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Department of Obstetrics and Gynecology, University of Tokyo, Tokyo 113-8655, Japan
Address all correspondence and requests for reprints to: Yutaka Osuga, Department of Obstetrics and Gynecology, University of Tokyo, Tokyo 113-8655, Japan. E-mail: yutakaos-tky{at}umin.ac.jp.
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Abstract |
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Introduction |
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In pursuit of as-yet-unidentified proinflammatory substances involved in the pathogenesis of endometriosis, we focused our attention on the thrombin system, i.e. thrombin and its specific receptor, protease-activated receptor (PAR) 1, because thrombin possesses a proinflammatory property (14) in addition to its well-known procoagulation activity. The generation of thrombin is indicated in endometriosis, given the disease is characterized by recurrent ectopic bleeding (15). Furthermore, thrombin is implicated to play a role in angiogenesis in decidualized endometrium (16) and, therefore, may participate in the pathogenesis of endometriosis, in which neovascularization is believed to be requisite for the progression of the disease. What is more, PAR1 is present in the endometrium (17). Viewed in this light, we hypothesized that the thrombin system could be part of the mechanisms by which endometriosis progress.
As a first step toward understanding a possible role of the thrombin system, we investigated the presence of PAR1 in human endometriotic tissues. In addition, we examined the effects of thrombin and a PAR1 agonist on the expression of IL-8, monocyte chemoattractant protein (MCP)-1, cyclooxygenase (COX)-2, and matrix metalloproteinases (MMPs), putative inflammatory molecules involved in the pathophysiology of endometriosis. We further asked whether thrombin and a PAR agonist have a mitogenic effect on endometriotic cells.
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Materials and Methods |
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Thrombin, type I collagenase, and antibiotics (mixture of penicillin, streptomycin, and amphotericin B) were purchased from Sigma (St. Louis, MO). DMEM/Ham’s F12 (DMEM/F12) medium was from Life Technologies (Rockville, MD). SFLLRN (Ser-Phe-Leu-Leu-Arg-Asp), a PAR1 agonist peptide, was from Bachem (Bubendorf, Switzerland). D-Phenylalanyl-L-prolyl-L arginine chloromethyl ketone (PPACK), a thrombin inhibitor, was from Calbiochem (La Jolla, CA). Nonimmune murine IgG2a and antibodies of proliferating cell nuclear antigen (PCNA), CD45, vimentin, and cytokeratin were from Dako (Kyoto, Japan). Charcoal/dextran-treated fetal bovine serum was from Hyclone (Logan, UT). Deoxyribonuclease I was from Takara (Tokyo, Japan).
Collection of endometriotic tissues
Tissue specimens were obtained from patients with endometriosis (n = 28) undergoing laparoscopy or laparotomy after obtaining written informed consent under a study protocol approved by the Institutional Review Board of the University of Tokyo. These patients had not received hormones or GnRH agonist for at least 3 months before surgery. Endometriotic tissue samples were obtained from the cyst wall of ovarian endometrioma under sterile conditions and transported to the laboratory on ice in DMEM/F12.
