June 2012
Volume 53, Issue 7
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Cornea  |   June 2012
Inhibition by Medroxyprogesterone Acetate of Interleukin-1β–Induced Collagen Degradation by Corneal Fibroblasts
Author Affiliations & Notes
  • Hongyan Zhou
    From the Department of Ophthalmology, Yamaguchi University Graduate School of Medicine, Ube City, Yamaguchi, Japan; and the
    Department of Ophthalmology, China–Japan Union Hospital, Jilin University, Changchun City, Jilin Province, China.
  • Kazuhiro Kimura
    From the Department of Ophthalmology, Yamaguchi University Graduate School of Medicine, Ube City, Yamaguchi, Japan; and the
  • Tomoko Orita
    From the Department of Ophthalmology, Yamaguchi University Graduate School of Medicine, Ube City, Yamaguchi, Japan; and the
  • Teruo Nishida
    From the Department of Ophthalmology, Yamaguchi University Graduate School of Medicine, Ube City, Yamaguchi, Japan; and the
  • Koh-Hei Sonoda
    From the Department of Ophthalmology, Yamaguchi University Graduate School of Medicine, Ube City, Yamaguchi, Japan; and the
  • Corresponding author: Kazuhiro Kimura, Department of Ophthalmology, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami-Kogushi, Ube City, Yamaguchi 755-8505, Japan; k.kimura@yamaguchi-u.ac.jp
Investigative Ophthalmology & Visual Science June 2012, Vol.53, 4213-4219. doi:10.1167/iovs.11-8822
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      Hongyan Zhou, Kazuhiro Kimura, Tomoko Orita, Teruo Nishida, Koh-Hei Sonoda; Inhibition by Medroxyprogesterone Acetate of Interleukin-1β–Induced Collagen Degradation by Corneal Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2012;53(7):4213-4219. doi: 10.1167/iovs.11-8822.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To examine the effect of medroxyprogesterone 17-acetate (MPA) on interleukin-1β (IL-1β)–induced collagen degradation by corneal fibroblasts.

Methods.: Rabbit corneal fibroblasts were cultured in three-dimensional collagen gels with or without MPA. Collagen degradation was determined by measurement of hydroxyproline after acid hydrolysis. The expression or activity of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) was evaluated by immunoblot analysis or gelatin zymography. The phosphorylation of mitogen-activated protein kinases (MAPKs) in corneal fibroblasts was examined by immunoblot analysis. Cell proliferation and viability were evaluated by measurement of bromodeoxyuridine incorporation and the release of lactate dehydrogenase, respectively.

Results.: MPA inhibited IL-1β–induced collagen degradation by corneal fibroblasts in a concentration- and time-dependent manner. MMP expression and activation as well as TIMP expression in corneal fibroblasts exposed to IL-1β were also inhibited by MPA. MPA had no effect on cell proliferation or viability. MPA inhibited the IL-1β–induced phosphorylation of p38 MAPK without affecting that of the MAPKs ERK or JNK. IL-1β–induced MMP expression and activation as well as collagen degradation were also blocked by the p38 MAPK inhibitor SB203580.

Conclusions.: MPA inhibited MMP expression and thereby suppressed collagen degradation by corneal fibroblasts induced by IL-1β. Furthermore, inhibition of p38 MAPK phosphorylation by MPA may contribute to its inhibition of collagen degradation.

