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Department of Gynecology and Obstetrics (L.C.G., G.H.F., K.I., I.S., B.I., L.-F.S., J.C.I.), Stanford University Medical Center, Stanford, California 94305; Endocrine Research Laboratory (C.A.C., L.B.), The Mayo Foundation, Rochester, Minnesota 55905; The Statens Serum Institut (M.C.), DK-2300 Copenhagen, Denmark; and Department of Molecular and Structural Biology (M.T.O., C.O.), University of Aarhus, DK-8000 Aarhus, Denmark
Address all correspondence and requests for reprints to: Linda C. Giudice, Ph.D., M.D., Division of Reproductive Endocrinology and Infertility, Center for Research on Women’s Health and Reproductive Medicine, Department of Gynecology and Obstetrics, Stanford University Medical Center, Stanford, California 94305-5317. E-mail: . giudice{at}stanford.edu
Abstract
The IGF family plays an important role in implantation and placental physiology. IGF-II is abundantly expressed by placental trophoblasts, and IGF binding protein (IGFBP)-4, a potent inhibitor of IGF actions, is the second most abundant IGFBP in the placental bed, expressed exclusively by the maternal decidua. Proteolysis of IGFBP-4 results in decreased affinity for IGF peptides, thereby enhancing IGF actions. In the current study, we have identified the IGFBP-4 protease and its inhibitor in human trophoblast and decidualized endometrial stromal cell cultures, and we have investigated their regulation in an effort to understand control of IGF-II bioavailability at the placental-decidual interface in human implantation. IGFBP-4 protease activity was detected in conditioned media (CM) from human trophoblasts and decidualized endometrial stromal cells using 125I-IGFBP-4 substrate. Identification of the IGFBP-4 protease as pregnancy-associated plasma protein-A (PAPP-A) was confirmed by specific immunoinhibition and immunodepletion of the IGFBP-4 protease activity with specific PAPP-A antibodies. The IGFBP-4 protease activity was IGF-II-dependent in trophoblast CM. In decidualized stromal CM, PAPP-A/IGFBP-4 protease activity was also IGF-II-dependent, but was evident only when IGF-II was added in molar excess of the predominant IGFBP in decidualized stromal cell CM, IGFBP-1, supporting bioavailable IGF-II as a key cofactor of IGFBP-4 proteolysis by PAPP-A. Cultured first and second trimester human trophoblasts (n = 5) secreted PAPP-A into CM with mean ± SEM levels of 172.4 ± 32.8 mIU/liter·105 cells, determined by specific ELISA. PAPP-A in trophoblast CM (n = 3) and did not change in the presence of IGF-II (1–100 ng/ml). Cultured human endometrial stromal cells (n = 4) secreted low levels of PAPP-A (6.25 ± 3.6 mIU/liter·105 cells). A physiological inhibitor of PAPP-A, the proform of eosinophil major basic protein (proMBP), was detected in trophoblast CM at levels of 1853 ± 308 mIU/liter·105 cells, determined by specific ELISA, and was nearly undetectable in CM of human endometrial stromal cells. Upon in vitro decidualization of endometrial stromal cells with progesterone, PAPP-A levels in CM increased nearly 9-fold without a concomitant change in proMBP. In contrast to the experiments with trophoblasts, IGF-II and the IGF analogues, Leu27 IGF-II, and Des (1–6) IGF-II, resulted in a dose-dependent decrease of PAPP-A levels in decidualized endometrial stromal CM by 70–90%, and a dose-dependent increase in proMBP of 14- to 41-fold. The data demonstrate conclusively that the IGF-II-dependent IGFBP-4 protease of human trophoblast and decidual origin is PAPP-A. Furthermore, the differential regulation of decidual PAPP-A and proMBP by insulin-like peptides supports a role for trophoblast-derived IGF-II as a paracrine regulator of these maternal decidual products that have the potential to regulate IGF-II bioavailability at the trophoblast-decidual interface. Overall, the data underscore potential roles for a complex family of enzyme (PAPP-A), substrate (IGFBP-4), inhibitor (proMBP), and cofactor (IGF-II) in the placental bed during human implantation.
