March 2017
Volume 58, Issue 3
Open Access
Cornea  |   March 2017
Extracellular Collagen Promotes Interleukin-1β–Induced Urokinase-Type Plasminogen Activator Production by Human Corneal Fibroblasts
Author Affiliations & Notes
  • Koji Sugioka
    Department of Ophthalmology, Kindai University Faculty of Medicine, Osaka-sayama City, Osaka, Japan
  • Aya Kodama-Takahashi
    Department of Ophthalmology, Kindai University Faculty of Medicine, Osaka-sayama City, Osaka, Japan
  • Koji Yoshida
    Department of Biomedical Engineering, Faculty of Biology-Oriented Science and Technology, Kindai University, Kinokawa City, Wakayama, Japan
  • Keiichi Aomatsu
    Department of Ophthalmology, Kindai University Faculty of Medicine, Osaka-sayama City, Osaka, Japan
  • Kiyotaka Okada
    Division of Basic Medical Science, Kindai University Faculty of Medicine, Osaka-sayama City, Osaka, Japan
  • Teruo Nishida
    Department of Ophthalmology, Yamaguchi University Graduate School of Medicine, Ube City, Yamaguchi, Japan
  • Yoshikazu Shimomura
    Department of Ophthalmology, Kindai University Faculty of Medicine, Osaka-sayama City, Osaka, Japan
  • Correspondence: Koji Sugioka, Department of Ophthalmology, Kindai University Faculty of Medicine, 377-2 Ohno-higashi, Osaka-sayama City, Osaka 589-8511, Japan; sugioka@med.kindai.ac.jp
Investigative Ophthalmology & Visual Science March 2017, Vol.58, 1487-1498. doi:10.1167/iovs.16-20685
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      Koji Sugioka, Aya Kodama-Takahashi, Koji Yoshida, Keiichi Aomatsu, Kiyotaka Okada, Teruo Nishida, Yoshikazu Shimomura; Extracellular Collagen Promotes Interleukin-1β–Induced Urokinase-Type Plasminogen Activator Production by Human Corneal Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2017;58(3):1487-1498. doi: 10.1167/iovs.16-20685.

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

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Abstract

Purpose: Keratocytes maintain homeostasis of the corneal stroma through synthesis, secretion, and degradation of collagen fibrils of the extracellular matrix. Given that these cells are essentially embedded in a collagen matrix, keratocyte–collagen interactions may play a key role in regulation of the expression or activation of enzymes responsible for matrix degradation including urokinase-type plasminogen activator (uPA), plasmin, and matrix metalloproteinases (MMPs). We examined the effect of extracellular collagen on the production of uPA by corneal fibroblasts (activated keratocytes) stimulated with the proinflammatory cytokine interleukin-1β (IL-1β).

Methods: Human corneal fibroblasts were cultured either on plastic or in a three-dimensional gel of type I collagen. Plasminogen activators were detected by fibrin zymography, whereas the IL-1 receptor (IL-1R) and MMPs were detected by immunoblot analysis. Collagen degradation by corneal fibroblasts was assessed by measurement of hydroxyproline in acid hydrolysates of culture supernatants.

Results: Collagen and IL-1β synergistically increased the synthesis and secretion of uPA in corneal fibroblasts. Collagen also upregulated IL-1R expression in the cells in a concentration-dependent manner. The conversion of extracellular plasminogen to plasmin, as well as the plasminogen-dependent activation of MMP-1 and MMP-3 and degradation of collagen apparent in three-dimensional cultures of corneal fibroblasts exposed to IL-1β, were all abolished by a selective uPA inhibitor.

Conclusions: Collagen and IL-1β cooperate to upregulate uPA production by corneal fibroblasts. Furthermore, IL-1β–induced collagen degradation by these cells appears to be strictly dependent on uPA expression and mediated by a uPA–plasmin–MMP pathway.

