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Endocrinology Vol. 141, No. 2 666-674
Copyright © 2000 by The Endocrine Society

ARTICLES

Human Placental Trophoblasts Secrete a Disintegrin Metalloproteinase Very Similar to the Insulin-Like Growth Factor Binding Protein-3 Protease in Human Pregnancy Serum1

Juan C. Irwin, Lii-Fang Suen, Bi-Hua Cheng, Robert Martin, Paul Cannon, Cheri L. Deal and Linda C. Giudice

Department of Gynecology and Obstetrics (J.C.I., L.-F.S., L.C.G.), Stanford University Medical Center, Stanford, California 94305-5317; Department Obstetrics & Gynecology (B.-H.C.), Kaohsiung Medical College Hospital, 80708 Taiwan; Roche Bioscience (R.M., P.C.), Palo Alto, California 94304; and Department of Pediatrics (C.L.D.), Hôpital Sainte-Justine, Montréal, Québec, H3T 1CS Canada

Address all correspondence and requests for reprints to: Linda C. Giudice, M.D., Ph.D., Department of Gynecology & Obstetrics, Stanford University Medical Center, Stanford, California 94305-5317. E-mail: giudice{at}stanford.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
During the course of human pregnancy, there is a marked increase in insulin-like growth factor (IGF) binding protein (IGFBP)-3 protease activity in maternal serum that is first evident at 6 weeks of gestation, persists through term, and returns to nonpregnancy levels by day 5 postpartum. This protease activity cleaves IGFBP-3 into smaller fragments that have markedly reduced affinity for the IGFs. To date, the precise identity and cellular origin of the pregnancy-associated serum IGFBP-3 protease have not been established. To investigate whether placental and/or decidual tissues, which uniquely develop during pregnancy, may be sources of the pregnancy-associated serum IGFBP protease, we examined the secretion of IGFBP-3 protease in vitro by isolated human cytotrophoblasts or fibroblasts from second trimester placentae and by in vitro decidualized human endometrial stromal cells. Cytotrophoblasts were either cultured alone, which favors aggregation and fusion, or cocultured with decidualized endometrial stromal cells, which favors differentiation to an invasive phenotype. IGFBP-3 protease activity was detected in trophoblast, but not in placental fibroblast or decidualized endometrial cultures, and was also present in trophoblast-endometrial cocultures. Western ligand blot and Western immunoblot analyses showed that most of the endogenous IGFBP-3 in trophoblast cultures was in the form of low molecular weight fragments with reduced IGF binding affinity. The substrate specificity of the trophoblast-derived protease was identical to that in pregnancy serum, showing activity against IGFBP-2, -3, and -4, but being inactive against IGFBP-1. IGFBP-3 proteolysis by both pregnancy serum and trophoblast conditioned medium showed a major peak of activity at neutral pH. The trophoblast-derived activity caused time- and temperature-dependent proteolysis of IGFBP-3 into fragments of identical size as those produced by pregnancy serum, and also shared its sensitivity to protease inhibitors: highly sensitive to EDTA and o-phenanthroline, partially sensitive to the serine protease inhibitors AEBSF and aprotinin, and insensitive to {alpha}2-antiplasmin, and to aspartic and cysteine protease inhibitors. IGFBP-3 proteolysis by both pregnancy serum and trophoblast conditioned medium was also insensitive to tissue inhibitor of metalloproteinase-1, precluding the involvement of the matrix metalloproteinases. In contrast, both the pregnancy serum- and trophoblast-derived proteases were preferentially inhibited by a hydroxamic acid derivative with selective activity against the disintegrin-metalloproteinase tumor necrosis factor-{alpha} converting enzyme. This study shows that placental trophoblasts produce an IGFBP-3 protease with characteristics very similar to the activity found in pregnancy serum and indicates these cells at the maternal-fetal interface are a potential source of the pregnancy-associated serum IGFBP-3 protease. The findings further suggest that the main IGFBP-3 protease activity in both pregnancy serum and trophoblast conditioned medium may correspond to a disintegrin-metalloproteinase type enzyme.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE INSULIN-LIKE growth factors (IGFs) are peptides structurally related to insulin that participate in the regulation of growth and differentiation in a variety of cells and tissues, by virtue of their mitogenic, metabolic, and antiapoptotic effects (1). The IGF system also comprises a family of soluble IGF binding proteins (IGFBPs) that serve as carriers and modulators of the biological actions of IGFs (2, 3, 4, 5). There are six distinct IGFBPs, all of which share structural homology and bind IGFs with equal or higher affinity than the cellular IGF receptors, thereby modulating the bioavailability of IGF peptides to target cells. In addition, at least two of the IGFBPs (IGFBP-1 and -3) are known to have IGF-independent effects which result from their direct interaction with cell surface receptor molecules (6, 7, 8). Another level of regulation in the IGF system is provided by a heterogeneous group of proteases that specifically cleave one or more of the IGFBPs (9, 10, 11). Proteolysis of IGFBPs brings about changes in their biological activity resulting in altered IGF and/or insulin ligand binding (12, 13, 14), and may also affect their IGF-independent actions (7, 8, 15).