Isolation, purification, and culture of endometriotic stromal cells (ESCs)
Primary ESC cultures were prepared according to the method described by Ryan et al. (18). More precisely, endometriotic tissues were dissected free from the underlying parenchyma, minced into small pieces, incubated in DMEM/F12 with type I collagenase (2.5 mg/ml) and deoxyribonuclease I (15U/ml) for 1–2 h at 37 C, and separated using serial filtration. Debris was removed by a 100-µm nylon cell strainer (Becton Dickinson, Lincoln Park, NJ), and some of epithelial glands were deprived by a 70-µm nylon cell strainer (Becton Dickinson). Stromal cells remaining in the filtrate were collected by centrifugation, resuspended in DMEM/F12, and plated onto 100-mm dishes (Iwaki, Asahi Technologies Co., Tokyo, Japan) and allowed to adhere at 37 C for 30 min, after which nonadhering epithelial cells and blood cells were removed with PBS rinses. The cells were cultured in DMEM/F12 reconstituted with 10% charcoal-stripped fetal bovine serum, penicillin (100 U/ml), streptomycin (100 µg/ml), and amphotericin B (250 ng/ml). When the cells became confluent, they were dissociated with 0.25% trypsin, harvested by centrifugation, replated in 6-well plates at 2 x 105 cells /well or 48-well plates at 1 x 104 cells /well, and kept at 37 C in a humidified 5% CO2-95% air environment, and allowed to grow to confluence. Purification of the stromal cell population was confirmed by immunocytochemical staining for the following antibodies: vimentin (stromal cells), cytokeratin (epithelial cells), and CD45 (monocytes and other leukocytes). The purity of the stromal cell was more than 98%, as judged by positive cellular staining for vimentin and negative cellular staining for cytokeratin and CD45.
Treatment of the cultured cells
When ESCs approached confluence, the medium was removed and replaced with fresh serum-free medium and antibiotics, and the cells were cultured for an additional 12–24 h. To evaluate the dose effects of thrombin, the cells were incubated with serum-free medium with different concentrations of thrombin (0, 0.1, 1, and 10 U/ml) or SFLLRN (300 µM) for 24 h. To evaluate the effects of PPACK, the cells were preincubated with PPACK (0.01, 0.1, and 1 µM) in serum-free medium for 30 min and then incubated with or without thrombin (10 U/ml) for a further 24 h. After the treatments, the conditioned media were collected, centrifuged, and stored at –80 C for ELISA.
The cells were cultured for 2 h in serum-free medium to study the effects of thrombin or SFLLRN on mRNA levels of IL-8, MCP-1, and COX-2. Similarly, the effects of thrombin and IL-8 on the expression of tissue factor (TF) mRNA, ESCs were incubated with thrombin (0.1, 1, and 10 U/ml) and IL-8 (100 ng/ml) in serum-free medium for 2 h.
RNA extraction; RT-PCR of PAR1 mRNA; and real-time quantitative PCR of IL-8, MCP-1, COX-2, and TF mRNA
RT-PCR and real-time quantitative PCR were performed as reported previously (19, 20). Total RNA was extracted from ESCs, using RNAeasy minikit (QIAGEN, Hilden, Germany). RT-PCR was performed using Rever Tra Dash (Toyobo, Tokyo, Japan). One microgram of total RNA was reverse transcribed in a 20-µl volume, and cDNA was amplified using oligonucleotide primers based on human PAR1 sequence. The human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers (Toyobo) were used to ensure RNA quality and amounts. PCR conditions for amplifications of PAR1 and GAPDH were 25 cycles at 98 C for 10 sec, 60 C for 2 sec, and 74 C for 20 sec. PCR products were analyzed by agarose gel electrophoresis with ethidium bromide.
To assess IL-8, MCP-1, COX-2, and TF mRNA expression, real-time quantitative PCR and data analysis were performed using a Light Cycler (Roche Diagnostic GmbH, Mannheim, Germany), according to the manufacturer’s instructions. Expression of IL-8, MCP-1, COX-2, and TF mRNA was normalized to RNA loading for each sample using GAPDH mRNA as an internal standard. PCR conditions of IL-8 for amplifications were 40 cycles at 95 C for 10 sec, 66 C for 8 sec, 72 C for 11 sec, followed by melting curve analysis. PCR conditions of MCP-1 for amplifications were 40 cycles at 95 C for 10 sec, 64 C for 7 sec, and 72 C for 10 sec, followed by melting curve analysis. PCR conditions of COX-2 for amplifications were 40 cycles at 95 C for 10 sec, 64 C for 10 sec, and 72 C for 12 sec, followed by melting curve analysis. PCR conditions of TF for amplifications were 40 cycles at 95 C for 10 sec, 64 C for 10 sec, and 72 C for 10 sec, followed by melting curve analysis.
