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(Investigative Ophthalmology and Visual Science. 2004;45:2998-3004.)
© 2004 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.04-0051
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Dexamethasone Inhibition of IL-1–Induced Collagen Degradation by Corneal Fibroblasts in Three-Dimensional Culture

Ying Lu,1 Ken Fukuda,2 Yang Liu,1 Naoki Kumagai,1 and Teruo Nishida1

1From the Departments of Biomolecular Recognition and Ophthalmology and 2Ocular Pathophysiology, Yamaguchi University School of Medicine, Yamaguchi, Japan.


   Abstract
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 Abstract
Methods
 Results
 Discussion
 References
 
PURPOSE. Corticosteroids regulate the functions of inflammatory cells. The purpose of the present study was to investigate the effect of dexamethasone on collagen degradation by corneal fibroblasts, an underlying cause of corneal ulceration.

METHODS. Rabbit corneal fibroblasts were cultured in three-dimensional gels of type I collagen and in the absence or presence of IL-1ß or dexamethasone. The extent of collagen degradation was determined by measurement of the amount of hydroxyproline generated by acid-heat hydrolysis of culture supernatants. The expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) was evaluated by immunoblot analysis, gelatin zymography, and reverse transcription and real-time polymerase chain reaction. The phosphorylation of mitogen-activated protein kinases (MAPKs) in corneal fibroblasts was assessed by immunoblot analysis.

RESULTS. Dexamethasone inhibited IL-1ß–induced collagen degradation by corneal fibroblasts in a dose-dependent manner. Both the synthesis and activation of MMPs and the expression of TIMPs were inhibited by dexamethasone, as was the activity of plasmin in culture supernatants. Dexamethasone also inhibited the IL-1ß–induced phosphorylation of the MAPKs extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), but not that of p38.

CONCLUSIONS. Dexamethasone exerted multiple effects on the MMP-TIMP system in corneal fibroblasts and thereby inhibited IL-1ß–induced collagen degradation by these cells. Inhibition of the IL-1ß–induced activation of ERK and JNK may contribute to these effects of dexamethasone.

The cornea is a transparent and avascular tissue, most of which is composed of the stroma. The corneal stroma in turn consists both of keratocytes and extracellular matrix (ECM), which is composed mostly of type I collagen. Corneal ulceration results from the destruction of collagen fibrils in the stroma, and the concentration of IL-1 in tear fluid is increased in individuals with corneal ulcer.1 Inhibition of IL-1 by an IL-1 receptor antagonist reduces the severity of mouse bacterial keratitis,2 indicating the importance of this cytokine in the pathogenesis of the melting of the corneal stroma. We have shown that culture of corneal fibroblasts in a three-dimensional gel of type I collagen induces the cells to elongate and to adopt a spindle-shaped morphology, with long processes that extend toward and form gap junctions with neighboring cells,3 similar to the arrangement of these cells apparent in vivo. Corneal fibroblasts both synthesize4 and degrade collagen fibrils, the latter action being achieved by the release of matrix-degrading enzymes such as matrix metalloproteinases (MMPs).5 We also have established an assay system with which to measure the collagenolytic activity of corneal fibroblasts in three-dimensional culture. With this system, we showed that IL-1, Pseudomonas aeruginosa elastase, and neutrophils each stimulate collagen degradation by corneal fibroblasts through different pathways.6 7 8 Interactions among inflammatory cells, resident cells, and the ECM thus probably play important roles in corneal ulceration.

Corticosteroids, such as dexamethasone and prednisone, regulate the functions of the immune system and are commonly used in the treatment of a wide variety of immune and inflammatory diseases. Some of the actions of these drugs appear to be mediated by direct effects on the traffic and functions of specific cell types involved in inflammatory responses, including neutrophils,9 macrophages,10 lymphocytes,11 and monocytes,12 whereas other actions seem attributable to more generalized effects on blood vessels, epithelial and endothelial regeneration, and fibroblast activity. Steroid administration is effective in the treatment of some cases of corneal ulceration. However, steroids also have adverse effects, including systemic complications such as osteoporosis13 as well as ocular complications such as recurrence of infection, increased intraocular pressure, and the development of posterior subcapsular cataract.14 In vitro studies have revealed direct effects of dexamethasone on proliferation, apoptosis,15 phagocytic activity,16 and collagenase expression17 in corneal fibroblasts.