The corneal stroma, which is largely responsible for the rigidity and transparency of the cornea, consists of keratocytes and an extracellular matrix (ECM) composed mostly of collagen type I and proteoglycans. In the healthy cornea, keratocytes form a three-dimensional network of cells scattered throughout the densely packed collagen fibrils.1 Although the total volume of keratocytes is only ∼2% to 3% of the overall volume of the stroma,2 keratocytes maintain homeostasis of the collagen matrix through synthesis, secretion, and degradation of the collagen fibrils. Keratocytes are usually quiescent cells with a slow turnover rate. Under pathologic conditions such as inflammation, infection, or injury, however, keratocytes differentiate into activated corneal fibroblasts or myofibroblasts, which contribute to corneal wound healing. The activated cells migrate to the wound edge and mediate tissue remodeling, with excessive collagen degradation by these cells sometimes leading to corneal ulceration.3 Under normal conditions and during wound healing, extracellular collagen not only maintains corneal stromal morphology but is also thought to regulate the structure and function of keratocytes. The shape of the corneal stromal cells in vitro has thus been found to be affected by the presence of an extracellular collagen matrix,4 whereas collagen has been shown to regulate cell movement and migration, cytoskeletal organization, and cell proliferation in other cell types.57 
A proteolytic cascade including urokinase-type plasminogen activator (uPA), plasmin, and matrix metalloproteinases (MMPs) plays a key role in the degradation of collagen fibrils.8,9 Cultured corneal fibroblasts synthesize and secrete uPA,10 which catalyzes the proteolytic conversion of plasminogen (inactive) to plasmin (active). Plasmin then activates MMPs that are responsible for collagen degradation.11,12 
Proinflammatory cytokines such as interleukin-1β (IL-1β), produced in association with tissue inflammation, infection, or injury, have been shown to promote the degradation of collagen by corneal fibroblasts.13,14 The concentration of IL-1β in tear fluid has also been found to be increased in patients with corneal ulceration, implicating this cytokine in the regulation of corneal stromal wound healing.15 Furthermore, IL-1β upregulates MMP expression in corneal fibroblasts in vitro,13 and it has been shown to regulate uPA expression in various cell types.1619 It thus induces transcriptional activation of the uPA gene and consequent accumulation of uPA mRNA and newly synthesized uPA protein in A549 human pulmonary epithelial cells.17 We have previously shown that keratocytes and leukocytes migrating to the edge of a corneal ulcer express uPA,20 and that uPA promotes leukocyte infiltration during corneal inflammation and is a key player in the inflammatory response in the cornea.21 
Collagen itself has been found to modulate the expression of genes related to collagen degradation in various cell types.2224 To provide insight into the role of cell-matrix interaction in collagen homeostasis in the corneal stroma during inflammation, we examined the effects of type I collagen and IL-1β on uPA expression, plasmin formation, as well as MMP-1 and MMP-3 production in three-dimensional cultures of human corneal fibroblasts embedded in a collagen gel. 
Methods
Cell Isolation and Culture
Human corneal fibroblasts were isolated from the corneoscleral rim of corneas obtained for corneal transplantation surgery from The Eye-Bank for Sight Restoration (New York, NY, USA). The human tissue was used in accordance with the tenets of the Declaration of Helsinki. The sclera, limbal region, and endothelial layer of each specimen were removed mechanically, and the remaining tissue was immersed in dispase (2 mg/mL in minimum essential medium [MEM]) (Sigma-Aldrich Corp., St. Louis, MO, USA) for 1 hour at 37°C. After removal of the epithelial sheet, the tissue was treated with collagenase A of Clostridium histolyticum (2 mg/mL in MEM) (Sigma-Aldrich Corp.) for 5 hours at 37°C to obtain a single-cell suspension. The isolated cells were cultured under 5% CO2 in air at 37°C in MEM supplemented with 10% fetal bovine serum (Gibco-BRL, Grand Island, NY, USA). The cells were harvested for experiments after four to six passages. 
Two-Dimensional Culture of Corneal Fibroblasts on Plates Coated With Fibronectin, Laminin, or Type I Collagen
Plastic culture plates were coated with fibronectin (Sigma-Aldrich Corp.), laminin (Sigma-Aldrich Corp.), or collagen type I (Nitta Gelatin, Osaka, Japan) as previously described.25 In brief, fibronectin, laminin, or collagen type I, each at 10 μg/mL (unless indicated otherwise) in phosphate-buffered saline (PBS), was added to culture plates and incubated for 1 hour at 37°C, after which the plates were washed with PBS. The plates were then similarly incubated with 1% bovine serum albumin (BSA) to prevent nonspecific adhesion of cells. The treated plates were washed with PBS three times before experiments. Plates coated with 1% BSA alone served as a control. 
Three-Dimensional Culture of Corneal Fibroblasts in a Collagen or Laminin Gel Matrix
