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Department of Obstetrics and Gynecology (Y.H., Y.O., O.Y., K.K., T.Y., T.H., E.N., T.A., A.N., O.T., Y.T.), University of Tokyo, Tokyo 113-8655; Department of Obstetrics and Gynecology (T.A.), Teikyo University, Tokyo 173-8605; and CREST (O.T.), Japan Science and Technology, Kawaguchi 332-0012, Japan
Address all correspondence and requests for reprints to: Dr. 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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B to the nucleus from the cytoplasm in LGC. Expression of PAR 1 mRNA was detected in LGC by RT-PCR analysis. These findings suggest that thrombin plays physiological roles in ovulation by enhancing the production of chemoattractive and gelatinolytic substances by granulosa cells by a mechanism involving PAR 1. |
Introduction |
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Thrombin receptor, a unique transmembrane-type receptor, was first cloned in 1991 (3). The receptor and three other related receptors discovered later are collectively called protease-activated receptors (PAR 1–4), which constitute a large family of seven-transmembrane G protein-coupled receptors (4, 5, 6). The PARs are activated by proteases such as thrombin and trypsin that cleave 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.
Recent studies have suggested a role for PAR 1 in a variety of biological events including coagulation, inflammation, chemotaxis, mitogenesis, apoptosis, and angiogenesis. Given the presence of the thrombin system in ovarian follicles and known diverse functions of PAR 1, thrombin may play multiple roles through activation of PAR 1 in the ovary. In the present study, we therefore examined the effect of thrombin on the expression of chemokines and matrix metalloproteinases, important mediators of inflammation, as well as expression of PARs in human luteinized granulosa cells (LGC).
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Materials and Methods |
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Thrombin, hyaluronidase, fetal bovine serum (FBS), DMEM, and antibiotics (mixture of penicillin, streptomycin, and amphotericin B) were purchased from Sigma (St. Louis, MO). SFLLRN was from Bachem (Bubendorf, Switzerland). PPACK and calphostin C were from Calbiochem (La Jolla, CA). Ficoll-Paque was from Amersham Biosciences (Uppsala, Sweden). Hirudin was from American Diagnostica (Greenwich, CT). Anti-nuclear factor B (NFB) (p65) antibody and fluorescein isothiocyanate-conjugated antirabbit secondary antibody were from Santa Cruz Biotechnology (Santa Cruz, CA).
Cell culture of human LGC
Follicular fluid with LGC was aspirated from patients undergoing ovarian stimulation for in vitro fertilization. The clinical reasons for in vitro fertilization in these patients were mainly male factor or tubal factor infertility. Patients with ovarian dysfunction were excluded from the study. The experimental procedure was approved by the institutional review board, and signed informed consent for use of granulosa cells was obtained from each patient. The protocol for ovarian stimulation was described previously (7, 8). To obtain LGC, follicular fluid was aspirated during oocyte pickup procedure. All the follicular aspirates from each patient were mixed and centrifuged at 200 x g for 5 min, followed by being resuspended in PBS with 0.2% hyaluronidase and incubated at 37 C for 30 min. The suspension was layered onto Ficoll-Paque and centrifuged at 150 x g for 30 min. The LGC recovered from the interface were treated with anti-CD45 monoclonal antibodies coupled with magnetic immunobeads (DynAl AS, Oslo, Norway) to remove white blood cells. Briefly, isolated LGC were suspended in 2 ml medium containing 2% FBS and incubated for 20 min at 4 C with anti-CD45 immunomagnetic beads. Then the suspension was placed into a magnetic test tube rack (DynAl) for 2 min at room temperature to remove immunobeads-bound white blood cells. With this method, remaining CD68 (a marker of monocyte/macrophages) positive cells were confirmed to be less than 1% in isolated LGC by immunostaining. Isolated LGC were resuspended in DMEM containing 10% FBS, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 250 ng/ml amphotericin B. The cells were plated onto 24-well plates at a density of 2 x 105 cells/ml and kept at 37 C in a humidified 5% CO2/95% air environment. After 24 h, culture media were replaced, and the cells were cultured for another 48 h before treatments.
Treatments of LGC
To evaluate the dose effects of thrombin, the media were replenished with serum-free medium with increasing concentrations of thrombin (0, 0.01, 0.1, 1, 10 U/ml) and LGC were incubated for 24 h. For time-course experiments, LGC were incubated with serum-free medium with or without 10 U/ml thrombin for 1, 2, 4, 8, 12, 24, 36, and 48 h. At 24 h, cell number was counted, and 5-bromo-2'-deoxyuridine uptake was determined using a cell proliferation ELISA system (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer’s instruction. In some experiments, estradiol (10-8 M) was added to see its effect on thrombin-induced reactions in LGC.
