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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 6 2897-2904
Copyright © 2004 by The Endocrine Society

Expression of Extracellular Matrix Metalloproteinase Inducer in Human Placenta and Fetal Membranes at Term Labor

Wei Li, Nadia Alfaidy and John R. G. Challis

Canadian Institutes of Health Research Group in Fetal and Neonatal Health and Development (W.L., J.R.G.C.), Departments of Physiology, Obstetrics and Gynecology, and Medicine, University of Toronto, Toronto, Ontario, Canada M5S 1A8; and Commissariat à l’Énergie Atomique, Département Réponse et Dynamique Cellulaires, Institut National de la Santé et de la Recherche Medicale

Address all correspondence and requests for reprints to: Dr. Wei Li, 1 King’s College Circle, Medical Sciences Building, Room 3344, Department of Physiology, University of Toronto, Toronto, Ontario, Canada M5S 1A8. E-mail: weisun.li{at}utoronto.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Matrix metalloproteinases (MMPs) are the main mediators of extracellular matrix (ECM) degradation during human parturition. However, the mechanisms involved in regulation of MMP production during parturition remain poorly understood. Recently, an extracellular matrix metalloproteinase inducer (EMMPRIN) has been shown to play a key role, as a local regulator, in stimulating MMP production in cancer systems. Whether EMMPRIN is expressed and stimulates MMP production in human placenta and fetal membranes is presently unknown. In this study, we investigated the expression of EMMPRIN at the levels of mRNA and protein in human term placenta and fetal membranes with or without labor. Western blot analysis showed that EMMPRIN protein was detected in term placenta and fetal membranes at two molecular masses of 40 and 65 kDa (glycosylated protein) and one of approximately 30 kDa (nonglycosylated protein). The ratio of 65 kDa EMMPRIN to total EMMPRIN significantly increased (P < 0.05) in term labor chorio-decidua and amnion compared with nonlabor chorio-decidua and amnion. Immunohistochemical analysis revealed that EMMPRIN was expressed in placental syncytiotrophoblast, amniotic epithelial cells, trophoblast cells of chorion laeve, and decidua parietalis. EMMPRIN was also detected at the mRNA level using RT-PCR in cultured placental syncytiotrophoblast, amniotic epithelial cells, and chorionic trophoblast cells. We conclude that human placenta and fetal membranes express EMMPRIN, with the potential to stimulate MMP production, thereby facilitating fetal membrane rupture and leading to detachment of the placenta and fetal membranes from the maternal uterus at the time of parturition.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
HUMAN PARTURITION (TERM and preterm) is the result of multistep processes that include myometrial contraction, cervical ripening, fetal membrane rupture, and detachment of the placenta and fetal membranes from the maternal uterus. Some of these processes require the degradation or remodeling of extracellular matrix (ECM) macromolecules by proteolytic enzymes. Among these proteinases, matrix metalloproteinases (MMPs) are particularly implicated because of their specific spectrum of substrates (1). Many studies have demonstrated that increased levels of MMPs in the human placenta, fetal membranes, and amniotic fluid are associated with term or preterm labor (2, 3, 4, 5, 6, 7, 8), indicating a role for MMPs in human parturition. It is known that complex regulatory mechanisms control MMP activity, including up- or down-regulation of expression by inducers/inhibitory factors, the proteolytic activation of catalytic activity, and direct inhibition through the activators and tissue inhibitors of MMPs (TIMPs). These factors determine the balance between free MMP and TIMP concentrations and net proteolytic activity (9, 10, 11, 12, 13, 14, 15). However, it remains unclear whether a local control of MMP production exists in the human placenta and fetal membranes.