Culture of corneal fibroblasts in a collagen gel was performed as previously described.4 In brief, type I collagen (Nitta Gelatin) was mixed with 10× MEM and neutralized with 0.2 M NaOH before the addition of corneal fibroblasts to a final density of 1 × 105 cells/mL and final collagen concentration of 2 mg/mL (unless indicated otherwise). A portion (300 μL) of the cell suspension was transferred to the wells of a 24-well tissue culture plate, which was then incubated at 37°C for 1 hour to allow gel formation. Laminin gel culture was performed with the use of Cultex 3-D Culture Matrix Laminin I (Trevigen, Cambridge, MA, USA). Minimum essential medium (300 μL) with various additions including recombinant human IL-1β (R&D Systems, Minneapolis, MN, USA), α2-antiplasmin, human plasminogen (Hyphen BioMed, Paris, France), and the selective uPA inhibitor uPA-STOP (N-a-[2,4,6-triisopropyl-phenylsulfonyl]-3-amidino-[L]-phenylalanine-4-ethoxycarbonyl-piperazide hydrochloride hemihydrate) (American Diagnostica, Stamford, CT, USA) was then added on top of each collagen or laminin gel. 
Fluorescence Microscopy
Cells cultured in collagen gels or on plastic (1 × 104 cells per well of a 24-well plate) for 24 hours were fixed for 10 minutes at room temperature with 3.7% formaldehyde in PBS, washed with PBS, permeabilized for 5 minutes with 0.1% Triton X-100 in PBS, and incubated at room temperature first for 30 minutes with PBS containing 1% BSA and then for 1 hour with Alexa Fluor 568–conjugated phalloidin (1:200 dilution in the same solution) (Thermo Fisher Scientific, Waltham, MA, USA) to stain F-actin. The cells were examined with a laser-scanning confocal microscope (Axiovert200M; Carl Zeiss, Tokyo, Japan). 
Sample Preparation for Fibrin Zymography and Immunoblot Analysis
Culture supernatants from cells maintained in two- or three-dimensional culture were collected for fibrin zymography and immunoblot analysis. The cells embedded in each gel or attached to culture plates were then pulverized in 200 μL extraction buffer (10 mM sodium phosphate buffer [pH 7.2], 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 0.2% NaN3), and the cell lysates were centrifuged at 17,000g for 5 minutes at 4°C. The resulting supernatants (10 μg of protein) were then also examined by fibrin zymography and immunoblot analysis. 
Fibrin Zymography
The production of uPA, as well as the conversion of plasminogen to plasmin in corneal fibroblast cultures, was examined by fibrin zymography as previously described.26 In brief, samples and molecular markers were subjected to electrophoresis on a 10% polyacrylamide gel containing bovine fibrinogen (0.55 mg/mL) (Sigma-Aldrich Corp.) and thrombin (0.056 NIH U/mL) (Sigma-Aldrich Corp.). The gel was washed with 2.5% Triton X-100 for 1 hour, incubated for 36 hours at 37°C in a reaction buffer containing 0.5 M glycine-HCl (pH 8.4), stained for 1 hour with Coomassie Blue R-250, and then destained with a solution comprising 30% methanol and 10% acetic acid. The intensity of bands corresponding to uPA was measured with the use of a LAS-1000 system (Fuji Film, Tokyo, Japan) calibrated with human standard uPA (0.1 IU). 
Immunoblot Analysis
Samples were subjected to SDS–polyacrylamide gel electrophoresis on 8% to 16% gradient gels. The separated proteins were transferred to a polyvinylidene difluoride membrane, which was then incubated for 1 hour at room temperature with 5% dried skim milk in PBS containing 0.1% Tween 20 (PBST) before exposure overnight at 4°C to antibodies specific for MMP-1 (R&D Systems), MMP-3 (R&D Systems), the IL-1 receptor (IL-1R) (R&D Systems), phosphorylated p65 (Cell Signaling, Danvers, MA, USA), phosphorylated p38 MAPK (mitogen-activated protein kinase) (Cell Signaling), phosphorylated JNK (c-Jun NH2-terminal kinase) (Cell Signaling), or phosphorylated ERK (extracellular signal–regulated kinase) (Cell Signaling). The membrane was then washed three times in PBST before detection of immune complexes with horseradish peroxidase–conjugated secondary antibodies and enhanced chemiluminescence reagents (GE Healthcare Bio-Sciences, Little Chalfont, UK). Band intensities were measured with the use of ImageJ software (http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA) and were normalized by that for β-actin. 
RNA Interference
Corneal fibroblasts were seeded in 35-mm dishes and cultured for 24 hours to ∼70% confluence before transfection for 48 hours with a small interfering RNA (siRNA) targeted to human IL-1R mRNA (Thermo Fisher Scientific) with the use of the Lipofectamine2000 reagent (Thermo Fisher Scientific). The effectiveness and specificity of IL-1R knockdown by the siRNA were confirmed by immunoblot analysis. 
Assay of Collagen Degradation
The collagenolytic activity of corneal fibroblasts was assessed as previously described by measurement of the amount of hydroxyproline generated from collagen fragments by acid hydrolysis.27 In brief, after removal of nondegraded collagen by ultrafiltration, culture supernatants of collagen gel incubations were subjected to hydrolysis for 24 hours at 110°C with 6 M HCl, and the amount of hydroxyproline in the hydrolysates was then measured by spectrophotometry. 
Statistical Analysis
Quantitative data are presented as means ± SEM and were analyzed with Student's unpaired t-test or Dunnett's multiple comparison test. A P value of <0.05 was considered statistically significant. 
Results
Effect of Collagen on the Morphology of Corneal Fibroblasts
We first examined the effect of culture conditions on the morphology of human corneal fibroblasts by staining the cells for F-actin with phalloidin. Cells cultured in a three-dimensional collagen gel manifested a spindle-like morphology (Fig. 1A). In contrast, those cultured in two dimensions on plastic became flattened and developed prominent stress fibers (Fig. 1B). 
Figure 1
 
