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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 12 6063-6072
Copyright © 2003 by The Endocrine Society

Identification and Characterization of Extracellular Matrix Metalloproteinase Inducer in Human Endometrium during the Menstrual Cycle in Vivo and in Vitro

Yutaka Noguchi, Takashi Sato, Michiko Hirata, Tetsuaki Hara, Koso Ohama and Akira Ito

Department of Biochemistry and Molecular Biology (Y.N., T.S., M.H., A.I.), School of Pharmacy, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo 192-0392, Japan; Bio-oriented Technology Research Advancement Institution (M.H.), Minato-ku, Tokyo 105-0001, Japan; and Department of Obstetrics and Gynecology (T.H., K.O.), Hiroshima University School of Medicine, Minami-ku, Hiroshima 734-8551, Japan

Address all correspondence and requests for reprints to: Takashi Sato, Department of Biochemistry and Molecular Biology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan. E-mail: satotak{at}ps.toyaku.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Extracellular matrix metalloproteinase inducer (EMMPRIN) participates in the breakdown of the extracellular matrix (ECM) by augmenting matrix metalloproteinase (MMP) expression. In the present study, we identified and characterized the menstrual cycle-dependent expression of EMMPRIN in human endometrium in vivo. At the proliferative phase of the menstrual cycle, EMMPRIN was detected in glandular epithelium of the basal layer in endometrium. In addition, at the superficial region of the functional layer, EMMPRIN was expressed in stroma but not glandular epithelium. At the secretory phase, EMMPRIN was found in both stroma and glandular epithelium of the functional layer and glandular epithelium of the basal layer. Furthermore, EMMPRIN colocalized with MMP-1/collagenase-1 in the glandular epithelium in vivo. Western blot analysis of tissue from the functional layer showed that EMMPRIN species with molecular weights of approximately 35 and 47 kDa were detected at the proliferative phase, whereas approximately 35- and 51-kDa EMMPRIN species were predominantly expressed at the secretory phase. In addition, the variant EMMPRIN molecules were found to differ in glycosylation. On the other hand, EMMPRIN was constitutively produced in primary cultured endometrial stromal and glandular epithelial cells. The production and glycosylation of EMMPRIN in the stromal cells were augmented by progesterone at the posttranscriptional and posttranslational stages, respectively. These results suggest for the first time that EMMPRIN is expressed in human endometrium during the menstrual cycle and that its expression and glycosylation are augmented by progesterone. Moreover, EMMPRIN may be involved in ECM breakdown at the interface between endometrial cells and ECM by using EMMPRIN-bound MMP-1.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
HUMAN ENDOMETRIUM IS a unique tissue that spatiotemporally undergoes cyclical tissue remodeling during the menstrual cycle, which is a requisite event for establishing and maintaining pregnancy (1). During the proliferative phase of the menstrual cycle, endometrial cells proliferate and grow from approximately 0.5 to 5 mm in height. By angiogenesis, spiral vessels extend to a point immediately below the epithelial membrane. These structural changes are controlled by an ovarian steroid hormone, estradiol. At the secretory phase, when progesterone reaches physiological levels, stromal edema occurs and many sinuous epithelial glands appear in the endometrium along with the infiltration of inflammatory cells such as large granular lymphocytes and macrophages. At the late secretory phase, the fall in concentration of progesterone is followed by the onset of menstruation, in which the functional layer of endometrium is shed into the uterine lumen and bleeding occurs (2, 3).

Throughout the normal menstrual cycle, the degradation and regeneration of the extracellular matrix (ECM) are crucial events to cause maintenance and disruption of the functional layer and of blood vessel integrity. As pivotal enzymes in the cycle, matrix metalloproteinases (MMPs) play important roles in tissue dissolution (1, 4). More than 20 MMPs with different structural and substrate characteristics have been identified and classified to five subfamilies; collagenases, gelatinases, stromelysins, membrane type-MMPs (MT-MMPs), and others (5). Human endometrium has been reported to temporally and spatially produce different sets of MMPs. In premenstrual and menstrual phases, MMP-1 and MMP-3 have been detected mostly in stromal cells in normal endometrium (6, 7). MMP-2 and MMP-9 have also been expressed in stroma and glandular epithelium through the menstrual cycle or at the secretory phase (6, 8, 9). Furthermore, MT1-MMP has been expressed in endometrial epithelium throughout the menstrual cycle (10, 11).

The expression of endometrial MMPs during the menstrual cycle is regulated by steroid hormones such as estradiol and progesterone (12, 13, 14, 15) and various cytokines and growth factors such as IL-1, TNF{alpha}, and TGFß (1, 4). In addition, endometrial stromal and epithelial cells produce tissue inhibitors of metalloproteinases (TIMPs) (16, 17), the production of which is also regulated by endocrine and paracrine factors (4), suggesting that the balance between MMPs and TIMPs is crucial for tissue remodeling in endometrium during the menstrual cycle. Recently, Zhang and Salamonsen (18) reported that both gelatinase and collagenase activities are present at discrete foci in endometrial tissues and increase at menstruation in vivo, suggesting that the locally augmented production and activation of endometrial MMPs may be more important in tissue breakdown during the menstrual cycle. Therefore, a unique mechanism for focal MMP expression may exist in endometrium during the cycle, but this remains unclear.

