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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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Recently protease-activated receptors (PARs) have attracted increasing attention in relation to certain proteases. PAR is a member of a group of seven transmembrane G protein-coupled receptors. On the activation of PARs, proteases such as thrombin and trypsin cleave at a point within the extracellular N-terminal domain and, thereby, unmask a new amino terminus that functions as a tethered ligand to bind back to the receptor (2, 3, 4). This property of PAR enables the usage of a specific agonist comprised of the amino-terminal peptides to study distinct PARs, e.g. a PAR2 agonist peptide (PAR2AP), SLIGKV, stimulates PAR2 specifically.
To date, four PARs have been discovered and characterized. PAR1, PAR3, and PAR4 are activated by thrombin, whereas PAR2 is activated by various proteases but not thrombin. Interestingly, recent findings that PAR2 is activated by various proteases such as trypsin, mast cell tryptase, neutrophil serine proteases, the cell membrane-anchored membrane-type serine protease 1, factor Xa, and the sperm protease acrosin (5, 6, 7, 8, 9, 10) imply its pleiotropic physiological roles.
Expression of PAR1, a likely regulator in the coagulation pathway, has been demonstrated in endometrial stromal cells (11). Furthermore, neutrophils and mast cells, possible activators of PAR2 residing in the endometrium, argue for colocalization of PAR2. In view of the involvement of these leukocytes in menstruation, we speculated that PAR2 may play a role in breakdown and repair of the tissue in the endometrium. Based on this assumption, we first explored the expression of PAR2 in the human endometrium in the present study. Second, the effects of activation of PAR2 on the expression of IL-8, matrix metalloproteinase (MMP)-7, and stem cell factor (SCF), which are molecules related to tissue remodeling, were examined using endometrial cell cultures. In addition, the effects of PAR2 activation on the proliferation of endometrial cells were determined.
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Materials and Methods |
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Type I collagenase and antibiotics (a 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). PAR2AP SLIGKV was from Bachem (Bubendorf, Switzerland). MAPK inhibitors SB202190, PD98059, and SP600125 were from Calbiochem (La Jolla, CA). Antirabbit antibodies of p38 MAPK, phospho-p38 MAPK, p42/44 MAPK, phospho-p42/44 MAPK, and phospho-stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) were from New England BioLabs (Beverly, MA). Antirabbit antibody of MMP-7 was from Chemicon International (Temecula, CA). Antirabbit horseradish peroxidase secondary antibody was from Amersham Biosciences (Little Chalfont, UK). Charcoal-stripped fetal bovine serum was from HyClone (Logan, UT). Deoxyribonuclease I was from Takara (Tokyo, Japan).
Sources of tissues
Endometrial tissues were obtained from a total of 50 patients (aged 40.5 ± 5.7 yr, mean ± SD) undergoing hysterectomy for uterine fibroid or adenomyosis without endometrial pathologies after obtaining written informed consent under a study protocol approved by the Institutional Review Board of University of Tokyo. Although the relatively high ages of the subjects in the reproductive age range and the pathologies of the myometrium may place some limitations on the present study, we used these samples due to the unavailability of endometrial tissue of healthy young women. All patients had regular menstrual cycles and had not received hormone therapy for at least 6 months before surgery. The specimens were dated according to the patients’ menstrual history and standard histological criteria by Noyes et al. (12) and were classified as early proliferative, midproliferative, late proliferative, early secretory, midsecretory, and late secretory phases. Moreover, decidual tissues without trophoblast contamination were obtained from three women by dilation and curettage during an operation for ectopic pregnancy. Endometriotic tissues were collected from three women during laparoscopic surgery for endometriosis. Twenty-eight endometrial tissues, three decidual tissues, and three endometriotic tissues were used for the extractions of mRNA. They were snap frozen in liquid nitrogen and stored at –80 C. Forty endometrial tissues were collected under sterile conditions and processed for the primary cell culture.