With the use of our model culture and assay systems, we have now investigated whether dexamethasone inhibits collagen degradation mediated by corneal fibroblasts in response to IL-1. Specifically, we investigated the possible effects of dexamethasone on the expression of MMPs and tissue inhibitors of metalloproteinases (TIMPs) in corneal fibroblasts cultured in three-dimensional collagen gels and on the activation of mitogen-activated protein kinases (MAPKs) in these cells in response to IL-1.


   Methods
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 Abstract
 Methods
Results
 Discussion
 References
 
Materials
Eagle’s minimum essential medium (MEM), fetal bovine serum (FBS), and trypsin-EDTA were obtained from Invitrogen-Gibco (Carlsbad, CA), 24- and 96-well culture plates and 60-mm cell culture dishes from Corning (Corning, NY), and native porcine type I collagen (acid solubilized) and 5x Dulbecco’s modified Eagle’s medium (DMEM) from Nitta Gelatin (Osaka, Japan). Bovine plasminogen, collagenase, dispase, dimethyl sulfoxide (DMSO), protease inhibitor cocktail, and dexamethasone were obtained from Sigma-Aldrich (St. Louis, MO); recombinant human IL-1ß and goat antibodies to human TIMP-1 or -2 from R&D Systems (Minneapolis, MN); and antibodies to human extracellular signal-regulated kinase (ERK), phospho-ERK, p38, phospho-p38, c-Jun N-terminal kinase (JNK), and phospho-JNK from Cell Signaling (Beverly, MA). Sheep antibodies to rabbit MMP-1 and -3 were kindly provided by Hideaki Nagase. Filters (Ultrafree-MC) were obtained from Millipore (Bedford, MA); synthetic substrate S-2251 for plasmin from Chromogenix (Milan, Italy); and nitrocellulose membranes and a chemiluminescence (ECL) kit from Amersham Pharmacia Biotech (Uppsala, Sweden). Kits for RNA purification (RNeasy Mini Kit) and PCR (QuantiTect SYBR Green PCR Kit) were obtained from Qiagen (Hilden, Germany), and a reverse transcription system was from Promega (Madison, WI). All media and reagents used for cell culture were endotoxin minimized.

Cell Isolation
Male Japanese albino rabbits (body mass, 2.0–2.5 kg) were obtained from Biotec (Saga, Japan). The study protocol 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.7 In brief, the endothelial layer of the cornea was removed mechanically, and the tissue was then incubated with dispase (2 mg/mL, in MEM) for 1 hour at 37°C. After mechanical removal of the epithelial sheet, the tissue was treated with collagenase (2 mg/mL, in MEM) at 37°C until a single-cell suspension was obtained. Isolated corneal fibroblasts were maintained under a humidified atmosphere of 5% CO2 at 37°C in MEM supplemented with 10% FBS. The cells were used for experiments after four to seven passages and were harvested at subconfluence, in the actively proliferating state. Dexamethasone did not exhibit cytotoxicity at any of the concentrations examined (data not shown).

Three-Dimensional Culture
Collagen gels were prepared as described.7 In brief, corneal fibroblasts were harvested by exposure to trypsin-EDTA, collected by centrifugation, and resuspended in serum-free MEM. Acid-solubilized type I collagen (3 mg/mL), 5x DMEM, reconstitution buffer (0.05 M NaOH, 0.26 M Na2CO3, and 0.2 M HEPES [pH 7.3]) and corneal fibroblast suspension (2.2 x 106 cells/mL in MEM) were mixed on ice in the 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 under 5% CO2 at 37°C, after which 0.5 mL of serum-free MEM containing test agents or plasminogen (60 µg/mL) was overlaid and the cultures were returned to the incubator for the indicated times. Dexamethasone was dissolved and diluted in DMSO; the final DMSO concentration was 0.2% in all cultures containing the steroid and the same amount of vehicle was added to control cultures.