To evaluate the effects of hirudin and PPACK (thrombin inhibitors) and calphostin C [protein kinase C (PKC) inhibitor], LGC were preincubated with serum-free medium with or without hirudin (10 U/ml), PPACK (1 µM), or calphostin C (300 µM) for 30 min, and then thrombin was added to the medium (10 U/ml) and incubated for another 24 h. In parallel, LGC were incubated with SFLLRN (PAR 1 agonist peptide, 300 µM) in serum-free medium for 24 h. After the treatments, the conditioned media were collected, centrifuged and stored at -80 C for subsequent analysis.
Measurement of IL-8 and macrophage chemoattractant protein-1 (MCP-1)
Concentrations of IL-8 and MCP-1 in conditioned culture media were measured using their specific ELISA kits (Quantikine; R&D systems, Minneapolis, MN) according to the manufacturer’s protocol.
Gelatinase activity assays
Gelatinase activities were measured by two methods. Gelatinase activity assay kit (Chemicon international, Inc., Temecula, CA) measures total gelatinase activity of the medium including, if present, gelatinase inhibitors. Gelatin zymography was used to measure activities of matrix metalloproteinase (MMP)-2 (gelatinase A) and MMP-9 (gelatinase B) in the medium separately. Gelatin Zymo electrophoresis kit (Yagai, Yamagata, Japan) containing a 7.5% polyacrylamide gel with 1 mg/ml gelatin was used for the assay according to the manufacturer’s instruction.
RNA extraction, RT-PCR of PARs mRNA, and real-time quantitative PCR of IL-8 and MCP-1 mRNA
Total RNA was extracted from LGC, 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 PAR 1, PAR 2, PAR 3, and PAR 4 sequence. The human glycerinaldehyde dehydrogenase (GAPDH) primers (TOYOBO) were used to ensure RNA quality and amounts. PAR 1 primers (sense, 5'-TGTGTACACCGGAGTGTTTGTAG-3'; antisense, 5'-ACTGTCATGAGCAAGATAGAGGC-3') were chosen to amplify a 264-bp fragment. PAR 2 primers (sense, 5'-CTGCATCTGTCCTCACTGGA-3'; antisense, 5'-ACAGAGAGGAGGTCAGCCAA-3') were chosen to amplify a 181-bp fragment. PAR 3 primers (sense, 5'-GGTGTGGGCAACAGTTTTCT-3'; antisense, 5'-TGCATTAAGTGTCCGGATGA-3') were chosen to amplify a 226-bp fragment. PAR 4 primers (sense, 5'-CAATGACAGTGACACCCTGG-3'; antisense, 5'-CATGTGACCATAGAGTGCGG-3') were chosen to amplify a 316-bp fragment. PCR conditions for amplifications of PAR 1, PAR 2, PAR 3, PAR 4, and GAPDH were 30 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 and MCP-1 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 and MCP-1 mRNA was normalized to RNA loading for each sample using GAPDH mRNA as an internal standard. IL-8 primers (sense, 5'-ACTTCCAAGCTGGCCGTGGCTCTCTTGGCA-3'; antisense, 5'-TGAATTCTCAGCCCTCTTCAAAAACTTCTC-3') were chosen to amplify a 295-bp fragment. MCP-1 primers (sense, 5'-TGGCTGTGTTTGCTTCTGTC-3'; antisense, 5'-TCTCACTGCCCTATGCCTCT-3') were chosen to amplify a 230-bp fragment. PCR conditions of IL-8 for amplifications were 50 cycles at 95 C for 15 sec, 55 C for 10 sec, and 72 C for 20 sec, followed by melting curve analysis. PCR conditions of MCP-1 for amplifications were 50 cycles at 95 C for 15 sec, 65 C for 6 sec, and 72 C for 16 sec, followed by melting curve analysis.
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).
Immunofluorescence detection of NFB translocation
Isolated LGC were cultured in 16-well chamber slides (Nunc, Naperville, IL) at a density of 5 x 103 cells/well at 37 C in a humidified 5% CO2/95% air environment. After 3 d of preincubation, LGC were treated with serum-free medium with thrombin (10 U/ml) for 10 min or SFLLRN (300 µM) for 15 min. To evaluate the effects of calphostin C, LGC were preincubated with serum-free medium with calphostin C (300 µM) for 30 min, and then thrombin (10 U/ml) was added to the medium and incubated for a further 10 min. Treated LGC were fixed with cold methanol/acetic acid (3:1) at -20 C for 20 min, washed twice with PBS, blocked for 20 min with 5% bovine serum in PBS, and incubated with anti-NFB (p65) antibody (2 µg/ml in 1.5% bovine serum in PBS) for 60 min. After two washes with PBS, the slides were incubated in fluorescein isothiocyanate-conjugated secondary antibodies (1:200 in 1.5% bovine serum) for 45 min. Then the slides were washed two more times and mounted with coverslips using VectaShield (Vector Laboratories, Burlingame, CA). Analysis of the specimens was performed using a fluorescence microscope (BX50; Olympus, Tokyo, Japan).