Extracellular matrix metalloproteinase inducer (EMMPRIN)/CD147 is a membrane-associated glycoprotein, which belongs to the immunoglobulin superfamily (19, 20), and is identical to human basigin (21) and human leukocyte activation-associated M6 antigen (22). EMMPRIN is highly expressed on the cell surface of various tumors (23, 24, 25, 26, 27) and stimulates peritumoral fibroblasts to produce proMMP-1, -2, and -3, and MT1- and MT2-MMPs (19, 20, 28). In addition to the MMP-inducible activity, Guo et al. (29) reported that proMMP-1 can be localized to the cell surface of human lung carcinoma LX-1 cells by binding to EMMPRIN. On the other hand, EMMPRIN has been expressed in normal human keratinocytes (30) and differentiated macrophages (31). In addition, Igakura et al. (32) reported that mouse basigin is involved in embryonic implantation in basigin-knockout mice. Therefore, it seems likely that EMMPRIN closely participates in remodeling of the tissue microenvironment under physiological conditions and plays an important role at least in successful implantation. However, it remains unclear whether EMMPRIN is expressed in human endometrium and whether EMMPRIN is involved in MMP expression associated with tissue remodeling in endometrium during the menstrual cycle.

In the present study, we found novel evidence that EMMPRIN is expressed in human endometrium in vivo and that its tissue distribution differs during different phases of the menstrual cycle. In addition, EMMPRIN was found to colocalize with MMP-1/collagenase-1 in human endometrium in vivo. Furthermore, the production and glycosylation of EMMPRIN were augmented by progesterone in primary cultures of human endometrial stromal cells. These results suggest that EMMPRIN may participate in endometrial ECM breakdown during the human menstrual cycle.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Specimens

Human endometrial tissues were obtained (with informed consent) from hysterectomy specimens from 27 nonpregnant patients aged 30–51 yr who had normally cycling menstrual cycles. The hysterectomies had been performed to treat benign diseases. Only histologically normal endometrial tissues were included in the study. Endometrial samples were dated with respect to the menstrual cycle using Noyes’ histological criteria (33): early (<d 7, n = 2), mid- (d 8–10, n = 3), and late (d 11–14, n = 8) proliferative phase; and early (d15–19, n = 5), mid- (d 20–24, n = 7), and late (d 25–28, n = 2) secretory phase. Of these 27 tissue samples, all of which were immediately frozen in liquid nitrogen for later assay, 17 were studied by RT-PCR and 15 by Western blot analysis. Some portions of each tissue sample were fixed in 4% paraformaldehyde for 60 min, dehydrated, and embedded in paraffin wax. Other portions were snap frozen in liquid nitrogen and stored at -80 C. The fixed or frozen tissues were sectioned into 4-µm thick sections for immunohistochemical analysis. Use of the specimens was approved by the institutional review committees at Hiroshima University Hospital, Hiroshima Memorial Hospital, and Hiroshima Prefectual Hospital, Japan.

Preparation of endometrial stromal cells and glandular epithelial cells

Primary cultures of human endometrial stromal and glandular epithelial cells were prepared from endometrium of six nonpregnant patients at the secretory phase (d 16–22) by the method developed by Satyaswaroop et al. (34) with some modifications. Minced endometrial tissues were incubated in 20 ml DMEM/F12 (Invitrogen, Carlsbad, CA) containing 300 IU/ml of collagenase type VI and 0.002% DNase (Sigma-Aldrich Fine Chemicals, St. Louis, MO) at 37 C for 60 min. The digested tissues were filtered using double layers of monofilament nylon mesh membranes (pore size: 37 and 105 µm). Stromal, epithelial, and blood cells passed through these membranes while undigested glandular tissues were trapped by the 37-µm pores.

For the preparation of stromal cells, the filtered-cell mixture was placed in 25-cm2 tissue culture flasks (BD Biosciences, Tokyo, Japan) and incubated for 30 min at 37 C. Adhering cells had the characteristics of stromal cells. In addition, we confirmed that these endometrial stromal cells caused augmentation of prolactin mRNA expression by administering progesterone (P4) in vitro as previously described (35, 36).

For the preparation of glandular epithelial cells, glandular tissues trapped by the 37-µm pore membrane were incubated in 20 ml DMEM/F12 containing 0.025% trypsin (Invitrogen) for 3 min at 37 C. Tissues were then supplemented with the same medium containing 10% heat-inactivated fetal bovine serum (FBS) (JRH Biosciences, Lenexa, KS) to terminate the trypsin digestion. When the digested cell mixture was placed in 25-cm2 tissue culture flasks and incubated for 30 min at 37 C, unadhered cells had the characteristics of glandular epithelial cells.