Assay of Collagenolytic Activity
Degraded collagen in culture supernatants was measured as previously described.7 8 In brief, the supernatants from collagen gel incubations were collected, and native collagen fibrils with a molecular size of more than 100 kDa were removed by ultrafiltration. The filtrate was then subjected to hydrolysis with 6 M HCl for 24 hours at 110°C. The amount of hydroxyproline in the hydrolysate was measured spectrophotometrically, and the amount of degraded collagen was expressed as micrograms of hydroxyproline per well.

Immunoblot Analysis
Immunoblot analysis of rabbit MMP-1, MMP-3, TIMP-1, and TIMP-2 was performed as described.7 Culture supernatants were subjected to SDS-polyacrylamide gel electrophoresis on a 10% gel under reducing conditions, and the separated proteins were then transferred electrophoretically to a nitrocellulose membrane. After blocking of nonspecific sites, the membrane was incubated with antibodies to MMP-1, to MMP-3, to TIMP-1, or to TIMP-2 and immune complexes were then detected with the use of secondary antibodies and enhanced chemiluminescence reagents. For immunoblot analysis of MAPKs, corneal fibroblasts (5 x 105 cells) were cultured in 60-mm dishes for 24 hours in MEM supplemented with 0.5% FBS and then for an additional 24 hours in serum-free medium. They were then treated with the indicated concentrations of dexamethasone for 6 hours before exposure to IL-1ß (0.1 ng/mL) for 30 minutes at 37°C. The cells were lysed in 100 µL of a solution containing 1% Nonidet P-40, 50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 1% protease inhibitor cocktail. Cell lysates (10 µg protein) were then subjected to immunoblot analysis, as described earlier, with antibodies to extracellular signal–regulated kinase (ERK), to p38, to c-Jun NH2-terminal kinase (JNK), or to phosphorylated forms of these MAPKs.

Gelatin Zymography
Gelatin zymography of culture supernatants was performed as described previously.7 In brief, culture supernatants (4 µL) were mixed with 2 µL of nonreducing SDS sample buffer (125 mM Tris-HCl [pH 6.8], 20% glycerol, 2% SDS, and 0.002% bromophenol blue) and fractionated by SDS-polyacrylamide gel electrophoresis 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, to promote recovery of protease activity, before incubation for 18 hours at 37°C in a reaction buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM CaCl2, and 1% Triton X-100. The gel was then stained with Coomassie brilliant blue.

Reverse Transcription and Quantitative Real-Time PCR Analysis
After culture for 12 hours, corneal fibroblasts were extracted from collagen gels by incubation with 0.01% collagenase for 30 minutes at 37°C. Total RNA was then isolated from the cells and subjected to reverse transcription. The abundance of MMP-1, -2, -3, and -9 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNAs was quantified by real-time polymerase chain reaction (PCR) with a thermocycler (LightCycler; Roche Molecular Biochemicals, Indianapolis, IN), as described previously.8 The sequences of the PCR primers for MMP-1, -2, -3, and -9 and GAPDH cDNAs were also as described previously;18 these primers yielded PCR products of the expected sizes of 649, 313, 306, 271, and 293 bp, respectively.

Measurement of Plasmin Activity
Plasmin activity was measured with the substrate S-2251, as described previously.6 In brief, culture supernatants (100 µL) were incubated at 37°C in the wells of a 96-well plate with 20 µL of 50 mM Tris-HCl (pH 7.4) containing 0.01% Triton X-100 and with 100 µL of 0.6 mM S-2251. The release of p-nitroanilide during 30 minutes was monitored by measurement of absorbance at 405 nm with a microplate reader.

Statistical Analysis
Data are expressed as the mean ± SEM. Statistical analysis was performed with the Dunnett multiple comparison test or Student’s unpaired t-test. P <0.05 was considered statistically significant.