THE IGF FAMILY plays an important role in implantation and placental physiology. In humans, IGF-II is abundantly expressed by trophoblasts invading into the maternal decidua (1), a process that involves matrix degradation by matrix metalloproteinases (2). The decidua expresses regulators of trophoblast invasion, including IGFBP-1 (1, 3, 4) and tissue inhibitor of metalloproteinase-3 (TIMP-3) (5). Our group has recently shown that IGF-II inhibits maternal decidual IGFBP-1 and TIMP-3 expression (6), supporting paracrine actions of trophoblast-derived IGF-II on decidual regulators of invasion. In addition to their effects on trophoblast invasion, insulin-like peptides regulate trophoblast metabolism and differentiation, including glucose metabolism, glucose and amino acid uptake (7, 8), and steroidogenesis (9, 10). IGFBP-4 is the second most abundant IGFBP in the maternal decidua (1) and is a potent inhibitor of IGF actions (11). We have described an IGF-II-dependent IGFBP-4 protease, of unknown identity, secreted by decidualized human endometrial stromal cells (12) and IGFBP-4 proteolysis by human trophoblast conditioned media (CM) (13). Recently, we have demonstrated that the IGFBP-4 protease secreted by human dermal fibroblasts is PAPP-A (14). PAPP-A has long been recognized as a placental product of unknown function (15), that is a useful first trimester marker for Down’s syndrome (16). In pregnancy, more than 99% circulates as a covalent, heterotetrameric 2:2 complex of 500 kDa with the proform of eosinophil major basic protein (proMBP) (17), a 50 kDa protein also of placental origin (18, 19), which functions as an inhibitor of the proteolytic activity of PAPP-A (20). PAPP-A contains an elongated zinc binding motif that is strictly conserved within the metzincins, a superfamily of zinc peptidases (21). Recently, it has been demonstrated that PAPP-A is an active enzyme for which IGFBP-4 is a substrate, and IGFBP-4 proteolysis by PAPP-A is IGF-dependent (14, 22). During human pregnancy in the maternal circulation, IGFBP-4 proteolysis and elevated levels of PAPP-A are detected early in gestation (23, 24, 25), although the identity and site of production of the circulating IGFBP-4 protease have been unclear. In the current study, we have identified the IGF-II-dependent IGFBP-4 protease in human trophoblast and decidualized endometrial stromal cells as PAPP-A, and we have investigated proMBP in these cells in an effort to understand IGF-II regulation in the placental bed during human implantation. In addition, we have investigated regulation of PAPP-A and proMBP by insulin-like peptides, because IGF-II is a major product of the invading cytotrophoblast, in an effort to investigate potential autocrine or paracrine regulation of PAPP-A and proMBP in the placental bed. The data demonstrate a complete enzyme (PAPP-A), inhibitor (pro-MBP), cofactor (IGF-II), and substrate (IGFBP-4) family at the trophoblast-decidual interface, whose function is hypothesized to regulate IGF-II bioavailability in the placenta for trophoblast metabolism, steroidogenesis, and during invasion in human implantation.
Materials and Methods
Tissue specimens
Tissue specimens were collected under a protocol approved by the Stanford University Panel on the Use of Human Subjects in Medical Research and in accordance with the guidelines of The Declaration of Helsinki. Human endometrial samples were collected from the Cooperative Human Tissue Network, Ohio State University (Columbus, OH), and human first (n = 1) and second (n = 4) trimester placentae were collected at the time of vacuum aspiration from subjects at Stanford University Hospital (Stanford, CA). Histologically normal endometrial tissue samples were obtained from cycling premenopausal patients (27–34 yr old) in the secretory phase of natural cycles, undergoing hysterectomy for benign reasons. All samples were collected in DMEM (Life Technologies, Inc., Grand Island, NY) and then transported to the laboratory and processed as described below.
