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Department of Obstetrics and Gynecology, Tottori University School of Medicine, Yonago 683-8504, Japan
Address all correspondence and requests for reprints to: Souichi Yoshida, M.D. Department of Obstetrics and Gynecology, Tottori University School of Medicine, Yonago 683-8504, Japan. E-mail: souichi{at}grape.med.tottori-u.ac.jp.
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Introduction |
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Recent studies suggest that hepatocyte growth factor (HGF) was significantly increased in the PF of women with endometriosis (6, 7). However, the roles of HGF in the pathogenesis of endometriosis have not yet been examined. HGF, originally characterized as a potent mitogen for adult hepatocytes (8), is known as a mesenchymal-derived pleiotropic growth factor that enhances cell proliferation and migration as well as morphogenic activity on various types of epithelial cells, usually as a paracrine factor (9, 10). The biological effect of HGF appears to bind a high affinity, membrane-spanning Met receptor that is the c-met protooncogene product (11). In the normal uterine endometrium, HGF produced by endometrial stromal cells promotes proliferation, migration, and lumen formation of endometrial epithelial cells (12). We also demonstrated that stromal-derived HGF enhances the invasive growth of endometrial carcinoma cells (13). On the other hand, overexpression of Met was observed in several types of malignant tumors, such as those of the uterine endometrium and ovary (14, 15). Although Met was predominantly expressed in cells of epithelial origin, its expression was also shown in some fibroblast and sarcoma cell lines in which HGF was endogenously expressed (16). Furthermore, adding HGF exogenously stimulates the invasive growth of human sarcoma cells and of Met-transformed NIH-3T3 cells (16, 17). These data suggest that HGF may also exert its effects on the cells of mesenchymal origin in both an autocrine and a paracrine manner, and the diverse role of HGF may help us to understand the invasive process of endometriosis.
In this context, we investigated whether the HGF/Met system is involved in the pathogenesis of endometriosis. First, we evaluated HGF concentrations in the PF of patients with or without endometriosis. Sources of HGF production were also examined. Second, we investigated Met expressions in endometrial and endometriotic stromal cells. Third, we examined whether endometrial and endometriotic stromal cells expressed a functional receptor for HGF and whether HGF enhances stromal cell proliferation and invasion. The invasive process might involve the plasminogen activator (PA) and matrix metalloproteinase (MMP) systems, which reportedly contribute to the pathogenesis of endometriosis (18, 19). Urokinase-type PA (uPA), one of the factors associated with the systems, catalyzes the conversion of plasminogen into plasmin, which degrades a variety of extracellular matrix proteins and activates MMPs. As recent reports suggest that the promotion of uPA production participates in HGF-induced cell motility and invasion (20, 21), we also determined whether increased uPA production is involved in HGF stimulation in endometrial and endometriotic stromal cell invasions.
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Materials and Methods |
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After obtaining informed consent, PF was collected from 37 Japanese women of reproductive age who underwent either laparoscopy during an infertility workup or laparoscopic cystectomy for ovarian chocolate cysts. None of the patients had received hormonal treatment before surgery. Of the 37 patients studied, 25 had pelvic endometriosis, and 12 were free of gynecological disease. Eleven PF specimens from patients with endometriosis were contaminated with blood. In the endometriosis patient group, no statistically significant difference in HGF concentrations was found between the samples contaminated with blood and those without blood among patients with similar stages of disease. No significant differences were observed in patient age between those with and those without endometriosis (31.8 ± 1.09 vs. 31.4 ± 1.67 yr). The amount of ascites was significantly increased in the patients with endometriosis compared with disease-free patients (25.2 ± 3.23 vs. 18.0 ± 4.37 ml; P < 0.01), as was indicated in previous reports (5). According to the criteria of the revised American Society of Reproductive Medicine (R-ASRM) score classification system, patients were classified in stage I (n = 7), stage II (n = 8), stage III (n = 5), or stage IV (n = 5). Of the 10 patients with stages III and IV, 7 had endometriotic cysts. PF was collected with a laparoscopic cannula immediately after introducing the laparoscope. The aspiration was performed completely under direct visualization from the posterior cul-de-sac, anterior vesicouterine fold, and iliac fossa. Fluid samples were centrifuged at 800 x g for 10 min at 4 C to separate the cell pellet and the supernatant. The cell-free supernatant was then stored at -70 C until assayed.