Statistical analysis
Data were evaluated using ANOVA with post hoc analysis (Fisher’s protected least significance) for multiple comparisons. P < 0.05 was accepted as significant.
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Results |
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We first conducted time-course experiments to see the effects of thrombin (10 U/ml) on the release of IL-8 and MCP-1 by LGC as a function of time up to 48 h in culture (Fig. 1
). An increase in IL-8 release was evident at 8 h. IL-8 levels were further increased with time in culture. A significant increase in MCP-1 level was observed at 24 h, followed by continued increases up to 48 h. The increase per hour appeared maximal at 24 h in both IL-8 and MCP-1. During the period, both the concentrations of IL-8 and MCP-1 in the media were significantly less in control cells, compared with thrombin-treated cells.
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, thrombin at 1 U/ml and more caused an increase in IL-8 release. An increase with thrombin at 10 U/ml was about 3-fold over the control. A significant increase in MCP-1 release was also observed with thrombin at 1 U/ml and more. Cell counts and 5-bromo-2'-deoxyuridine uptake at 24 h were substantially the same whether or not the cells were treated with thrombin (data not shown).
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shows the effect of estradiol (10-8 M) on thrombin-induced secretion of IL-8 and MCP-1 by LGC. Estradiol up-regulated the thrombin-induced secretion of IL-8 and MCP-1, but estradiol alone had no effect on basal secretion of the cytokines in the control cells.
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). In the same condition, an increase in the amount of MCP-1 mRNA was 5.9-fold over the control.
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To elucidate the mechanism of thrombin-induced secretion of IL-8 and MCP-1 by LGC, the effects of PAR 1-specific agonist, the thrombin inhibitor, and the PKC inhibitor were examined. As shown in Fig. 5, the PAR 1-specific agonist, SFLLRN, stimulated the secretion of IL-8 by 2 times and MCP-1 by 2.4 times, compared with the control. Concomitant addition of the thrombin inhibitors, hirudin, and PPACK, with thrombin completely eliminated the stimulatory effect of thrombin on the release of IL-8 and MCP-1. Likewise, the PKC inhibitor, calphostin C, abrogated the stimulatory effect of thrombin on IL-8 and MCP-1 secretion.
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Gelatinase activities of conditioned media of LGC cultured with SFLLRN and thrombin with or without thrombin inhibitors were measured. Both thrombin and SFLLRN increased the activity by 13.6 and 3.5 times, respectively, over the control (Fig. 6). Addition of hirudin or PPACK in combination with thrombin inhibited thrombin-induced gelatinase activity. Gelatin zymography (Fig. 7) showed that conditioned medium of untreated LGC contained 92 kDa pro-MMP-9 and 72 kDa pro-MMP-2. Thrombin increased the production of 92 kDa pro-MMP-9. Moreover, thrombin induced the production of 62 kDa MMP-2, the active form. These effects were inhibited by hirudin and PPACK. SFLLRN also increased 92-kDa pro-MMP-9 production, whereas SFLLRN was without effect in the production of 62 kDa MMP-2.
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As shown in Fig. 8, by use of specific primers to amplify human PAR 1, PAR 2, PAR 3, and PAR 4 mRNA, PCR products revealed the bands for mRNAs corresponding to PAR 1, PAR 2, PAR 3, and PAR 4. The cDNA fragment samples obtained from the RT-PCR experiments were sequenced and found to be identical to known sequences (data not shown).
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B activation in LGC
NFB, when activated, translocates from the cytoplasm to the nucleus, resulting in its binding to the promoter sequences of IL-8 and MCP-1 genes and enhancing their transcriptions. As shown in Fig. 9, NFB, stained positive for fluorescence, was present in the nucleus as well as the cytoplasm in LGC treated with thrombin (10 U/ml) for 10 min, but NFB was observed only in the cytoplasm in nontreated LGC. Calphostin C (300 µM) completely blocked the thrombin-induced nuclear translocation of NFB. SFLLRN also induced the translocation of NFB to the nucleus.