Effect of culture conditions on the morphology of human corneal fibroblasts. Cells cultured for 24 hours either in a collagen matrix (A) or on plastic (B) were stained for F-actin with fluorescently labeled phalloidin (green) and examined with a fluorescence microscope. Data are representative of three separate experiments. Scale bars: 50 μm.
Figure 1
 
Effect of culture conditions on the morphology of human corneal fibroblasts. Cells cultured for 24 hours either in a collagen matrix (A) or on plastic (B) were stained for F-actin with fluorescently labeled phalloidin (green) and examined with a fluorescence microscope. Data are representative of three separate experiments. Scale bars: 50 μm.
Effects of IL-1β and ECM Proteins on uPA Production by Corneal Fibroblasts in Two-Dimensional Culture
To examine the effects of IL-1β and ECM components on the expression of uPA in corneal fibroblasts, we cultured the cells for 24 hours on plastic coated with BSA either alone or together with fibronectin, laminin, or collagen type I as well as in the absence or presence of IL-1β. In the absence of IL-1β, fibrin zymography revealed that the amount of uPA in cell lysates (cell-associated uPA) was significantly increased for cells cultured on laminin-BSA compared with those cultured on BSA alone (Fig. 2). In the presence of IL-1β (5 ng/mL), the expression of uPA in cells cultured on laminin-BSA or collagen-BSA was significantly increased as compared with that in those cultured on BSA alone, with the effect of collagen being significantly greater than that of laminin (Fig. 2). The addition of IL-1β significantly increased uPA expression only for the cells cultured on collagen-BSA. These results thus indicated that laminin upregulated cell-associated uPA abundance regardless of the absence or presence of IL-1β, whereas collagen and IL-1β induced a marked synergistic increase in uPA expression. 
Figure 2
 
Fibrin zymographic analysis of the effects of IL-1β and ECM proteins on cell-associated uPA abundance in corneal fibroblasts in two-dimensional culture. Cells were cultured for 24 hours on plates coated with BSA either alone or together with fibronectin (FN), laminin (LN), or collagen and in the absence or presence of IL-1β (5 ng/mL), after which the amount of uPA in cell lysates was examined by fibrin zymography. Representative results (left) and quantitative data (means ± SEM) from three independent experiments (right) are shown. *P < 0.05 (Dunnett's test). NS, not significant.
Figure 2
 