Introduction
Keratocytes in the corneal stroma become activated and undergo transformation into corneal fibroblasts in response to corneal injury or inflammation. The activation of keratocytes by inflammatory cytokines and infiltrating cells contributes to the recovery of corneal homeostasis. 1 Corneal fibroblasts undergo proliferation and upregulate the synthesis of matrix metalloproteinases (MMPs) that mediate remodeling of the corneal stroma. We have also previously shown that neutrophils, pseudomonal elastase, and the proinflammatory cytokine interleukin (IL)–1β promote collagen degradation by corneal fibroblasts by stimulating MMP synthesis. 2,3 MMPs are zinc-dependent endopeptidases that cleave various extracellular matrix proteins. 4 Their synthesis is induced by cytokines as well as by cell–matrix and cell–cell interactions. 5 In addition to their beneficial effects, MMPs have been shown to complicate corneal wound healing after refractive surgery as well as to play a role in corneal diseases such as keratoconus, bacterial keratitis, and ulceration. 69  
Progesterone is a female hormone produced by the ovary and plays a key role in the menstrual cycle and pregnancy. It is also administered in hormone replacement therapy in women. Progesterone was shown to retard uterine involution and the associated loss of collagen in rats. 10 Medroxyprogesterone 17-acetate (MPA) is a synthetic progestin and inhibits MMP expression induced by proinflammatory cytokines such as tumor necrosis factor–α in decidual cells or endometrial stromal cells. 1113 Sex hormones are also thought to have numerous effects on the structure and function of ocular tissues. 14 Progesterone receptors have thus been detected in human corneal epithelial, stromal, and endothelial cells. 14 Moreover, the administration of MPA has been shown to prevent or markedly delay ulceration of the alkali-injured rabbit cornea. 15  
In addition to MMPs, proinflammatory cytokines such as IL-1β are implicated in the pathogenesis of various corneal diseases. 7,16,17 We have previously shown that the MMP inhibitor galardin and the immunosuppressant triptolide inhibit IL-1β–induced collagen degradation by corneal fibroblasts. 18,19 We have now examined whether MPA might inhibit collagen degradation by rabbit corneal fibroblasts exposed to IL-1β. We also investigated the possible effects of MPA on MMP expression and signaling pathways in these cells. 
Methods
Materials
Eagle's minimum essential medium (MEM), Dulbecco's phosphate-buffered saline (DPBS), dispase, antibiotic–antimycotic mixture, and trypsin-EDTA were obtained commercially (Invitrogen-Gibco, Grand Island, NY); native porcine type 1 collagen (acid solubilized), 5× Dulbecco's modified Eagle's medium (DMEM), and collagen reconstitution buffer were commercially supplied (Nitta Gelatin, Osaka, Japan); other materials used were fetal bovine serum (FBS; JRH Biosciences, Lenexa, KS); bovine plasminogen as well as collagenase, protease inhibitor cocktail, and MPA (Sigma-Aldrich, St. Louis, MO); and SB203580 (Merck Millipore, Darmstadt, Germany). Recombinant human IL-1β and goat polyclonal antibodies to human TIMP-1 and TIMP-2 were obtained commercially (R&D Systems, Minneapolis, MN); other materials used were mouse monoclonal antibodies to rabbit MMP-1 and MMP-3 (Daiichi Fine Chemicals, Toyama, Japan); antibodies to p38 mitogen-activated protein kinase (MAPK), to phosphorylated p38 MAPK (Thr180, Tyr182), to c-Jun NH2-terminal kinase (JNK), to phosphorylated JNK (Thr183, Tyr185), to extracellular signal-regulated kinase 1 or 2 (ERK1/2), and to phosphorylated ERK1/2 (Thr202, Tyr204) (Cell Signaling, Beverly, MA); an enhanced chemiluminescence (ECL) kit as well as horseradish peroxidase (HRP)–conjugated secondary antibodies (GE Healthcare, Piscataway, NJ); culture plates (24- and 96-well) and 60-mm cell culture dishes (Corning Inc., Corning, NY); Coomassie brilliant blue and gelatin (Bio-Rad, Hercules, CA); a cytotoxicity assay (CytoTox 96Non-Radioactive; Promega, Madison, WI); and a cell proliferation assay based on a colorimetric enzyme-linked immunosorbent assay for bromodeoxyuridine (BrdU; Roche, Basel, Switzerland). All media and reagents used for cell culture were endotoxin minimized. 
Cell Isolation
Male Japanese albino rabbits (body weight, 2.0 to 2.5 kg) were commercially obtained (Biotec, Saga, Japan). This study adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and was approved by the Animal Experimental Committee of Yamaguchi University School of Medicine. Rabbit corneal fibroblasts were isolated and maintained as described previously. 3 In brief, the enucleated eye was washed with DPBS containing an antibiotic–antimycotic mixture, the endothelial layer of the excised cornea was removed mechanically, and the remaining corneal tissue was incubated with dispase (2 mg/mL, in MEM) for 1 hour at 37°C. After mechanical removal of the epithelial sheet, the remaining tissue was treated with collagenase (2 mg/mL, in MEM) at 37°C until a single-cell suspension of corneal fibroblasts was obtained. The isolated corneal fibroblasts were cultured under a humidified atmosphere of 5% CO2 at 37°C in 60-mm culture dishes containing MEM supplemented with 10% FBS. The rabbit corneal fibroblasts used in the present study were positive for vimentin and negative for α–smooth muscle actin, as shown previously for human cells. 20,21 Proliferating cells were harvested for experiments at the subconfluent stage after four to six passages in monolayer culture. 
Three-Dimensional Culture
Collagen gels were prepared as described. 3 In brief, corneal fibroblasts were harvested by exposure to trypsin-EDTA followed by centrifugation at 15,000g for 5 minutes, and were then suspended in serum-free MEM. Acid-solubilized collagen type I (3 mg/mL), 5× DMEM, collagen reconstitution buffer (0.05 M NaOH, 0.26 M Na2CO3, 0.2 M HEPES [pH 7.3]), and corneal fibroblast suspension (2.2 × 106 cells/mL in MEM) were mixed on ice at a volume ratio of 7:2:1:1. The resultant mixture (0.5 mL) was added to each well of a 24-well culture plate and allowed to solidify in an incubator containing 5% CO2 at 37°C, after which 0.5 mL of serum-free MEM containing test reagents and plasminogen (60 μg/mL) was overlaid and the cultures were returned to the incubator for 36 hours. 
Assay of Collagenolytic Activity
Collagen degradation was measured as previously described. 19 In brief, the supernatants from collagen gel incubations were collected, and native collagen fibrils with a molecular size of >100 kDa were removed by ultrafiltration. The filtrate was subjected to hydrolysis with 6 M HCl for 24 hours at 110°C, and the amount of hydroxyproline in the hydrolysate was determined by measurement of absorbance at 558 nm with a spectrophotometer. 
Immunoblot Analysis
Immunoblot analysis of MMP-1 and MMP-3 (as well as TIMP-1 and TIMP-2) was performed as described previously. 3 In brief, culture supernatants from collagen gel incubations were subjected to SDS–polyacrylamide gel electrophoresis (PAGE) on a 10% gel, and the separated proteins were transferred electrophoretically to a nitrocellulose membrane. Nonspecific sites of the membrane were blocked and then incubated with primary antibodies. Immune complexes were detected with the use of HRP-conjugated secondary antibodies and ECL reagents. Immunoblot analyses of total or phosphorylated forms of ERK, p38 MAPK, or JNK were also performed as described previously. 22 Cells (5 × 105 per well of a 24-well plate) were cultured for 24 hours in unsupplemented MEM and then incubated first for 12 hours with or without MPA and then for 30 minutes in the additional absence or presence of IL-1β (0.1 ng/mL). The cells were lysed in a solution containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 5 mM NaF, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM Na3VO4, and 1% protease inhibitor cocktail, after which the cell lysates (10 μg of protein) were then subjected to immunoblot analysis. 
Gelatin Zymography
Gelatin zymography was performed as described previously. 3 In brief, culture supernatants (8 μL) from collagen gel incubations were mixed with 4 μL of nonreducing SDS sample buffer (125 mM Tris-HCl [pH 6.8], 20% glycerol, 2% SDS, 0.002% bromophenol blue), and 5 μL of the resulting mixture was subjected to SDS–PAGE in the dark at 4°C on a 10% gel containing 0.1% gelatin. The gel was then washed with 2.5% Triton X-100 for 1 hour before incubation for 18 hours at 37°C in a reaction mixture containing 50 mM Tris-HCl (pH 7.5), 5 mM CaCl2, and 1% Triton X-100. The gel was finally stained with Coomassie brilliant blue. 
Cell Proliferation Assay
Cells (2 × 104 per well) seeded in a 96-well plate were incubated in MEM with or without MPA for 24 hours, with BrdU added to the culture medium for the final 2 hours. The medium was then removed, and the cells were processed for colorimetric detection of incorporated BrdU by measurement of absorbance at 370 nm with a microplate reader. 
Cytotoxicity Assay
Cells (2 × 104 per well) seeded in a 96-well plate were incubated in MEM with or without MPA for 24 hours. The amount of lactate dehydrogenase (LDH) released into the culture medium was then measured with the use of an assay kit as described previously. 23 Absorbance at 490 nm was measured with a microplate reader. 
Statistical Analysis
Data are presented as means ± SEM and were analyzed with Dunnett's multiple comparison test or Student's unpaired t-test. A value of P < 0.05 was considered statistically significant. 
Results
Inhibitory Effect of MPA on IL-1β–Induced Collagen Degradation by Corneal Fibroblasts
We previously showed that the proinflammatory cytokine IL-1β markedly increased the extent of collagen degradation by cultured corneal fibroblasts. 18,19,24 To examine the effect of the synthetic progestin MPA on IL-1β–induced collagen degradation in three-dimensional cultures of rabbit corneal fibroblasts, we incubated the cells for 36 hours with various concentrations of MPA (0.1 nM to 1 μM) in the absence or presence of IL-1β (0.1 ng/mL). The stimulatory effect of IL-1β was inhibited by MPA in a concentration-dependent manner (Fig. 1A), with the inhibition being significant at MPA concentrations of ≥0.1 μM. Investigation of the time course of IL-1β–induced collagen degradation in the absence or presence of MPA (1 μM) revealed that the inhibitory effect of MPA was time dependent and was significant at 36 and 48 hours (Fig. 1B). 
Figure 1. 
 