Isolation, purification, and culture of endometrial stromal cells and epithelial cells
The isolation and culture of human endometrial stromal cells (ESCs) and epithelial cells (EECs) were described previously (13, 14). Fresh endometrial biopsy specimens collected in a sterile medium were rinsed to remove blood cells. The tissues were minced into small pieces and incubated in DMEM/F12, containing 0.25% type I collagenase and 15 U/ml deoxyribonuclease I, for 60 min at 37 C. The resultant dispersed endometrial cells were separated by filtration through a 40-µm nylon cell strainer (Becton Dickinson, Lincoln Park, NJ). The endometrial epithelial glands that remained intact were retained by the strainer, whereas the dispersed ESCs passed through the strainer into the filtrate. The ESCs in the filtrate were collected by centrifugation and resuspended in phenol-red free DMEM/F12 containing 10% charcoal-stripped fetal bovine serum, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 µg/ml amphotericin B. The ESCs were plated in a 100-mm culture plate and kept at 37 C in a humidified 5% CO2/95% air atmosphere. At the first passage, the cells were plated at a density of 2 x 105 cells/well into 12-well culture plates for the RT-PCR, Western blotting, and ELISA experiments. The cells that reached confluence in 2 or 3 d were used for the experiments.
EECs were collected by backwashing the strainer with DMEM/F12, plated in a 100-mm plate, and incubated at 37 C for 30 min to allow contaminated stromal cells to attach to the plate wall. The nonattached epithelial cells were recovered and cultured in the culture medium as described above at a density of 2 x 105 cells/well into 12-well culture plates. The cells that reached confluence in 2 or 3 d were used for the experiments.
The purity of both the stromal and epithelial cell preparations was more than 95%, as judged by positive cellular staining for vimentin and cytokeratin, respectively.
Treatment of cell cultures
When the ESCs and EECs were approaching confluence, the complete media were removed and replaced with fresh media and antibiotics, and the cells were cultured for an additional 12–24 h. To evaluate the dose effects of PAR2AP, the wells were replenished with serum-free media with different concentrations of PAR2AP, and the cells were incubated for 24 h. For time-course experiments, ESCs and EECs were incubated with serum-free medium with PAR2AP (300 µM) for different periods up to 24 h. To evaluate the effects of the MAPK inhibitors, the cells were preincubated with MAPK inhibitors for 1 h before PAR2AP treatment. After the treatments, the conditioned media were collected, centrifuged, and stored at –80 C for subsequent analysis.
To assess the effects of PAR2AP on the expression of IL-8 and SCF mRNA, the cells incubated with different concentrations of PAR2AP in serum-free medium for 2 h were harvested, snap frozen, and stored at –80 C.
RNA extraction, RT-PCR of PAR2 mRNA, and real-time quantitative PCR of PAR2, IL-8, and SCF mRNA
RT-PCR and real-time quantitative PCR were performed as we reported previously (14, 15, 16). Total RNA was extracted from the endometrial tissues, decidual tissues, endometriotic tissues, ESCs, and EECs, using an 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 total volume of 20 µl, and cDNA was amplified using oligonucleotide primers based on the human PAR2 sequence. The human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers (Toyobo) were used to ensure the quality and amounts of RNA. PAR2 primers (sense, 5'-CTGCATCTGTCCTCACTGGA-3'; antisense, 5'-ACAGAGAGGAGGTCAGCCAA-3') were chosen to amplify a 181-bp fragment. PCR conditions for the amplifications of PAR2 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 PAR2, IL-8, and SCF mRNA expression, real-time quantitative PCR and data analysis were performed using a LightCycler (Roche Diagnostic GmbH, Mannheim, Germany), according to the manufacturer’s instructions. Expression of PAR2, IL-8, and SCF mRNA was normalized