Cell culture and treatments

Endometrial stromal cells (1 x 106 cells) or glandular epithelial cells (1 x 106 cells) were seeded in 25-cm2 culture flasks and cultured in DMEM/F12 with 10% FBS, 2 mM L-glutamine, 20 mM HEPES, and 100 ng/ml gentamicin (Sigma) for 3 d to complete cell adhesion. Next, the cells were maintained in the same fresh medium supplemented with P4 (1 x 10-6 M) or 17ß-estradiol (E2) (1 x 10-6 M) (Sigma) for 24 h. After washing cells with serum-free DMEM/F12, the treatment of cells with P4 and E2 in the same serum-free culture medium was carried out for another 24 h to monitor direct hormonal effects as previously described (37, 38). Otherwise, to eliminate endogenous hormonal effects, endometrial stromal cells (1 x 106 cells) or glandular epithelial cells (1 x 106 cells) were seeded in 35-mm culture dishes (BD Biosciences) and cultured for 3 d to complete cell adhesion. Next, the cells were maintained every 2 d in 10% FBS/DMEM/F12 with exogenous P4 (3.2 x 10-7 M) and/or E2 (3.7 x 10-9 M) for 7 d and then were similarly treated with the same concentration of P4 and E2 in serum-free DMEM/F12 for another 24 h. The concentrations and the molar ratio of P4 and E2 used corresponded to their levels in peripheral blood at the secretory phase during menstruation (39). Moreover, to investigate whether the actions of E2 are mediated through its receptor in freshly prepared endometrial cells, confluent stromal and glandular epithelial cells in 25-cm2 culture flasks were pretreated with a high-affinity E2 receptor antagonist, ICI 182,780 [7{alpha}-(9[4,4,5,5,5,-pentafluoropemtyl]sulfinyl)-estra-1,3,5[10]-triene-3,17ß-diol] (40 x 10-6 M) (Tocris, Ellisville, MO) (40, 41) for 1 h and then treated with E2 (1 x 10-6 M) and the combination of E2 (1 x 10-6 M) and P4 (1 x 10-6 M) in the presence of the antagonist for 24 h under serum-free conditions. Human uterine cervical carcinoma SKG-II cells (42) (kindly provided by Prof. S. Hirakawa, Toho University School of Medicine, Tokyo, Japan) were cultured to confluence in DMEM with 10% FBS, 200 U/ml penicillin, and 200 µg/ml streptomycin.

Immunohistochemistry

Formalin-fixed, paraffin-embedded sections of human endometrium were deparaffinized in xylene and rehydrated by routine methods and then incubated in methanol with 0.3% H2O2 for 10 min at room temperature to block endogenous peroxidase activity. For the localization of endometrial EMMPRIN, immunohistochemical staining was performed using a Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA). After blocking nonspecific reactivity with diluted normal horse serum, sections were incubated with mouse anti-(human CD147)antibody (10 µg/ml) (PharMingen, San Diego, CA) for 1 h at room temperature and then exposed to biotinylated antimouse IgG for 30 min at room temperature, followed by incubation with avidin-biotin complex reagent for 30 min at room temperature. Reaction products were visualized using 3-amino-9-ethylcarbazole in the presence of 0.003% H2O2, and sections were counterstained with hematoxylin before clearing and mounting.

For colocalization of EMMPRIN and MMP-1, thawed-frozen tissue samples were cut into 4-µm-thick sections, placed on glass slides, and then fixed with acetone for 30 min at -20 C. After rinsing sections with PBS, the slides were incubated with fluorescein isothiocyanate fluorescein isothiocyanate-conjugated mouse anti-(human CD147)IgG (5 µg/ml) (Ancell Co., Bayport, MN) and a sheep polyclonal antibody of human proMMP-1 (30 times dilution) (kindly provided by Prof. H. Nagase) in 1% BSA/PBS for 1.5 h at room temperature. The sections were washed with PBS and then incubated with tetramethylrhodamine isothiocyanate tetramethylrhodamine isothiocyanate-conjugated donkey anti-(sheep IgG) (2.5 µg/ml) (Chemicon International, Temecula, CA) in 1% BSA/PBS for 1.5 h at room temperature for detecting MMP-1. After washing with PBS, counterstaining was performed with 4'6-diamidino-2-phenylindole (Sigma). Negative control sections were processed by substituting nonimmune serum for the primary antibody. Sections were viewed with a fluorescence and differential interference microscope (Olympus Optical Co., Tokyo, Japan), and the colocalization of EMMPRIN and MMP-1 in endometrium was shown by merging the colors using a computer.