IGF peptides, analogs, and IGFBP-4
Recombinant human IGF-II was from Bachem (Torrance, CA). IGF analogs were obtained from GroPep Pty. Ltd. (Adelaide, Australia). Long R3IGF-I, a recombinant IGF-I analog, with an arginine for glutamate substitution at position 3 and a 13-amino acid extension peptide at the N terminus, does not bind to the type 2 IGF receptor. Its affinity for the type 1 IGF receptor is similar to native IGF-I, but it has three orders of magnitude lower affinity for IGFBPs (26). Consequently, it has approximately 10-fold increased potency, compared with IGF-I, as a result of its increased bioavailability. The IGF-II analog, Des (1–6) IGF-II, lacks the N-terminal hexapeptide and binds, compared with native IGF-II, with 2.6-fold lower affinity to the type 2 IGF receptor and with 2-fold lower affinity to the type 1 IGF receptor. However, its affinity for the IGFBPs is reduced more than 300-fold, overall resulting in a biological potency similar to IGF-II (27). Leu (27) IGF-II has a 500-fold lower affinity for the type I IGF receptor and about 50% decreased binding affinity for the IGFBPs (26). Human IGFBP-4 was kindly provided by Dr. S. Mohan (Loma Linda, CA). In some experiments, C-terminally c-myc tagged IGFBP-4 (21, 22) was used.
Cell cultures
Cytotrophoblasts were isolated from first and second trimester placentae as described (3), by sequential digestion with collagenase-hyaluronidase-DNase, and trypsin-EDTA-DNase, followed by Percoll gradient centrifugation. Isolated cytotrophoblasts were washed with DMEM, and viable cells counted by Trypan blue exclusion. Trophoblasts were collected by centrifugation, washed, and plated on fibronectin-coated wells at 5 x 105 cells per well. Trophoblasts were cultured for up to 6 d in the absence of peptides, or in the presence of various concentrations of IGF-II (13, 26, 65, and 130 nM), Des (1–6) IGF-II (7, 30, and 75 nM), insulin (10 and 100 ng/ml), or Long R3IGF-I (6 nM). Every 2 d, the culture medium was renewed, the CM was collected and centrifuged, and the supernatant was stored at -80 C for further analysis.
Endometrial tissue was subjected to collagenase digestion and stromal cells separated from epithelium, cultured and passaged as previously described (28, 29). Cells isolated from different subjects (n = 3) were used at passages 1–4 for these studies. Stromal cells were grown to confluence in multi (2 cm2) well trays (Costar, Cambridge, MA) in DMEM supplemented with 5 µg/ml insulin and 10% charcoal-stripped FBS. Confluent cultures, in triplicate, were then decidualized in vitro with 10 nM E2, 1 µM progesterone (P) and 20 ng/ml EGF in serum-free medium (75% DMEM, 25% MCDB-104, 50 µg/ml ascorbic acid, 1 mg/ml BSA, 5 µg/ml transferrin) for 10 d in the absence of IGF peptides, and then cultured for an additional 6 d in the absence of peptides, or in the presence of various concentrations of IGF-II (13, 26, 65, 130 nM), or Des (1–6) IGF-II (7, 30, 75 nM), or with Long R3IGF-I (6 nM). Every 2 d the culture medium was renewed, the CM was collected and centrifuged, and the supernatant stored at -80 C for further analysis. Cells were also treated with 1 mM 8-Br-cAMP to effect decidualization, as described (29). CM were collected 24 and 48 h after treatment and were processed and stored as described above.
IGFBP-4 protease assay
IGFBP-4 proteolysis was assayed as described previously (14, 21), by incubating samples at 37 C for 6 h with [125I]-IGFBP-4. Reaction products were separated by nonreducing 7.5–15% gradient SDS-PAGE and visualized by autoradiography. In some experiments, PAPP-A polyclonal or monoclonal antibodies (30) or nonspecific rabbit IgG were used in conjunction with Protein G/Protein-A-agarose (Oncogene Science, Inc., Cambridge, MA) to immunoprecipitate IGFBP-4 protease activity, and the supernatants were then used in the protease assay (14). In other experiments, incubation was performed in the presence of inhibitory PAPP-A polyclonal or monoclonal antibodies (30).
PAPP-A, ProMBP, and IGFBP-1 ELISAs
To measure PAPP-A, a sandwich biotin-tyramide amplified ELISA was performed using PAPP-A polyclonal antibodies (30, 31) for capturing and a collection of PAPP-A monoclonal antibodies (32) for detection. Assays were calibrated with the World Health Organization’s International Reference Standard for pregnancy proteins (78/610). ProMBP was measured in a similar assay using polyclonal antibodies (30, 31) for capturing and proMBP monoclonal antibodies (30, 32) for detection. Levels of PAPP-A and proMBP are expressed in milliunits per liter. By definition, term pregnancy serum contains 100 U/liter of both proteins. At term, the molar level of proMBP exceeds the molar level of PAPP-A by approximately 4-fold (25).