Isolation and culture of the cells
Endometrial tissues were obtained from the uteri of cycling premenopausal women who underwent hysterectomy for uterine leiomyoma (n = 22) during the proliferative (n = 8) or secretory (n = 14) phase. Because an increase in HGF secretion by eutopic endometrial stromal cells in women with endometriosis had been demonstrated in previous reports (22, 23), endometrial specimens from the leiomyoma patients associated with endometriosis were excluded from this study. The chocolate cyst lining of the ovaries of patients with endometriosis (n = 21) was the source of endometriotic tissue collected during the follicular (n = 6) or luteal (n = 15) phase. Patients had received no hormonal treatment before surgery. The menstrual cycle phase was determined by measuring serum estradiol and progesterone levels as well as by histological examination. We used the endometrial and endometriotic stromal cells derived from both proliferative and secretory phases in the experiments to detect Met gene and protein expressions. In the other experiments we used stromal cells isolated from the endometrium and endometriotic tissues in the late secretory phase. The endometrium in the late secretory phase was used as a model of stromal cells in endometrial fragments present in retrograde menstruum.
Stromal cells were collected from endometrial and endometriotic tissue as previously described (24). The tissues were minced and digested with 0.5% collagenase in DMEM and Ham’s F-12 (1:1, vol/vol) at 37 C for 60 min. The dispersed cells were filtered through a 70-µm pore size nylon mesh to remove the undigested tissue pieces containing the glandular epithelium. The filtered fraction was separated further from epithelial cell clumps by differential sedimentation. The medium containing stromal cells was filtered through 40-µm pore size nylon mesh. Final purification was achieved by allowing stromal cells, which attach rapidly to plates, to adhere selectively to culture dishes for 30 min at 37 C in 5% CO2 in air. Nonadherent epithelial cells were removed. Stromal cells were cultured in DMEM/Ham’s F-12 supplemented with 100 IU/ml penicillin G, 50 mg/ml streptomycin, 2.5 µg/ml amphotericin B, and 10% fetal bovine serum (FBS; vol/vol). The purity of isolated stromal cells was assessed by immunocytochemical staining, using antibodies against cytokeratin, vimentin, and factor VIII as markers of epithelial, stromal, and endothelial cells, respectively. The results showed that the purity of stromal cells was more than 98%. Furthermore, we confirmed that cultured endometriotic stromal cells had the ability to secrete prolactin when incubated with medroxyprogesterone acetate, as previously described (25). In our preliminary study using ELISA, we observed no significant differences in HGF production between endometrial stromal cells isolated from specimens with leiomyoma and those without. For the analysis of gene and protein expressions of HGF and c-met, we used stromal cells derived from both proliferative and secretory phases. The results are shown in Figs. 2
and 3. In the other experiments, we used stromal cells of monolayer culture from late secretory samples after the first passage.
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Peritoneal tissues were obtained from the posterior cul-de-sac of surgical specimens of four patients with endometriosis. After removing adipose tissue and washing in PBS, peritoneal tissues were minced and used as samples.
The Ishikawa cells (Ishikawa 3-H-12 no. 56), a well differentiated endometrial carcinoma cell line, which we received as a gift from Dr. Masato Nishida (Kasumigaura National Hospital, Ibaragi, Japan), were maintained in Eagle’s MEM in the presence of 10% FBS. Because this cell line expresses Met receptor, but not HGF (13), we used it as a control of Met expression.
Collection of the supernatant of endometrial and endometriotic stromal cells
Endometrial and endometriotic stromal cells (primary culture, and first and second passages) derived from each of five specimens were plated in a 100-mm dish. The media were changed to remove unattached cells after 24-h incubation. Cultures were allowed to proliferate until subconfluence (5–7 d), with the medium containing 10% FBS exchanged every 48 h. Then cells were incubated in 5 ml phenol red-free medium supplemented with 2 mg/ml BSA (Sigma-Aldrich Corp., St. Louis, MO) for 24 h at 37 C.
ELISA
The concentrations of HGF in PF samples and in the supernatants of stromal cells were determined by an ELISA kit (Genzyme, Cambridge, MA). The sensitivity of the assay is 10 ng/ml, and it is linear for concentrations of 0.1–10 ng/ml. Undetectable HGF levels (<0.1 ng/ml) were assumed to be equal to 0.1 ng/ml.