The primer sequences are shown in Table 1
. Each PCR product was purified with a QIAEX II gel extraction kit (QIAGEN), and their identities were confirmed using an ABI PRISM 310 genetic analyzer (Applied Biosystems, Foster City, CA).
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Concentrations of IL-8 and MCP-1 in conditioned culture media were measured using respective specific ELISA kits (Quantikine; R&D Systems, Minneapolis, MN) according to the manufacturer’s protocol. Data were standardized by total protein of cell lysates.
Gelatin zymography
Gelatin zymography, used to measure activities of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) in the medium separately were performed as we reported previously (19) using gelatin zymo electrophoresis kit (Yagai, Yamagata, Japan), according to the manufacturer’s instructions. The zymogram was scanned, and densitometric analysis of bands was performed using National Institutes of Health image software.
Immunocytochemical staining for PCNA
Isolated ESCs were cultured in 48-well plate at a density of 1 x 104 cells/well at 37 C in a humidified 5% CO2-95% air environment. After 24 h of preincubation, the complete medium was removed and replaced with fresh serum-free medium and antibiotics, and the cells were cultured for an additional 24 h. Thereafter, ESCs were treated with serum-free medium with thrombin (10 U/ml) or SFLLRN (300 µM) for 24 h. To evaluate the effects of PPACK, ESCs were preincubated with serum-free medium with PPACK (1 µM) for 30 min, and then thrombin (10 U/ml) was added to the medium and incubated for 24 h.
The cells were fixed with cold methanol/acetone (1:1) at –20 C for 20 min and washed twice with PBS. The fixed cells were treated with 3% hydrogen peroxide for 5 min to eliminate endogenous peroxidase. After blocking with 1.5% horse serum for 20 min, the cells were incubated with a mouse monoclonal antibody to PCNA (1:200) for 20 min at room temperature. Control cells were incubated with nonimmune murine IgG2a, the concentration of which was adjusted to that of the primary antibody. The cells were then incubated with biotinylated horse antimouse IgG, followed by avidin peroxidase using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA). The chromogenic reaction was carried out with diaminobenzidine (Vector Laboratories). The replacement of the specific primary antibody with nonimmune murine IgG2a resulted in a lack of positive immunostaining.
Immunostained cells were analyzed, in a blinded fashion, without knowledge of the treatment group. The PCNA labeling index was determined by observing more than 1000 nuclei for each experimented sample and was used for evaluating the proliferating activity of the cells.
5-Bromo-2'-deoxyuridine (BrdU) proliferation assay
The BrdU proliferation assay was performed as previously reported (21, 22). The effects of thrombin and SFLLRN on the proliferation of ESCs were examined not only by PCNA staining but also by measuring BrdU incorporation into DNA using the Biotrak cell proliferation ELISA system (Amersham Biosciences, Little Chalfont, UK) according to the manufacturer’s instructions. Briefly, ESCs were seeded into Falcon 96-multiwell plates (Becton Dickinson) at a density of 5 x 103 cells/well in 100 µl of the culture medium. After 24 h, 100 µl BrdU solutions were added and incubated at 37 C for an additional 2 h. After removing the culture medium, the cells were fixed and the DNA denatured by the addition of 200 µl/well fixative. The peroxidase-labeled anti-BrdU bound to the BrdU incorporated in the newly synthesized, cellular DNA. The immune complexes were detected by the subsequent substrate reaction, and the resultant color was read at 450 nm in the DigiScan microplate reader (ASYS Hitech GmbH, Eugendorf, Austria).
Statistical analysis
Data were evaluated using Student’s t test and ANOVA with post hoc analysis for multiple comparisons. P < 0.05 was accepted as significant.