Fibrin zymographic analysis of the effects of IL-1β and ECM proteins on cell-associated uPA abundance in corneal fibroblasts in two-dimensional culture. Cells were cultured for 24 hours on plates coated with BSA either alone or together with fibronectin (FN), laminin (LN), or collagen and in the absence or presence of IL-1β (5 ng/mL), after which the amount of uPA in cell lysates was examined by fibrin zymography. Representative results (left) and quantitative data (means ± SEM) from three independent experiments (right) are shown. *P < 0.05 (Dunnett's test). NS, not significant.
Effects of IL-1β and Collagen on uPA Production by Corneal Fibroblasts in Three-Dimensional Culture
We next examined the effect of IL-1β on the expression of uPA by measurement of uPA in culture supernatants (secreted uPA) and in cell lysates (cell-associated uPA) for corneal fibroblasts cultured in collagen gels or on plastic. In the absence of IL-1β, uPA was barely detectable in culture supernatants by fibrin zymography regardless of the culture condition (Fig. 3A). In the presence of IL-1β (5 ng/mL), however, the amount of uPA in culture supernatants was markedly greater for cells embedded in collagen gels than for those maintained on plastic, with IL-1β itself having little effect on uPA release from cells cultured on plastic (Fig. 3A). A similar synergistic effect of collagen and IL-1β was apparent for cell-associated uPA (Fig. 3B). The effects of IL-1β on the amounts of both secreted (Fig. 4A) and cell-associated (Fig. 4B) uPA in collagen gel cultures were concentration dependent, as was the ability of collagen to enhance these effects of IL-1β (Fig. 5). These results thus showed that IL-1β increases both the synthesis and secretion of uPA in corneal fibroblasts embedded in a collagen matrix. 
Figure 3
 
Fibrin zymographic analysis of the effects of IL-1β and collagen on secreted and cell-associated uPA abundance in corneal fibroblast cultures. Cells were cultured for 24 or 48 hours in a collagen matrix or on plastic and in the absence or presence of IL-1β (5 ng/mL), after which the amounts of uPA in culture supernatants (A) and in cell lysates (B) were examined by fibrin zymography. Markers for tissue plasminogen activator (tPA) and uPA were included in the analysis. Representative results (left) and quantitative data (means ± SEM) from three independent experiments (right) are shown. *P < 0.05 (unpaired Student's t-test).
Figure 3
 
Fibrin zymographic analysis of the effects of IL-1β and collagen on secreted and cell-associated uPA abundance in corneal fibroblast cultures. Cells were cultured for 24 or 48 hours in a collagen matrix or on plastic and in the absence or presence of IL-1β (5 ng/mL), after which the amounts of uPA in culture supernatants (A) and in cell lysates (B) were examined by fibrin zymography. Markers for tissue plasminogen activator (tPA) and uPA were included in the analysis. Representative results (left) and quantitative data (means ± SEM) from three independent experiments (right) are shown. *P < 0.05 (unpaired Student's t-test).
Figure 4
 
Concentration dependence of the effects of IL-1β on uPA secretion and cell-associated uPA abundance in corneal fibroblasts. Cells were cultured in collagen gels for 24 hours in the presence of the indicated concentrations of IL-1β, after which the amounts of uPA in culture supernatants (A) and in cell lysates (B) were examined by fibrin zymography. Representative results (left) and quantitative data (means ± SEM) from three independent experiments (right) are shown. *P < 0.05 versus the corresponding value for cells incubated in the absence of IL-1β (Dunnett's test).
Figure 4
 
Concentration dependence of the effects of IL-1β on uPA secretion and cell-associated uPA abundance in corneal fibroblasts. Cells were cultured in collagen gels for 24 hours in the presence of the indicated concentrations of IL-1β, after which the amounts of uPA in culture supernatants (A) and in cell lysates (B) were examined by fibrin zymography. Representative results (left) and quantitative data (means ± SEM) from three independent experiments (right) are shown. *P < 0.05 versus the corresponding value for cells incubated in the absence of IL-1β (Dunnett's test).
Figure 5
 
