous IGF-II significantly increased during the first and second trimesters and reached a plateau by the third trimester. However, in the absence of IGF-II, IGFBP-4 proteolysis by pregnancy serum was only observed after prolonged incubation. IGF-II dose dependently increased IGFBP-4 proteolysis by pregnancy serum, with maximal stimulation observed at a concentration of 0.7 mol/L relative to IGFBP-4. In contrast, IGF-II at an equimolar dose had little effect on proteolysis of recombinant human IGFBP-3, whereas excess IGF-II reproducibly inhibited recombinant human IGFBP-3 proteolysis by pregnancy serum. Although IGF-II enhanced IGFBP-4 proteolysis, results from N-terminal sequence and mass spectrometric analyses of IGFBP-4 proteolytic fragments demonstrate that the cleavage site (Met¹³⁵-Lys¹³⁶) in human IGFBP-4 was not altered by IGF-II. Deletion of the residues 121–141 containing this cleavage site blocked IGFBP-4 proteolysis. These findings demonstrate that the increase in IGFBP-4 proteolysis during pregnancy was accounted for mainly by the IGF-II-dependent IGFBP-4 proteolysis. Because IGFBP-4 is a potent inhibitor of IGF actions, it can be speculated that the pregnancy-induced IGFBP-4 proteases may play an important role in regulating fetal growth.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
THE INSULIN-LIKE growth factors (IGFs) are important growth factors that act both systemically and locally to promote proliferation and differentiation in a variety of cell types (1, 2, 3, 4, 5). Administration of IGFs exhibits growth-promoting effects in various tissues, including bone growth in experimental animal models (6) and somatic growth in GH receptor-deficient children (7). In contrast, IGF-I gene knockout in mice led to growth retardation (8). The biological activity of IGFs is dependent not only on the amount of IGF produced, but also on the concentrations of the six high affinity IGF-binding proteins (IGFBPs) in body fluids that modulate IGF actions and bioavailability (1, 2, 3, 4, 5). In serum, 75- 80% of IGF circulates as a 150- to 200-kDa complex, and the remaining IGFs circulate as a 50-kDa complex. Less than 1% of IGF is known to circulate as a free form (5). The 150- to 200-kDa complex primarily consists of IGF, IGFBP-3, and the acid-labile subunit (ALS) (5, 9, 10). However, recent studies demonstrate that IGFBP-5 also forms a ternary complex with ALS and IGFs (11, 12). The size of this ternary complex would preclude it from crossing the vascular endothelial barrier into the extravascular space (5). Thus, the IGF/IGFBP-3/ALS complex must be dissociated for IGFs to be delivered to target tissues. The 50-kDa complex consists of IGF and one of the remaining five IGFBPs (5). Although these smaller IGF/IGFBP complexes can cross the vascular endothelium, they must also be dissociated in the extracellular compartment to release IGFs to interact with cell surface receptors to elicit a biological response. One of the mechanisms by which the IGFBP/IGF complex can be disrupted is through the proteolysis of IGFBPs by proteases. In this regard, various IGFBP proteases have been reported to be present in serum, especially in pregnancy serum (13, 14, 15, 16), and in the conditioned medium (CM) of cell cultures (17, 18, 19, 20, 21, 22, 23). It is believed that proteolysis of IGFBPs by proteases in the circulation and extracellular space plays an important role in increasing the local concentrations of free IGFs when they are needed.

Among the six IGFBPs, IGFBP-4 is the only IGFBP that consistently and potently inhibits IGF-II actions in a variety of cell types under a number of experimental conditions (24, 25, 26, 27, 28, 29, 30). Therefore, degradation of IGFBP-4 by IGFBP-4 protease appears to be particularly important in modulating IGF actions. Previous studies from our laboratory and others have shown that specific IGF-II-dependent IGFBP-4 proteases are produced and secreted by many cell types in vitro (17, 18, 19, 20, 21, 22, 23). The physiological significance of IGFBP-4 protease in regulating IGFBP-4 availability and thus the mitogenic activity of IGFs is emphasized by the following findings. 1) The IGF-II-dependent IGFBP-4 protease cleaves IGFBP-4 into fragments that bind to IGFs with little or no affinity and thus do not inhibit IGF-induced cell proliferation (27, 28, 31). 2) IGFBP-4 analogs that are resistant to IGFBP-4 protease exhibited higher potency in blocking IGF-I- or IGF-II-induced cell proliferation (27, 31, 32). However, the physiological role and regulation of IGFBP-4 protease in vivo are poorly understood. Recently, Kubler and co-workers reported that a 50-kDa serum metalloprotease induced by human pregnancy was able to cleave IGFBP-3, -4, and -5 (33). During the preparation of this manuscript, Lawrence et al. (34) reported that pregnancy-associated plasma protein A (PAPP-A), a protein previously purified from human pregnancy serum (35), was identical to the IGF-II-dependent IGFBP-4 protease produced by human fibroblasts. In addition, proteolysis of other IGFBPs, such as IGFBP-2, IGFBP-3, and IGFBP-5, is increased during pregnancy (13, 14, 15, 16, 33). Therefore, down-regulation of IGFBP availability via specific proteolysis may play an important role in increasing the local concentrations of free IGFs, which are essential for normal fetal growth.