A search for tumor cell-derived MMP-inducing factors led to the discovery and characterization of CD147/extracellular matrix metalloproteinase inducer (EMMPRIN) (16, 17). This protein is a glycoprotein with a molecular mass of 44–66 kDa due to different degrees of glycosylation of the native protein (~30 kDa). EMMPRIN contains two extracellular Ig domains, a transmembrane domain and a 39-amino acid cytoplasmic domain (17, 18, 19, 20). Various independent laboratories have discovered the EMMPRIN protein, naming it basigin (18), OX-47 (21), neurothelin (22), M6 antigen (19), and HT7 antigen (23). It is widely expressed in tumor cells and may play a role in the invasion and metastasis of cancer cells by stimulating tumor cells themselves or nearby fibroblasts to secrete increased amounts of MMPs through direct cell-cell contact or paracrine/autocrine release (16, 24, 25, 26, 27). In addition, EMMPRIN is expressed not only in cancer cells but also in nontumor tissues (28, 29, 30, 31). Although EMMPRIN expression in normal tissue cells is relatively weak, it might be up-regulated in relation to a physiological and pathological role in tissue remodeling under particular conditions. The fact that mice lacking the gene for CD147/EMMPRIN/basigin had defects in embryogenesis, spermatogenesis, and female fertilization suggested that this protein might have multiple roles in the reproductive system (28, 29).

At present, there are no studies that examine the expression of EMMPRIN or its control and relationship to the expression of MMPs in human placenta and fetal membrane during labor. Therefore, we hypothesized that EMMPRIN would be expressed in specific cell types in the human placenta and fetal membranes and that this expression would change with parturition. Therefore, we performed immunoblotting, immunohistochemistry, and RT-PCR analysis on human placenta and fetal membrane tissues obtained from patients at term gestation in the presence or absence of labor to reveal the intrauterine expression and to clarify the cell origin of EMMPRIN.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Collection of fetal membranes and placenta

Placentas were collected from normal term (>37 weeks of gestation) pregnancies after elective cesarean delivery (nonlabor, n = 16) or spontaneous vaginal delivery (labor, n = 16) from Mount Sinai Hospital, Toronto. Patients who had multiple gestation, preeclampsia, or induction of labor or who had not been followed up routinely in the perinatology division at the Mount Sinai Hospital were excluded. None of the patients had received any prostaglandin synthesis inhibitors or corticosteroids. Patient consent and ethical approval were obtained before tissue collection in accordance with the Canadian Tri-Council guidelines and the regulations of Mount Sinai Hospital, Toronto, and the University of Toronto.

Cell purification and culture

Placental and chorionic trophoblast cell cultures. Placental and chorionic trophoblast cells were prepared using a modification of the method of Kliman et al. (33), as described previously (34). Briefly, term human placenta (n = 5) and chorion tissue (n = 5) were obtained from uncomplicated pregnancies after elective cesarean section in the absence of labor. The placental tissue was pooled and digested with 0.125% trypsin (Sigma, St. Louis, MO) and 0.02% DNase I (Sigma) in DMEM (Life Technologies, Inc., Grand Island, NY) containing 0.1% BSA, 0.005% gentamicin, and 0.01% streptomycin three times for 30 min each. The chorion with adherent decidua was peeled away from the amnion and digested three times for 60 min each time with DMEM as above, containing 0.2% collagenase (Sigma) without DNase I. The placental or chorio-decidual cells were loaded onto a 5–75% Percoll (Sigma) gradient at step increments of 5% Percoll and then centrifuged at 37 C at 2500 x g for 20 min to separate different cell types. Cytotrophoblasts between the density markers of 1.049 and 1.062 g/ml were collected, and 0.5 ml of 106 cells/ml per well (for immunostaining) in eight-well chamber slides (Labtek; Nunc, Naperville, IL) or 107 cells/well (Western blotting and RT-PCR) were plated in six-well plates in DMEM culture medium containing 10% fetal calf serum (Life Technologies, Inc.). The cells were cultured for 3 d at 37 C in 5% CO2-95% O2. Under these conditions, the placental cells aggregate to form a syncytium, whereas the chorionic trophoblast cells form clumps or remain as single cells. Cultures were immunostained to determine the proportion of cytokeratin- (an epithelial cell marker) or vimentin- (a fibroblast cell marker) positive cells and were counterstained with Carazzi’s hematoxylin.