   Results
Top
 Abstract
 Methods
 Results
Discussion
 References
 
Inhibitory Effect of Dexamethasone on Collagen Degradation by Rabbit Corneal Fibroblasts
We first examined the effect of dexamethasone on collagen degradation by corneal fibroblasts. The cells were incubated in collagen gels for 48 hours in the presence of plasminogen, in the absence or presence of IL-1ß (0.1 ng/mL), and in the presence of various concentrations of dexamethasone (0.01 to 1.0 nM). Consistent with our previous observations,6 IL-1ß markedly increased the extent of collagen degradation by corneal fibroblasts. Dexamethasone inhibited in a dose-dependent manner the collagen degradation by corneal fibroblasts apparent in the absence or presence of IL-1ß, with IL-1ß–induced collagen degradation being almost completely inhibited at a dexamethasone concentration of 1 nM (Fig. 1) .



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  		FIGURE 1. Dose-dependent inhibition by dexamethasone of collagen degradation by corneal fibroblasts. Cells were cultured in collagen gels for 48 hours in the presence of plasminogen, in the absence ({circ}) or presence (•) of IL-1ß (0.1 ng/mL), and in the presence of the indicated concentrations of dexamethasone, after which the amount of degraded collagen was determined. Data are expressed as micrograms of hydroxyproline (HYP) per well and are the mean ± SEM of triplicates from an experiment that was repeated three times with similar results. *P < 0.001 (Dunnett test) versus the corresponding value for cells cultured without dexamethasone.

 
Effects of Dexamethasone on the Expression of MMPs and TIMPs by Corneal Fibroblasts
To investigate the mechanism by which dexamethasone inhibits collagen degradation by corneal fibroblasts, we first examined its effects on the expression of MMPs in these cells by immunoblot analysis and gelatin zymography. Corneal fibroblasts were again cultured in collagen gels for 48 hours in the presence of plasminogen, in the absence or presence of IL-1ß (0.1 ng/mL), and in the presence of various concentrations of dexamethasone. Immunoblot analysis with antibodies to rabbit MMP-1 revealed that the culture supernatant of cells incubated without IL-1ß and dexamethasone contained relatively small amounts of 61- and 57-kDa immunoreactive proteins corresponding to proMMP-1 as well as of 49- and 45-kDa immunoreactive proteins corresponding to active MMP-1 (Fig. 2) . Culture of cells in the presence of IL-1ß resulted in an increase in the intensity of the bands corresponding to proMMP-1 and active MMP-1. In the absence or presence of IL-1ß, dexamethasone induced a dose-dependent decrease in the abundance of active MMP-1 and an increase in that of proMMP-1.



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  		FIGURE 2. Effects of dexamethasone on the expression of MMPs and TIMPs by corneal fibroblasts. Cells were cultured in collagen gels for 48 hours in the presence of plasminogen, in the absence or presence of IL-1ß (0.1 ng/mL), and in the presence of the indicated concentrations of dexamethasone (Dex). The culture supernatants were then subjected either to immunoblot analysis with antibodies to MMP-1 (top panel), MMP-3 (second panel), TIMP-1 (fourth panel), or TIMP-2 (bottom panel) or were subjected to gelatin zymography (third panel). Data are representative of three independent experiments. The positions of MMPs and TIMPs are indicated on the right and the molecular size standards are shown on the left.

 
Immunoblot analysis with antibodies to rabbit MMP-3 did not detect either proMMP-3 or active MMP-3 in the culture supernatants of cells incubated in the absence of IL-1ß (Fig. 2) . In the presence of IL-1ß, such analysis revealed 57- and 45-kDa immunoreactive proteins corresponding to pro and active forms of MMP-3, respectively. The addition of 0.01 nM dexamethasone to IL-1ß–treated cells induced a decrease in the abundance of active MMP-3 and an increase in the abundance of proMMP-3 in the culture supernatant. Neither the pro nor the active form of MMP-3 was detected in the culture supernatant of IL-1ß–treated cells incubated in the presence of 1.0 nM dexamethasone.