The purpose of this study was to systematically determine the IGFBP-4 proteolytic activity throughout human pregnancy and determine whether IGFBP-4 proteolytic activity induced by pregnancy can be enhanced by IGF-II. Our data suggest that the dramatic increase in IGFBP-4 proteolysis by serum during pregnancy was accounted for mainly by IGF-II-dependent IGFBP-4 proteases and that the pregnancy-induced IGFBP-4 protease(s) cleaves IGFBP-4 at a site identical to that recognized by the IGF-dependent IGFBP-4 protease produced by human osteoblasts.

	Materials and Methods

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Reagents

Recombinant human IGF-I and IGF-II were obtained from Bachem (Torrance, CA). ¹²⁵I was purchased from NEN Life Science Products (Boston, MA). Recombinant 6xHis-tagged wild-type human (h) IGFBP-4 and IGFBP-4 mutant were expressed in Escherichia coli and purified sequentially by nickel-agarose affinity and IGF-I-agarose affinity chromatography as previously described (30, 31). The wild-type IGFBP-4 containing five residues from the signal peptide and the entire mature IGFBP-4 sequence was designated 6xHis-BP-4(-5/237). The IGFBP-4 analog lacking residues His¹²¹ to Pro¹⁴² was designated 6xHis-BP-4(121–142). Human osteoblast (hOB)-CM was prepared as previously described (31).

Serum collection

Serum samples from nonpregnant and pregnant Korean women were collected in Soon Chun Hyang Hospital according to the approved research protocol, shipped on dry ice to the United States, and stored at -80 C before use. Eighteen pregnant Korean women (5 in the first trimester, 10 in the second trimester, and 3 in the third trimester), 12 postpartum women, and 6 nonpregnant women were included in this study. All subjects were age matched and free of other diseases (Table 1).

Serum concentrations of IGFBP-4 were quantitated by RIA, as previously described (36).

IGFBP-4 protease assay

The IGFBP-4 protease assay was conducted by incubating the recombinant IGFBP-4 with serum in the presence or absence of IGF-II. Assay buffer, unless noted otherwise, contained 150 ng IGFBP-4 peptide, 3 µL of 10-fold diluted serum, 100 ng IGF-II or vehicle (20 mmol/L acetic acid), and 8 µL DMEM with 0.5 mmol/L CaCl₂. After a 17-h incubation at 37 C, the samples were mixed with SDS-PAGE loading buffer, separated on 10% SDS-PAGE gels, transferred to nitrocellulose membrane, and subjected to either IGFBP-4 immunoblot or IGF-II ligand blot analysis, as previously described (36, 37). For quantitation of IGFBP-4 protease activity, the amount of uncleaved IGFBP-4 was determined by directly counting the radioactivity in the bands. The IGFBP-4 protease activity was expressed as the percentage of IGFBP-4 cleaved compared to the total IGFBP-4 added to the assay buffer.

N-Terminal amino acid sequence analysis

Two micrograms of 6xHis-BP-4(-5/237) were digested with 1 µL undiluted trimester serum in the presence of 0.5 µg IGF-II or vehicle. DMEM containing 500 µmol/L CaCl₂ was added to a final volume of 50 µL. After 40 h of digestion, samples were mixed with mercaptoethanol (5 µL) and SDS-PAGE loading buffer, separated on 12% SDS-PAGE gel, and transferred to a nitrocellulose membrane (Problott, PE Applied Biosystems, Foster City, CA). After Coomassie blue staining, the 14-kDa band was cut out and subjected to N-terminal amino acid sequencing using the Edman degradation procedure in the Peptide Analysis Laboratory at the California Institute of Technology (Pasadena, CA).