Isolation and culture of amniotic epithelial cell. Term human placenta with attached fetal membranes were collected immediately after elective caesarean section (n = 7). The amnion was peeled from the chorion, cut approximately 2 cm from the placenta disk, and washed in PBS (Dulbecco’s PBS, pH 7.5; Life Technologies, Inc./BRL, Burlington, Ontario, Canada). To obtain amniotic epithelial cells, the amnions were isolated as described previously with a little modification (35). The whole amnion was cut into five pieces, and then the tissues were treated with 0.2% trypsin and incubated at 37 C with shaking. The supernatant of the first time period (15 min) was discarded, and cells from the second digestion period (20–30 min) were used for cell culture. After isolation, amniotic epithelial cells were filtered through 100-µm nylon mesh and were pelleted by centrifugation at 2500 x g for 10 min. The pellets were suspended and washed in DMEM medium. Amniotic epithelial cell suspensions (0.4 ml/well of 106 cells/ml for immunostaining and 107/well for RT-PCR) were plated in eight-well chamber slides and in six-well plates in DMEM medium supplemented with 10% fetal calf serum (Life Technologies, Inc.) and antibiotics (1000 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.23 µg/ml Amphotericin; Sigma). The cells were maintained in culture at 37 C in 5% CO2-95% O2 for 3 d. Cultures were immunostained to determine the proportion of cytokeratin- (an epithelial cell marker) or vimentin- (a fibroblast cell marker) positive cells and were counterstained with Carazzi’s hematoxylin.

Western blotting for EMMPRIN

Preparation of protein from placenta and fetal membranes. Tissue samples (placental villi and fetal membranes obtained at 38–40 wk of gestation) were minced into small pieces and homogenized on ice for 1 min in radioimmunoprecipitation assay lysis buffer [50 mM Tris-HCl, pH 7.5; 150 mM NaCl, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 100 µM sodium orthovanadate (Sigma), 1% (vol/vol) Triton X-100 (Fisher Chemicals, Fairlawn, NJ), and complete Mini EDTA-free protease inhibitors (Roche Molecular Biochemical, Dorval, Quebec, Canada)]. Homogenates were centrifuged at 4 C at 15,000 x g for 15 min, and supernatants were collected. The extracts were stored at –20 C until further analysis.

Western blotting. Tissue extracts were adjusted to 60 µg of total protein per sample in Laemmli sample buffer. The samples were incubated for 15 min at 55 C and applied to the 10% sodium dodecyl sulfate-polyacrylamide gel. After electrophoresis, the gels were blotted onto a nitrocellulose membrane. The membrane was blocked with 10% blocking solution (5% skim milk powder in PBS-Tween 20) at 4 C overnight. Then the membranes were treated as follows: incubation with antibody to human EMMPRIN (Santa Cruz Biotechnology Inc., Santa Cruz, CA) at 1:200 dilution in blocking solution for 1 h at room temperature; incubation with horseradish peroxidase-labeled antimouse IgG (1:5000; Amersham Pharmacia Biotech, Uppsala, Sweden) in blocking solution for 1 h at room temperature; and incubation with enhanced chemiluminescence (Amersham Pharmacia Biotech) for 1 min. Blotting without the primary antibody and with the primary antibody preincubated with blocking peptide specific for the anti-EMMPRIN antibody (Santa Cruz Biotechnology Inc.) were used as negative controls. EMMPRIN was identified in a mixed protein sample from placenta, amnion, and chorio-decidua, and this sample was run on each blot as a positive control. ß-actin protein was detected as an internal control. Finally, blots were exposed to x-ray film (Eastman Kodak Co., Rochester, NY). The intensity of the protein signal was quantified using Duo Scan Transparency Scanner and NIH Image 6.1 software (National Institutes of Health, Bethesda, MD).