The existence of the physiologic phenomenon of IGFBP proteolysis was first realized with the discovery of IGFBP-3 protease activity in the serum of pregnant women (16, 17), mice (18), and rats (19). In humans, this activity is detected as early as 6 weeks of gestation and persists throughout pregnancy, returning to nearly undetectable levels of nonpregnancy serum by day 5 post partum. The IGFBP protease activity in pregnancy serum was originally identified and characterized based on its activity against IGFBP-3, the major carrier of IGFs in adult human serum. However, pregnancy serum has also been found to be proteolytically active against IGFBP-2, -4, and -5 (16, 17, 18, 19, 20), suggesting that proteolysis may account for the virtual absence of intact IGFBP-2, -3, and -4 in the peripheral circulation during pregnancy, detected by ligand blotting techniques (16, 17). In contrast, IGFBP-1 appears to be resistant to this protease(s) (16, 17).

The pregnancy-associated serum IGFBP protease cleaves IGFBP-3 into smaller fragments that have markedly reduced affinity for the IGFs. The proteolysis of circulating IGFBPs during pregnancy causes a net reduction of the total IGF binding capacity of serum (16, 17), resulting in higher levels of free circulating IGFs and a corresponding increase of IGF bioactivity of pregnancy serum (12). These observations support an important role for this protease in the physiologic changes of the maternal IGF system during pregnancy. However, to date, the precise molecular identity of the pregnancy-associated serum IGFBP-3 protease has not been established. Furthermore, the origin of this protease is also unknown. In view of the temporal pattern in which IGFBP-3 proteolytic activity appears in maternal serum, it is possible that the pregnancy-associated serum protease may be produced by placental and/or uterine decidual tissues, which uniquely develop during pregnancy and are shed after delivery. In the present study, we have investigated isolated human placental cells and decidualized endometrial cells as potential sources of the pregnancy-associated serum IGFBP-3 protease. Our findings show that placental trophoblast cells produce a neutral IGFBP-3 protease with characteristics very similar to the activity found in pregnancy serum, suggesting these cells at the maternal-fetal interface are a source of the pregnancy-associated serum IGFBP-3 protease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissues
The present study was approved by the Stanford University Committee on the Use of Human Subjects in Medical Research. Tissue and blood samples were obtained after informed consent, in accordance with the guidelines of The Declaration of Helsinki. Samples (n = 8) from second trimester placentae (gestational ages 12–20 weeks) were obtained immediately after vacuum aspiration and collected in DMEM (Life Technologies, Inc., Grand Island, NY). Histologically normal endometrium (n = 3) was obtained at the time of endometrial biopsy or hysterectomy and collected in DMEM. Blood samples were obtained by phlebotomy from normally cycling women and from pregnant women admitted at term to the Stanford University Hospital Labor and Delivery suite. Blood was collected in Vacutainer SST tubes for serum separation (Becton Dickinson and Co., Rutherford, NJ), centrifuged at 3000 rpm for 10 min, and serum decanted. Nonpregnancy serum samples were pooled, aliquoted, and stored at -80 C until analyzed, as were the term pregnancy samples.

Cell cultures
Placental trophoblast cultures were established according to the method of Fisher et al. (21), as described previously (22). Briefly, cytotrophoblasts were isolated from second trimester placentae by sequential collagenase-hyaluronidase-DNase, and trypsin-EDTA-DNase digestions, followed by Percoll gradient centrifugation. Isolated cytotrophoblasts (105/2 cm2) were plated on 16-mm diameter culture wells coated with 2–5 µg/cm2 human plasma fibronectin (Roche Molecular Biochemicals, Indianapolis, IN), and cultured in serum-free medium [75% DMEM (Life Technologies, Inc.), 25% MCDB-104, 50 µg/ml ascorbic acid, 1 mg/ml RIA grade BSA, 10 µg/ml human transferrin (all from Sigma, St. Louis, MO)]. As shown by immunocytochemistry, cell cultures prepared by this procedure consist predominantly (>90%) of cytokeratin-positive cytotrophoblasts. Trophoblast cultures contained no macrophages, <1% vascular cells positive for smooth muscle actin, and <2% vimentin-positive fibroblasts (22). Identity of the cultured trophoblast cells was further confirmed by their ability to produce human CG and progesterone (22). Placental villous core fibroblasts were isolated and cultured as described by Fisher et al. (21). Endometrial stromal cell cultures established and passaged as previously described (23), were decidualized in vitro for 14 days with estradiol, progesterone, epidermal growth factor, and insulin in the above serum-free medium, as described (24). These cultures were used because they are inherently free of trophoblastic elements that may contaminate pregnancy decidual tissue. Isolated cytotrophoblasts (105/2 cm2) were then plated on the decidualized multilayers and cocultured for up to 15 days in the above stromal cell medium. The villous cytotrophoblasts constitute a stem cell population of the trophoblastic lineage that aggregate and fuse in vitro when cultured on a plastic substrate (25, 26) or differentiate to an invasive phenotype when cultured on a Matrigel matrix (27, 28) or on decidualized endometrial stromal multilayers (22). Considering the possibility that IGFBP-3 production may be characteristic of a particular trophoblast subpopulation, placental cytotrophoblasts were cultured on fibronectin-coated plastic substrate that induces aggregation and fusion or on decidualized endometrial stromal multilayers which results in an invasive phenotype. Experiments were conducted under serum-free conditions and avoided the use of Matrigel, because both serum and Matrigel are known to contain proteases that could complicate the interpretation of these studies. Two-day conditioned media were collected from trophoblast, fibroblast, and endometrial stromal cultures, as well as from the co-cultures, then clarified by centrifugation, aliquoted, and stored at -80 C until analyzed.