Semiquantification of mRNA levels by RT-PCR

Cytoplasmic RNA (3 µg) isolated from human endometrial tissues was subjected to the synthesis of first-strand cDNA by Moloney-murine leukemia virus reverse transcriptase, RNase inhibitor (Roche Diagnostics, Tokyo, Japan) and oligo(dT)12–18 primer (Invitrogen) for 1 h at 37 C. One-tenth of the cDNA generated from the reverse transcription reaction was used for PCR amplification using specific primers for human EMMPRIN, proMMP-1, proMMP-3, TIMP-3, and human glyceraldehyde-3 phosphate dehydrogenase (GAPDH). Sense and antisense primers used were as follows. Human EMMPRIN: sense, 5'-GAATTCGAATCATGGCGGCTGCG-3' (-11 to 12 bp) (underlined; designed EcoRI) and antisense, 5'-TCGGGGCGGCCGCCTCAGGAA-3' (804–824 bp) (underlined; designed NotI) (20); human proMMP-1: sense, 5'-GGTGATGAAGCAGCCCAG-3' (323–340 bp) and antisense, 5'-CAGTAGAATGGGAGAGTC-3' (742–759 bp) (43); human proMMP-3: sense, 5'-AGTGGAAATGAAGAGTCTTC-3' (37–56 bp) and antisense, 5'-GTCACCTCTTCCCAGACT-3' (460–467 bp) (44); human TIMP-3: sense, 5'-CTACACCATCAAGCAGATGAAGATG-3' (253–277 bp) and antisense, 5'-GCTCAGGGGTCTGTGGCATTGATG-3' (686–709 bp) (45) and human GAPDH: sense, 5'-CCACCCATGGCAAATTCCATGGCA-3' (213–235 bp) and antisense, 5'-TCTAGACGGCAGGTCAGGTCCACC-3' (786–809 bp) (43). PCR was performed at 92 C for 40 sec, 56 C for 40 sec, and 72 C for 1 min with 28–32 cycles (for EMMPRIN and GAPDH) and 32–36 cycles (for proMMPs and TIMP-3), under which these genes were linearly amplified. The amplified PCR products were analyzed on 1% agarose gels and visualized by ethidium bromide staining. Furthermore, we confirmed cDNA sequences of the amplified PCR products by ligating them into pGEM-T Easy vector (Promega, Madison, WI) as described previously (46).

Western blot analysis for EMMPRIN

Endometrial tissues, stromal cells, epithelial cells, and SKG-II cells were homogenized in a lysis buffer: 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 1 mM EDTA, 10 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride. After centrifugation at 800 x g for 5 min at 4 C, the cells were resuspended in the same buffer, sonicated six times at 4 C for 10 sec, and then centrifuged at 8000 x g for 15 min at 4 C. Proteins in the resulting supernatant (50–100 µg) were precipitated by mixing with trichloroacetic acid (final concentration: 3.3%) and then resolved in SDS-PAGE sample buffer containing 2-mercaptoethanol (47). The proteins separated on 10% acrylamide gel were electrotransferred onto a nitrocellulose membrane. The membranes were reacted with mouse anti-(human CD147)antibody (PharMingen), which was then complexed with horseradish peroxidase-conjugated goat anti-(mouse IgG)IgG (Sigma). Immunoreactive EMMPRIN was visualized with enhanced chemiluminescence-Western blotting detection reagents (Amersham Biosciences, Tokyo, Japan) according to the manufacturer’s instructions. The relative amounts of those proteins were quantified by densitometric scanning using an Image Analyzer LAS-1000 plus (Fuji Photo Film Co., Tokyo, Japan).

Detection of the N-oligosaccharide chain of EMMPRIN by N-glycosidase

The homogenate (50 µg) of endometrial tissues and SKG-II cells was incubated with 10 U/ml N-glycosidase F (Roche) for 30 min at 37 C and then subjected to Western blot analysis for EMMPRIN using 15% acrylamide gel as described above.

Determination of prostaglandin (PG) F2{alpha} in the culture medium

PGF2{alpha} in the culture medium from primarily cultured human endometrial stromal and glandular epithelial cells was determined using commercial enzyme immunoassay kits (Cayman Chemical Co., Ann Arbor, MI) according to the manufacture’s instructions.

Statistical analysis

Data are presented as mean ± SD and were analyzed by ANOVA and unpaired t test. P < 0.05 was considered to indicate a statistically significant difference.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of EMMPRIN in human endometrium during the menstrual cycle

To clarify whether EMMPRIN was expressed in human endometrium during the menstrual cycle, immunohistochemical analysis was performed. As shown in Fig. 1BGo, at the proliferative phase, EMMPRIN was expressed in glandular epithelium in the basal layer and in the deeper region of the functional layer. In addition, EMMPRIN in the glandular epithelium was found to localize to both cytoplasm and the cell membrane of epithelial cells (Fig. 1CGo). However, there was no signal in luminal epithelium and glandular epithelium in the superficial region of the functional layer (Fig. 1AGo). On the other hand, stromal cells in the superficial region of the functional layer expressed EMMPRIN in the cytoplasm (Fig. 1AGo), whereas stromal cells were negative in the deeper functional and basal layers (Fig. 1BGo). In addition, there was no signal in endometrial myometrium (Fig. 1BGo).



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  		FIG. 1. Expression of EMMPRIN in human endometrium. Human endometrial tissues at the proliferative (A–C) and secretory phases (D–F) were subjected to immunohistochemical staining for EMMPRIN as described in Materials and Methods. Three independent experiments using endometrial tissues from different donors were reproducible, and typical data are shown. FL, Functional layer; BL, basal layer; MY, uterine myometrium; and LE, luminal epithelium. Bars, 50 µm (A, B, D, and E) and 10 µm (C and F).