to RNA loading for each sample using GAPDH mRNA as an internal standard. The PAR2 primers were the same primers as mentioned above. PCR conditions of PAR2 for amplifications were 40 cycles at 95 C for 15 sec, 64 C for 10 sec, 72 C for 10 sec, followed by melting curve analysis. IL-8 primers (sense, 5'-ACTTCCAAGCTGGCCGTGGCTCTCTTGGCA-3'; antisense, 5'-TGAATTCTCAGCCCTCTTCAAAAACTTCTC-3') were chosen to amplify a 295-bp fragment. PCR conditions of IL-8 for amplifications were 40 cycles at 95 C for 15 sec, 64 C for 10 sec, 72 C for 12 sec, followed by melting curve analysis. SCF primers (sense, 5'-CAGCCAAGTCTTACAAGGGC-3'; antisense, 5'-TAAATGAGACCCAAGTCCCG-3') were chosen to amplify a 378-bp fragment. PCR conditions of SCF for amplifications were 40 cycles at 95 C for 15 sec, 67 C for 10 sec, 72 C for 13 sec, followed by melting curve analysis. Standardization of the data was performed by subtracting the signal threshold cycles (CT) of the internal standard (GAPDH) from the CT of PAR2, IL-8, and SCF. Each PCR product was purified with a QIAEX II gel extraction kit (Qiagen, Tokyo, Japan), and their identities were confirmed using an ABI PRISM 310 genetic analyzer (Applied Biosystems, Foster City, CA).
Measurement of IL-8
Concentrations of IL-8 in conditioned culture media were measured using its specific ELISA kit (Quantikine; R&D Systems, Minneapolis, MN) according to the manufacturer’s protocol. Data were standardized by the total protein of cell lysates.
Western blotting
Cultured cells were homogenized in lysis buffer containing 50 mM Tris/HCl (pH 6.8), 2% sodium dodecyl sulfate, 10% glycerol, 50 mM dithiothreitol, and 0.1% bromophenol blue and diluted to 1 mg total protein per milliliter. Samples were resolved by 10% SDS-PAGE. Proteins were blotted onto a nitrocellulose membrane and incubated with rabbit antibodies to total p38 MAPK (1:1000), phospho-specific p38 MAPK (1:1000), total p42/44 MAPK (1:1000), phospho-specific p42/44 MAPK (1:1000), total SAPK/JNK (1:1000), phospho-specific SAPK/JNK (1:1000), or MMP-7 (1:1000) as primary antibodies and antirabbit horseradish peroxidase antibody (1:1000) as a secondary antibody. Immune complexes were visualized by use of an ECL Western blotting system (Amersham Biosciences). Densitometric analysis of bands on developed x-ray films was performed using National Institutes of Health image.
Cell proliferation assay
The cell proliferation assay was performed as we have reported previously (17). The effect of PAR2AP on the proliferation of ESCs was examined by measuring 5-bromo-2'-deoxyuridine (BrdU) incorporation into DNA using the Biotrak cell proliferation ELISA system (Amersham Biosciences) according to the manufacturer’s instructions. Briefly, ESCs were seeded into Falcon 96-multiwell plates (Becton Dickinson) at a density of 1 x 104 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 into 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 ANOVA with post hoc analysis (Fisher’s protected least significance) for multiple comparisons and Mann-Whitney’s U test for paired comparisons. P < 0.05 was accepted as significant.
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Results |
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PAR2 mRNA was detected in human ESCs and EECs with some interindividual variation by RT-PCR analysis (Fig. 1
). The cDNA fragment samples obtained from the RT-PCR experiments were sequenced and found to be identical with known sequence (data not shown). According to real-time quantitative PCR analysis, PAR2 mRNA expression levels in EECs (0.22 ± 0.07-fold of the GAPDH mRNA level, mean ± SEM, n = 13) appeared to be slightly higher than those in ESCs (0.15 ± 0.07).
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). The expression levels of PAR2 mRNA appeared to be low from the midproliferative phase to the midsecretory phase and increased from the late secretory phase to the early proliferative (menstrual) phase. The expression levels were also increased in the decidual tissues.