Protease assays
IGFBP-3 protease activity was assayed using as substrates either radioiodinated IGFBP-3 (29), or the nonlabeled recombinant protein. Accordingly, either 50–100 ng of recombinant nonglycosylated human IGFBP-3 (a gift from Dr. A. Sommer, Celtrix Pharmaceuticals, Inc. Santa Clara, CA), or 10,000–30,000 cpm 125I-labeled recombinant glycosylated human IGFBP-3 (Diagnostics Systems Laboratories, Inc. Webster, TX) were incubated, for various periods of time as indicated, with 1–5 µl term pregnancy or nonpregnancy sera, 10–50 µl conditioned medium, 2 mU purified human plasmin (Calbiochem, San Diego, CA), or 0.12–1.2 pmol stromelysin-1 [matrix metalloproteinase-3 (MMP-3), purified from serum-free media of IL-1-stimulated human dermal fibroblasts with an antistromelysin antibody column]. Following incubation, samples were heated 5 min at 95 C in SDS-sample buffer and the reaction products resolved by 12% nonreducing SDS-PAGE. When using the nonradioactive substrate, samples were subsequently analyzed by Western immunoblotting as described below. With the radiolabeled substrate, following electrophoresis, gels were dried and exposed to radiographic film. Relative band intensities were quantified by scanning densitometry using a PDI Desk Top Scanner (Protein DNA ImageWare Systems, Huntington Station, NY). For inhibitor studies, the following protease inhibitors were added as indicated at the beginning of the incubation period: 5 mM EDTA, 2 mg/ml aprotinin, 5 mM 1,10-phenanthroline, 10 µM E64, 1.5 µM pepstatin, 6 mg/ml soybean trypsin inhibitor (all from Sigma), 5 mIU/ml {alpha}2-antiplasmin (Calbiochem), 4 mM Pefablock (Roche Molecular Biochemicals), 0.2–5 µM recombinant human TIMP-1, or 0.3–1000 µM of the synthetic inhibitors 103158, 113456 (30), or 160281 (Roche Bioscience, Palo Alto, CA). All three synthetic inhibitors have similar broad specificity for the matrix metalloproteinases (IC50 ranging from 0.054 to 82 nM for collagenases, gelatinases, and stromelysins). However, the membrane-bound disintegrin metalloproteinase tumor necrosis factor-{alpha} converting enzyme (TACE), is selectively inhibited by 160281 (IC50 290 nM in a cell-based assay) compared with 103158 (IC50 >10,000 nM), and 113456 (IC50 36,000 nM). Additional samples were incubated with 1% (vol/vol) of the various solvents used to prepare inhibitor stock solutions (methanol, dimethylsulfoxide, PBS) as vehicle controls. To assess the pH dependence of IGFBP-3 proteolysis, samples of serum or conditioned medium were adjusted to pH 2–10 in with either HCl or NaOH before the addition of the IGFBP-3 substrate. In substrate specificity studies, samples of term pregnancy serum (3 µl), conditioned medium (50 µl), or purified plasmin (10 mU) were incubated with 100 ng of either IGFBP-1 purified from human amniotic fluid (Diagnostics Systems Laboratories, Inc.), recombinant nonglycosylated human IGFBP-3, recombinant nonglycosylated human IGFBP-4 (Austral Biologicals, San Ramon, CA), or with 50 ng recombinant human IGFBP-2 (Diagnostics Systems Laboratories, Inc.) for 24 h at 37 C. Following incubation, samples were heated 5 min at 95 C in SDS-sample buffer and analyzed by Western ligand blotting to assess IGFBP-1, -3, and -4 proteolysis, or by Western immunoblotting to assess IGFBP-2 and IGFBP-3 proteolysis.

Western blots
Western ligand blotting and immunoblotting were performed essentially as described previously (31). Briefly, samples of serum (2 µl), amniotic fluid (0.5 µl), seminal plasma (5 µl), conditioned media (75 µl), or protease assay incubations, were heated 5 min at 95 C in SDS-sample buffer, subjected to 12% nonreducing SDS-PAGE, and electroblotted onto nitrocellulose. For Western ligand blotting, according to the method of Hossenlopp et al. (32), membranes were incubated with 0.5 x 106 cpm of each [125I]IGF-I and [125I]IGF-II overnight at 48 C, washed, dried, and exposed to radiographic film for 4–8 days. For Western immunoblotting, primary rabbit antisera to human IGFBP-2 (Upstate Biotechnologies Inc., Lake Placid, NY), or human IGFBP-3 (a gift from Dr. R. G. Rosenfeld, Portland, OR) were used diluted 1:1000, and secondary peroxidase-conjugated donkey antibodies to rabbit immunoglobulins (Amersham Pharmacia Biotech, Arlington Heights, IL) were diluted 1:2000. Membranes were blocked with 5% nonfat milk in TBST (0.15 M NaCl, 0.01 M Tris, pH 7.4, 0.1% Tween-20) for 2 h, then incubated with primary antiserum followed by the secondary conjugated antibodies. Incubations were 1 h at room temperature, and followed by three washes with TBST. Antigen-antibody complexes were visualized by enhanced chemiluminescence (ECL) and exposure to radiographic film, using ECL reagents from Amersham Pharmacia Biotech according to manufacturer’s instructions.