RT-PCR
Total RNA was extracted from each sample by the guanidium thiocyanate method. RT of RNA from these cells into cDNA and PCR amplification for HGF and c-met were performed using the GeneAmp RNA PCR Core Kit (PerkinElmer, Branchburg, NJ). For PCR analysis, the following specific primers for human HGF, human c-met and glycerol-3-phosphate dehydrogenase (GAPDH) as an internal control were used; HGF: sense, 5'-TCA CGA GCA TGA CAT GAC TCC-3'; and antisense, 5'-AGC TTA CTT GCA TCT GGT TCC-3'; c-met: sense, 5'-GGT TGC TGA TTT TGG TCT TGC-3'; and antisense, 5'-TTC GGG TTG TAG GAG TCT TCT-3'; and GAPDH: sense, 5'-ACC ACA GTC CAT GCC ATC AC-3'; and antisense, 5'-TCC ACC ACC CTG TTG CTG TA-3'. The distances between primers, including the primers, were 303, 262, and 450 bp, respectively. The PCR products were transferred to a nylon membrane and hybridized with internal biotin-labeled probes of HGF (5'-CAC ATG GAC AAG ATT-3') and c-met (5'-CGT CCT CTG GGA GCT-3') (27). The products on the membrane were detected by chemiluminescence. These procedures were previously described in detail (28).
Real-time RT-PCR
The levels of Met gene (c-met) transcriptions were compared between endometrial and endometriotic stromal cells by real-time quantitative RT-PCR. PCR reactions were performed in the GeneAmp 5700 Sequence Detection System (PE Applied Biosystems, Foster City, CA). As previously described, the assay used SYBER Green I dye (SYBER Green assay, PE Applied Biosystems, Foster City, CA) (13). Primers were chosen with the assistance of the computer program Primer Express (PE Applied Biosystems) and were located over two different exons as follows: c-met: sense, 5'-TTG GAT CAG GAC CAT GTG AGG-3'; and antisense, 5'-TCC ACG ACC AGG AAC AAT GA-3'; and GAPDH: sense, 5'-GAA GGT GAA GGT CGG AGT C-3'; and antisense, 5'-GAA GAT GGT GAT GGG ATT TC-3'. Normalization to the GAPDH expression level was performed for each sample. The c-met/GAPDH ratio was calculated and reported as the mean of triplicate assays.
Western blot analysis
Endometrial and endometriotic stromal cells from the proliferative and secretory phases and peritoneal tissues processed as described above were lysed with ice-cold lysis buffer containing 50 mM Tris-HCl (pH 7.6), 125 mM NaCl, 0.1% sodium dodecyl sulfate, 0.1% Nonidet P-40, 1 mM dithiothreitol, 50 mM NaF, and 1x Complete Protease Inhibitor Cocktail (Roche, Ingelheim, Germany). The lysate was centrifuged, and supernatant was prepared. The protein concentration of the supernatant was measured by Bradford assay. An endometrial cancer cell line, Ishikawa cell lysate, was used for the positive control. Eighty micrograms of protein sample were resolved by electrophoresis on a 4–20% gradient polyacrylamide gel. These separated protein samples were then electroblotted onto a nitrocellulose membrane before blocking for 60 min in PBS containing 2.5% skimmed milk. The membrane was then probed with anti-c-Met ß-chain antibodies (rabbit polyclonal; IBL, Gumma, Japan) at a dilution of 1:20 (5 µg/ml), followed by a peroxidase-conjugated secondary antibody (1:2000). Protein bands were visualized using an enhanced chemiluminescence system (Amersham Pharmacia Biotech, Little Chalfont, UK). The protein band size was determined using a Full Range Rainbow (Amersham Pharmacia Biotech) molecular weight marker.
To determine how HGF affects the production of uPA, endometrial and endometriotic stromal cells were cultured for 12 h in serum-free DMEM/Ham’s F-12. Then the medium was changed to serum-free DMEM/Ham’s F-12 with or without 50 ng/ml HGF. After a further 24-h incubation, cell lysates were collected and processed as described above. In this experiment the membrane was probed with anti-uPA antibodies (Gt X Hu Urokinase, Chemicon International, Temecula, CA) at a dilution of 1:500.