Gelatin zymography of culture supernatants obtained after incubation of corneal fibroblasts for 48 hours without IL-1ß and dexamethasone revealed three major bands of 89, 65, and 57 kDa, corresponding to an intermediate form of MMP-9, proMMP-2, and active MMP-2, respectively (Fig. 2) . Dexamethasone induced a dose-dependent decrease in the amounts of the intermediate form of MMP-9 and of active MMP-2. Culture of cells in the presence of IL-1ß resulted in an increase in the intensity of the bands corresponding to proMMP-2 and active MMP-2, the disappearance of the band corresponding to the intermediate form of MMP-9, and the appearance of bands at 92 and 77 kDa corresponding to proMMP-9 and active MMP-9, respectively. Dexamethasone inhibited the effect of IL-1ß on the gelatinolytic band corresponding to active MMP-2. At a dexamethasone concentration of 0.1 nM, the proMMP-9 band was no longer apparent, and the intermediate form of MMP-9 reappeared. At a steroid concentration of 1.0 nM, neither pro nor active MMP-9 was detected, and the intensity of the band corresponding to the intermediate form was increased.

We also examined the effects of dexamethasone on the expression of TIMPs in corneal fibroblasts. Immunoblot analysis with antibodies to TIMP-1 revealed that the culture supernatant of cells maintained in collagen gels for 48 hours in the absence of IL-1ß and dexamethasone contained a 28-kDa immunoreactive protein corresponding to TIMP-1 (Fig. 2) . Culture of the cells with IL-1ß (0.1 ng/mL) did not affect the intensity of the TIMP-1 band, whereas dexamethasone induced a dose-dependent decrease in the abundance of TIMP-1 in the culture supernatants of cells incubated in the absence or presence of IL-1ß.

Immunoblot analysis with antibodies to TIMP-2 revealed that the culture supernatant of cells incubated in the absence of IL-1ß and dexamethasone contained a 21-kDa immunoreactive protein corresponding to TIMP-2 (Fig. 2) . Neither IL-1ß nor dexamethasone alone affected the intensity of the TIMP-2 band. In the presence of IL-1ß, however, dexamethasone induced a dose-dependent decrease in the abundance of TIMP-2.

Inhibitory Effect of Dexamethasone on MMP Synthesis by Corneal Fibroblasts
To investigate whether dexamethasone inhibits the synthesis of MMPs by corneal fibroblasts, we cultured the cells in collagen gels for 48 hours in the absence of plasminogen, in the absence or presence of IL-1ß (0.1 ng/mL), and in the presence of various concentrations of dexamethasone and then subjected the culture supernatants to immunoblot analysis. Such analysis with antibodies to MMP-1 revealed that the culture supernatant of cells maintained without IL-1ß and dexamethasone contained a relatively small amount of proMMP-1 (Fig. 3A) . Culture of cells in the presence of IL-1ß resulted in an increase in the intensity of the bands corresponding to proMMP-1. In the absence or presence of IL-1ß, dexamethasone induced a dose-dependent decrease in the abundance of proMMP-1 in culture supernatants.



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  		FIGURE 3. Effects of dexamethasone on the synthesis of MMP-1 and -3 by corneal fibroblasts. (A) Cells were cultured in collagen gels for 48 hours in the absence of plasminogen, in the absence or presence of IL-1ß (0.1 ng/mL), and in the presence of the indicated concentrations of dexamethasone. The culture supernatants were then subjected to immunoblot analysis with antibodies to either MMP-1 (top panel) or MMP-3 (bottom panel). Data are representative of three independent experiments. (B) Cells were cultured in collagen gels for 12 hours in the absence of plasminogen and in the absence or presence of IL-1ß (0.1 ng/mL) or dexamethasone (1.0 nM), as indicated. The amounts of MMP-1 mRNA (left) and MMP-3 mRNA (right) in the cells were then determined by reverse transcription and real-time PCR. Data are normalized on the basis of the abundance of GAPDH mRNA, are expressed in arbitrary units, and are the mean ± SEM of results in three experiments. *P < 0.001 (Student’s t-test) versus the corresponding value for cells cultured without IL-1ß; {dagger}P < 0.001 (Student’s t-test) versus the corresponding value for cells cultured without dexamethasone.

 
Immunoblot analysis with antibodies to MMP-3 revealed that proMMP-3 was not detectable in the culture supernatant of cells incubated in the absence of IL-1ß and dexamethasone (Fig. 3A) . ProMMP-3 was detected, however, in the culture supernatant of cells incubated in the presence of IL-1ß, and dexamethasone inhibited this effect of IL-1ß in a dose-dependent manner.