Statistics
Statistical analysis was conducted using the StatView 4.5 software (Abacus Concepts, Inc., Berkeley, CA). One- or two-way ANOVA were used for statistical evaluation of densitometric data. Statistical significance of the differences between individual group means were determined by post hoc testing using Fisher’s protected least significant difference (PLSD), with significance set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Initial experiments examined whether isolated cells from the placenta-decidual interface produced IGFBP-3 proteolytic activity. Cytotrophoblasts were isolated from second trimester placentae (n = 8), and endometrial stromal cells from nonpregnant endometrium were induced to undergo decidual differentiation in vitro by treatment with ovarian steroids (23, 24), as described in Materials and Methods. As shown in Fig. 1Go, radiolabeled IGFBP-3 remained unchanged after a 5 h incubation with culture medium that had not been in contact with cells (lanes a and b). Incubation with term pregnancy serum (lanes c and d), but not with nonpregnancy serum (lanes e and f), resulted in proteolysis of IGFBP-3 with the generation of cleavage products of 18, 22, and 30 kDa. Conditioned medium from 16-week placental trophoblasts cultured alone (lanes g and h) or cocultured with decidualized cells (lanes k and l) also proteolyzed IGFBP-3 into 18-, 22-, and 30-kDa fragments. However, conditioned medium from the decidualized cells alone (lanes i and j) had no detectable IGFBP-3 protease activity. Because trophoblast cultures may contain placental villous core fibroblast contaminants, whether the latter might be a source of IGFBP-3 protease was also investigated. These results showed that conditioned medium from 15-week placental fibroblast cultures (lanes m and n) had no detectable IGFBP-3 protease activity, whereas trophoblast cultures derived from two different 18-week placentae (lanes o-r) consistently produced IGFBP-3 protease. Identical results were obtained using placental fibroblasts isolated from a 12-week gestation (data not shown).



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  		Figure 1. IGFBP-3 protease activity in conditioned media from isolated human placental cytotrophoblasts, decidualized endometrial stromal cells, endometrial-trophoblast cocultures, and placental fibroblasts. [125I]IGFBP-3 was mixed with culture medium that had not been in contact with cells (M, lanes a and b), term-pregnancy serum, (PS, lanes c and d), nonpregnancy serum (NS, lanes e and f), conditioned medium from cultures of cytotrophoblast isolated from a 16-week placenta (T, lanes g and h), conditioned medium from decidualized endometrial stromal cells either alone (D, lanes i and j) or cocultured with 16-week placental cytotrophoblasts (D+T, lanes k and l), conditioned medium from fibroblasts isolated from a 20-week placenta (PF, lanes m and n), or cytotrophoblast cultures established from two different 18-week placentae (T1 and T2, lanes o through r). Samples were either mixed immediately with SDS-sample buffer and heated 5 min at 95 C (0 h, lanes a, c, e, g, i, k, m, o, q), or incubated 5 h at 37 C (5 h, lanes b, d, f, h, j, l, n, p, r), before being processed for electrophoresis and autoradiography. Mr (kilodaltons) for intact IGFBP-3 (BP-3) and IGFBP-3 proteolytic fragments (*) are indicated on the left margin. Differences in the intensities of intact IGFBP-3 bands in lanes a–i and m–r are compounded effects of differences in the total number of counts in the reaction mix (all of which is loaded onto the gel), and the use of 20 vs. 15 lanes for SDS-PAGE.

 
Conditioned media from trophoblast cultures derived from three different placentae were analyzed by Western ligand blotting to detect all IGF binding proteins (Fig. 2AGo) and by Western immunoblotting to detect immunoreactive IGFBP-3 (Fig. 2BGo). Nonpregnancy serum, seminal plasma, and amniotic fluid were used as internal controls. Nonpregnancy serum (lane a) showed the prominent 37/43 kDa IGFBP-3 doublet, as well as major 32 kDa IGFBP-2, and 24 kDa IGFBP-4 bands (Fig. 2AGo), and contained both intact and proteolyzed immunoreactive IGFBP-3 (Fig. 2BGo). Seminal plasma (lane b) containing only IGFBP-2 and -4 (33) served as a negative control for immunoreactive IGFBP-3, whereas amniotic fluid (lane c) served as a positive control for IGFBP-1 (34), and contains immunoreactive IGFBP-3 only in proteolyzed form (35, 36). Conditioned media from placental fibroblasts (lane d) showed IGFBPs comigrating with IGFBP-3, -2, and -4 (Fig. 2AGo), and intact, but not proteolyzed, immunoreactive IGFBP-3 (Fig. 2BGo), consistent with the absence of IGFBP-3 protease activity (Fig. 1Go). Trophoblast conditioned media (lanes e-g) contained variable amounts of intact IGFBP-3, ranging from abundant to barely detectable, as evidenced by both IGF binding activity (Fig. 2AGo) and immunoreactivity (Fig. 2BGo). However, in all cases immunoreactivity corresponded predominantly to proteolyzed IGFBP-3, consistent with endogenous IGFBP-3 proteolysis in trophoblast cultures (Fig. 1Go).



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  		Figure 2. Evidence of endogenous IGFBP-3 proteolysis in trophoblast but not in placental fibroblast cultures. A, Western ligand blot analysis of samples of nonpregnancy serum (NS, lane a), seminal plasma (SP, lane b), amniotic fluid (AF, lane c), conditioned medium from a 20-week placental fibroblasts culture (PF, lane d), or conditioned media from cytotrophoblast cultures established from (n = 3) different placentae, gestational ages 18 (T1, lane e), 18 (T2, lane f), and 21 (T3, lane g) weeks. B, Same membrane shown in panel A, subsequently analyzed by Western immunoblotting with an antibody to IGFBP-3. Mr (kilodaltons) for the various IGFBPs and the IGFBP-3 proteolytic fragments are indicated on the left margin. Similar results were obtained in n = 2 additional experiments.