Immunohistochemistry for EMMPRIN

Placenta and fetal membrane tissues. Placenta and fetal membrane tissues (nonlabor, n = 6; and labor, n = 6) were fixed by immersion in 4% paraformaldehyde and 0.2% glutaraldehyde in 70 mM phosphate buffer (pH 7.0) at 4 C overnight. Fixed tissues were then washed with PBS and stored in 90% ethanol before being embedded in paraffin wax. The paraffin blocks were sectioned at 6 µm for immunohistochemistry. Staining was performed according to the protocol described with the Vector ABC kit (Vector Laboratories, Inc., Burlingame, CA). Tissue sections were deparaffinized in xylene, rehydrated in serial gradient alcohol solution, and washed in PBS. Antigen retrieval on these sections was performed by microwave irradiation for 10 min in sodium citrate (10 mM, pH 6.0). Endogenous peroxide activity was blocked with 0.3% hydrogen peroxide in absolute methanol for 30 min at room temperature. The sections were then treated according to the following procedures. First, sections were incubated with 10% normal horse serum (that served as a blocking agent for nonspecific binding) in PBS for 1 h at room temperature. After blotting excess serum, the sections were incubated overnight at 4 C with a mouse monoclonal antibody against human EMMPRIN (CD147/neurothelin; BD Biosciences, Mississauga, Ontario, Canada) diluted 1:100 in PBS containing 1% BSA. Sections were then washed and incubated with biotinylated horse antimouse IgG at room temperature for 2 h, followed by incubation with avidin-biotin-peroxidase complex for 2 h at room temperature and incubation with a combination of 3,3'-diaminobenzidine tetrahydrochloride (Sigma) for 2–10 min at room temperature. Finally, the slides were counterstained with hematoxylin, dehydrated, cleared in xylene, and mounted. As a negative staining control, the tissue section was treated in an identical manner with the omission of primary antibody.

Analysis of cultured cells. The placental syncytiotrophoblast, amniotic epithelium, and chorionic trophoblast cells were fixed in cold acetone-ethanol (1:1) mixed solution for 10 min at 4 C. These cells were then treated according to the protocol described earlier for the staining.

RT-PCR

Extraction of total RNA. Placental syncytiotrophoblast, amniotic epithelial, and chorionic trophoblast cells in six-well plates were dispersed mechanically by scraping for 1 min and then incubated for 5 min in the presence of 0.5 ml/well Trizol. The rest of the procedures followed the manufacturer’s instructions (Invitrogen Corp., Carlsbad, CA), except that the incubation with isopropyl alcohol was performed at 4 C for 20 min. The RNA concentration and purity of each sample were determined by measuring the absorbency at 260 nm and evaluating the ratio of the absorbencies at 260/280 (optical density > 1.8). Samples were then stored at –80 C in diethylpyrocarbonate-treated water.

RT-PCR. One hundred nanograms of total RNA per sample were used for running RT-PCR by using QIAGEN OneStep RT-PCR Kit (Qiagen, Valencia, CA) in a 50 µl reaction with added 0.6 µM of target-specific primer (human EMMPRIN: sense, 5'-GGC-CAG-AAA-ACG-GAG-TTC-AA-3' and antisense, 5'-GCG-CTT-CTC-GTA-GAT-GAA-GA-3'), producing a 492-bp band. The primer was synthesized by ACGT Corp. (Toronto, Ontario). The reaction was performed according to the protocol instruction. First, reverse transcription was performed at 50 C for 30 min; second, initial PCR activation was done at 95 C for 15 min; and finally, three-step cycling PCR amplification was run for 25 cycles of 1 min of denaturation at 95 C, 1 min of annealing at 60 C, and 1 min of extension at 72 C, with 10 min of extra extension used for the last cycle. Amplifications performed without RNA were used as negative PCR control. PCR products were electrophoresed in 1.2% agarose gels, stained with 1 µg/ml ethidium bromide, and visualized under UV light.

Statistical analysis

Results are expressed as mean ± SEM for the number of different tissues (patients) studied. Statistical analysis of the differences between groups was performed using the Student’s t test. The criterion for significance was P < 0.05. Calculations were carried out using SigmaStat (Jandel Scientific Software, San Rafael, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of EMMPRIN protein in term placenta, chorio-decidua, and amnion