Reverse transcription and real-time PCR revealed that culture of corneal fibroblasts in collagen gels for 12 hours with IL-1ß (0.1 ng/mL) resulted in an 11.4-fold increase in the amount of MMP-1 mRNA compared with that present in cells cultured in the absence of this cytokine (Fig. 3B) . Dexamethasone (1.0 nM) had no significant effect on the basal abundance of MMP-1 mRNA but significantly inhibited the effect of IL-1ß on the amount of MMP-1 mRNA by ~80%. Similarly, IL-1ß induced an 8.4-fold increase in the amount of MMP-3 mRNA in corneal fibroblasts, and dexamethasone inhibited this effect of IL-1ß by ~80%.

Gelatin zymography of culture supernatants obtained after incubation of corneal fibroblasts in collagen gels for 48 hours in the absence of plasminogen, IL-1ß, and dexamethasone revealed the presence of proMMP-9, proMMP-2, and active MMP-2 (Fig. 4A) . Culture of cells in the presence of IL-1ß (0.1 ng/mL) resulted in an increase in the intensity of the bands corresponding to proMMP-9, proMMP-2, and active MMP-2. Dexamethasone reduced the amount of proMMP-9 in the absence or presence of IL-1ß, but it had no effect on the abundance of proMMP-2 or active MMP-2.



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  		FIGURE 4. Effects of dexamethasone on the synthesis of MMP-2 and -9 by corneal fibroblasts. (A) Cells were cultured in collagen gels for 48 hours in the absence of plasminogen, in the absence or presence of IL-1ß (0.1 ng/mL), and in the presence of the indicated concentrations of dexamethasone. The culture supernatants were then subjected to gelatin zymography. Data are representative of three independent experiments. (B) Cells were cultured in collagen gels for 12 hours in the absence of plasminogen and in the absence or presence of IL-1ß (0.1 ng/mL) or dexamethasone (1.0 nM), as indicated. The amounts of MMP-2 mRNA (left) and MMP-9 mRNA (right) in the cells were then determined by reverse transcription and real-time PCR. Data are normalized on the basis of the abundance of GAPDH mRNA, are expressed in arbitrary units, and are the mean ± SEM of results of three experiments. *P < 0.001 (Student’s t-test) versus the corresponding value for cells cultured without IL-1ß; {dagger}P < 0.001 (Student’s t-test) versus the corresponding value for cells cultured without dexamethasone.

 
Reverse transcription and real-time PCR showed that neither IL-1ß (0.1 ng/mL) nor dexamethasone (1.0 nM) had a significant effect on the abundance of MMP-2 mRNA in corneal fibroblasts cultured in collagen gels for 12 hours (Fig. 4B) . IL-1ß induced a 5.2-fold increase in the abundance of MMP-9 mRNA, and dexamethasone significantly inhibited this effect of IL-1ß (~70%).

We also examined whether dexamethasone exerts a direct effect on MMP activity. Dexamethasone (0.01–1.0 nM) had no effect on the activity of recombinant human MMP-1 (data not shown). Similarly, the MMP-1 activity present in culture supernatants of corneal fibroblasts incubated for 48 hours in collagen gels with plasminogen and IL-1ß (0.1 ng/mL) was not inhibited by dexamethasone. These results thus demonstrate that dexamethasone does not inhibit MMP-1 activity directly.

Inhibitory Effect of Dexamethasone on Plasmin Activity in Culture Supernatants
To investigate whether dexamethasone inhibits the activation of MMPs by plasmin, we examined the possible effect of culture of corneal fibroblasts with this steroid on the activity of plasmin present in culture supernatants. Earlier, we have shown that the addition of plasminogen is important for collagen degradation by corneal fibroblasts in our culture system.6 Plasminogen activator mediates the conversion of plasminogen to plasmin, which then activates latent MMPs. The plasmin activity in culture supernatants of cells incubated in the absence or presence of IL-1ß was inhibited by dexamethasone in a dose-dependent manner (Fig. 5) , whereas dexamethasone did not inhibit plasmin activity directly (data not shown).