Mass spectrometric analysis

To determine the molecular weights of the IGFBP-4 proteolytic fragments, 800 ng 6xHis-BP-4(-5/237) were digested with 0.25 µL third trimester serum or 5 µL of 50-fold concentrated hOB-CM in the presence of 200 ng IGF-II or vehicle for 17 h at 37 C. To remove the high molecular weight protein contaminants, digested samples were subjected to ultrafiltration using a membrane with a 50-kDa exclusion limit. To increase the recovery of the IGFBP-4 proteolytic fragments, filters were washed twice with 100 µL 75% acetonitrile 0.1% trifluoroacetic acid. The samples were dried under negative pressure, resuspended in 3 µL water, loaded to reverse phase chips, and subjected to mass spectrometric analysis on surface-enhanced laser desorption and ionization (SELDI Protein Biosystem, Ciphergen, Inc., Palo Alto, CA).

Statistical analysis

The differences in serum concentrations of IGFBP-4 and IGF-II and IGFBP-4 protease activity among groups were analyzed by ANOVA, followed by multiple comparison. The data were expressed as the mean ± SEM.

	Results

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IGFBP-4 protease activity in the sera from nonpregnant and pregnant women at various stages

To systematically study the changes in serum IGFBP-4 proteolytic activity during pregnancy, recombinant IGFBP-4 was incubated with serum in the presence of IGF-II, and the degradation of IGFBP-4 was visualized after immunoblot or IGF-II ligand blot analysis of the digested samples. To monitor the background of IGF-II binding to endogenous IGFBPs, serum from each stage of pregnancy was incubated under identical conditions except for the absence of recombinant IGFBP-4. As shown in Fig. 1, no detectable IGF-II-binding activity around 31 kDa was observed from 0.3 µL serum alone under the experimental conditions used in this study. Therefore, the 31-kDa [¹²⁵I]IGF-II-labeled band represented the undigested recombinant IGFBP-4, 6xHis-BP-4(-5/237), which migrated approximately 5 kDa slower than the native IGFBP-4, mainly due to the presence of the 6xHis tag (30, 31). As revealed by both the IGF-II ligand blot (Fig. 1B) and the IGFBP-4 immunoblot (Fig. 1C), IGFBP-4 proteolysis by the nonpregnancy serum was negligible. Degradation of IGFBP-4 by serum increased dramatically during the first and second trimesters and peaked by the third trimester of pregnancy.

View larger version (50K): [in this window] [in a new window]   Figure 1. Proteolysis of IGFBP-4 by nonpregnancy and pregnancy sera. To prepare a pooled sample for each group, an equal volume of serum from each individual sample was mixed. The pooled serum samples were used to perform protease assays. The assay mixture contained 150 ng 6xHis-BP-4(-5/237) peptide, 100 ng IGF-II, 10 µL DMEM containing 0.5 mmol/L CaCl2, and 3 µL concentrated hOB-CM or 3 µL of 10-fold diluted serum. Water was added to adjust the final volume to 20 µL. Two sets of protease assays were prepared. After 17 h of incubation, the reaction was terminated by adding SDS-PAGE loading buffer containing no reducing agent. One digestion set was subjected to separation on 10% gel by SDS-PAGE followed by Coomassie blue staining (A). The second digestion set was split into two equal aliquots and subjected to IGF-II ligand blot analysis (B) and IGFBP-4 immunoblot analysis (C), respectively. Under nonreducing conditions, 6xHis-BP-4(-5/237) migrated as a 31-kDa protein (19 ). The data shown in this experiment were confirmed in a duplicate experiment.

The activity of IGFBP-4 protease produced by a variety of cell types, including osteoblasts, is largely dependent on the presence of IGF-II (17, 19, 27, 31). To determine whether IGFBP-4 protease(s) in the pregnancy serum depends on IGF-II for maximal activity, we determined the rate of IGFBP-4 proteolysis by nonpregnancy and pregnancy sera in the presence or absence of IGF-II. As shown in Fig. 2A, nonpregnant serum cleaved little or no IGFBP-4 regardless of IGF-II supplementation. Under both conditions (presence or absence of IGF-II), IGFBP-4 degradation by serum increased dramatically during the first and second trimesters. However, the rate of IGFBP-4 proteolysis was markedly enhanced when IGF-II was added to the protease assays. Figure 2B shows quantitative data from additional experiments in which IGFBP-4 protease activity was determined using individual samples. IGFBP-4 protease activity in the presence of IGF-II showed a significant increase during the first trimester and reached a plateau by the second trimester. IGFBP-4 protease activity measured in the absence of IGF-II also increased with pregnancy, reaching a maximum by the third trimester (P < 0.001). During the third trimester, although serum IGFBP-4 protease activity was numerically higher in the presence of IGF-II than in the absence of IGF-II, the difference was not statistically significant.