 
At the secretory phase, EMMPRIN was detected in glandular epithelium of both basal and functional layers and in stroma of the functional layer (Fig. 1Go, D and E). However, there was still no detection of EMMPRIN in luminal epithelium (Fig. 1DGo), stroma of the basal layer, and myometrium (Fig. 1EGo). It was of interest that EMMPRIN was predominantly localized to the cell membrane of glandular epithelial cells. In addition, the localization was observed at the site of the basement membrane and intercellular region of epithelial cells but not at the site of the glandular lumen (Fig. 1FGo). Furthermore, endometrial stromal and glandular epithelial cells of the functional layer produced MMP-1 (Fig. 2BGo), which colocalized with EMMPRIN in glandular epithelium of the endometrium (Fig. 2CGo). These results suggest for the first time that EMMPRIN is expressed in human endometrium, and its distribution and localization differ during the menstrual cycle.



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  		FIG. 2. Colocalization of EMMPRIN and MMP-1 on glandular epithelium of human endometrium. Human endometrial tissues at the secretory phase were reacted with fluorescein isothiocyanate-conjugated mouse anti-(human CD147)IgG (A) and sheep anti-(human proMMP-1)antibody (B) and then reacted with tetramethylrhodamine isothiocyanate-conjugated donkey anti-(sheep IgG)IgG. C, Merged staining of EMMPRIN and MMP-1. D, Counterstaining with 4'6-diamidino-2-phenylindole. Three independent experiments using endometrial tissues from different donors were reproducible, and typical data are shown. Bar, 50 µm.

 
Characterization of endometrial EMMPRIN

As shown in Fig. 3Go, EMMPRIN mRNA was detected in human endometrium at both proliferative (Fig. 3AGo) and secretory phases (Fig. 3BGo), and there was no significant difference in the level of its mRNA between phases. Western blot analysis showed the menstrual cycle-dependent variation of EMMPRIN proteins in human endometrial tissues: approximately 35- and 47-kDa species at the proliferative phase and approximately 35- and 51-kDa species at the secretory phase were detected (Fig. 4AGo). In addition, the 51-kDa EMMPRIN had the same mobility as that from human uterine cervical carcinoma SKG-II, in which EMMPRIN is expressed on the cell surface and augmented MMP production (our unpublished observations). However, these bands disappeared upon substitution of the first antibody with nonimmune mouse IgG (data not shown), indicating that the immunological bands corresponded to EMMPRIN. Furthermore, the amount of 51-kDa EMMPRIN increased relative to the 47-kDa EMMPRIN in endometrium at the secretory phase (P < 0.05) (Fig. 4BGo). Thus, these results suggest the menstrual cycle-dependent regulation of EMMPRIN expression in human endometrium in vivo.



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  		FIG. 3. Expression of EMMPRIN, proMMPs, and TIMP-3 mRNAs in human endometrium. Cytoplasmic RNAs (3 µg) isolated from human endometrial tissues at the proliferative (P) (A) and secretory phases (S) (B) were subjected to RT-PCR for EMMPRIN, proMMP-1, proMMP-3, TIMP-3, and GAPDH mRNA as described in Materials and Methods. A, Sample tissues consisted of two early (lanes 1 and 2), three mid- (lanes 4, 6, and 9), and four late (lanes 3, 5, 7, and 8) phases. B, Sample tissues consisted of three early (lanes 10–12), four mid- (lanes 13–15 and 17), and one late (lane 16) phases.

 


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  		FIG. 4. Characterization of EMMPRIN in human endometrium. A, Plasma membrane fractions (50 µg) from human endometrial tissues at the proliferative (P) and secretory phases (S) and human uterine cervical carcinoma SKG-II cells were subjected to Western blot analysis for EMMPRIN as described in Materials and Methods. Sample tissues consisted of two mid- (lanes 3 and 5) and five late (lanes 1, 2, 4, 6, and 7) proliferative phases and two early (lanes 8 and 9), four mid- (lanes 10, 12, 13, and 15), and two late (lanes 11 and 14) secretory phases. B, The relative amounts of 51- and 41-kDa EMMPRIN (A) were quantified by densitometric scanning, and then the expression of 51-kDa EMMPRIN relative to 41-kDa EMMPRIN was expressed as the mean ± SD. *, Significantly different from tissues at proliferative phase (P < 0.05).

 
Next, to confirm whether the variant EMMPRIN contributed to differences in glycosylation because of a membrane-bound glycoprotein of EMMPRIN (19, 20), the deletion of oligosaccharide chains of EMMPRIN by N-glycosidase F was performed. As shown in Fig. 5Go, immunological bands with 35, 47 and 51 kDa in endometrium at the proliferative and secretory phases were entirely shifted to a single band with approximately 30 kDa. In addition, the motility of N-glycosidase-treated endometrial EMMPRIN was the same as that for SKG-II cells (Fig. 5Go). Therefore, these results suggested that EMMPRIN mRNA is constitutively expressed in human endometrium through the proliferative and secretory phases and that human endometrium expresses variant EMMPRIN with differences in its glycosylation occurring in a menstrual cycle-dependent manner.