IGFBP-1 levels were assayed in duplicate in 2-d CM from triplicate cultures. IGFBP-1 enzyme-linked immunospectrophotometric assay (ELISA) kits from Diagnostics Systems Laboratories, Inc. (Webster, TX) were used, as previously described (6).
Data and statistical analyses
Mean levels ± SEM of PAPP-A or ProMBP levels in CM of duplicate wells were calculated and normalized to total cell count. The 
2 test was used to compare the mean levels of PAPP-A or proMBP within each treatment group using the commercially available software package SPSS, with P < 0.05 taken as significant.
Results
Identification of the IGFBP-4 protease as PAPP-A in human trophoblasts
Human first and second trimester cytotrophoblasts were isolated and cultured, as described (3). These cells are about 98% pure by immunocytochemical staining for cytokeratin, vimentin, and CD68 (3). To investigate IGFBP-4 protease activity in CM of these cells, 2-d CM from cultured trophoblasts were incubated with [125I]-IGFBP-4 and IGF-II, which resulted in loss of intact IGFBP-4 (24 kDa nonreducing, 32 kDa reducing SDS-PAGE) and the generation of fragments of 18- and 14-kDa (Fig. 1
), similar to those reported previously (14, 33). Inclusion of the PAPP-A polyclonal antibody (30) in the assay, but not nonspecific rabbit IgG, completely inhibited the IGFBP-4 protease activity in the CM (Fig. 1A). In other experiments, IGFBP-4 proteolytic activity was immunodepleted from the CM, using specific PAPP-A polyclonal antibody (Fig. 1B). Note that proteolytic activity was not appreciably elicited in the absence of IGF-II.
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We previously reported that decidualized human endometrial stromal cells contain an IGF-II-dependent IGFBP-4 protease (12). In the current study, we first investigated whether the IGFBP-4 proteolytic activity was the result of the decidualization process. Human endometrial stromal cells were decidualized in vitro with P, after E2 priming, or were not treated with P and E2 (nondecidualized). Figure 2A shows that CM from nondecidualized, but not from decidualized, endometrial stromal cells contains an IGF-II-dependent IGFBP-4 protease. The surprising finding, different from our earlier observation of IGFBP-4 protease activity in decidualized stromal cells (12), prompted us to revisit the physiology in the placental bed. Therein, decidual cells secrete high levels of IGFBP-1 (1), as do endometrial stromal cells decidualized in vitro (34, 35). The IGF-II-dependent IGFBP-4 protease in media conditioned by decidualized endometrial stromal cells was evident when IGF-II was added to the CM in 1.5-fold molar excess over the IGFBP-1 in the medium (Fig. 2B). Note that proteolytic activity was not appreciably elicited in the absence of IGF-II.
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Immunoinhibition and immunodepletion studies further confirmed the identities of the endometrial stromal-derived IGFBP-4 protease as PAPP-A (Fig. 3). Inclusion of the PAPP-A polyclonal antibody in the assay, but not nonspecific rabbit IgG, completely inhibited the IGFBP-4 protease activity in the CM (Fig. 3A), and in other experiments, IGFBP-4 proteolytic activity was effectively immunodepleted from CM, using specific PAPP-A monoclonal antibody (Fig. 3B). It should be noted that upon decidualization, only with a 1.5-fold molar excess of IGF-II over IGFBP-1 was the IGFBP-4 protease activity identified, and this was inhibited and immunodepleted with PAPP-A antibodies, but not with IgG controls (Fig. 3).
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Levels of PAPP-A and proMBP in CM from human trophoblasts and endometrial stromal cells were determined by ELISA. Table 1 shows the levels of PAPP-A in media conditioned by human trophoblasts and human endometrial stromal cells. PAPP-A was secreted by trophoblasts cultured in defined medium. PAPP-A was detectable in CM from nondecidualized endometrial stromal cells and increased about 9-fold upon decidualization with P after E2-priming. Similar results were observed when 1 mM 8-Br-cAMP was used to effect stromal decidualization (data not shown). ProMBP was found in high levels in CM from trophoblasts, but was barely detectable in CM from endometrial stromal cells, whether or not they were decidualized (Table 1).