Concentration- and time-dependent inhibition by MPA of IL-1β–induced collagen degradation by corneal fibroblasts. (A) Cells were cultured in collagen gels with or without IL-1β (0.1 ng/mL) and in the presence of the indicated concentrations of MPA for 36 hours, after which the amount of degraded collagen was determined. Data are expressed as micrograms of hydroxyproline (HYP) per well and are means ± SEM of quadruplicates from an experiment that was repeated three times with similar results. *P < 0.05 (Dunnett's test) versus the corresponding value for cells cultured without MPA. (B) Cells were cultured for the indicated times in collagen gels in the absence or presence of IL-1β (0.1 ng/mL) and MPA (1 μM), as indicated. The amount of degraded collagen was then determined. Data are means ± SEM of quadruplicates from an experiment that was repeated three times with similar results. *P < 0.05 (Student's t-test) versus the corresponding value for cells incubated with IL-1β alone.
Figure 1. 
 
Concentration- and time-dependent inhibition by MPA of IL-1β–induced collagen degradation by corneal fibroblasts. (A) Cells were cultured in collagen gels with or without IL-1β (0.1 ng/mL) and in the presence of the indicated concentrations of MPA for 36 hours, after which the amount of degraded collagen was determined. Data are expressed as micrograms of hydroxyproline (HYP) per well and are means ± SEM of quadruplicates from an experiment that was repeated three times with similar results. *P < 0.05 (Dunnett's test) versus the corresponding value for cells cultured without MPA. (B) Cells were cultured for the indicated times in collagen gels in the absence or presence of IL-1β (0.1 ng/mL) and MPA (1 μM), as indicated. The amount of degraded collagen was then determined. Data are means ± SEM of quadruplicates from an experiment that was repeated three times with similar results. *P < 0.05 (Student's t-test) versus the corresponding value for cells incubated with IL-1β alone.
Effects of MPA on MMP Expression and Activity
We next examined the effects of MPA on MMP abundance or activity in culture supernatants of corneal fibroblasts incubated in the presence of IL-1β by immunoblot analysis and gelatin zymography. The cells were thus cultured in collagen gels for 36 hours with IL-1β (0.1 ng/mL) and in the presence of various concentrations of MPA (0.1 nM to 1 μM). Consistent with our previous results, the amounts of active or pro forms of MMP-1, MMP-2, MMP-3, and MMP-9 in the culture supernatants were increased by exposure to IL-1β. 24 Immunoblot analysis showed that MPA inhibited the IL-1β–induced increase in the amounts of both the active and pro forms of MMP-1 and MMP-3 in a concentration-dependent manner (Fig. 2A). Moreover, gelatin zymography revealed that the abundance of the active forms of MMP-9 and MMP-2 was decreased by MPA in a concentration-dependent manner (Fig. 2B). We also examined the effect of MPA on TIMP-1 and TIMP-2 abundance in culture supernatants of IL-1β–treated corneal fibroblasts by immunoblot analysis. IL-1β increased the amounts of TIMP-1 and TIMP-2 in a manner sensitive to inhibition by MPA (Fig. 3). All of these inhibitory effects of MPA appeared maximal at a concentration of 1 μM. 
Figure 2. 
 