Western blotting analysis. The antibody directed against EMMPRIN recognized several bands between 25–75 kDa in labor or nonlabor placental villi, amnion, and chorio-decidua tissue extracts (Figs. 1Go and 2GoGo). To confirm the specificity of these bands for EMMPRIN, we used a blocking peptide for anti-EMMPRIN antibody and also omitted the primary antibody in some blotting. The results showed that all of these bands disappeared (Fig. 1Go). The biochemical characterization of EMMPRIN is consistent with previous reports (18). It suggests that EMMPRIN in human placenta and fetal membranes is composed of proteins with different degrees of glycosylation. For the characterization of EMMPRIN ex-pression in different tissues, we found 40–55 kDa bands (glycosylated EMMPRIN) in all tissue extracts, whereas an additional band at around 30 kDa (expected to be nonglycosylated EMMPRIN) was detected in placenta (Fig. 2AGoGo, L). This was usually very weak and was rarely seen in chorio-decidua tissue or amnion tissue extracts. The 65-kDa higher molecular mass band (glycosylated EMMPRIN) was present in chorio-decidua and amnion tissue extracts (Fig. 2Go, B and C, H), but this was rarely seen in placenta tissue extracts. There was a tendency for this 65-kDa molecular mass EMMPRIN to be higher in labor chorio-decidua and amnion tissue extracts than in nonlabor extracts. To evaluate the changes of EMMPRIN protein expression in placenta, chorio-decidua, and amnion between nonlabor and labor states, we compared the ratios of relative optical density (ROD) [total optical density of EMMPRIN (T)/optical density of ß-actin or optical density of 65-kDa EMMPRIN (H)/total optical density of EMMPRIN (T)] (Fig. 2GoGo, B and C). We found that the differences in the ROD ratio of H/T chorio-decidua obtained in labor were significantly higher than in nonlabor chorio-decidua and amnion (P < 0.05), although there were no significant differences in the ROD ratio of T/ß-actin between nonlabor and labor placenta, chorio-decidua, and amnion (P > 0.05).



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  		FIG. 1. Identification by Western blotting of EMMPRIN in a mixed protein sample from placenta, amnion, and chorio-decidua from patients at term pregnancy using a specific antibody. Lane 1, EMMPRIN antibody alone; lane 2, EMMPRIN antibody plus specific EMMPRIN antibody blocking peptide; lane 3, omission of EMMPRIN antibody.

 


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  		FIG. 2. Expression of EMMPRIN in term human placenta (A), chorio-decidua (B), and amnion (C). Examples of Western blotting are shown for five different placentas in each group (labor and nonlabor). Histograms show densitometric analysis of the Western blot; bars represent the relative amounts of EMMPRIN. Data are the means ± SEM for the same nine patients in each group; *, P < 0.05 labor vs. term nonlabor.

 


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  		FIG. 2A. Continued

 
Immunohistochemical staining. Representative photomicrographs demonstrating the immunolocalization of EMMPRIN in placenta and fetal membranes are shown in Fig. 3Go. There were no apparent differences in localization of EMMPRIN in tissues collected at elective caesarean section (nonlabor) when compared with those with spontaneous vaginal delivery (labor). In the placenta, EMMPRIN was expressed in the syncytiotrophoblast layer (Fig. 3AGo), whereas no immunoreactive cells were present in the villous core. In fetal membranes, EMMPRIN-positive staining was found in amniotic epithelium, trophoblast cell layer of chorion leave, reticular layer of chorion leave, and decidua (Fig. 3Go, C and C1–C3) but not in the amniotic mesenchymal cell layer. Negative control sections were without staining (for representative sections, see Fig. 3Go, B and D).



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  		FIG. 3. Immunolocalization of EMMPRIN in term human placenta (A) and fetal membranes (C). The brown staining indicates the presence of immunoreactive EMMPRIN. B and D are representative negative control sections of placenta and fetal membranes. C1, C2, and C3 are representative of selected areas in the section of fetal membranes (C). s, Syncytiotrophoblast layer; e, amniotic epithelium; m, amniotic mesenchymal layer; r, reticular layer of chorionic laeve; t, trophoblast cell layer of chorionic layer; d, decidua.

 
To examine the cellular expression of EMMPRIN further, we separated amniotic epithelial cells and generated relatively purified preparations of chorion and placental trophoblast cells. After 96 h of culture, these cells approached confluence. The analysis of identification showed that the separated amniotic epithelial cells were more than 95% cytokeratin positive and vimentin negative, and about 95% of placental cells and 90% of chorionic cells were cytokeratin positive. Immunohistochemical analysis showed that positive immunoreactive staining for EMMPRIN was present in cytokeratin-positive cells (Fig. 4Go: EMMPRIN, A, E, and I; cytokeratin, B, F, and J; vimentin, C, G, and K; and negative control, D, H, and L). These results reveal that placental syncytiotrophoblast, chorionic trophoblast, and amniotic epithelial cells expressed EMMPRIN protein. These results were consistent with the results of immunohistochemistry in placenta and fetal membrane tissues.