Immunoprecipitation
Immunoprecipitations were performed to detect phosphorylated Met. At the first passage, stromal cells were plated in 100-mm culture dishes and maintained as described above. The subconfluent stromal cells were preincubated in serum-free medium for 24 h. After incubating in fresh medium for 20 min, the culture media were changed to those containing HGF (50 ng/ml) and those without HGF for 15 min. After washing three times with ice-cold PBS, stromal cells were lysed with lysis buffer. One milligram of each lysate was incubated with agarose conjugated anti-c-Met rabbit polyclonal antibody, C-28 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) overnight at 4 C. The immunocomplexes were washed three times with lysis buffer, then boiled for 5 min in sodium dodecyl sulfate sample buffer. The protein samples were processed as described above, except using 1% Tween 20 containing Tris-buffered saline [10 mM Tris-HCl (pH 7.5) and 100 mM NaCl] supplemented with 2% BSA for the blocking buffer. The membrane was then incubated overnight at 4 C with 0.5 µg/ml polyclonal antibody against the phosphorylated form of Met (rabbit anti-c-Met [pYpYpY1230/1234/1235] phosphospecific antibody (BioSource International, Camarillo, CA) that reacts with Met phosphorylation of Tyr1230, Tyr1234, and Tyr1235. Phosphorylation of Met on those tyrosine residues has been shown to be important in receptor activation. After incubation with a secondary antibody, protein bands were analyzed using the enhanced chemiluminescence system.
Cell proliferation assay
The effects of recombinant human HGF (R&D Systems, Minneapolis, MN) and a monoclonal antibody against HGF (antihuman HGF; R&D Systems) on endometrial and endometriotic stromal cells were examined. The cells were trypsinized and plated at a density of 2 x 103/well in a 96-well plate with DMEM/Ham’s F-12 serum-free medium supplemented with 2 mg/ml BSA in combination with various concentrations of HGF (0–50 ng/ml) and a monoclonal antibody against HGF (0–10 µg/ml; antihuman HGF; R&D Systems). Nonimmune mouse IgG1 
(Cosmo Bio, Tokyo, Japan) was also added solely or simultaneously with HGF. Each plate had one control column (six wells) containing medium that was free of HGF. After 72-h incubation, DNA synthesis and cell proliferation were measured. To examine DNA synthesis, 5-bromo-2'-deoxyuridine (Brdu) incorporation was assessed with a kit [cell proliferation ELISA, Brdu (colorimetric), Roche, Tokyo, Japan] according to the manufacturer’s instructions. The proliferation of each stromal cell was determined spectrophotometrically by measuring the incorporation of tetrazolium dye [3-[4,5-dimethlthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay]. The MTT assay used in this study was a previously described system (24). The ratio to the mean value of control (percent control) was used for comparison. Results were presented as the mean ± SE of triplicate assays.
Matrigel invasion chamber assay
Biocoat Matrigel invasion chambers (Nippon BD Biosciences, Tokyo, Japan) were used. The endometrial and endometriotic stromal cells were inoculated in the inner cup coated with Matrigel at a density of 10 x 104 cells/ml in a volume of 0.5 ml serum-free DMEM/Ham’s F-12 containing 2 mg/ml BSA. The same medium with the inner cup was placed in 24-well chambers. HGF (0–50 ng/ml) was added to the lower compartment. To neutralize the specific effects of HGF, various concentrations of a monoclonal antibody against HGF (0–10 µg/ml) were added to the serum-free medium in a 24-well chamber with or without HGF. Nonimmune mouse IgG1 was used as the control. In another set of experiments, PA inhibitor-1 (PAI-1; 0–100 ng/ml; Molecular Innovations, Southfield, MI), one of the specific inhibitors of uPA, was added to the serum-free medium in a 24-well chamber. Both stromal cells were then cultured for 48 h. The cell penetration was quantified by staining the 8-µm pore size membrane with a Diff-Quick stain kit (Green Cross, Kobe, Japan). The number of penetrating cells per filter (x100) was counted. Each concentration of reagents was added to each of the three wells, and the results were reported as the means of the three membranes. The assay was repeated at least three times, and similar results were obtained. The mean cell number per 1 cm2 was used for comparison.
Statistical analysis
To compare the values obtained with various treatments vs. the controls, results were analyzed using one-way ANOVA, followed by Fisher’s protected least significant difference test. The data are expressed as the mean ± SEM. A level of P < 0.05 was accepted as indicating statistical significance.