Concentration dependence of the ability of collagen to enhance the effects of IL-1β on uPA secretion and cell-associated uPA abundance in corneal fibroblasts. Cells were cultured for 24 hours in gels formed by the indicated concentrations of collagen and in the presence of IL-1β (5 ng/mL), after which the amounts of uPA in culture supernatants (A) and in cell lysates (B) were examined by fibrin zymography. Representative results (left) and quantitative data (means ± SEM) from three independent experiments (right) are shown. *P < 0.05 versus the corresponding value for cells incubated in a gel formed by collagen at 0.02 mg/mL (Dunnett's test).
Figure 5
 
Concentration dependence of the ability of collagen to enhance the effects of IL-1β on uPA secretion and cell-associated uPA abundance in corneal fibroblasts. Cells were cultured for 24 hours in gels formed by the indicated concentrations of collagen and in the presence of IL-1β (5 ng/mL), after which the amounts of uPA in culture supernatants (A) and in cell lysates (B) were examined by fibrin zymography. Representative results (left) and quantitative data (means ± SEM) from three independent experiments (right) are shown. *P < 0.05 versus the corresponding value for cells incubated in a gel formed by collagen at 0.02 mg/mL (Dunnett's test).
We next compared the effects of collagen or laminin on uPA expression in corneal fibroblasts between two- and three-dimensional culture conditions. In the presence of IL-1β, the amount of cell-associated uPA was significantly greater for cells exposed to collagen in three-dimensional culture than in two-dimensional culture, whereas it did not differ significantly between the two culture conditions for cells exposed to laminin (Fig. 6). These results thus suggested that a three-dimensional environment specifically increased the ability of collagen to upregulate uPA expression in the presence of IL-1β. 
Figure 6
 
Fibrin zymographic analysis of the effects of laminin and collagen on cell-associated uPA abundance in corneal fibroblasts in two- or three-dimensional cultures. Cells were cultured for 24 hours in the presence of IL-1β (5 ng/mL) and either on plates coated with collagen-BSA or laminin-BSA or in collagen or laminin gels, after which the amount of uPA in cell lysates was examined by fibrin zymography. Representative results (left) and quantitative data (means ± SEM) from three independent experiments (right) are shown. *P < 0.05 (Dunnett's test).
Figure 6
 
Fibrin zymographic analysis of the effects of laminin and collagen on cell-associated uPA abundance in corneal fibroblasts in two- or three-dimensional cultures. Cells were cultured for 24 hours in the presence of IL-1β (5 ng/mL) and either on plates coated with collagen-BSA or laminin-BSA or in collagen or laminin gels, after which the amount of uPA in cell lysates was examined by fibrin zymography. Representative results (left) and quantitative data (means ± SEM) from three independent experiments (right) are shown. *P < 0.05 (Dunnett's test).
Effect of Collagen on IL-1R Expression in Corneal Fibroblasts
To investigate how collagen enhances the effect of IL-1β on uPA production in corneal fibroblasts, we examined the expression of IL-1R in these cells. Immunoblot analysis revealed that culture of the cells in a matrix formed by various concentrations of collagen increased the abundance of IL-1R in a concentration-dependent manner (Fig. 7A). Similar to its effect on uPA expression (Fig. 6), collagen increased IL-1R expression to a significantly greater extent in three-dimensional culture than in two-dimensional culture (Fig. 7B). Furthermore, the concentration dependence of the upregulation of IL-1R expression by collagen (Fig. 7A) was similar to that for the enhancement by collagen of IL-1β–induced uPA expression in these cells (Fig. 5), suggesting that collagen promotes this action of IL-1β by increasing IL-1R expression. 
Figure 7
 
Effect of collagen on IL-1R expression in corneal fibroblasts. (A) Cells were cultured for 24 hours in gels formed by the indicated concentrations of collagen, after which cell lysates were subjected to immunoblot analysis with antibodies to IL-1R and to β-actin (loading control). Representative results (left) and quantitative data for the IL-1R/β-actin ratio (means ± SEM) from three independent experiments (right) are shown. *P < 0.05 versus the value for collagen at 0.02 mg/mL (Dunnett's test). (B) Cells were cultured for 24 hours either on plates coated with collagen (2 mg/mL) and BSA or in collagen gels (2 mg/mL), after which cell lysates were subjected to immunoblot analysis as in (A). Representative results (top) and quantitative data (means ± SEM) from three independent experiments (bottom) are shown. *P < 0.05 versus collagen-BSA (unpaired Student's t-test). (C) Cells were cultured in collagen gels in the absence or presence of α2-antiplasmin (50 μg/mL) for 24 hours, after which cell lysates were analyzed as in (A). Representative results (top) and quantitative data (means ± SEM) from three independent experiments (bottom) are shown.
Figure 7
 