Effects of MPA on IL-1β–induced MMP-1, MMP-2, MMP-3, and MMP-9 release by corneal fibroblasts. Cells were cultured in collagen gels in the absence or presence of IL-1β (0.1 ng/mL) and with the indicated concentrations of MPA for 36 hours, after which the culture supernatants were subjected to immunoblot analysis with antibodies to MMP-1 or to MMP-3 (A) or to gelatin zymography for analysis of MMP-2 and MMP-9 (B). Similar results were obtained in three separate experiments.
Figure 2. 
 
Effects of MPA on IL-1β–induced MMP-1, MMP-2, MMP-3, and MMP-9 release by corneal fibroblasts. Cells were cultured in collagen gels in the absence or presence of IL-1β (0.1 ng/mL) and with the indicated concentrations of MPA for 36 hours, after which the culture supernatants were subjected to immunoblot analysis with antibodies to MMP-1 or to MMP-3 (A) or to gelatin zymography for analysis of MMP-2 and MMP-9 (B). Similar results were obtained in three separate experiments.
Figure 3. 
 
Effects of IL-1β and MPA on TIMP-1and TIMP-2 release by corneal fibroblasts. Cells were cultured in collagen gels in the absence or presence of IL-1β (0.1 ng/mL) and with the indicated concentrations of MPA for 36 hours, after which the culture supernatants were subjected to immunoblot analysis with antibodies to TIMP-1 or to TIMP-2. Similar results were obtained in three separate experiments.
Figure 3. 
 
Effects of IL-1β and MPA on TIMP-1and TIMP-2 release by corneal fibroblasts. Cells were cultured in collagen gels in the absence or presence of IL-1β (0.1 ng/mL) and with the indicated concentrations of MPA for 36 hours, after which the culture supernatants were subjected to immunoblot analysis with antibodies to TIMP-1 or to TIMP-2. Similar results were obtained in three separate experiments.
Effects of MPA on Cell Proliferation and Viability
We examined whether MPA might affect the proliferation of rabbit corneal fibroblasts. Incubation of the cells with MPA at 0.1 or 1 μM for 24 hours had no effect on cell proliferation (Fig. 4). Similarly, measurement of LDH release revealed that MPA at 0.1 or 1 μM had no cytotoxic effect on corneal fibroblasts (Fig. 5). 
Figure 4. 
 
Lack of effect of MPA on the proliferation of corneal fibroblasts. Cells were cultured for 24 hours in MEM either in the absence (negative control) or presence of 0.1 or 1 μM MPA or with 10% FBS (positive control). Cell proliferation was evaluated by measurement of BrdU incorporation with a colorimetric assay. Data are means ± SEM of quadruplicates from an experiment that was repeated three times with similar results. *P < 0.05 vs. positive control (Dunnett's test).
Figure 4. 
 
Lack of effect of MPA on the proliferation of corneal fibroblasts. Cells were cultured for 24 hours in MEM either in the absence (negative control) or presence of 0.1 or 1 μM MPA or with 10% FBS (positive control). Cell proliferation was evaluated by measurement of BrdU incorporation with a colorimetric assay. Data are means ± SEM of quadruplicates from an experiment that was repeated three times with similar results. *P < 0.05 vs. positive control (Dunnett's test).
Figure 5. 
 
Lack of a cytotoxic effect of MPA on corneal fibroblasts. Cells were incubated for 24 hours in MEM and in the absence (negative control) or presence of 0.1 or 1 μM MPA, after which the culture supernatants were assayed for LDH activity with a colorimetric assay. The amount of LDH released from cells by a cell lysis solution was determined as a positive control. Data are means ± SEM from three independent experiments. *P < 0.05 vs. positive control (Dunnett's test).
Figure 5. 
 
Lack of a cytotoxic effect of MPA on corneal fibroblasts. Cells were incubated for 24 hours in MEM and in the absence (negative control) or presence of 0.1 or 1 μM MPA, after which the culture supernatants were assayed for LDH activity with a colorimetric assay. The amount of LDH released from cells by a cell lysis solution was determined as a positive control. Data are means ± SEM from three independent experiments. *P < 0.05 vs. positive control (Dunnett's test).
Effect of MPA on MAPK Signaling Pathways in Corneal Fibroblasts
We previously showed that IL-1β induced the activation of MAPK signaling pathways in corneal fibroblasts. 25 We therefore examined the effect of MPA on MAPK signaling in corneal fibroblasts stimulated with IL-1β. Cells were exposed to MPA (1 μM) for 12 hours and then incubated in the additional presence of IL-1β (0.1 ng/mL) for 30 minutes. Immunoblot analysis showed that the IL-1β–induced phosphorylation of p38 MAPK was inhibited by MPA, whereas that of ERK or JNK was not affected (Fig. 6). 
Figure 6. 
 