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Results |
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PAR1 mRNA was detected as a predicted band size of 264 bp, in cultured ESCs, based on a RT-PCR analysis (Fig. 1). The sequence of cDNA fragment obtained from the RT-PCR experiment was identical with that documented before.
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The addition of thrombin (1 and 10 U/ml) and SFLLRN (300 µM) for 2 h significantly increased the expression of IL-8 mRNA in ESC (Fig. 2A). Thrombin (10 U/ml) and SFLLRN (300 µM) also induced significant increase in MCP-1 mRNA expression in ESC (Fig. 2B).
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, A and B).
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, C and D, treatment with thrombin at concentrations of both 1 and 10 U/ml for 24 h induced significant increases in the amounts of IL-8 and MCP-1 released into the culture media. The increases in IL-8 and MCP-1 release with thrombin at 10 U/ml were 2.7- and 1.8-fold over the control, respectively. Thrombin inhibitor, PPACK, dose-dependently suppressed the thrombin-induced release of IL-8. PPACK also suppressed the thrombin-induced MCP-1 secretion. SFLLRN, a PAR1 agonist, stimulated the release of IL-8 and MCP-1 by 2- and 1.4-fold, respectively, compared with the control. Effect of PAR1 activation on COX-2 mRNA expression in ESCs
As shown in Fig. 4, treatments of ESCs with thrombin at 1 and 10 U/ml and SFLLRN increased COX-2 mRNA expressions 7-, 9-, and 11-fold over the control, respectively.
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As illustrated in Fig. 5, thrombin and IL-8 induced the expression of TF mRNA. Increases in TF mRNA with thrombin at 10 U/ml and IL-8 were 2.9- and 2.7-fold over the control, respectively.
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Gelatin zymography (Fig. 6, A and B) showed that conditioned medium of untreated ESCs contained 92 kDa pro-MMP-9 and 72 kDa pro-MMP-2. Thrombin significantly increased the production of 92 kDa pro-MMP-9. Moreover, thrombin significantly induced the production of 62 kDa MMP-2, the active form. These effects were eliminated by PPACK, whereas SFLLRN did not stimulate the production of pro-MMP-9 and 62 kDa MMP-2.
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Immunocytochemical staining demonstrated that PCNA-positive nuclei of ESCs increased by the addition of SFLLRN (300 µM, Fig. 7B) and thrombin (10 U/ml, Fig. 7C), compared with control cultures (Fig. 7A). The effects of thrombin were abrogated by PPACK (Fig. 7D). Replacement of the primary antibody with murine IgG2a showed a lack of positive immunostaining in nuclei of ESCs (Fig. 7F; negative control). These findings were statistically verified by counting PCNA-positive rates of the treated cells (Fig. 7G).
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Effects of thrombin and SFLLRN on DNA synthesis were determined in ESCs (Fig. 8). Thrombin at 10 U/ml and SFLLRN at 300 µM significantly increased the BrdU incorporation into DNA. The levels were 134 and 126% of the control, respectively.
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Discussion |
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MCP-1 and IL-8 are chemokines, concentrations of which are increased in the peritoneal fluid of women with endometriosis (23, 24, 25). In addition, they are expressed in endometriotic lesions, and their expressions are increased with IL-1ß treatment (7, 26, 27). These findings imply that MCP-1 and IL-8 are involved in the pathogenesis of endometriosis. MCP-1 recruits and activates macrophages, which are supposed to play a central role in the pathophysiology of endometriosis. Pleiotropic functions of IL-8, such as chemoattraction and activation of neutrophils, angiogenesis, stimulation of proliferation, and survival of endometrial cells, could contribute to the development of endometriosis. Thus, the finding that activated PAR1 stimulated productions of MCP-1 and IL-8 in endometriotic cells suggests that the thrombin system might work in regulating the production of autocrine substances responsible for the development of endometriosis.