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Results |
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HGF concentrations in PF were significantly higher in patients with endometriosis than in those without endometriosis (2.4 ± 0.19 vs. 1.7 ± 0.14 ng/ml; P < 0.05; Fig. 1A). The total amount of HGF (volume x concentration) was substantially different between patients with and without endometriosis (57.8 ± 8.4 vs. 30.2 ± 8.0 ng; P < 0.05). A positive correlation was seen between the HGF concentrations in PF and R-ASRM scores (r = 0.556; P = 0.003; Fig. 1B). Both endometriotic lesion and adhesion scores in the R-ASRM classification correlated with HGF levels (endometriotic lesion scores: r = 0.492; P = 0.012; adhesion scores: r = 0.474; P = 0.016). No significant difference was observed in HGF concentrations between the proliferative and secretory phases, consistent with a previous report (6).
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To determine the sources of HGF in PF, HGF gene expressions were examined in endometriotic stromal cells, peritonea, and peritoneal macrophages by RT-PCR and Southern blot analysis (Fig. 2
). HGF transcriptions were detected in endometriotic stromal cells and peritonea (lanes 2 and 3) as well as in endometrial stromal cells (lane 1), which were shown in our previous study (13). In contrast, only a faint expression was observed in the peritoneal macrophages (lane 4).
Met receptor expressions in endometrial and endometriotic stromal cells
Endometrial and endometriotic stromal cells were analyzed for expression of the Met gene (c-met) by RT-PCR (Fig. 3A). The expected 252-bp bands were detected in both stromal cells (lanes 2 and 3). The Ishikawa endometrial carcinoma cell line served as a positive control (lane 1). We then quantified c-met expression levels in stromal cells isolated from each menstrual phase by real-time RT-PCR (Fig. 3B). Endometriotic stromal cells expressed higher levels of c-met than did endometrial stromal cells in each phase (2.7-fold in the proliferative phase and 3.6-fold in the secretory phase). The c-met transcriptions of both stromal cells were increased in the proliferative phase compared with the secretory phase (3.7-fold in the endometrial stromal cells; 2.7-fold in the endometriotic stromal cells).
We also determined Met protein expressions by Western blot analysis (Fig. 3C). As seen in Ishikawa cells (positive control), 145-kDa Met protein along with its unprocessed precursor (170 kDa) were detected in both stromal cells. Similar to the gene transcriptions, endometriotic stromal cells expressed more abundant Met than did endometrial stromal cells in each phase (lanes 3 and 5). Met expressions of both stromal cells were decreased in the secretory phase.
Measurement of HGF production in isolated stromal cells
As endometrial and endometriotic stromal cells produce HGF, we determined HGF concentrations in the culture supernatants of those stromal cells after 24-h incubation. The production of HGF was also tested after the cell passage. The ELISA showed that endometrial and endometriotic stromal cells had similar HGF productions when the total HGF secretions per cells (concentration x volume/cell numbers) were calculated (Fig. 4). The ability to produce HGF in both stromal cells decreased after the first passages, but the differences were not statistically significant. We used stromal cells after the first passage in the following experiments.
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Binding of HGF to Met induces autophosphorylation at tyrosine residues and initiates a cascade of intracellular events. As HGF and Met were coexpressed in endometrial and endometriotic stromal cells, we next examined Met phosphorylation in response to endogenous and exogenous HGF. Stromal cell lysates were immunoprecipitated with an anti-Met antibody and immunoblotted with an antibody against phosphorylated Met (Fig. 5). Tyrosine residues of the 145-kDa ß-chain of Met were spontaneously phosphorylated by endogenous HGF (lanes 3 and 5). The addition of exogenous HGF induced an increase in Met autophosphorylation in both stromal cells (lanes 4 and 6), whereas Met activation in Ishikawa cells was detected only after adding exogenous HGF (lanes 1 and 2). These findings suggest that the Met receptor expressed in endometrial and endometriotic stromal cells was functional and activated by both endogenous and exogenous HGF.
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To evaluate the effects of HGF on stromal cell proliferation, we measured cell growth and DNA synthesis by means of MTT assay and Brdu ELISA. The exogenously added HGF did not affect the growth of either stromal cell (Fig. 6A). To examine the effects of endogenous HGF, endometrial and endometriotic stromal cells were cultured in serum-free medium supplemented with various concentrations of anti-HGF antibody. We observed a modest, but statistically significant, reduction in the growth of endometrial stromal cells (10.8%) and endometriotic stromal cells (14.7%) in the presence of 10 µg/ml concentrations of the antibody (Fig. 6B). We obtained similar results in DNA synthesis of each stromal cell in response to HGF and an anti-HGF antibody (data not shown). These results suggest that endogenous HGF modestly stimulates endometrial and endometriotic stromal cell proliferation via an autocrine pathway. The failure to detect effects of exogenous HGF could be the result of saturating endogenous HGF levels for cell proliferation.