View larger version (44K): [in this window] [in a new window]   Figure 2. Serum IGFBP-4 protease activity in the presence and absence of IGF-II. A, IGF-II ligand blot analysis of IGFBP-4 digested with serum. Proteolysis of the wild-type IGFBP-4, 6xHis-BP-4(-5/237) by pooled serum in the absence of IGF-II (vehicle) or presence of IGF-II. Protease assay was carried out as described in Fig. 1. Digested sample was subjected to IGF-II ligand blot analysis. B, Percent degradation of IGFBP-4 by nonpregnancy and pregnancy sera. IGFBP-4 proteolysis was performed using subpooled serum samples (n = 3–4) as described in Fig. 1. For quantitation of IGFBP-4 protease activity, the amount of intact IGFBP-4 was determined by directly counting the radioactivity in the bands. IGFBP-4 protease activity was expressed as the percent degradation of the added IGFBP-4. In the absence of IGF-II (solid triangle), values (mean ± SEM) labeled without the same uppercase letter were different from each other (P < 0.05). In the presence of IGF-II (solid circle), values (mean ± SEM) labeled without the same lowercase letter were differentfrom each other (P < 0.05). Addition of IGF-II significantly (P < 0.05) increased IGFBP-4 protease activity in the first and second trimester sera.

View larger version (64K): [in this window] [in a new window]   Figure 3. Effects of varying amounts of serum (A) and IGF-II (B) on IGFBP-4 proteolysis. In A, 150 ng 6xHis-BP-4(-5/237) peptide were incubated with various amounts of third trimester serum as indicated in the presence of 100 ng IGF-II or vehicle under the buffer conditions described in Fig. 1. After 3.5-h incubation at 37 C, the digested samples were subjected to IGF-II ligand blot analysis as previously described (35 ). In B, 150 ng 6xHis-BP-4(-5/237) was incubated with 0.1 µL third trimester serum in the presence of various amounts of IGF-II. After 3.5-h incubation at 37 C, the digested samples were subjected to IGF-II ligand blot analysis.

View larger version (39K): [in this window] [in a new window]   Figure 4. Effect of IGF-II on IGFBP-3 proteolysis by human nonpregnancy and pregnancy sera. Forty nanograms of rhIGFBP-3 were incubated with 2 µL undiluted nonpregnancy or pregnancy serum in the presence of 10 or 100 ng IGF-II or vehicle under the buffer conditions described in Fig. 1. After 23 h of incubation at 37 C, the digested samples were subjected to IGF-II ligand blot analysis. The dose of serum and the incubation time were chosen based on a time-course and dose-response experiment. A similar experiment was performed twice. The upper arrow indicates the endogenous glycosylated IGFBP-3 present in the serum. The lower arrow indicates the exogenously added nonglycosylated rhIGFBP-3.

As determination of the proteolytic site will provide important information on the nature of the proteases, we sought to localize the cleavage site in IGFBP-4 recognized by the pregnancy-induced IGFBP-4 proteases and determine whether addition of IGF-II alters the cleavage site. To localize the cleavage site, 6xHis-BP-4(-5/237) peptide was digested with third trimester pregnancy serum in the presence or absence of IGF-II. Consistent with the data shown in Figs. 2 and 3, addition of IGF-II enhanced IGFBP-4 proteolysis. In both conditions, two proteolytic fragments of 24 and 14 kDa were visualized on SDS-PAGE gels under reducing conditions (Fig. 5A). As the 24-kDa band bound to IGF with reduced affinity (32), it represented the N-terminal portion of the 6xHis-BP-4(-5/237). Therefore, the 14-kDa bands were cut out and subjected to N-terminal amino acid sequence analysis. It was found that Lys¹³⁶ was the N-terminus of the 14-kDa IGFBP-4 fragment, suggesting that the pregnancy-induced IGFBP-4 protease cleaved IGFBP-4 between Met¹³⁵ and Lys¹³⁶ (Fig. 5B). This cleavage site was not altered by IGF-II (Fig. 5B). To determine whether the IGFBP-4 proteases induced by pregnancy cleaved IGFBP-4 at additional sites, 6xHis-BP-4(-5/237) digested with third trimester serum or hOB-CM was subjected to mass spectrometric analysis. As a small volume of serum (0.25 µL) did not contain substantial amounts of proteins with molecular masses in the range of 15–20 kDa, it was possible to directly determine the masses of IGFBP-4 fragments without extensive protein purification. To avoid overloading the reverse phase sample application chips, the high molecular mass proteins in the serum (mainly albumin) were removed by ultrafiltration. As shown in Table 2, the observed masses of the two proteolytic fragments were consistent with the expected molecular masses. This result indicated that the N- and C-terminal proteolytic fragments contained 135 N-terminal and 102 C-terminal mature IGFBP-4 residues, respectively. The addition of IGF-II did not alter the mass of either fragment. These results clearly defined Met¹³⁵-Lys¹³⁶ as the primary cleavage site recognized by the pregnancy-induced IGFBP-4 protease(s) in serum, and IGF-II did not alter the cleavage site, although it enhanced IGFBP-4 proteolysis.