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PAR2AP-induced mRNA expression and protein secretion of IL-8 by ESCs and EECs
ESCs incubated with 30 µM PAR2AP for 2 h exhibited a 6-fold increase in IL-8 mRNA expression over the control (Fig. 3A), whereas 300 µM PAR2AP was needed to elicit a significant (4-fold) increase in IL-8 mRNA in EECs (Fig. 3B).
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, C and D) by both ESCs and EECs. Minimal effective concentrations to produce statistically significant increases in IL-8 secretion were 30 and 300 µM in ESCs and EECs, respectively. Time-course experiments showed that PAR2AP (300 µM) increased IL-8 secretion in ESCs and EECs with time, up to 24 h in culture (Fig. 3, E and F). Significant increases were seen at 12 h for ESCs and 4 h for EECs. PAR2AP-induced MAPK phosphorylation in ESCs
We determined the phosphorylation of three MAPKs (p38 MAPK, p42/44 MAPK, and SAPK/JNK) by PAR2AP in cultured ESCs (Fig. 4). Increases in phosphorylation of all the MAPKs were apparent as early as 5 min. The phosphorylation levels appeared to reach a maximal level at 15 min.
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To investigate the intracellular mechanism of PAR2AP-induced secretion of IL-8 by ESCs and EECs, effects of MAPK inhibitors were examined. As depicted in Fig. 5, A and B, the addition of inhibitors for p38 MAPK, p42/44 MAPK, and SAPK/JNK significantly suppressed the PAR2AP-induced increase in IL-8 secretion by ESCs and EECs.
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The effect of PAR2AP on DNA synthesis was determined in ESCs (Fig. 6). PAR2AP at 3–300 µM increased the BrdU incorporation into DNA significantly, the highest level (193% of the control) being observed at 30 µM.
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The proliferative effects of PAR2AP on ESCs were studied with inhibitors for p38 MAPK, p42/44 MAPK, and SAPK/JNK. The proliferative effects of PAR2AP on ESCs were not observed with the addition of each of the three inhibitors (Table 1).
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As shown in Fig. 7A, Western blotting demonstrated the presence of the active form of MMP-7, epithelial cell-specific MMP, and its latent form, pro-MMP-7, in EECs at 18 and 28 kDa, respectively. Densitometric analysis revealed that the active form of MMP-7 was increased significantly by stimulation with PAR2AP at 3–300 µM, the highest level being observed at 30 µM (Fig. 7B).
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PAR2AP at concentrations ranging from 3 to 300 µM increased SCF mRNA expression in cultured ESCs (Fig. 8). A significant increase (2.2-fold of the control) was observed with PAR2AP at 30 µM.
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Discussion |
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We observed that PAR2 mRNA in endometrial tissues increased around the menstrual phase and in early pregnancy. It can be speculated that the increased PAR2 expression renders the tissue more sensitive to injury by up-regulating cellular responses to proteases and, thereby, contributes to tissue repair during menstruation.
A remarkable increase in the number of leukocytes in the endometrium around menstruation implies that molecules secreted by the resident leukocytes such as mast cells and neutrophils could be possible activators for PAR2. Neutrophils have PAR2-activating serine proteases such as proteinase 3, human leukocyte elastase, and cathepsin G, all of which are stored in the cytoplasmic azurophil granules (5). In view of the potent chemotactic effect of IL-8 for neutrophils, PAR2-induced IL-8 secretion, in turn, stimulates neutrophil mobilization to the endometrium, thus possibly forming the self-stimulation system operative in the menstrual phase. In addition, endometrial mast cells, possibly involved in the tissue remodeling of the endometrium, undergo activation and degranulation before and during menstruation, leading to the secretion of tryptase, a PAR2 activator (18). Interestingly, the data presented here showed that PAR2 activation increases SCF expression in ESCs. As SCF stimulates proliferation, maturation, and chemoattraction of mast cells (19, 20, 21), mast cell-induced PAR2 activation may, the other way around, potentiate the biological actions of mast cells.