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The effects of HGF on the invasive capacity of stromal cells were examined using the Matrigel invasion chamber assay (Fig. 7). The endometriotic stromal cells constitutively had more invasive activity (3.3-fold more than endometrial stromal cells). The addition of HGF in the lower chamber significantly increased the number of penetrated cells in a dose-dependent manner. In the presence of 50 ng/ml HGF, the invasive abilities of endometrial and endometriotic stromal cells were enhanced 5.3 and 3.3 times more than those in the absence of HGF. The specificity of HGF action was confirmed by the complete inhibition of HGF-stimulated cell invasion by adding HGF-neutralizing antibody (Fig. 7). Moreover, we tested the effects of adding anti-HGF antibody alone on endometrial and endometriotic stromal cell invasion. The HGF-neutralizing antibody dose-dependently reduced both stromal cell invasions, whereas nonimmune mouse IgG had no influence (Fig. 7C). Collectively, these results indicate that HGF stimulates the invasiveness of endometrial and endometriotic stromal cells in autocrine and paracrine pathways.
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The invasive process might involve the activation of a local extracellular proteolysis system. To confirm the above result, we next examined HGF effects on the production of uPA, which is an activator of this system. The lysates of the cells treated with or without 50 ng/ml HGF were analyzed by means of Western blotting to determine whether increased uPA production participated in a mechanism of HGF stimulation of stromal cell invasion (Fig. 8A). Basal 53-kDa uPA expression was observed in endometrial and endometriotic stromal cells (lanes 1 and 3). Adding HGF induced increases in uPA production in both stromal cells (lanes 2 and 4). The results also indicated that endometriotic stromal cells, regardless of HGF treatment, expressed higher levels of uPA than did endometrial stromal cells.
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). The results for PAI-1 were similar to the effects of HGF-neutralizing antibody, which were shown in Fig. 7. |
Discussion |
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Studies have reported that levels of several cytokines and growth factors are elevated in the PF of women with endometriosis, implying that these factors may play a part in the development and progression of this disease (5). We demonstrated here that HGF is another factor that is higher in the PF of endometriosis patients. This finding is consistent with prior reports suggesting that HGF concentrations in PF were higher in patients with endometriosis than in those without endometriosis (6, 7). We showed that the sources of HGF would be endometriotic stromal cells and peritoneum rather than peritoneal macrophages, which have been reported as the main sources of several cytokines in PF. Furthermore, of the 10 patients in the advanced stage of endometriosis, seven had endometriotic cysts. As endometriotic cysts are surrounded by large numbers of endometriotic stromal cells, endometriotic cysts are likely to be one of the main sources of HGF in PF. However, we did not exclude the contributions of possible HGF-producing cells, such as ovarian surface epithelium and ovarian stromal cells, which expressed HGF (27, 29). In addition, eutopic endometrial stromal cells of patients with endometriosis reportedly produced higher levels of HGF than those without endometriosis (22, 23). Endometrial stromal cells in retrograde menstruation may also produce HGF in PF.
We and other researchers previously demonstrated that several cytokines, such as IL-6, TNF
, and prostaglandins, were increased in the PF of endometriosis patients (5). These factors, known as HGF inducers, may stimulate HGF production in the pelvic cavity of women with endometriosis. The cytokine network, which stimulates the mutual production of factors, may operate in the peritoneal cavity of patients with endometriosis.
The Met/HGF receptor is predominantly expressed in the epithelial cells of adult and embryonic tissues, whereas HGF is produced in the surrounding mesenchymal cells. Consequently, research interest has focused on the paracrine signaling system in which mesenchymal cells produce HGF and exert its effects after binding to the receptor expressed on epithelial cells. In this study, however, we observed Met expression in endometrial and endometriotic stromal cells. Some earlier studies also demonstrated the expression of Met in cells of mesenchymal origin, such as ovarian stromal cells, fibroblasts, smooth muscle, and sarcoma cells (16, 27). Therefore, the Met expression did not seem to be restricted to epithelial cells, and it is likely that Met expressed in mesenchymal cells modulates those cell functions in physiological and pathological conditions. Furthermore, we observed an alteration of the Met expression level in endometrial and endometriotic stromal cells during menstrual phases. Because the changes in Met expression were observed at both mRNA and protein levels, it seems that certain factors may regulate Met gene transcription during menstrual cycles.