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  		FIG. 4. Expression of EMMPRIN in cultured and characterized human placental and fetal membrane cells. Placental syncytiotrophoblast cells (A–D), chorionic trophoblast cells (E–H), and amniotic epithelial cells (I–L). EMMPRIN (A, E, and I), cytokeratin (B, F, and J), vimentin (C, G, and K), and negative control (D, H, and L). Original magnification, x400.

 
Expression of EMMPRIN mRNA in cultured cells from term placenta and fetal membranes

To investigate expression of EMMPRIN mRNA in cultured placental syncytiotrophoblast, amniotic epithelial, and chorionic trophoblast cells, we used RT-PCR with specific primers and total RNA prepared from these cell types from tissue obtained at elective caesarean section (n = 3). Amniotic epithelial (Fig. 5Go, lane 2), placental syncytiotrophoblast (lane 3), and chorionic trophoblast (lane 4) cells expressed 492-bp EMMPRIN. This expression corresponded with the results of Western blotting and immunohistochemistry (see Western blotting analysis and Immunohistochemical staining sections). There was no signal in the negative control (lane 5).



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  		FIG. 5. Expression of EMMPRIN mRNA in cultured human placental and fetal membranes. Lane 1, Marker; lane 2, placental syncytiotrophoblast; lane 3, amniotic epithelial cells; lane 4, chorionic trophoblast cells; and lane 5, negative control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we show clearly the presence of EMMPRIN in human term placenta and fetal membranes. EMMPRIN protein was detected by immunohistochemistry mainly in syncytiotrophoblast from placenta villi, amnion epithelium, chorion laeve trophoblast, and decidua parietalis. EMMPRIN mRNA transcripts were detected in placental syncytiotrophoblast, amniotic epithelial cells, and chorionic trophoblast cells by RT-PCR. By Western blot, we found different molecular mass bands corresponding to EMMPRIN in placenta and fetal membranes. These immunoreactive forms included one at around 30 kDa, several around 40–55 kDa, and one around 65 kDa bands. These results are consistent with those of Miyauchi et al. (18). These authors pointed out that the molecular mass of glycosylated basigin was largely in the range between 43 kDa and 66 kDa, whereas that of the peptide portion with a putative signal sequence was inferred to be about 30 kDa. Additionally, previous experiments have indicated that N-linked oligosaccharide chains may contribute approximately 50% of the size of mature CD147 because the high-molecular mass band was shifted to approximately 28 kDa after treatment with endoglycosidase F and N-glycanase (19, 20). Thus, these differences in molecular mass of EMMPRIN are likely due to varying extents of glycosylation because the protein backbone of EMMPRIN corresponds to an approximate molecular mass of 27 kDa. These data, taken together, suggest that human placenta and fetal membrane express glycosylated EMMPRIN to varying extents.

It is noteworthy that the highly glycosylated 65-kDa EMMPRIN increased in fetal membranes at term after labor compared with nonlabor fetal membranes, whereas the total amount of EMMPRIN protein did not change significantly. This observation raises the possibility of labor-associated changes in posttranslational processing, for example, glycosylation of EMMPRIN. Gao et al. (32) found that bacterially produced recombinant EMMPRIN (~29 kDa) and recombinant EMMPRIN from Chinese hamster ovary cells transfected with EMMPRIN cDNA (29–45 kDa) were inactive in stimulating MMP production by human fibroblasts. However, approximately 58-kDa EMMPRIN produced by Chinese hamster ovary cells transfected with EMMPRIN cDNA significantly stimulated MMP-1, -2, and -3 productions. Furthermore, a recent study showed that purified deglycosylated EMMPRIN treated with tunicamycin, an inhibitor of N-glycanase, failed to induce MMP-1 or MMP-2 but, instead, antagonized the MMP-1-inducing activity of purified native CD147 (24). These results suggest that maturation and N-link glycosylation of CD147 are essential for the capability to stimulate MMPs. Taken together with our results, this implies that the degree of glycosylation of EMMPRIN may be critical for the effects of EMMPRIN in human parturition. Although these results suggest that glycosylation is important for functional activity, the significance of differing extents of glycosylation in the human placenta and fetal membranes remains to be evaluated.