 
Because the proteolytic activity of pregnancy serum is not restricted to IGFBP-3 and affects other IGFBPs, substrate specificity of the of the trophoblast IGFBP protease was investigated, comparing the proteolytic activity of trophoblast conditioned medium and pregnancy serum on IGFBP-1, -2, -3, and -4 substrates (Fig. 3Go). Following incubation, the integrity of IGFBP-1 (panel A), -3 (panel B), and -4 (panel C) were assessed by Western ligand blotting, and that of IGFBP-2 (because of limited availability) by immunoblotting (panel D). All IGFBPs were stable under the assay conditions, remaining unchanged after incubation with culture medium that had not been in contact with cells, (lanes a and b). Incubation with trophoblast conditioned medium (lanes c and d) or pregnancy serum (lanes e and f) resulted in degradation of IGFBP-2, -3, and -4, as evidenced by a marked reduction of IGFBP-3 (panel B), and -4 (panel C) bands in Western ligand blots, and the generation of lower molecular weight immunoreactive IGFBP-2 cleavage products (panel D). The presence of endogenous proteolyzed IGFBP-2 in pregnancy serum results in the detection of immunoreactive proteolytic fragments before incubation (panel D, lane e). Purified plasmin (lanes g and h), included as positive control, also proteolyzed IGFBP-2, -3, and -4. In contrast, IGFBP-1 (panel A), was consistently resistant to all three proteases, remaining unchanged under conditions which resulted in proteolysis of the other IGFBPs.



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  		Figure 3. Substrate specificity of IGFBP proteolytic activity in pregnancy serum and trophoblast conditioned medium. Purified human IGFBP-1 (BP-1, panel A), recombinant nonglycosylated human IGFBP-3 (BP-3, panel B), recombinant nonglycosylated human IGFBP-4 (BP-4, panel C), or recombinant human IGFBP-2 (BP-2, panel D), were mixed with culture medium that had not been in contact with cells (M, lanes a and b), or with trophoblast conditioned medium (T-CM, lanes c and d), term-pregnancy serum (PS, lanes e and f), or purified plasmin (lanes g and h). Samples were analyzed before (0 h, lanes a, c, e, g) and after incubation at 37 C for 24 h (24 h, lanes b, d, f, h) to determine IGFBP proteolysis. IGFBP-1, -3, and -4 proteolysis were assessed by Western ligand blotting (panels A–C), and IGFBP-2 proteolysis by Western immunoblotting (panel D). Positions of intact (BP-2) and proteolyzed (*) IGFBP-2 are indicated on the left margin in panel D.

 
The trophoblast protease was further characterized kinetically and with regard to temperature and pH optimum, using the radiolabeled IGFBP-3 assay (Fig. 4Go). IGFBP-3 proteolysis by trophoblast conditioned medium and pregnancy serum followed a similar time course, being close to maximal after 8 h of incubation (Fig. 4AGo), and both activities were optimal at 37 C (Fig. 4BGo). Both trophoblast conditioned medium and pregnancy serum displayed optimal IGFBP-3 protease activity at neutral pH, (Fig. 4CGo). Furthermore, at neutral pH, the trophoblast-derived activity proteolyzed IGFBP-3 into fragments of identical size as those produced by pregnancy serum (compare lanes k–n in Fig. 4Go, D and E). However, trophoblast conditioned medium showed, in addition, a discrete peak of activity at pH 3 (Fig. 4CGo). IGFBP-3 proteolysis by trophoblast conditioned medium at acid pH corresponded to a clearly distinct activity that produced a distinct profile of cleavage products with an additional larger fragment of 34 kDa (Fig. 4EGo, lanes g and h).



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  		Figure 4. Time-, temperature-, and pH-dependent IGFBP-3 proteolysis by pregnancy serum and trophoblast conditioned medium. Term-pregnancy serum (PS, open circles) or trophoblast conditioned medium (T-CM, solid circles) were incubated with [125I]IGFBP-3: (A) at 37 C and neutral pH for the indicated time, (B) at neutral pH for 5 h at the indicated temperature, or (C–E) at 37 C for 5 h at the indicated pH. Following incubation samples were analyzed by electrophoresis and autoradiography as described in Fig. 1Go. The extent of proteolysis was assessed by scanning densitometry of the radiograms. Values represent the band density of proteolyzed [125I]IGFBP-3 (after subtraction of background determined from respective time 0 samples run in parallel) expressed as a percentage of total band density (intact plus proteolyzed [125I]IGFBP-3) for each sample. Data are the mean ± SEM (bars) from n = 2 experiments. Representative radiograms illustrating pH-dependent IGFBP-3 proteolysis by term-pregnancy serum and trophoblast conditioned medium are shown in panels D and E, respectively. Term-pregnancy serum (PS, panel D) or trophoblast conditioned medium (T-CM, panel E) were incubated with [125I]IGFBP-3 at 37 C for 5 h at the indicated pH (lanes f–o). Time 0 samples (lane e) shown for term-pregnancy serum (PS, panel D) and trophoblast conditioned medium (T-CM, panel E) are at neutral pH. In both panels, time 0 (lanes a and c), and 5 h incubations (lanes b and d) of negative controls include [125I]IGFBP-3 mixed with culture medium that had not been in contact with cells (BP-3, lanes a and b), and nonpregnancy serum, (NS, lanes c and d). Mr (kilodaltons) for intact IGFBP-3 and proteolytic fragments (*) are indicated in the left margin.