View larger version (48K): [in this window] [in a new window]   Figure 5. N-Terminal amino acid sequence analysis of the C-terminal IGFBP-4 fragment. 6xHis-BP-4(-5/237) was digested with third trimester serum in the presence of IGF-II or vehicle as described in Materials and Methods. Digested samples were separated on 12% SDS-PAGE gel under reducing conditions, transferred to a nitrocellulose membrane (Blott, PE Applied Biosystems), and stained with Coomassie blue (A). The 14-kDa bands shown in A were cut out and subjected to N-terminal amino acid sequencing using the Edman degradation procedure (B).

View larger version (53K): [in this window] [in a new window]   Figure 6. Proteolysis of the 6xHis-BP-4(-5/237) and 6xHis-BP-4(121–141) by hOB-CM and pregnancy serum. One hundred and fifty nanograms of either 6xHis-BP-4(-5/237) or 6xHis-BP-4(121–141) were digested with 3 µL of 50-fold concentrated hOB-CM or 3 µL of 10-fold diluted third trimester serum in the presence of 100 ng IGF-II. After 17 h of digestion, the samples were subjected to IGF-II ligand blot analysis. The data shown in this experiment were confirmed in a duplicate experiment.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

During pregnancy, rapid fetal growth obviously increases the need for growth-promoting hormones such as IGFs. As the mitogenic activity of IGFs at the local cellular level depends on the concentrations of free IGFs that are able to interact with their cell surface receptors, proteolysis of IGFBPs by specific proteases is believed to be important in maintaining the concentrations of free IGFs during pregnancy. Results from our studies revealed that IGFBP-4 protease activity was essentially undetectable in nonpregnancy serum, but dramatically increased during the first and second trimesters of pregnancy and remained highly active during the third trimester. Despite a noticeable decrease in IGFBP-4 proteolysis by postpartum serum compared with third trimester serum observed in some of our experiments, this decrease was not statistically significant. The lack of a significant decrease in IGFBP-4 proteolysis by postpartum sera was apparently due to the fact that postpartum sera were collected only 2–3 days after delivery (postpartum serum was not readily obtainable once the mothers were discharged from the hospital, usually within 2–3 days). Importantly, our results showed for the first time that the addition of exogenous IGF-II enhanced IGFBP-4 proteolysis by the pregnancy serum. Under modified assay conditions (i.e. reduced digestion time and/or amount of pregnancy serum), the majority of IGFBP-4 was cleaved in the presence of IGF-II, whereas little IGFBP-4 was degraded in the absence of IGF-II. These data suggest that the increase in IGFBP-4 proteolysis during pregnancy was mainly contributed to by the IGF-II-dependent IGFBP-4 proteolysis.

In contrast to the stimulatory effect of IGF-II on IGFBP-4 proteolysis, the proteolysis of IGFBP-3 by either nonpregnancy or pregnancy serum was not affected by IGF-II at a dose that is sufficient to stimulate IGFBP-4 proteolysis. Excess IGF-II appears to inhibit IGFBP-3 proteolysis, an observation that was not seen with IGFBP-4. These data together with our recent finding that the partially purified IGF-II-dependent IGFBP-4 protease from hOB-CM did not cleave IGFBP-3 (Qin et al., unpublished data) suggest that the pregnancy-induced IGFBP-4 protease is different from the pregnancy-increased IGFBP-3 protease.