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IGF-II, PAPP-A, and proMBP are major products of the human trophoblast (1, 18, 19). Because autocrine regulation of PAPP-A and/or proMBP by IGF-II may contribute to control of IGF-II bioavailability in the placental bed, we investigated the effects of IGF-II on trophoblast-derived PAPP-A and proMBP (Table 1
). In addition, because trophoblast-derived IGF-II affects decidual cell function in the placental bed, regulating trophoblast invasion, we investigated whether IGF-II regulates decidualized endometrial stromal cell production of PAPP-A and/or proMBP. We investigated different IGF analogs to determine whether such regulation occurs via the type 1 or type 2 IGF receptor. In particular, we investigated the actions of IGF-II, Des (1–6) IGF-II (an analog of IGF-II with markedly decreased affinity for IGFBPs and about equal potency to IGF-II at the type 1 and type 2 IGF receptors) (27), Long R3IGF-I (an IGF-I analog that does not bind to the type 2 IGF receptor), and Leu (27) IGF-II, which has 500-fold lower affinity for the type 1 IGF receptor and binds to the type 2 receptor with approximately equal affinity compared with IGF-II. Figure 4 shows the dose response of PAPP-A and proMBP levels (mean ± SEM) in decidualized stromal cell CM in response to IGF-II, Des (1–6), and Leu (27) IGF-II. [PAPP-A and proMBP levels in trophoblast CM were not appreciably affected by IGF-II (Table 1) or IGF analogs (not shown).] PAPP-A and proMBP levels in trophoblast CM were not appreciably affected by IGF-II (Table 1). IGF-II treatment of decidualized endometrial stromal cells resulted in a significant dose-dependent decrease of PAPP-A and a significant increase in proMBP secreted into CM. Leu (27) IGF-II treatment resulted in a significant dose-dependent decrease in PAPP-A and a trend for an increase in proMBP in CM of these cells. The results with Des (1–6) IGF-II are similar to those obtained with native IGF-II, although statistical significance was not achieved. We also observed, in experiments with decidualized stromal cells, significant inverse correlations between PAPP-A levels and concentrations of IGF-II and IGF analogs and positive correlations between proMBP levels with various treatment dosages applying a Pearson’s correlation test (data not shown). Long R3IGF-I (10 ng/ml) action on decidualized endometrial stromal cells resulted in 90% inhibition of PAPP-A and a 20-fold increase in proMBP in CM (data not shown). Together, these results support IGF action on PAPP-A and proMBP levels being mediated through the type 1 IGF receptor and action through the type 2 receptor, as well.
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In the current study, we have demonstrated that human trophoblasts secrete an IGFBP-4 protease with IGFBP-4 cleavage patterns identical to those produced by the IGFBP-4 protease secreted by human decidualized endometrial stroma (12) and dermal fibroblasts and osteoblasts (14, 36, 37, 38). In addition, we have identified the IGFBP-4 protease in media conditioned by first and second trimester human trophoblast and human endometrial stromal cells as PAPP-A using both immunoinhibition and immunodepletion analysis with PAPP-A polyclonal and monoclonal antibodies. PAPP-A has long been recognized as a placental product of unknown function (15) that increases in the maternal circulation throughout human pregnancy and is a useful first trimester marker for Down’s syndrome (16). In pregnancy, more than 99% circulates as a covalent, heterotetrameric 2:2 complex of 500 kDa with its inhibitor, proMBP (17, 20). The data reported herein on secretion and identification of the IGFBP-4 protease as PAPP-A in trophoblasts are strongly supported by in situ hybridization studies using term placentae, demonstrating PAPP-A mRNA expression in syncytiotrophoblasts and extravillous cytotrophoblasts (19). In addition, immunohistochemical studies with first trimester and term placentae have also localized PAPP-A to syncytiotrophoblasts, extravillous trophoblasts, and the extracellular matrix in placental septae (19, 39). Early immunohistochemical studies on the cellular localization of PAPP-A were conflicting, likely due to different antisera used and the fact that available polyclonal antibodies to PAPP-A contained antibodies to both PAPP-A and proMBP (17). They demonstrated localization only to the apical surface of the syncytiotrophoblasts of early (6 wk gestation) as well as late (term) placentae (15, 40, 41, 42) or localization to the syncytiotrophoblast and the extravillous, intermediate (invading) trophoblast (43, 44). Subsequent immunofluorescence studies, using proMBP-absorbed anti-PAPP-A polyclonal antibodies, localized immunoreactive PAPP-A to the extravillous trophoblast in the anchoring villi, as well as to the syncytiotrophoblast layer in the floating villi (19). No other tissue elements, such as villous stroma or fetal endothelial cells, showed positive staining.