Effect of MPA on IL-1β–induced MAPK phosphorylation in corneal fibroblasts. (A) Cells were incubated in the absence or presence of MPA (1 μM) for 12 hours and then in the additional absence or presence of IL-1β (0.1 ng/mL) for 30 minutes. Cell lysates were then prepared and subjected to immunoblot analysis with antibodies to total or phosphorylated (P-) forms of ERK, p38 MAPK, or JNK. (B) The intensity of the phosphorylated ERK, p38 MAPK, and JNK bands in immunoblots similar to that shown in (A) was determined by densitometry and normalized by that of the corresponding total ERK, p38 MAPK, and JNK bands. Data are expressed relative to the value for cells incubated without addition and are means ± SEM from three independent experiments. *P < 0.05 (Dunnett's test).
Figure 6. 
 
Effect of MPA on IL-1β–induced MAPK phosphorylation in corneal fibroblasts. (A) Cells were incubated in the absence or presence of MPA (1 μM) for 12 hours and then in the additional absence or presence of IL-1β (0.1 ng/mL) for 30 minutes. Cell lysates were then prepared and subjected to immunoblot analysis with antibodies to total or phosphorylated (P-) forms of ERK, p38 MAPK, or JNK. (B) The intensity of the phosphorylated ERK, p38 MAPK, and JNK bands in immunoblots similar to that shown in (A) was determined by densitometry and normalized by that of the corresponding total ERK, p38 MAPK, and JNK bands. Data are expressed relative to the value for cells incubated without addition and are means ± SEM from three independent experiments. *P < 0.05 (Dunnett's test).
Effects of SB203580 on IL-1β–Induced Collagen Degradation, MMP Expression and Activity, and MAPK Signaling
We next examined the effect of the p38 MAPK inhibitor SB203580 on IL-1β–induced collagen degradation. Similar to the effect of MPA, SB203580 (10 μM) significantly inhibited the collagen degradation by corneal fibroblasts induced by IL-1β (Fig. 7). We also examined the effect of SB203580 on MMP expression and activity in culture supernatants of corneal fibroblasts incubated in the presence of IL-1β. Immunoblot analysis showed that SB203580 inhibited the IL-1β–induced increase in the amounts of both the active and pro forms of MMP-1 and MMP-3 (Fig. 8A). Gelatin zymography also revealed that the abundance of the active forms of MMP-9 and MMP-2 was decreased by SB203580 (Fig. 8A). Finally, we examined the effect of SB203580 on the phosphorylation of p38 MAPK, ERK, and JNK in corneal fibroblasts stimulated with IL-1β. Cells were exposed to SB203580 for 1 hour and then incubated in the additional presence of IL-1β for 30 minutes. Immunoblot analysis showed that the IL-1β–induced phosphorylation of ERK and JNK was potentiated by SB203580, whereas that of p38 MAPK was not affected (Fig. 8B). 
Figure 7. 
 
Inhibition by MPA and SB203580 of IL-1β–induced collagen degradation by corneal fibroblasts. Cells were cultured in collagen gels with or without IL-1β (0.1 ng/mL) and in the absence or presence of MPA (1 μM) and SB203580 (10 μM) for 36 hours, after which the amount of degraded collagen was determined. Data are means ± SEM of quadruplicates from an experiment that was repeated three times with similar results. *P < 0.05 (Dunnett's test) versus the value for cells incubated with IL-1β alone.
Figure 7. 
 
Inhibition by MPA and SB203580 of IL-1β–induced collagen degradation by corneal fibroblasts. Cells were cultured in collagen gels with or without IL-1β (0.1 ng/mL) and in the absence or presence of MPA (1 μM) and SB203580 (10 μM) for 36 hours, after which the amount of degraded collagen was determined. Data are means ± SEM of quadruplicates from an experiment that was repeated three times with similar results. *P < 0.05 (Dunnett's test) versus the value for cells incubated with IL-1β alone.
Figure 8. 
 
Effects of SB203580 on IL-1β–induced MMP release and MAPK phosphorylation in corneal fibroblasts. (A) Cells were cultured in collagen gels in the absence or presence of IL-1β (0.1 ng/mL) and SB203580 (10 μM) for 36 hours, after which the culture supernatants were subjected to immunoblot analysis with antibodies to MMP-1 or to MMP-3 or to gelatin zymography for analysis of MMP-2 and MMP-9. (B) Cells were incubated in the absence or presence of SB203580 (10 μM) for 1 hour and then in the additional absence or presence of IL-1β (0.1 ng/mL) for 30 minutes. Cell lysates were then prepared and subjected to immunoblot analysis with antibodies to total or phosphorylated (P-) forms of ERK, p38 MAPK, or JNK. All data are representative of three independent experiments.
Figure 8. 
 