During preparation of this manuscript, Lawrence et al. (34) reported that a 200-kDa glycoprotein previously purified from human pregnancy serum, designated PAPP-A (35), was responsible for the IGFBP-4 protease activity in human fibroblast-CM. Similar to the IGFBP-4 proteases produced by cells in culture (17, 19, 27, 31), the activity of PAPP-A is largely dependent on the presence of exogenous IGF-II (34). Although the cleavage site in IGFBP-4 recognized by PAPP-A was not determined, indirect evidence suggests that PAPP-A cleaves IGFBP-4 between Met¹³⁵ and Lys¹³⁶ based on the observation that the IGF-II-dependent IGFBP-4 protease produced by human fibroblasts, which is identical to PAPP-A, cleaves IGFBP-4 between Met¹³⁵-Lys¹³⁶ (27). Our data clearly demonstrate that the pregnancy-induced IGFBP-4 proteases cleaved IGFBP-4 between Met¹³⁵ and Lys¹³⁶. Taken together, these findings suggest that PAPP-A induced by pregnancy contributes to the IGF-II-dependent IGFBP-4 proteolysis induced during pregnancy. However, whether multiple IGF-II-dependent IGFBP-4 proteases that cleave IGFBP-4 at the same site (Met¹³⁶-Lys¹³⁶) are induced during pregnancy needs to be further studied.

Although our data clearly demonstrate that IGF-II-dependent IGFBP-4 proteolysis was predominant in pregnancy-induced IGFBP-4 proteolysis, proteolysis of exogenously added IGFBP-4 was observed after prolonged incubation with pregnancy serum. As 0.3 µL pregnancy serum contained less than 0.3 ng total IGFs, which is far below the concentration required for protease activation, the proteolysis of IGFBP-4 by pregnancy serum in the absence of exogenous IGF-II could not be explained by the possible activation of IGF-II-dependent IGFBP-4 protease by endogenous IGFs in serum. These data raise the possibility that the IGF-II-independent IGFBP-4 proteases may also be induced during pregnancy. In previous studies, matrix metalloproteases (MMPs) such as MMP-1 and MMP-3, are suggested to be increased during pregnancy (38). Recently, Kubler et al. reported that increased IGFBP-3, -4, and -5 proteolysis by human pregnancy serum was associated with a novel 50-kDa metalloprotease (33). These pregnancy-associated proteases are able to cleave not only IGFBP-4 but also other IGFBPs (33, 38). It is possible that these nonspecific proteases may contribute to the IGF-II-independent IGFBP-4 proteolysis observed in our studies. Alternatively, the IGF-II-dependent IGFBP-4 protease may also cleave IGFBP-4 in the absence of IGF-II but with a lower efficiency based on the finding that IGFBP-4 was cleaved at the same site by pregnancy serum in both the presence and absence of IGF-II. However, this hypothesis needs to be confirmed by future studies using specific blocking antibodies for IGF-II-dependent or IGF-II-independent IGFBP-4 proteases.

Although the potency of the pregnancy-induced IGFBP-4 protease is sufficient to degrade all of the endogenously produced IGFBP-4 based on our data, the concentrations of IGFBP-4 in the serum determined by RIA did not correlate to the IGFBP-4 protease activity. We believe that this lack of correlation was due to the fact that the polyclonal IGFBP-4 antibody used for RIA recognized both the intact and the proteolytic IGFBP-4 fragments. Measurements of both total (intact and fragments) and intact IGFBP-4 may prove useful to monitor IGFBP-4 proteolysis in body fluids under different physiological and pathological conditions.

In summary, the increase in IGFBP-4 proteolytic activity during pregnancy was accounted for mainly by the IGF-II-dependent and, to a lesser extent, the IGF-II-independent IGFBP-4 proteolysis. IGF-II enhances IGFBP-4 proteolysis by pregnancy serum, but did not alter the cleavage site (Met¹³⁵-Lys¹³⁶) in hIGFBP-4. It is conceivable that the IGFBP-4 proteases, in addition to other IGFBP proteases induced by pregnancy, may play a critical role in maintaining the availability of free IGFs in the fetal tissues and, consequently, normal fetal growth.

	Acknowledgments

 
We thank Joe-Rung Aroon and Daniel Bruch for excellent technical support, and the Medical Media Development Department at the J. L. Pettis V.A. Medical Center for illustrations. We also thank Ms. Carol Farrell for secretarial help.

	Footnotes

 
¹ This work was supported by NIH Grants R03-DE-12142–01 and R0–3-AR-45081–01 (to X.Q.) and R01-AR-31062 (to S.M.), and Loma Linda University seed money grants (to X.Q.).

Received June 16, 1999.

Revised September 30, 1999.

Accepted October 11, 1999.

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