 
The pregnancy serum and the trophoblast-derived neutral IGFBP-3 protease activities had superimposable inhibitor profiles (Fig. 5Go). Both were strongly inhibited by the metal ion chelators EDTA and o-phenanthroline, which is characteristic of metalloproteinases. Partial inhibition was observed with AEBSF and aprotinin which inhibit serine protease activity, but the specific inhibitor of plasmin, {alpha}2-antiplasmin, which also inhibits plasminogen activators, was ineffective. The aspartic protease inhibitor pepstatin, and the cysteine protease inhibitor E64 were also without effect on neutral IGFBP-3 proteolysis by either pregnancy serum or trophoblast conditioned medium. The trophoblast acid protease was selectively inhibited by the cysteine protease inhibitor E64 (data not shown).



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  		Figure 5. Pregnancy serum and trophoblast neutral IGFBP-3 protease inhibitor profiles. Term-pregnancy serum (PS, hatched bars, panel A) or trophoblast conditioned medium (T-CM, solid bars, panel B) were incubated with [125I]IGFBP-3 at neutral pH for 5 h at 37 C without further additions (Control), or in the presence of the following inhibitors: 10 µM E64, 5 mM EDTA, 1.5 µM pepstatin, 4 mM AEBSF, 2 mg/ml aprotinin, 5 mM 1,10-phenanthroline (o-Phenan), or 5 mIU/ml {alpha}2-antiplasmin (a2-AP). Vehicle control samples were incubated with 1% (vol/vol) of the methanol (MeOH) or PBS solvents used to prepare inhibitor stock solutions. Following incubation samples were analyzed by electrophoresis and autoradiography as described in Fig. 1Go. Proteolysis was assessed by scanning densitometry as described in Fig. 4Go and is expressed as a percentage of the mean value of the respective controls incubated without inhibitors. Data are the mean ± SEM (bars) of two experiments, except for trophoblast conditioned medium with {alpha}2-antiplasmin which corresponds to one experiment. *, Significantly different from respective control group (P < 0.01).

 
Matrix metalloproteinases (MMPs) constitute a multigene family of zinc-requiring enzymes involved in processes of tissue remodeling and breakdown that degrade components of the extracellular matrix and are inhibited by specific tissue inhibitors of metalloproteinases (TIMPs) (37, 38). The MMPs have been implicated in IGFBP proteolysis in various systems (9, 11), including rat pregnancy serum (39) and media conditioned by human fibroblasts (40), smooth muscle cells (41), or murine osteoblasts (42). Because the MMPs are widely distributed in biological fluids and also produced by a variety of cells in culture, we investigated the possible contribution of MMPs to IGFBP-3 proteolysis by pregnancy serum and/or trophoblast conditioned medium. To this end, TIMP-1 and the synthetic metalloproteinase inhibitors 103158, 113456 (30), and 160281 were investigated as potential inhibitors of IGFBP-3 proteolysis by pregnancy serum and trophoblast conditioned medium. All three synthetic inhibitors have similar broad specificity for MMPs (IC50 ranging from 0.054 to 82 nM for collagenases, gelatinases, and stromelysins). However, 160281 selectively inhibits (IC50 290 nM in a cell-based assay) the membrane-bound disintegrin metalloproteinase TACE (43), which is insensitive to both 103158 (IC50 >10,000 nM), and 113456 (IC50 36,000 nM). In these experiments, stromelysin-1 (MMP-3) was included as a positive control for MMP-dependent IGFBP-3 proteolysis (40). As shown in Fig. 6Go, IGFBP-3 proteolytic activities of pregnancy serum (panel A) and trophoblast conditioned medium (panel B) were not affected by 0.2 µM TIMP-1 (lane c), or 3 µM of either 113456 (lane e) or 103158 (lane f), but were both inhibited by the nonspecific metalloproteinase inhibitor 1,10-phenanthroline (lane g). In comparison, IGFBP-3 proteolysis by stromelysin-1 (panel C) was completely inhibited by TIMP-1 (lane c), and also by the inhibitors 113456 (lane e) or 103158 (lane f), as well as by 1,10-phenanthroline (lane g). Additional experiments (Fig. 6DGo) showed that IGFBP-3 protease activity in pregnancy serum was insensitive to even higher concentrations of TIMP-1 (up to 5 µM), and 113456 (1 mM). In contrast, at 1 mM, the TACE-specific inhibitor 160281 significantly inhibited the IGFBP-3 proteases activity of pregnancy serum (59 ± 5% of control; P < 0.05). The trophoblast-derived IGFBP-3 protease also displayed selective sensitivity to the TACE-specific inhibitor, being significantly inhibited (51 ± 11% of control; P < 0.01) with 10–100 µM 160281, while being resistant to similar concentrations of 113456 (Fig. 6DGo). (These observed differences may reflect the complex nature of serum wherein, among its numerous components, a variety of molecules may have the capacity to bind or sequester 160281.) A differential sensitivity to TACE-specific inhibitor 160281 (IC50 10–100 µM) compared with 113456 (IC50 100–500 µM) was consistently observed with conditioned media from trophoblast cultures derived from two different placentae.