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  		FIG. 5. Differential glycosylation of EMMPRIN in human endometrium during the menstrual cycle. Plasma membrane fractions (50 µg) from human endometrial tissues at the proliferative (P) and secretory (S) phases and from human uterine cervical carcinoma SKG-II cells were incubated with (+) or without (-) N-glycosidase F (10 U/ml) for 24 h at 37 C and then subjected to Western blot analysis for EMMPRIN as described in Materials and Methods. Four independent experiments using endometrial tissues from different donors were reproducible, and typical data are shown.

 
Production and glycosylation of EMMPRIN are regulated by P4 and E2 in human endometrial stromal and glandular epithelial cells

Our finding that in vivo localization and glycosylation of EMMPRIN were altered in human endometrium during the menstrual cycle suggested that the production and glycosylation of EMMPRIN might be regulated by sex hormones. To test this hypothesis, we first examined the effects of P4 and E2 on the production of EMMPRIN in cultured primary stromal and glandular epithelial cells, which were prepared from the functional layer of human endometrium at the secretory phase. As shown in Fig. 6Go, both stromal and epithelial cells were found to produce EMMPRIN with approximately a 51-kDa molecular mass. When stromal and epithelial cells were treated with E2 (1 x 10-6 M) and P4 (1 x 10-6 M), the production of EMMPRIN in stromal cells was augmented by P4 (1.60 ± 0.20-fold, P < 0.01) but not E2 (Fig. 6AGo, left panel). In addition, the expression of stromal EMMPRIN mRNA was not modulated by these treatments (Fig. 6BGo, left panel). Furthermore, neither E2 nor P4 influenced the mRNA expression and production of EMMPRIN in epithelial cells (Fig. 6Go, A and B, right panels). Thus, these results suggest that the production of EMMPRIN in human endometrial stromal cells is posttranscriptionally augmented by P4. Moreover, the hormonal regulation of EMMPRIN production in human endometrium may differ between stromal and epithelial cells.



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  		FIG. 6. Hormonal regulation of the expression of EMMPRIN in primarily cultured human endometrial stromal and glandular epithelial cells. Human endometrial stromal (left panels) and glandular epithelial cells (right panels) in primary culture were maintained in DMEM/F12 medium supplemented with 10% FBS for 3 d and then pretreated with E2 (1 x 10-6 M) and P4 (1 x 10-6 M) for 24 h. After pretreatment, the cells were treated with the same concentration of E2 and P4 in serum-free DMEM/F12 medium for another 24 h. The harvested plasma membrane fractions (50 µg) and isolated RNA (3 µg) were subjected to Western blot analysis for EMMPRIN (A) and RT-PCR for EMMPRIN and GAPDH mRNAs (B and C), respectively. Four independent experiments were reproducible, and typical data are shown.

 
Next, we examined the effects of physiological levels of P4 and E2 on the expression of EMMPRIN in endometrial stromal cells. When the cells derived from the secretory phase were treated with P4 (3.2 x 10-7 M) and E2 (3.7 x 10-9 M), the concentrations of which corresponded to their levels in blood at the secretory phase (39), two immunological bands with 47- and 51-kDa sizes were detected in untreated stromal cells (Fig. 7Go). This result indicated that an appearance of the low-molecular-weight form of EMMPRIN resulted from the elimination of the endogenous effect of sex hormones through in vitro culture. In addition, although the appearance of 47-kDa EMMPRIN was not prevented by E2 treatment, P4 was found to interfere with the appearance of 47-kDa EMMPRIN. A similar interference was observed in stromal cells treated with both E2 and P4. Therefore, these results suggest for the first time that the glycosylation of EMMPRIN is regulated by P4 in human endometrial stromal cells.



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  		FIG. 7. Augmentation of glycosylation of EMMPRIN by progesterone in primary cultured human endometrial stromal cells. Human endometrial stromal cells in primary culture were maintained in DMEM/F12 medium supplemented with 10% FBS for 3 d and then pretreated with E2 (3.7 x 10-9 M) and/or P4 (3.2 x 10-7 M) every 2 d for 7 d. After the pretreatment, the cells were treated with the same concentration of E2 and/or P4 in the serum-free medium for another 24 h. The harvested plasma membrane fractions (50 µg) were subjected to Western blot analysis for EMMPRIN. Four independent experiments were reproducible, and typical data are shown.