Decidualized endometrial stromal cells are abundant in first trimester decidua and less abundant at term, and our data herein demonstrate that PAPP-A is indeed secreted by this cell type. Supporting the latter are previous studies demonstrating immunoreactive PAPP-A in decidua (19) and secreted by term decidual explants (45). However, previous immunohistochemical studies used polyclonal antibodies that recognize both PAPP-A and proMBP because they were raised against PAPP-A purified from pregnancy serum in which PAPP-A exists in a covalent 2:2 complex with proMBP. In the current study, we used monoclonal antibodies to PAPP-A that conclusively demonstrate the identity of the IGFBP-4 protease secreted from human trophoblasts and endometrial stroma as PAPP-A. Nonetheless, previous studies are supportive of our observation that decidualized endometrial stromal cells secrete PAPP-A in culture.
The IGFBP-4 protease activity in trophoblast CM was IGF-dependent, as has been observed previously (36, 37, 46). In decidualized stromal CM, the IGFBP-4 protease activity was also IGF-dependent, but was evident only when IGF-II was added in molar excess of the predominant IGFBP in decidualized stromal cell CM, IGFBP-1. These data support bioavailable IGF-II as a key cofactor of IGFBP-4 proteolysis by PAPP-A and a complex regulatory system in the placental bed for IGF-II regulation. In addition, they raise the issue of relative affinities of IGFBP-1 and IGFBP-4 for IGF-II in the placental bed and microenvironments in the decidua in which relative expression of these two IGFBPs may differ.
ProMBP is also of placental origin. Intense immunoreactivity has been observed in septal areas surrounding the extravillous trophoblast in anchoring villi (19, 47). In situ hybridization confirmed proMBP mRNA expression exclusively in the extravillous trophoblast of the anchoring villous and not in any other cell type of the placenta (18, 19). Of interest is the observation that when immunofluorescence staining or hybridization intensities have been quantified, both demonstrate greater expression of PAPP-A in the chorionic villi and greater proMBP expression in the anchoring villi (19). In the current study, molar levels of proMBP in media conditioned by human cytotrophoblasts were found to be approximately an order of magnitude greater than the levels of PAPP-A. Finding high levels of proMBP, a physiological inhibitor of PAPP-A, in trophoblast CM is surprising because this CM contains active PAPP-A enzyme. In maternal serum, PAPP-A and proMBP levels are each 100 IU/liter (125 nM PAPP-A and 500 nM proMBP) (25). PAPP-A enzymatic activity is detected due to less than 1% of circulating PAPP-A being uncomplexed with proMBP (20). To what extent PAPP-A is complexed with proMBP in these cultures is not known and is currently being investigated in our laboratories. However, the high levels of proMBP in trophoblast CM suggest that inhibition of PAPP-A enzymatic activity by proMBP is likely to be important in the placental-decidual interface. Alternatively, proMBP may have additional functions (i.e. in addition to being an inhibitor of PAPP-A enzymatic activity) in placental physiology, because it has been shown to bind angiotensinogen and complement C3 (24).
PAPP-A is measurable in the plasma of nonpregnant women, suggesting that tissues other than the placenta produce the protein. Although it is produced by the corpus luteum (48), circulating levels of PAPP-A do not change during the menstrual cycle (49). However, endometrial PAPP-A levels are up-regulated in the secretory phase and correlate with circulating P levels (49, 50). In addition, immunoreactive PAPP-A has been described in decidualizing endometrial stromal cells and in uterine fluid, paralleling levels in the endometrium during the menstrual cycle (50, 51). The data reported herein highly support the IGFBP-4 protease, PAPP-A, as being regulated by P during the process of stromal decidualization. Interestingly, in early (and late) pregnancy, the decidua contains 3–4 times more PAPP-A than does the trophoblast (52), and, in vitro, trophoblasts and decidual explants secrete PAPP-A (45). We have demonstrated that human endometrial stromal cells secrete PAPP-A, which increases 9-fold upon decidualization effected by P or cAMP. Thus, the data presented herein unequivocally demonstrate that both the decidua and the trophoblast produce PAPP-A and that the trophoblast secretes proMBP when cultured in defined medium. These culture systems are useful models to investigate further the function(s) of PAPP-A and proMBP in endometrial and placental physiology, as well as trophoblast invasion and migration.