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  		Figure 6. Effects of matrix metalloproteinase inhibitors on IGFBP-3 proteolysis by term-pregnancy serum, trophoblast conditioned medium, and stromelysin-1 (panels A–C). Term-pregnancy serum (PS, panel A), trophoblast conditioned medium (T-CM, panel B), or recombinant activated human stromelysin-1 (MMP-3, panel C), mixed with recombinant nonglycosylated IGFBP-3 at neutral pH with no further addition are shown before (t0, lane a), or after 20 h incubation at 37 C (controls "C", lane b). Additional samples were incubated 20 h at 37 C in the presence of 0.2 µM TIMP-1 (TIMP, lane c), 1% vol/vol DMSO vehicle (V, lane d), 3 µM of the inhibitors 113456 (lane e) or 103158 (lane f), or 5 mM 1,10-phenanthroline (o-Ph, lane g). Samples were analyzed by Western immunoblotting with an antihuman IGFBP-3 antibody to reveal intact and proteolyzed IGFBP-3. Results were confirmed in at least two experiments for all inhibitors except 1031568. D, Quantitative assessment of the effects of metalloproteinase inhibitors on IGFBP-3 proteolysis by term-pregnancy serum, and trophoblast conditioned medium. Term-pregnancy serum (PS, hatched bars) or trophoblast conditioned medium (T-CM, solid bars) were incubated with recombinant nonglycosylated IGFBP-3 at neutral pH for 20 h at 37 C in the presence of TIMP-1 (0.2–5 µM), or of the inhibitors 113456 or 160281. The latter inhibitors were added at a final concentration of 1 mM to pregnancy serum, and 10–100 µM to conditioned media. Following incubation samples were analyzed by Western immunoblotting as described in Fig. 2Go. Proteolysis was assessed by scanning densitometry as described in Fig. 4Go, and is expressed as a percentage of the mean value of the respective controls incubated with vehicle alone. Data are the mean ± SEM (bars) of at least two experiments. *, Significantly different (P < 0.05) from respective control and 113456 groups. {dagger}, Significantly different (P < 0.01) from respective control and 113456 groups.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study shows that isolated human placental trophoblasts produce a neutral IGFBP-3 protease with characteristics very similar to the activity found in pregnancy serum and provides the first experimental evidence suggesting that the pregnancy-associated serum IGFBP-3 protease may be produced by a specific cell type from the utero-placental unit. As a putative trophoblast-derived product, the IGFBP-3 protease would be expected to appear in early pregnancy following the development of the trophoblast and persist throughout gestation, but be cleared from the circulation in the absence of intrauterine gestational tissues postpartum, analogous to placental steroids. Indeed, during the course of pregnancy, IGFBP-3 proteolytic activity is detected in maternal serum from 6 weeks gestation to term and disappears by day 5 postpartum (16, 17, 44). Therefore, the temporal pattern in which IGFBP-3 proteolytic activity is detected in maternal serum is consistent with a trophoblastic origin. Furthermore, in a recent study IGFBP-3 protease activity in pregnancy serum was found to increase with gestational age and with fetal number (45), indicating a direct correlation between placental tissue mass and maternal serum IGFBP-3 protease activity. These clinical observations, together with our in vitro findings, strongly support the placental trophoblast as a major source of the IGFBP-3 protease in the maternal circulation.

While the current study suggests that the pregnancy-associated serum IGFBP-3 protease may be one of several trophoblast products normally present in the maternal circulation, the question arises as to which trophoblast subpopulation(s) may produce it. In the present study we found that IGFBP-3 protease activity was equally produced when cytotrophoblasts were cultured alone [which results in aggregation and fusion (25, 26)] or when they were cocultured with decidualized endometrial stromal cells [which favors differentiation to an extravillous invasive phenotype (22)]. These results suggest that both the syncytiotrophoblast and the extravillous trophoblast may be potential sources of IGFBP-3 protease at the maternal-fetal interface. Anatomically, both the villous syncytiotrophoblast and the extravillous endovascular trophoblast lie in direct contact with maternal blood, and their secreted products have immediate access to the maternal circulation.

The neutral IGFBP-3 protease activity of trophoblast conditioned medium has striking similarities with the activity displayed by unfractionated pregnancy serum. Both share the same IGFBP substrate specificity, generate the same cleavage product profile, and display similar sensitivity to specific protease inhibitors. Inhibitor studies characterized the main IGFBP-3 protease activities in unfractionated pregnancy serum and trophoblast conditioned medium, primarily as a metalloproteinase, with partial inhibition by the serine protease inhibitors AEBSF and aprotinin. In a previous study, Bang and Fielder reported the isolation of two distinct IGFBP-3 proteases from human serum (46). One protease was found in both pregnancy and nonpregnancy sera and corresponded to plasmin. The other protease was found exclusively in pregnancy serum, and its molecular identity remains undetermined. IGFBP-3 protease activity is notably absent from fetal serum (47, 48). In the current study, the specific inhibitor {alpha}2-antiplasmin caused no appreciable inhibition of IGFBP-3 proteolysis (Fig. 5Go, and data not shown), suggesting a balance favoring plasmin inhibitors in the unfractionated pregnancy serum samples tested and indicating minimal contribution of plasmin activity to IGFBP-3 proteolysis under the assay conditions. Similarly, we found that {alpha}2-antiplasmin had no effect on IGFBP-3 proteolysis by trophoblast-conditioned medium. These results suggest that the activities in pregnancy serum and trophoblast conditioned medium may correspond to the pregnancy-specific protease previously identified by Bang and Fielder (46).