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  		FIGURE 5. Inhibitory effect of dexamethasone on the activity of plasmin in culture supernatants of corneal fibroblasts. Cells were cultured in collagen gels for 48 hours in the presence of plasminogen, in the absence ({circ}) or presence (•) of IL-1ß (0.1 ng/mL), and in the presence of the indicated concentrations of dexamethasone, after which the activity of plasmin in the culture supernatants was determined. Data are expressed in absorbance units and are the mean ± SEM of triplicate results of an experiment that was repeated three times with similar results. *P < 0.00,001 (Dunnett test) versus the corresponding value for cells cultured without dexamethasone.

 
Effects of Dexamethasone on MAPK Phosphorylation in Corneal Fibroblasts
The activation of the MAPKs ERK, p38, and JNK by IL-1ß peaked after stimulation of corneal fibroblasts for 20 to 30 minutes (data not shown). We therefore examined the possible effects of dexamethasone on the activation of MAPKs by exposing serum-deprived cells to various concentrations of dexamethasone for 6 hours before treatment with IL-1ß (0.1 ng/mL) for 30 minutes at 37°C. Immunoblot analysis revealed that the abundance of ERK, p38, and JNK was not affected by IL-1ß or dexamethasone (Fig. 6) . Dexamethasone inhibited the IL-1ß–induced phosphorylation (activation) of ERK and JNK but not that of p38.



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  		FIGURE 6. Effects of dexamethasone on MAPK phosphorylation in corneal fibroblasts. Cells were cultured for 24 hours in MEM supplemented with 0.5% FBS and then for an additional 24 hours in serum-free medium. They were then incubated with the indicated concentrations of dexamethasone for 6 hours before treatment with IL-1ß (0.1 ng/mL) for 30 minutes. Cell lysates were then subjected to immunoblot analysis with antibodies to ERK, p38, or JNK or to phosphorylated forms (p-) of these MAPKs. Data are representative of results in three independent experiments.

 

   Discussion
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 Abstract
 Methods
 Results
 Discussion
References
 
We have shown that dexamethasone inhibits IL-1ß–induced collagen degradation by corneal fibroblasts. Dexamethasone inhibited not only the synthesis of MMPs by these cells at both the protein and mRNA levels, but also the activation of these enzymes. It did not inhibit the enzymatic activity of MMPs directly. The expression of TIMPs was also inhibited by dexamethasone. Furthermore, this steroid inhibited the IL-1ß–induced phosphorylation of ERK and JNK, but not that of p38, in corneal fibroblasts.

The MMP family comprises at least 23 secreted or membrane-bound zinc-dependent endopeptidases that break down components of the ECM.19 MMPs are synthesized and secreted as inactive proenzymes that are activated by serine proteinases, such as plasmin, in the extracellular space. Cultured corneal fibroblasts produce MMP-1, -2, -3, and -9.20 In the present study, dexamethasone inhibited the IL-1ß–induced synthesis of MMP-1, -3, and -9 by corneal fibroblasts in three-dimensional cultures. These results are consistent with the previous observation that dexamethasone inhibits the synthesis of MMPs in corneal fibroblasts cultured on plastic.20 The plasminogen–plasmin system has been implicated in the initiation and perpetuation of collagen degradation in the cornea.21 In the present study, dexamethasone inhibited both plasmin activity and the conversion of proMMPs to MMPs in culture supernatants of corneal fibroblasts. These results suggest that dexamethasone may inhibit not only the synthesis of MMPs by corneal fibroblasts but also the activation of these enzymes and thereby inhibit collagen degradation in the cornea stroma.