The expression of IL-8, a chemokine that stimulates chemotaxis of neutrophils, is low during the midproliferative to midsecretory phase and high around menstruation (22, 23, 24). Thus, it is conceivable that the chemokine may be responsible for leukocyte accumulation in the endometrium around the time of menstruation. Furthermore, IL-8 in the uterus is suggested to play unique roles in endometrial angiogenesis, apoptosis, proliferation, and differentiation (25), which are crucial events for the endometrium in preparation for implantation. Proinflammatory cytokines, such as IL-1ß and TNF
, are known to stimulate IL-8 production in endometrial cells (26, 27). The present study suggests that activators of PAR2, like these cytokines, may influence the endometrial function through enhancing IL-8 expression in the endometrium. PAR2 mediated IL-8 secretion has also been reported in other cells, such as respiratory epithelial cells (28) and keratinocytes (29), in which PAR2 activation is supposed to be implicated in homeostasis and inflammatory processes.
Restoration of the disrupted endometrium is an integral part of the tissue modification during the menstrual cycle. The present study also showed that PAR2 activation stimulated the proliferation of ESCs and MMP-7 activation in EECs. MMP-7, matrilysin, is an epithelial-specific MMP expressed in the perimenstrual and proliferative phase but not the early to midsecretory phase (30). The expression pattern implies possible roles of the enzyme in tissue breakdown and regeneration of the endometrium. Similarly, proliferation of the endometrial cells is an indispensable event to repair the endometrium undergoing shedding during menstruation. Therefore, our findings on cell proliferation and MMP-7 activation by PAR2 may further support the notion that PAR2 activation is involved in the regeneration of the endometrium.
PAR2 activation appears to be mediated via different sets of MAPKs, depending on the cell type (31, 32, 33). In the present study, PAR2 activation stimulated the phosphorylation of all the MAPKs studied in ESCs as reported in blood eosinophils (32). This observation may explain the pleiotropic effects of PAR2, considering that the MAPK signal transduction pathways are among the most widespread mechanisms of eukaryotic cell regulation. Interestingly, all the MAPK inhibitors inhibited PAR2-dependent IL-8 secretion from ESCs and EECs. In addition, the PAR2-induced proliferation of ESCs was not observed with these inhibitors. These findings suggest that all the three MAPKs are involved in the signal transduction in the endometrium.
The present findings may also possibly implicate PAR2 in endometriosis, a disease characterized by the growth of cells mimicking endometrial cells, moving to their ultimate location possibly via retrograde menstrual blood. In view of the elevation of SCF concentrations in the peritoneal fluid of women with endometriosis (34) and the presence of mast cells in endometriotic tissue (35, 36), PAR2 may be activated by mast cell tryptase in endometriotic cells. In view of the inflammatory responses and proliferation of endometriotic cells observed in endometriosis, it can be speculated that PAR2 activation participates in the pathogenesis of endometriosis.
The effects of PAR2 in the endometrium demonstrated in the present study imply the importance of PAR2 in the fecundity of humans. On the other hand, studies on PAR2-deficient mice showed that their fertilities were comparable with those of wild-type mice and heterozygous mice (37, 38). Because rodents do not undergo menstruation, PAR2 may have less significance in the endometrium for reproduction in mice.
In summary, the present study demonstrated that PAR2 activation induces IL-8 secretion, cell proliferation, MMP-7 activation, and SCF expression in endometrial cells. Together with the findings that PAR2 expression is high around menstruation and in early pregnancy, PAR2 may be assumed to be important in regulating endometrial remodeling and functions.
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Footnotes |
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Abbreviations: BrdU, 5-Bromo-2'-deoxyuridine; CT, threshold cycle; DMEM/F12, DMEM/Ham’s F12; EEC, endometrial epithelial cell; ESC, endometrial stromal cell; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; JNK, c-Jun N-terminal kinase; MMP, matrix metalloproteinase; PAR, protease-activated receptor; PAR2AP, PAR2 agonist peptide; SAPK, stress-activated protein kinase; SCF, stem cell factor.
Received April 18, 2004.
Accepted November 23, 2004.
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