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Discussion |
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B, an intracellular signal for PAR 1. These findings suggest possible physiological roles of the thrombin-PAR 1 system in the human ovaries. Ovulation is known to be an inflammatory process, in which leukocytes infiltrate into extravascular spaces of the follicle. IL-8 and MCP-1, prototype chemokines expressed in the follicles (9), are suggested to play a pivotal role in ovulation by stimulating migration and activation of leukocytes during the periovulatory period (10). Migrated leukocytes are also suggested to play roles in both luteogenesis and luteolysis (11). IL-8 may promote neovascularization of the corpus luteum with its angiogenic properties. Thus, the findings of thrombin-stimulated secretion of IL-8 and MCP-1 by LGC imply that thrombin affect ovulation and luteal functions through increased expression of the cytokines. Estradiol-induced up-regulation of these effects may further support the notion because granulosa cells are normally exposed to locally produced estradiol in vivo.
We have shown that PAR 1-specific agonist, like thrombin, stimulated the production of IL-8 and MCP-1 and that thrombin inhibitors, which inhibit the thrombin-induced activation of PAR 1, suppressed thrombin-induced IL-8 and MCP-1 production by LGC. These data suggest that thrombin-induced production of IL-8 and MCP-1 by granulosa cells is mediated by PAR 1. In addition, the finding that the inhibitor of PKC, an intracellular mediator of PAR 1 (12, 13), suppressed the effects of thrombin is in agreement with the above notion. The mechanism of thrombin action to stimulate IL-8 and MCP-1 secretion is not exclusive for granulosa cells but may work in monocytes and endothelial cells (14, 15, 16).
During the ovulatory process, the dissolution of the granulosa cell basement membrane and fragmentation of the extracellular matrix of the follicular wall precede rupture of the follicle. MMP-2 and MMP-9, members of the gelatinase family, which cleave type IV collagen, the principal component of the basement membrane, are present in the follicle of rats (17), sheep (18), and humans (19, 20), with gelatinase activity increasing during the periovulatory period (21, 22). In view of the stimulatory effects of thrombin on gelatinase activity, it seems that thrombin could play a role in the tissue degradation occurring during the ovulatory process by producing and activating gelatinases.
NFB has been shown to mediate a signal of activated PAR 1 in endothelial cells and vascular smooth muscle cells (23, 24). Activated NFB translocates from the cytoplasm to the nucleus and binds to the elements, leading to subsequent gene transcription (25). IL-8 and MCP-1 contain NFB-binding elements in their promoter regions (26, 27). In the present study, thrombin and PAR 1 agonist stimulated translocation of NFB to the nucleus in LGC, which was inhibited by the PKC inhibitor. Collectively, we surmise that PAR 1 stimulates IL-8 and MCP-1 production by mediating thrombin action in LGC.
Gelatin zymography analysis showed that LGC treated with thrombin produced 62 kDa MMP-2 and increased amounts of 92 kDa pro-MMP-9. These effects of thrombin were abrogated by the thrombin inhibitor hirudin. On the other hand, although SFLLRN, a specific activator of PAR 1 increased gelatinase activity, the stimulatory effect was limited to 92 kDa pro-MMP-9, compared with thrombin. Thus, it may be that the thrombin-induced production of 62 kDa MMP-2 cannot be completely explained by a mechanism involving PAR 1 activation.
In addition to PAR 1, the expressions of PAR 2, PAR 3, and PAR 4 in LGC were shown in this study. Because thrombin is known to activate PAR 3 and PAR 4, the observed weak agonistic effects of SFLLRN for IL-8 secretion and MMP activation may imply the involvement of these PARs in the thrombin action.
In summary, the present study demonstrated biological actions of thrombin and its signal transduction pathway in human granulosa cells, thus implicating the thrombin system in the ovulatory process.
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Acknowledgments |
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Footnotes |
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B, nuclear factor B; PAR, protease-activated receptor; PKC, protein kinase C. Received November 13, 2002.
Accepted May 12, 2003.
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References |
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B and potentiates endothelial cell activation by TNF. J Immunol 159:5620–5628[Abstract]
B through thrombin receptor and results in proliferation of vascular smooth muscle cells: role of thrombin in atherosclerosis and restenosis. Ann N Y Acad Sci 811:429–436[Medline]
B activity. Annu Rev Immunol 18:621–663[CrossRef][Medline]
B sites and NF-B/Rel subunit specificity. J Biol Chem 272:31092–31099This article has been cited by other articles:
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) or presence (
) of thrombin (10 U/ml) for indicated hours at 37 C in 5% CO2. At the end of the incubation period, the conditioned medium was collected and assayed for concentrations of IL-8 and MCP-1 by ELISA. Values are the mean ± SEM of quadruplicate cultures. A, *, P < 0.05; **, P < 0.005; ***, P < 0.0001 (all vs. control). B, *, P < 0.0005; **, P < 0.0001 (both vs. control).