It is striking that PAPP-A is decreased and proMBP is induced by IGF-II added to cultures of decidualized human endometrial stromal cells. If this in vitro observation can be extrapolated in vivo to the placental bed, it is possible that the IGF-II secreting trophoblast may regulate IGFBP-4 protease activity in its local environment by paracrine actions on the decidua. That IGF-II has no effect on trophoblast-derived PAPP-A and proMBP secretion suggests that constitutive production of PAPP-A and proMBP by the trophoblast may be important for controlling decidual IGFBP-4 proteolysis, and any further regulation on IGFBP-4 proteolysis can be elicited by paracrine down-regulation of PAPP-A and induction of proMBP in the decidua by trophoblast-derived IGF-II. The data herein suggest that IGF-II likely regulates PAPP-A and proMBP in decidualized endometrial stromal cells via the type 1 IGF receptor and also via the type 2 receptor. A recent study has demonstrated that IGF-II stimulates trophoblast invasion in vitro by signaling through the type 2 receptor in the trophoblast (53). Whether similar signaling occurs in the decidualized endometrial stromal cell is not certain, although the data herein suggest signaling through both the type 1 and type 2 receptors.
The IGF family plays an important role in implantation and placental physiology. Because IGF-II is abundantly expressed by placental trophoblasts and has effects on trophoblast function and trophoblast invasion (53) as well as on maternal modulators of invasion (6), regulation of this growth factor in the placental bed is important for trophoblast homeostasis and invasion. Because IGFBP-4, a potent inhibitor of IGF actions, is abundantly expressed by the maternal decidua (1), our findings that human trophoblasts secrete an IGF-dependent IGFBP-4 protease and identification of this protease as PAPP-A underscore potential autocrine and paracrine interactions involving trophoblast regulation of maternally derived (i.e. decidual) IGFBP-4. That PAPP-A is also secreted by decidualized endometrial stroma is not unexpected, because there is often redundancy among decidual and trophoblast products (54). PAPP-A levels increase in the maternal circulation as pregnancy progresses (16). Our data strongly support the placental and decidual origins of the IGFBP-4 protease in the maternal circulation (23, 24) that was recently identified as PAPP-A (20, 55). The roles of this complex family of enzyme (PAPP-A), substrate (IGFBP-4), inhibitor (proMBP), and cofactor (IGF-II), in the placental bed during human implantation warrant further investigation.
Acknowledgments
We acknowledge Ms. Isabel Bourelle for her assistance in the preparation of this manuscript.
Footnotes
1 Present address for J.C.I.: Berlex Laboratories, Richmond, California. 
This research was supported as a Collaborative Research Initiative of the Specialized Cooperative Centers Program in Reproductive Research through cooperative agreement U54 HD31398 (to L.C.G.), the Mayo Foundation (to C.A.C.), the Danish Medical Research Council (to C.O.), and the Novo Nordic Foundation (to C.O.).
Abbreviations: CM, Conditioned media; IGFBP, IGF binding protein; P, progesterone; PAPP-A, pregnancy-associated plasma protein-A; proMBP, proform of eosinophil major basic protein; TIMP-3, tissue inhibitor of metalloproteinase-3.
Received October 8, 2001.
Accepted January 27, 2002.