Effects of SB203580 on IL-1β–induced MMP release and MAPK phosphorylation in corneal fibroblasts. (A) Cells were cultured in collagen gels in the absence or presence of IL-1β (0.1 ng/mL) and SB203580 (10 μM) for 36 hours, after which the culture supernatants were subjected to immunoblot analysis with antibodies to MMP-1 or to MMP-3 or to gelatin zymography for analysis of MMP-2 and MMP-9. (B) Cells were incubated in the absence or presence of SB203580 (10 μM) for 1 hour and then in the additional absence or presence of IL-1β (0.1 ng/mL) for 30 minutes. Cell lysates were then prepared and subjected to immunoblot analysis with antibodies to total or phosphorylated (P-) forms of ERK, p38 MAPK, or JNK. All data are representative of three independent experiments.
Discussion
We have shown that MPA inhibited IL-1β–induced collagen degradation by rabbit corneal fibroblasts in a concentration- and time-dependent manner. MPA also suppressed the expression and activation of MMPs in corneal fibroblasts exposed to IL-1β in a concentration-dependent manner. It had no effect on the proliferation or viability of corneal fibroblasts, although the IL-1β–induced phosphorylation of p38 MAPK in corneal fibroblasts was inhibited by MPA. These results thus suggest that MPA inhibits IL-1β–induced collagen degradation by corneal fibroblasts by preventing the upregulation of the expression and activity of MMPs. 
Progesterone was previously shown to inhibit MMP expression in endometrial cells and explant cultures of the endometrium. 26,27 MPA also inhibits MMP-1, -2, -3, and -9 expression in human uterine endometrium, endometrial stromal cells, or other cell types. 12,2830 We have now shown that MPA inhibits IL-1β–induced collagen degradation by corneal fibroblasts as well as MMP expression and activation in these cells. A similar inhibitory effect of MPA on collagen degradation mediated through inhibition of MMP expression has been demonstrated in various cell types. 30,31  
We also found that IL-1β induced the expression of TIMP-1 and TIMP-2 in corneal fibroblasts. TIMP-1 and -2 bind directly to MMP-2 and MMP-9, respectively, and thereby inhibit their activation. 32 The ratio of MMPs to TIMPs is thus thought to determine the rate of extracellular matrix degradation. 33 MPA inhibited the IL-1β–induced expression of both MMPs and TIMPs in corneal fibroblasts as well as IL-1β–induced collagen degradation by these cells. The downregulation of MMP expression by MPA thus appeared to dominate the corresponding effect on TIMP expression with regard to the extent of collagen degradation. 
The female sex hormones progesterone and estrogen regulate metabolism of the extracellular matrix by various cell types. 34,35 Receptors for estrogen and progesterone have been detected in corneal epithelial, stromal, and endothelial cells. 14,36 The physiology of the eye may be affected by changes in sex steroid homeostasis associated with aging or disease. 37 Our present observations that MPA inhibits IL-1β–induced collagen degradation by corneal fibroblasts as well as MMP expression and activation in these cells suggest that female sex hormones may maintain the collagen content of the corneal stroma under physiologic or pathologic conditions. 
Glucocorticoids are the primary physiological antiinflammatory hormones in mammals. Dexamethasone exerts potent antiinflammatory effects by repressing the expression of proinflammatory genes. 38,39 We previously showed that dexamethasone inhibits MMP expression and activation associated with IL-1β–induced collagen degradation by corneal fibroblasts. 24,25 Dexamethasone also inhibited the IL-1β–induced phosphorylation of JNK in these cells. 25 We have now shown that MPA inhibited the phosphorylation of p38 MAPK induced by IL-1β in corneal fibroblasts. Cross talk between glucocorticoid and estrogen receptors contributes to repression of proinflammatory gene expression in human osteosarcoma cells. 40 Dexamethasone and MPA may inhibit MMP expression and activation through modulation of different intracellular signaling pathways, which also may engage in cross talk, in corneal fibroblasts. 
IL-1β induces the phosphorylation of MAPKs in corneal fibroblasts. MPA inhibited the IL-1β–induced phosphorylation of p38 MAPK but not that of ERK or JNK in these cells. Moreover, the p38 MAPK inhibitor SB203580 blocked IL-1β–induced collagen degradation by corneal fibroblasts. We previously showed that dexamethasone blocks the IL-1β–induced phosphorylation of JNK, but not that of p38 MAPK, in corneal fibroblasts. 24 IL-1β also activates the nuclear factor-kappaB (NF-κB) signaling pathway in corneal fibroblasts. 16,41 Triptolide, a traditional Chinese medicine, inhibits the production of inflammatory cytokines and chemokines and exhibits antiinflammatory activity in various immune cell types. 42 Triptolide also inhibits IL-1β–induced collagen degradation by corneal fibroblasts. 19 In addition, triptolide and dexamethasone suppress the IL-1β–induced activation of NF-κB signaling in corneal fibroblasts. 43 These observations suggest that cross talk between MAPK and NF-κB signaling pathways may also contribute to IL-1β–induced collagen degradation by corneal fibroblasts. 
The p38 MAPK inhibitor SB203580 blocked IL-1β–induced collagen degradation by corneal fibroblasts without affecting the IL-1β–induced phosphorylation of p38 MAPK. Moreover, the IL-1β–induced phosphorylation of ERK and JNK was potentiated by SB203580. SB203580 inhibits the catalytic activity of p38 MAPK by binding to the adenosine triphosphate binding pocket, but it does not inhibit phosphorylation of the enzyme. 44 SB203580 was previously shown to induce the phosphorylation of JNK through activation of the MAPK kinases MKK4 and MKK7 in human lung alveolar epithelial cells and microvascular endothelial cells. 45 It also upregulated the phosphorylation of ERK in Madin–Darby canine kidney cells. 46 These results suggest that direct inhibition of the p38 MAPK signaling pathway by SB203580 is associated with activation of ERK and JNK signaling in corneal fibroblasts. We found that SB203580 and MPA inhibited IL-1β–induced collagen degradation by corneal fibroblasts to similar extents. The IL-1β–induced phosphorylation of p38 MAPK, but not that of ERK or JNK, was also inhibited by MPA in these cells. Moreover, whereas SB203580 promoted IL-1β–induced phosphorylation of ERK and JNK in corneal fibroblasts, it inhibited the expression and activation of MMPs elicited by IL-1β in these cells. The activity of p38 MAPK may thus play a dominant role in the regulation of MMP expression by IL-1β in corneal fibroblasts. 
Although dexamethasone and triptolide inhibit IL-1β–induced collagen degradation by corneal fibroblasts, 19,24 they also suppress the immune system, which may increase the risk of infection or exacerbate an existing infection. 4749 In contrast, MPA does not have an immunosuppressive action as potent as that of azathioprine or corticosteroids. 50 MPA inhibited the IL-1β–induced phosphorylation of p38 MAPK in corneal fibroblasts without affecting that of ERK or JNK. These observations suggest that MPA may be more suitable for potential clinical use to inhibit collagen degradation with minimal side effects. 
Various cytokines and chemokines contribute to the pathogenesis of corneal inflammation. IL-1β produced by resident corneal fibroblasts or infiltrated cells such as neutrophils induces collagen degradation by promoting the production of MMPs by corneal fibroblasts. 18 IL-1 receptor antagonist (IL-1ra) has been shown to have beneficial effects on corneal inflammatory conditions such as those associated with corneal transplantation, 51 ketatitis, 52 and alkali injury. 53 In the present study, we show that MPA inhibits IL-1β–induced collagen degradation by corneal fibroblasts and that this effect is associated with suppression of the expression or activation of MMP-1, MMP-2, MMP-3, and MMP-9 as well as with inhibition of p38 MAPK signaling. Our results thus suggest that MPA is a potential drug for the treatment of corneal inflammation. 
References
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Footnotes
 Disclosure: H. Zhou, None; K. Kimura, None; T. Orita, None; T. Nishida, None; K.-H. Sonoda, None
Figure 1. 
 