Recently, IGFBP-3 proteases with approximate Mr of 79, 50, 30, and 22 kDa were identified in pregnancy serum by IGFBP zymography (20, 49), suggesting that multiple enzymes may participate in IGFBP-3 proteolysis by pregnancy serum. Of these, the 50-kDa protease was inhibited by the metalloproteinase inhibitor o-phenanthroline, whereas the 30-kDa species was sensitive to serine protease inhibitors. Only the 50-kDa enzyme also had IGFBP-4 protease activity. The 50-kDa species was partially characterized as a novel soluble disintegrin metalloproteinase, insensitive to TIMP but sensitive TAPI (20), a hydroxamic acid-based TACE inhibitor (43). Consistent with these recent findings, the current study has demonstrated that IGFBP-3 proteolysis by pregnancy serum and trophoblast conditioned medium is sensitive to both metalloproteinase and serine protease inhibitors, the main activity corresponding to a metalloproteinase. Both the serum and trophoblast proteases in the current study are not only insensitive to TIMP-1 and, therefore, distinct from the MMPs, but are also differentially inhibited by a metalloproteinase inhibitor with selective activity against the disintegrin metalloproteinase TACE. Moreover, both fluids had IGFBP-4 protease activity, further suggesting the putative 50-kDa soluble disintegrin metalloproteinase may be also present in trophoblast-conditioned medium. The substrate specificity of the pregnancy serum disintegrin metalloproteinase for extracellular matrix proteins is unknown. However, some disintegrin metalloproteinases are known to degrade extracellular matrix proteins (43). Therefore, the local production of an enzymatically active disintegrin metalloproteinase by the trophoblast at the maternal-fetal interface may be relevant to processes of invasion and tissue remodeling during placental development, as well as increasing bioavailable IGFs at the maternal-fetal interface and in the maternal circulation (vide infra).

The question arises as to whether the trophoblast disintegrin metalloproteinase is identical to TACE. The current study revealed active proteolysis of endogenous IGFBP-3 in trophoblast cultures that we have recently shown express high levels of TIMP-3 messenger RNA (50), suggesting that the trophoblast-derived IGFBP-3 protease is not inhibited by TIMP-3. TACE has been found to be inhibited by TIMP-3, but not TIMP-1, -2, and -4 (51). Together, these observations suggest the trophoblast-derived protease is not identical to TACE. Furthermore, TACE has not been found in a soluble form (52), and thus it is unlikely to be identical to the trophoblast-derived or circulating IGFBP-3 protease activity described herein.

The physiological significance of IGFBP proteolysis in pregnancy can only be surmised at present. The proteolysis of circulating IGFBPs during pregnancy causes a net reduction of the total IGF binding capacity of serum (16, 17), resulting in higher levels of circulating free IGFs, and a corresponding increase of IGF bioactivity in pregnancy serum (12). These changes may be important for the maternal adjustment to the physiological demands of pregnancy and to provide increased availability of systemic IGF for placental growth and function. The presence of an IGFBP protease in serum may also reflect a primary tissue process that locally modulates IGF-independent actions of IGFBP-3, rather than a systemic response. IGFBP-3 has IGF-independent effects that result from direct interaction with cell surface receptor molecules (7, 8). Specific binding of IGFBP-3 to surface receptors in cultured cells results in IGF-independent growth inhibition, but interaction of IGFBP-3 with cell surface receptors is inhibited by IGF binding (7, 8). IGFBP-3 proteolytic fragments can retain their direct growth inhibitory effect (15) but, because they have reduced affinity for IGFs, their interaction with cell surface receptors will be less sensitive to inhibition through IGF binding, compared with intact IGFBP-3. In addition, IGFBP-3 proteolytic fragments have been shown to bind insulin with high affinity and thereby block insulin receptor binding and cellular actions (14). This mechanism may provide a means of regulating insulin action at the placental-decidual interface. This is particularly germane to the regulation of decidual IGFBP-1. At the maternal-fetal interface, the maternal decidua produces abundant IGFBP-1 (53) which, as shown in the current study, is resistant to serum- and trophoblast-derived IGFBP proteases. Therefore, it is unlikely that IGFBP-3 proteolysis will have a major impact on local IGF bioavailability in the placental bed. However, IGFBP-3 proteolytic fragments may play an important role in providing local protection to decidual cells from the effects of insulin, which is a potent inhibitor of decidual IGFBP-1 production (54).

This study demonstrates that isolated human placental trophoblast cells produce a neutral IGFBP-3 protease with characteristics very similar to those of the activity found in pregnancy serum, and implicates these cells at the maternal-fetal interface as a source of the pregnancy-associated serum IGFBP-3 protease. The findings further suggest that the main IGFBP-3 protease activity in both pregnancy serum and trophoblast conditioned medium corresponds to a disintegrin-metalloproteinase type enzyme. Identification of this enzyme (or enzyme complex) awaits further molecular characterization.


   Acknowledgments
 
The authors gratefully acknowledge Dr. A. Sommer (Celtrix Pharmaceuticals, Inc., Santa Clara, CA), for recombinant human IGFBP-3, and Dr. R. G. Rosenfeld, Department of Pediatrics, Oregon Health Sciences University (Portland, OR) for IGFBP-3 antibodies.


   Footnotes
 
1 This work was supported by NIH Grants HD-25220-08 and HD-31398 (to L.C.G.) from the NIH Specialized Cooperative Centers Program in Reproductive Research. Back

Received June 9, 1999.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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