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S. Glerup, S. Kloverpris, L. S. Laursen, F. Dagnaes-Hansen, S. Thiel, C. A. Conover, and C. Oxvig Cell Surface Detachment of Pregnancy-associated Plasma Protein-A Requires the Formation of Intermolecular Proteinase-Inhibitor Disulfide Bonds and Glycosaminoglycan Covalently Bound to the Inhibitor J. Biol. Chem., January 19, 2007; 282(3): 1769 - 1778. [Abstract] [Full Text] [PDF]
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 V. E. Murphy, R. Smith, W. B. Giles, and V. L. Clifton Endocrine Regulation of Human Fetal Growth: The Role of the Mother, Placenta, and Fetus Endocr. Rev., April 1, 2006; 27(2): 141 - 169. [Abstract] [Full Text] [PDF]
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S. Glerup, H. B. Boldt, M. T. Overgaard, L. Sottrup-Jensen, L. C. Giudice, and C. Oxvig Proteinase Inhibition by Proform of Eosinophil Major Basic Protein (pro-MBP) Is a Multistep Process of Intra- and Intermolecular Disulfide Rearrangements J. Biol. Chem., March 18, 2005; 280(11): 9823 - 9832. [Abstract] [Full Text] [PDF]
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 K. Hayashi, K. D Carpenter, T. H Welsh Jr, R. C Burghardt, L. J Spicer, and T. E Spencer The IGF system in the neonatal ovine uterus Reproduction, March 1, 2005; 129(3): 337 - 347. [Abstract] [Full Text] [PDF]
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 S. C.J.P. Gielen, E. E. Hanekamp, L. J. Blok, F. J. Huikeshoven, and C. W. Burger Steroid-Modulated Proliferation of Human Endometrial Carcinoma Cell Lines: Any Role for Insulin-like Growth Factor Signaling? Reproductive Sciences, January 1, 2005; 12(1): 58 - 64. [Abstract] [PDF]
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 Q.-P. Qin, S. Kokkala, J. Lund, N. Tamm, L.-M. Voipio-Pulkki, and K. Pettersson Molecular Distinction of Circulating Pregnancy-Associated Plasma Protein A in Myocardial Infarction and Pregnancy Clin. Chem., January 1, 2005; 51(1): 75 - 83. [Abstract] [Full Text] [PDF]
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H. B. Boldt, K. Kjaer-Sorensen, M. T. Overgaard, K. Weyer, C. B. Poulsen, L. Sottrup-Jensen, C. A. Conover, L. C. Giudice, and C. Oxvig The Lin12-Notch Repeats of Pregnancy-associated Plasma Protein-A Bind Calcium and Determine Its Proteolytic Specificity J. Biol. Chem., September 10, 2004; 279(37): 38525 - 38531. [Abstract] [Full Text] [PDF]
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 A. De Luca, M. De Falco, V. Fedele, L. Cobellis, A. Mastrogiacomo, V. Laforgia, I. L. Tuduce, M. Campioni, D. Giraldi, M. G. Paggi, et al. The Serine Protease HtrA1 Is Upregulated in the Human Placenta During Pregnancy J. Histochem. Cytochem., July 1, 2004; 52(7): 885 - 892. [Abstract] [Full Text] [PDF]
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F. W. Anthony, D. D. Mukhtar, M. A. Pickett, and I. T. Cameron Progesterone Up-Regulates WT1 mRna and Protein, and Alters the Relsative Expression of WT1 Transcripts in Cultured Endometrial Stromal Cells Reproductive Sciences, December 1, 2003; 10(8): 509 - 516. [Abstract] [PDF]
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M. T. Overgaard, E. S. Sorensen, D. Stachowiak, H. B. Boldt, L. Kristensen, L. Sottrup-Jensen, and C. Oxvig Complex of Pregnancy-associated Plasma Protein-A and the Proform of Eosinophil Major Basic Protein. DISULFIDE STRUCTURE AND CARBOHYDRATE ATTACHMENT SITES J. Biol. Chem., January 17, 2003; 278(4): 2106 - 2117. [Abstract] [Full Text] [PDF]
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L. S. Laursen, M. T. Overgaard, K. Weyer, H. B. Boldt, P. Ebbesen, M. Christiansen, L. Sottrup-Jensen, L. C. Giudice, and C. Oxvig Cell Surface Targeting of Pregnancy-associated Plasma Protein A Proteolytic Activity. REVERSIBLE ADHESION IS MEDIATED BY TWO NEIGHBORING SHORT CONSENSUS REPEATS J. Biol. Chem., November 27, 2002; 277(49): 47225 - 47234. [Abstract] [Full Text] [PDF]
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 I. Y. C. Sun, M. T. Overgaard, C. Oxvig, and L. C. Giudice Pregnancy-Associated Plasma Protein A Proteolytic Activity Is Associated with the Human Placental Trophoblast Cell Membrane J. Clin. Endocrinol. Metab., November 1, 2002; 87(11): 5235 - 5240. [Abstract] [Full Text] [PDF]
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