Concentration- and time-dependent inhibition by MPA of IL-1β–induced collagen degradation by corneal fibroblasts. (A) Cells were cultured in collagen gels with or without IL-1β (0.1 ng/mL) and in the presence of the indicated concentrations of MPA for 36 hours, after which the amount of degraded collagen was determined. Data are expressed as micrograms of hydroxyproline (HYP) per well and are means ± SEM of quadruplicates from an experiment that was repeated three times with similar results. *P < 0.05 (Dunnett's test) versus the corresponding value for cells cultured without MPA. (B) Cells were cultured for the indicated times in collagen gels in the absence or presence of IL-1β (0.1 ng/mL) and MPA (1 μM), as indicated. The amount of degraded collagen was then determined. Data are means ± SEM of quadruplicates from an experiment that was repeated three times with similar results. *P < 0.05 (Student's t-test) versus the corresponding value for cells incubated with IL-1β alone.
Figure 1. 
 
Concentration- and time-dependent inhibition by MPA of IL-1β–induced collagen degradation by corneal fibroblasts. (A) Cells were cultured in collagen gels with or without IL-1β (0.1 ng/mL) and in the presence of the indicated concentrations of MPA for 36 hours, after which the amount of degraded collagen was determined. Data are expressed as micrograms of hydroxyproline (HYP) per well and are means ± SEM of quadruplicates from an experiment that was repeated three times with similar results. *P < 0.05 (Dunnett's test) versus the corresponding value for cells cultured without MPA. (B) Cells were cultured for the indicated times in collagen gels in the absence or presence of IL-1β (0.1 ng/mL) and MPA (1 μM), as indicated. The amount of degraded collagen was then determined. Data are means ± SEM of quadruplicates from an experiment that was repeated three times with similar results. *P < 0.05 (Student's t-test) versus the corresponding value for cells incubated with IL-1β alone.
Figure 2. 
 
Effects of MPA on IL-1β–induced MMP-1, MMP-2, MMP-3, and MMP-9 release by corneal fibroblasts. Cells were cultured in collagen gels in the absence or presence of IL-1β (0.1 ng/mL) and with the indicated concentrations of MPA for 36 hours, after which the culture supernatants were subjected to immunoblot analysis with antibodies to MMP-1 or to MMP-3 (A) or to gelatin zymography for analysis of MMP-2 and MMP-9 (B). Similar results were obtained in three separate experiments.
Figure 2. 
 
Effects of MPA on IL-1β–induced MMP-1, MMP-2, MMP-3, and MMP-9 release by corneal fibroblasts. Cells were cultured in collagen gels in the absence or presence of IL-1β (0.1 ng/mL) and with the indicated concentrations of MPA for 36 hours, after which the culture supernatants were subjected to immunoblot analysis with antibodies to MMP-1 or to MMP-3 (A) or to gelatin zymography for analysis of MMP-2 and MMP-9 (B). Similar results were obtained in three separate experiments.
Figure 3. 
 
Effects of IL-1β and MPA on TIMP-1and TIMP-2 release by corneal fibroblasts. Cells were cultured in collagen gels in the absence or presence of IL-1β (0.1 ng/mL) and with the indicated concentrations of MPA for 36 hours, after which the culture supernatants were subjected to immunoblot analysis with antibodies to TIMP-1 or to TIMP-2. Similar results were obtained in three separate experiments.
Figure 3. 
 
Effects of IL-1β and MPA on TIMP-1and TIMP-2 release by corneal fibroblasts. Cells were cultured in collagen gels in the absence or presence of IL-1β (0.1 ng/mL) and with the indicated concentrations of MPA for 36 hours, after which the culture supernatants were subjected to immunoblot analysis with antibodies to TIMP-1 or to TIMP-2. Similar results were obtained in three separate experiments.
Figure 4. 
 
Lack of effect of MPA on the proliferation of corneal fibroblasts. Cells were cultured for 24 hours in MEM either in the absence (negative control) or presence of 0.1 or 1 μM MPA or with 10% FBS (positive control). Cell proliferation was evaluated by measurement of BrdU incorporation with a colorimetric assay. Data are means ± SEM of quadruplicates from an experiment that was repeated three times with similar results. *P < 0.05 vs. positive control (Dunnett's test).
Figure 4. 
 