COX is the key enzyme in prostaglandin biosynthesis. In this study, the expression of COX-2 has been shown in endometriotic tissues. Because COX-2 is involved in inflammation, this finding is in line with the notion that COX-2 is involved in the pathogenesis of endometriosis (28, 29) and further points to a possible role of PAR1 in endometriosis.
Interestingly, an emerging concept is that a feed-forward loop to enhance inflammation is important in the pathogenesis of endometriosis (30). The concept has been supported by the finding that IL-1ß, a potent proinflammatory cytokine secreted by macrophages, stimulates endometriotic stromal cells to produce regulated upon activation, normal T cell expressed and secreted, which further mobilizes macrophages (11). In the present study, IL-8 as well as thrombin stimulated the expression of TF in endometriotic cells. Increased TF expression may stimulate coagulation cascade distal to thrombin production, which subsequently enhances IL-8 production through PAR1 activation in endometriotic lesions. Viewed this way, it may be that PAR1 activation could link inflammation with coagulation, thus conferring self-sustaining mechanisms for the progression of endometriosis.
MMPs play a role in breakdown and remodeling of tissues. Increased expression of MMP-2 and MMP-9 in endometriotic tissues has been reported, implicating its involvement in the pathogenesis of endometriosis (31, 32, 33). Interestingly, we observed that thrombin increased the production of pro-MMP-9 and MMP-2 but not PAR1 agonist SFLLRN in endometriotic cells. We have shown that MMPs are differently regulated by thrombin and PAR1 agonist in the granulosa cells (19). It has been suggested that MMP-2 is activated by activated protein C, which is induced by thrombin binding to thrombomodulin (34, 35). Thrombin, thus, may exert its diverse effects in endometriosis in both PAR1-dependent and PAR1-independent manners.
Another notable finding in the present study is that PAR1 activation resulted in the stimulation of ESC proliferation. Proliferation-stimulating effects of thrombin through PAR1 activation have been reported in various nonneoplastic and neoplastic cells (36, 37, 38, 39). Our findings suggest that PAR1 activation may stimulate the progress of endometriosis.
Assuming that thrombin activation promotes the development of endometriosis, inhibition of PAR1 is a promising therapeutic approach for the treatment of the disease. Because a PAR1 antagonist is specific for the cellular actions of thrombin and does not interfere with fibrin generation, it is expected to have less adverse effects on bleeding than the currently available thrombin inhibitors. In fact, chemicals with selective antagonistic effects on PAR1 did not perturb coagulation parameters in animal models (40, 41). Hence, it would be interesting to examine whether these substances have the potential for endometriosis therapy.
ESCs used in the present study were derived from the ovarian endometrioma, which might represent different pathogenetic entities from peritoneal and deep nodular endometriosis. Nevertheless, in light of the thesis that recurrent bleeding is a common feature of the progressive disease in all three entities (15) and that prothrombin in blood is able to generate thrombin concentrations of 130–160 U/ml (42), it may be feasible to speculate that thrombin would also be implicated in peritoneal and deep nodular endometriosis.
In summary, the present study demonstrated that PAR1 activation stimulated the expression of IL-8, MCP-1, and COX-2 and the proliferation of endometriotic cells. These findings suggest that the thrombin system may evoke inflammatory and mitogenic responses of endometriotic cells and thereby be involved in the pathogenesis of endometriosis.
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Footnotes |
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Abbreviations: BrdU, 5-Bromo-2'-deoxyuridine; COX, cyclooxygenase; ESC, endometriotic stromal cell; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MCP, monocyte chemoattractant protein; MMP, matrix metalloproteinase; PAR1, protease-activated receptor 1; PCNA, proliferating cell nuclear antigen; PPACK, D-phenylalanyl-L-prolyl-L arginine chloromethyl ketone; TF, tissue factor.
Received March 12, 2004.
Accepted March 2, 2005.
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B site in the proximal promoter. J Clin Endocrinol Metab 86:4759–4764This article has been cited by other articles:
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