An imbalance between the activities of MMPs and TIMPs has been implicated in the pathogenesis of corneal ulceration.22 The TIMP family of specific inhibitors of active MMPs comprises TIMP-1, -2, -3, and -4.19 Cultured corneal fibroblasts produce TIMP-1 and -2, which bind directly to the hemopexin domains of MMP-9 and -2, respectively.23 The IL-1–induced downregulation of TIMP-1 expression in chondrocytes was potentiated by dexamethasone.24 Dexamethasone also reduced the amounts of TIMP-1 and -2 mRNAs in gingival fibroblasts.25 In our study, dexamethasone inhibited the expression of TIMP-1, but not that of TIMP-2, in nonstimulated corneal fibroblasts. The abundance of both TIMP-1 and -2 was reduced by dexamethasone in IL-1ß–stimulated corneal fibroblasts, however. These results suggest that the expression of TIMP-1 and -2 is regulated differentially in corneal fibroblasts. The physiological or pathologic relevance of these effects of dexamethasone on TIMP expression remains to be determined. Although dexamethasone inhibited the expression of both MMPs and TIMPs in corneal fibroblasts, it inhibited collagen degradation by these cells, suggesting that the effects on MMPs may be more biologically significant than are those on TIMPs. The ratio of MMPs to TIMPs is an important factor in various biological activities, including cell migration, angiogenesis, and remodeling of the ECM.26 Dexamethasone inhibited MMP-2 secretion and increased TIMP-2 secretion in smooth muscle cells and inhibited the migration of these cells in vitro.27 The ratio of MMPs to TIMPs in corneal fibroblasts may also be an important determinant of the balance between collagen degradation and collagen synthesis. The organization and movement of cell–matrix adhesion sites have been shown recently to correlate with force generation by corneal fibroblasts cultured on a fibrillar collagen matrix.28 The effects of dexamethasone on the mobility and migration of corneal fibroblasts remain to be determined.

The MAPK cascade is a pivotal intracellular signaling module activated by cytokine receptors. Our results revealed that IL-1ß induced the activation of the MAPKs ERK, p38, and JNK in corneal fibroblasts, consistent with previous observations with other types of fibroblasts such as chondrocytes.29 IL-1 induces activation of the MMP-1 gene promoter in corneal fibroblasts and this effect is mediated by the transcription factors nuclear factor–{kappa}B and activator protein (AP)–1).17 ERK activity is required for AP-1 activation.30 Furthermore, JNK initiates MMP gene transcription by phosphorylating and increasing the transactivation potential of the AP-1 subunits c-Jun and ATF2,31 although c-Jun is not a substrate of p38.32 In our study, dexamethasone inhibited the IL-1ß–induced phosphorylation of ERK and JNK, but not that of p38. Dexamethasone also inhibits MAPK activation in chondrocytes.33 The glucocorticoid receptor has been detected in corneal fibroblasts.15 These various observations thus provide insight into the complex mechanisms that underlie the inhibition by dexamethasone of the collagenolytic activity of corneal fibroblasts. They are consistent with a central role for MAPKs (especially ERK and JNK) as glucocorticoid-sensitive mediators of IL-1 actions that are dependent on AP-1 or other transcription factors.

Topical application of corticosteroids is widely used for the treatment of ocular inflammation. Corticosteroids modulate various aspects of neutrophil function including adhesion, migration, phagocytosis, and the oxidative burst.9 Our results show that, in addition to its inhibitory effects on inflammatory cells, dexamethasone inhibits the collagenolytic activity of resident corneal fibroblasts. This latter action of dexamethasone appears to be attributable to multiple effects on the MMP-TIMP system, at least some of which may be mediated at the level of ERK and JNK activation.


   Footnotes
 
Supported by Grants in Aid for Scientific Research A-14207070; TN) and C-14571674; NK), a Grant in Aid for Young Scientists B-14770959; KF) from the Japan Society for the Promotion of Science, and by an unrestricted grant (YLu) from the Japan National Society for the Prevention of Blindness.

Submitted for publication January 19, 2004; revised March 1, 2004; accepted March 9, 2004.

Disclosure: Y. Lu, None; K. Fukuda, None; Y. Liu, None; N. Kumagai, None; T. Nishida, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Teruo Nishida, Department of Biomolecular Recognition and Ophthalmology, Yamaguchi University School of Medicine, 1-1-1 Minami-Kogushi, Ube City, Yamaguchi 755-8505, Japan; tnishida{at}yamaguchi-u.ac.jp.


   References
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 Abstract
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 Discussion
 References
 

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