Lack of effect of MPA on the proliferation of corneal fibroblasts. Cells were cultured for 24 hours in MEM either in the absence (negative control) or presence of 0.1 or 1 μM MPA or with 10% FBS (positive control). Cell proliferation was evaluated by measurement of BrdU incorporation with a colorimetric assay. Data are means ± SEM of quadruplicates from an experiment that was repeated three times with similar results. *P < 0.05 vs. positive control (Dunnett's test).
Figure 5. 
 
Lack of a cytotoxic effect of MPA on corneal fibroblasts. Cells were incubated for 24 hours in MEM and in the absence (negative control) or presence of 0.1 or 1 μM MPA, after which the culture supernatants were assayed for LDH activity with a colorimetric assay. The amount of LDH released from cells by a cell lysis solution was determined as a positive control. Data are means ± SEM from three independent experiments. *P < 0.05 vs. positive control (Dunnett's test).
Figure 5. 
 
Lack of a cytotoxic effect of MPA on corneal fibroblasts. Cells were incubated for 24 hours in MEM and in the absence (negative control) or presence of 0.1 or 1 μM MPA, after which the culture supernatants were assayed for LDH activity with a colorimetric assay. The amount of LDH released from cells by a cell lysis solution was determined as a positive control. Data are means ± SEM from three independent experiments. *P < 0.05 vs. positive control (Dunnett's test).
Figure 6. 
 
Effect of MPA on IL-1β–induced MAPK phosphorylation in corneal fibroblasts. (A) Cells were incubated in the absence or presence of MPA (1 μM) for 12 hours and then in the additional absence or presence of IL-1β (0.1 ng/mL) for 30 minutes. Cell lysates were then prepared and subjected to immunoblot analysis with antibodies to total or phosphorylated (P-) forms of ERK, p38 MAPK, or JNK. (B) The intensity of the phosphorylated ERK, p38 MAPK, and JNK bands in immunoblots similar to that shown in (A) was determined by densitometry and normalized by that of the corresponding total ERK, p38 MAPK, and JNK bands. Data are expressed relative to the value for cells incubated without addition and are means ± SEM from three independent experiments. *P < 0.05 (Dunnett's test).
Figure 6. 
 
Effect of MPA on IL-1β–induced MAPK phosphorylation in corneal fibroblasts. (A) Cells were incubated in the absence or presence of MPA (1 μM) for 12 hours and then in the additional absence or presence of IL-1β (0.1 ng/mL) for 30 minutes. Cell lysates were then prepared and subjected to immunoblot analysis with antibodies to total or phosphorylated (P-) forms of ERK, p38 MAPK, or JNK. (B) The intensity of the phosphorylated ERK, p38 MAPK, and JNK bands in immunoblots similar to that shown in (A) was determined by densitometry and normalized by that of the corresponding total ERK, p38 MAPK, and JNK bands. Data are expressed relative to the value for cells incubated without addition and are means ± SEM from three independent experiments. *P < 0.05 (Dunnett's test).
Figure 7. 
 
Inhibition by MPA and SB203580 of IL-1β–induced collagen degradation by corneal fibroblasts. Cells were cultured in collagen gels with or without IL-1β (0.1 ng/mL) and in the absence or presence of MPA (1 μM) and SB203580 (10 μM) for 36 hours, after which the amount of degraded collagen was determined. Data are means ± SEM of quadruplicates from an experiment that was repeated three times with similar results. *P < 0.05 (Dunnett's test) versus the value for cells incubated with IL-1β alone.
Figure 7. 
 
Inhibition by MPA and SB203580 of IL-1β–induced collagen degradation by corneal fibroblasts. Cells were cultured in collagen gels with or without IL-1β (0.1 ng/mL) and in the absence or presence of MPA (1 μM) and SB203580 (10 μM) for 36 hours, after which the amount of degraded collagen was determined. Data are means ± SEM of quadruplicates from an experiment that was repeated three times with similar results. *P < 0.05 (Dunnett's test) versus the value for cells incubated with IL-1β alone.
Figure 8. 
 
Effects of SB203580 on IL-1β–induced MMP release and MAPK phosphorylation in corneal fibroblasts. (A) Cells were cultured in collagen gels in the absence or presence of IL-1β (0.1 ng/mL) and SB203580 (10 μM) for 36 hours, after which the culture supernatants were subjected to immunoblot analysis with antibodies to MMP-1 or to MMP-3 or to gelatin zymography for analysis of MMP-2 and MMP-9. (B) Cells were incubated in the absence or presence of SB203580 (10 μM) for 1 hour and then in the additional absence or presence of IL-1β (0.1 ng/mL) for 30 minutes. Cell lysates were then prepared and subjected to immunoblot analysis with antibodies to total or phosphorylated (P-) forms of ERK, p38 MAPK, or JNK. All data are representative of three independent experiments.
Figure 8. 
 
Effects of SB203580 on IL-1β–induced MMP release and MAPK phosphorylation in corneal fibroblasts. (A) Cells were cultured in collagen gels in the absence or presence of IL-1β (0.1 ng/mL) and SB203580 (10 μM) for 36 hours, after which the culture supernatants were subjected to immunoblot analysis with antibodies to MMP-1 or to MMP-3 or to gelatin zymography for analysis of MMP-2 and MMP-9. (B) Cells were incubated in the absence or presence of SB203580 (10 μM) for 1 hour and then in the additional absence or presence of IL-1β (0.1 ng/mL) for 30 minutes. Cell lysates were then prepared and subjected to immunoblot analysis with antibodies to total or phosphorylated (P-) forms of ERK, p38 MAPK, or JNK. All data are representative of three independent experiments.
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