The present results show that the Met expressed in endometrial and endometriotic stromal cells was autophosphorylated by endogenous and exogenous HGF. Endogenous HGF, but not exogenous HGF, promotes stromal cell proliferation. On the other hand, both endogenous and exogenous HGF enhance stromal cell invasion. The failure to detect the effects of exogenous HGF in the proliferation assay could be the result of saturating endogenous HGF levels for each cell proliferation. However, as shown in Fig. 4, HGF levels in the supernatants of endometrial and endometriotic stromal cell cultures were rather low compared with HGF concentrations reported to exert a mitogenic effect on the various types of cells. We needed rather high concentrations of the anti-HGF antibody to neutralize the endogenous HGF in both proliferation and invasion assays. Because HGF is a heparin-biding growth factor, studies have suggested that HGF is concentrated and protected from proteolytic degradations by binding to a heparin sulfate-modified CD44 at the cell surface (30). Consequently, it is likely that a part of HGF secreted from endometrial and endometriotic stromal cells may be stored on cell surfaces and may maintain those cell functions as a survival factor in an autocrine pathway.
We demonstrated for the first time that Met activation promotes endometrial and endometriotic stromal cell invasion by means of Matrigel invasion assay, suggesting that HGF enhances the degradation systems of extracellular matrix (ECM) and stimulates cell motility. We also showed that HGF induces uPA production of endometrial and endometriotic stromal cells. This finding, together with prior studies, supports the idea that promotion of uPA production participates in HGF-induced cell motility and invasion (20, 21). Because uPA activates pro-HGF (31) and MMPs, uPA may be associated with HGF-induced stromal cell invasion, not only by degrading ECM but also by activating pro-HGF and MMPs.
A controversy has arisen from Sampson’s implantation theory concerning whether shed endometrial tissue can attach and invade through the intact mesothelial surface of the peritoneum. Endometrial fragments may adhere only at places where the epithelial layer of the peritoneum is damaged and the ECM is exposed (32, 33). On the other hand, recent studies clearly show that stromal cells of endometrial fragments attach and rapidly invade through the intact mesothelial lining (2, 3). The inconsistent findings may be reconciled with the morphological changes in mesothelial cells (34). During peritoneal implantation of cancer cells, mesothelial cells become hemispherical and separate from each other in response to factors released by cancer cells and host peritoneal fibroblasts (35, 36). The alteration of mesothelial morphology results in exposure of underlying ECM and promotes cancer cell attachment. The cells isolated from menstrual effluent also had a similar effect on mesothelial morphology without causing apoptosis and necrosis (37, 38). Moreover, HGF was reported to affect mesothelial morphology on the peritoneal dissemination of cancer cells (36). Collectively, HGF increased in PF and, produced by endometrial stromal cells, may induce critical changes in the morphology of mesothelial cells, then enhance endometrial cell attachment and invasion.
Distinct biological characteristics between endometrial and endometriotic stromal cells have been proposed. We previously demonstrated the different responses of those cells to the IL-6 family of cytokines (24, 39). In the present study we also observed differences in these stromal cells in Met expression levels, including the degree of its alteration in menstrual phases and in the spontaneous invasive abilities. These findings propose that endometriotic cells have characteristics that differ from those of endometrial cells. However, comparisons between eutopic and ectopic endometrial stroma are limited in this investigation. We used ectopic stromal cells of ovarian endometriosis and eutopic stromal cells derived from patients without endometriosis. To be exact, experiments using ectopic stroma of pelvic endometriosis and eutopic stroma obtained from the same patients may be required.
Recently, an HGF antagonist, called HGF/NK4 (NK4) has been proposed for therapeutic use. NK4, produced by proteolytic digestion of HGF, binds to the Met/HGF receptor, but does not induce tyrosine phosphorylation of Met. NK4 competitively inhibits biological events driven by HGF, such as the invasion of distinct types of tumor cells (40, 41). Although the possibility that NK4 can function as a therapeutic agent for subjects with cancer warrants ongoing studies (42, 43), involvement of the HGF/Met system in the development and progression of endometriosis may indicate that HGF antagonists may be used in the prevention and treatment of endometriosis.
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Acknowledgments |
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Footnotes |
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Received May 21, 2003.
Accepted November 6, 2003.
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