EMMPRIN was previously referred to as tumor cellderived collagenase stimulatory factor. Using a multicellular culture system, it has been postulated that EMMPRIN induces MMP expression by cell-cell interaction or in a paracrine/autocrine manner. Both native and recombinant purified CD147 may induce the production of MMP-1, MMP-2, MMP-3, and MT1-MMP by fibroblasts and breast cancer cells, and EMMPRIN antibodies inhibited MMP production, consistent with CD147-dependent regulation of MMPs (24, 26, 32, 33). A recent study has shown that EMMPRIN antibody inhibited not only MMP-1 and MMP-2 but also MMP-9 activity in multidrug-resistant cancer cells, providing direct evidence that EMMPRIN is involved in the regulation of MMP-9 production (27). Interestingly, in vitro studies have demonstrated that although EMMPRIN induced MMP expression, it did not influence basal expression of TIMPs (31, 32). It is suggested strongly that an imbalance of active/inactive MMP production results from EMMPRIN action on cancer systems. Furthermore, Taylor et al. (25) have found that the EMMPRIN-induced MMP-2 production is mediated through the activation of a phospholipase A2/5-lipoxygenase pathway in fibroblasts. Lim et al. (36) investigated the signaling pathway mediating CD147 stimulation of fibroblast MMP-1 production and determined that protein tyrosine kinase and p38 MAPK were involved in the response. Thus, the glycoprotein may use distinct signaling pathways in the regulation of different MMPs. At present, despite this body of observations, the precise mechanism of action is largely unclear.

Most studies of MMPs and TIMPs have emphasized the key role of MMPs in the breakdown of ECM that ultimately leads to rupture of the fetal membranes and detachment of the placenta from maternal uterus at human parturition. A marked increase in expression of several MMPs (MMP-1, -2, -3, and -9) in placenta and fetal membranes or amniotic fluid occurs just after the onset and during parturition in association with a significant decrease in the expression of TIMPs (TIMP-1, -2, -3, and -4) (2, 3, 4, 5, 6, 7, 8, 9). As a result, a major alteration in the balance between enzymes and their inhibitors occurs that is in favor of tissue degradation. In addition, it is believed that alteration of this balance represents the final common pathway by which different regulators control MMP activity. Earlier studies have shown that a range of extracellular regulators can alter the expression and activity of MMPs in various cell systems. In human placenta and fetal membranes, it is likely that members of hormonal, cytokine signaling cascades and TIMPs contribute to alterations in MMP expression and activity. Locally produced TIMPs have been show to inhibit the activity of secreted MMPs by the formation of a 1:1 complex with MMP. IL-10 eliminated lipopolysaccharide induction of MMP-2 and MMP-9 in amniochorion (10), whereas TNF-{alpha}, relaxin, prostaglandin F2{alpha}, and prostaglandin E2 stimulated the release of MMP-1, -3, and -9 from human fetal membranes and cultured chorionic cells with a concomitant decrease in TIMPs expression (11, 12, 13, 14). It was expected that EMMPRIN would play a fundamental role in various physiological and pathological processes because of its broad distribution and effects on MMP production (28, 29, 30, 31). Because EMMPRIN is expressed by the human placenta and fetal membranes and the levels of glycosylated EMMPRIN increase selectively in association with labor, we suggest that EMMPRIN may mediate MMP action at that time. Previous studies (6, 37) have shown that placental syncytiotrophoblasts, chorion trophoblasts, and amnion epithelium are also major sites of MMP expression, consistent with the distribution of EMMPRIN in these tissues. Thus, changes in EMMPRIN expression may indirectly influence MMP action to enhance tissue degradation, leading to further rupture of the fetal membranes and detachment of placenta and fetal membrane from uterus. Future studies involving the use of purified EMMPRIN and cultured placental and fetal membrane cells are required to elucidate fully these steps in EMMPRIN-mediated MMP regulation.


   Footnotes
 
Abbreviations: ECM, Extracellular matrix; EMMPRIN, extracellular matrix metalloproteinase inducer; MMP, matrix metalloproteinase; ROD, relative optical density; T, total optical density of EMMPRIN; TIMP, tissue inhibitor of matrix metalloproteinase.

Received November 26, 2003.

Accepted March 4, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Woessner Jr JF 1991 Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J 5:2145–2154[Abstract]
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