 
Hormonal regulation of the gene expression of proMMPs and TIMP-3 and the production of PGF2{alpha} in human endometrium in vivo and in vitro

As shown in Fig. 3Go, in human endometrium, mRNA expression of proMMP-1 and proMMP-3 was more detectable at the proliferative phase than at the secretory phase. In contrast, the expression of endometrial TIMP-3 mRNA was predominant at the secretory phase (Fig. 3BGo). On the other hand, in cultured human endometrial stromal cells, E2 treatment (1 x 10-6 M) resulted in a significant decrease in the level of proMMP-1 mRNA in stromal cells (P < 0.01), but there was less of a decrease in the level of proMMP-3 mRNA (Table 1Go). In addition, P4 treatment (1 x 10-6 M) suppressed the expression of both stromal proMMP-1 and -3 mRNAs (P < 0.001 and P < 0.05, respectively). Further suppression of proMMP-1 mRNA expression was observed following combined treatment of E2 and P4 (P < 0.001). In contrast, TIMP-3 mRNA expression in stromal cells was found to increase with E2 or P4 treatment (P < 0.01 and P < 0.001, respectively) and by their combination (P < 0.05). However, although the expression of epithelial proMMP-3 was suppressed by combined treatment of E2 and P4 (P < 0.001), the expression of either proMMP-1 or TIMP-3 mRNA was not altered by P4 and/or E2. Furthermore, as shown in Table 2Go, the production of PGF2{alpha} in glandular epithelial cells was augmented by E2 (P < 0.01) and to a lesser extent by the combined treatment of E2 and P4 (P < 0.05) but not by P4 alone. In addition, neither E2 nor P4 altered the level of PGF2{alpha} in stromal cells.


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  		TABLE 1. Hormonal regulation of proMMPs-1 and -3 and TIMP-3 mRNA expression in primary cultured human endometrial stromal and glandular epithelial cells

 

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  		TABLE 2. Hormonal regulation of PGF2{alpha} production in primary cultured human endometrial stromal and glandular epithelial cells

 
Moreover, because previous reports have shown no effect of E2 alone or E2 and P4 together on the expression of proMMPs (48, 49) and TIMPs (14), respectively, in cultured human endometrial stromal cells, we investigated whether the effects of E2 on the expression of stromal TIMP-3 mRNA and the production of epithelial PGF2{alpha} were mediated through E2 receptors. As shown in Table 3Go, the augmented expression of TIMP-3 mRNA and production of PGF2{alpha} by E2 alone and the combined treatment of E2 and P4 were found to be abolished by administering an E2 receptor antagonist, ICI 182,780 (40 x 10-6 M), indicating that the regulation of proMMP and TIMP-3 expression and PGF2{alpha} production by E2 is mediated through E2 receptors in primary cultured endometrial cells. These results, therefore, suggest that in vivo regulation of proMMPs-1 and -3 and TIMP-3 mRNA expression can be attributed to changes in the level of E2 and P4 during the menstrual cycle. Furthermore, it is likely that hormonal regulation of the expression of proMMPs, TIMP-3 and PGF2{alpha} in human endometrium differs between stromal and glandular epithelial cells.


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  		TABLE 3. Effects of E2-receptor antagonist on the hormonal augmentation of TIMP-3 mRNA expression and PGF2{alpha} production in primary cultured human endometrial cells

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
MMPs play important roles in tissue remodeling of human endometrium during the menstrual cycle (1, 4), and their expression and activation are regulated by sex hormones (12, 13, 14, 15) and cytokines (1, 4). Recently, Igakura et al. (32) reported that basigin, the mouse homolog of EMMPRIN, participates in implantation and spermatogenesis in basigin-knockout mice. In addition, Li et al. (50) reported that recombinant mouse basigin augments the production of MMPs-1 and -3 in human foreskin fibroblasts. We demonstrated here that EMMPRIN is expressed in human endometrium, and its tissue distribution is altered from the proliferative to secretory phases during the normal menstrual cycle. In addition, EMMPRIN was found to colocalize with MMP-1 in glandular epithelium. Furthermore, we demonstrated that in addition to proMMP-1, activated MMP-1 binds to EMMPRIN and shows collagenolytic activity on the cell surface (our unpublished observations). Therefore, these results provide novel evidence that EMMPRIN is involved in the tissue remodeling of human endometrium during the menstrual cycle by controlling MMP-1 activity along with its known MMP-inducing action (19, 20).

Concerning the regulation of EMMPRIN expression, recent reports by Xiao et al. (51, 52) indicated that the expression of basigin in endometrium and embryo of rat and mouse is E2 dependent during the periimplantation period in vivo. In the present study, the production of EMMPRIN in human endometrial stromal cells was not affected by E2 but was posttranscriptionally augmented by P4. We also confirmed that the augmented expression of EMMPRIN is observed in decidualized stromal cells in vivo (data not shown). Endometrial decidualization is a reproductive event associated with morphogenetic and functional changes of endometrial stromal cells for maintaining pregnancy, which is dependent on a sustained increase in the level of P4 (1, 2). Therefore, it is strongly suggested that the expression of EMMPRIN in human endometrium is regulated by P4, the regulation of which differs from that for rat and mouse. Furthermore, our finding that the production of EMMPRIN did not correlate with its mRNA level in P4-treated stromal cells suggests the possibility that the expression of EMMPRIN may be regulated by P4 at the posttranscriptional level.

EMMPRIN is a transmembrane glycoprotein with N-linked oligosaccharides (19, 20), and 40- to 60-kDa forms of EMMPRIN, which differ in glycosylation, have been characterized in various tumor cells (23, 27). In normal human endometrium, three types of EMMPRIN were detected, of which two variants with 47 and 51 kDa showed menstrual cycle-dependent expression in human endometrium in vivo. In addition, these variant molecules were found to differ in glycosylation, suggesting that oligosaccharides of EMMPRIN may be associated with its biological functions in human endometrium. In this regard, it has been reported that the deletion of oligosaccharides in EMMPRIN causes the loss of its MMP-inducible activity (53, 54). Moreover, sugar moieties of glycoproteins such as growth factor receptors have been involved in eliciting their biological activities including the binding affinity of ligands (55, 56). Therefore, we speculate that the difference in glycosylation of EMMPRIN at the proliferative and secretory phases may influence its biological activities for augmenting MMP production and binding secreted MMP-1, and its tissue localization in human endometrium. Further experiments will be required for addressing functions of oligosaccharides in EMMPRIN.

Aplin et al. (57) reported that P4 augments glycosylation of secreted and membrane-bound glycoproteins in glandular epithelium of human endometrium. In the present study, P4 was found to enhance the production of EMMPRIN and increase its glycosylation in human endometrial stromal cells but not in endometrial glandular epithelial cells. In addition, hormonal regulation of gene expression of proMMP-1, proMMP-3, and TIMP-3 and the production of PGF2{alpha} differed between endometrial stromal and glandular epithelial cells, a finding that is similar to those of previous reports (14, 48, 58). Therefore, these results suggest that the regulatory mechanism of EMMPRIN expression in epithelial cells is substantially different from that in stromal cells. In this regard, we demonstrated that the expression of EMMPRIN in human carcinoma cells is augmented by cell-cell or cell-ECM interactions in a coculture model with normal human fibroblasts (our unpublished observations). Therefore, we suggest that the expression of EMMPRIN in human endometrial glandular epithelial cells may be regulated by cell-cell or cell-ECM interaction.

In endometrium, an increase in the level of P4 by luteinization results in remodeling of the functional layer, in which MMP-mediated ECM degradation is observed (1, 2). However, Marbaix et al. (12, 13) reported that P4 suppresses the expression and activation of proMMPs-1, -2, and -9 in tissue culture of human endometrium. Lockwood et al. (48) also reported that P4 decreases the production of proMMP-1 in human endometrial stromal cells. In this study, the gene expression of proMMP-1 and proMMP-3 was suppressed by P4 and E2, whereas TIMP-3 mRNA expression was augmented by both hormones in human endometrial stromal cells. Therefore, these observations seem to be contradictory to the in vivo phenomenon that MMPs are involved in ECM remodeling of human endometrium during the menstrual cycle. Recently, Zhang and Salamonsen (18) reported that the amounts of active MMPs, both collagenase and gelatinase, are increased at menstruation, and the pattern of MMP activity is at discrete foci within the stroma, consistent with the focal breakdown of endometrium in vivo. Taken together with our findings that P4 increases the production of EMMPRIN, which may in turn augment the expression of MMPs in peripheral cells, and active MMP-1 binds EMMPRIN on the cell surface and causes the pericellular degradation of type-I collagen (our unpublished observations), we strongly suggest that EMMPRIN contributes to tissue remodeling of endometrium during the menstrual cycle by focally up-regulating not only MMP production but also collagenolytic activity on the cell surface by binding MMP-1, even when the level of P4 is dominant in the secretory phase in vivo.

In conclusion, we demonstrated that EMMPRIN was expressed in human endometrium, and its in vivo expression and localization were menstrual cycle-dependent. In addition, EMMPRIN colocalized with MMP-1 in human endometrium during the menstrual cycle in vivo. Furthermore, the production and glycosylation of EMMPRIN in endometrial stromal cells were augmented by P4, the regulatory mechanism of which differed from that in glandular epithelial cells. Therefore, these results strongly suggest that EMMPRIN is a focal regulatory factor for endometrial ECM degradation by controlling the expression of MMPs, which may be attributed to cyclic tissue remodeling of human endometrium during the menstrual cycle.


   Acknowledgments
 
We thank Prof. H. Nagase of the Kennedy Institute of Rheumatology, The Imperial College London (London, UK) for generously providing sheep anti-(human proMMP-1)antibody; Prof. S. Hirakawa (Toho University School of Medicine, Tokyo, Japan) for providing SKG-II cells; and Ms. N. Akimoto, T. Ota, and A. Takahashi for their excellent technical assistance.


   Footnotes
 
This work was supported by the Bio-oriented Technology Research Advancement Institution.

Abbreviations: E2, 17ß-Estradiol; ECM, extracellular matrix; EMMPRIN, extracellular matrix metalloproteinase inducer; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3 phosphate dehydrogenase; MMP, matrix metalloproteinase; MT-MMP, membrane-type MMP; P4, progesterone; PG, prostaglandin; TIMP, tissue inhibitor of metalloproteinases.

Received March 14, 2003.

Accepted August 30, 2003.


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