Effect of collagen on IL-1R expression in corneal fibroblasts. (A) Cells were cultured for 24 hours in gels formed by the indicated concentrations of collagen, after which cell lysates were subjected to immunoblot analysis with antibodies to IL-1R and to β-actin (loading control). Representative results (left) and quantitative data for the IL-1R/β-actin ratio (means ± SEM) from three independent experiments (right) are shown. *P < 0.05 versus the value for collagen at 0.02 mg/mL (Dunnett's test). (B) Cells were cultured for 24 hours either on plates coated with collagen (2 mg/mL) and BSA or in collagen gels (2 mg/mL), after which cell lysates were subjected to immunoblot analysis as in (A). Representative results (top) and quantitative data (means ± SEM) from three independent experiments (bottom) are shown. *P < 0.05 versus collagen-BSA (unpaired Student's t-test). (C) Cells were cultured in collagen gels in the absence or presence of α2-antiplasmin (50 μg/mL) for 24 hours, after which cell lysates were analyzed as in (A). Representative results (top) and quantitative data (means ± SEM) from three independent experiments (bottom) are shown.
To investigate whether collagen itself or products of collagen degradation by plasmin induced the upregulation of IL-1R expression, we cultured corneal fibroblasts in collagen gels in the absence or presence of α2-antiplasmin. The presence of α2-antiplasmin had no effect on the expression of IL-1R (Fig. 7C), suggesting that native collagen is responsible for the observed upregulation of IL-1R. 
We next examined whether IL-1R mediates the effect of IL-1β on the expression of uPA in corneal fibroblasts cultured in a collagen gel with the use of an siRNA specific for human IL-1R mRNA. Transfection of the cells with this siRNA resulted in marked depletion of IL-1R (Fig. 8A). The IL-1β–induced upregulation of cell-associated uPA was significantly attenuated in cells transfected with the IL-1R siRNA compared with mock-transfected cells (Fig. 8B), suggesting that IL-1R is required for the effect of IL-1β on uPA expression in corneal fibroblasts cultured in a collagen gel. 
Figure 8
 
Inhibition of the stimulatory effect of IL-1β on uPA expression in corneal fibroblasts by knockdown of IL-1R. (A) Corneal fibroblasts were transfected with an siRNA specific for IL-1R mRNA or were subjected to mock transfection, after which the cells were cultured for 24 hours and then subjected to immunoblot analysis for quantitation of IL-1R abundance normalized by that of β-actin. Data are means ± SEM from three independent experiments. *P < 0.05 versus mock transfection (unpaired Student's t-test). (B) Cells transfected as in (A) were cultured in collagen gels for 24 hours in the absence or presence of IL-1β (5 ng/mL), after which cell lysates were subjected to immunoblot analysis with antibodies to IL-1R and to β-actin as well as to fibrin zymography for detection of uPA. Representative results (left) and quantitative data (means ± SEM) from three independent experiments (right) are shown. *P < 0.05 (unpaired Student's t-test).
Figure 8
 
Inhibition of the stimulatory effect of IL-1β on uPA expression in corneal fibroblasts by knockdown of IL-1R. (A) Corneal fibroblasts were transfected with an siRNA specific for IL-1R mRNA or were subjected to mock transfection, after which the cells were cultured for 24 hours and then subjected to immunoblot analysis for quantitation of IL-1R abundance normalized by that of β-actin. Data are means ± SEM from three independent experiments. *P < 0.05 versus mock transfection (unpaired Student's t-test). (B) Cells transfected as in (A) were cultured in collagen gels for 24 hours in the absence or presence of IL-1β (5 ng/mL), after which cell lysates were subjected to immunoblot analysis with antibodies to IL-1R and to β-actin as well as to fibrin zymography for detection of uPA. Representative results (left) and quantitative data (means ± SEM) from three independent experiments (right) are shown. *P < 0.05 (unpaired Student's t-test).
Effects of IL-1β on MAPK and NF-κB Signaling in Collagen Gel Cultures of Corneal Fibroblasts
We examined whether MAPK or nuclear factor–κB (NF-κB) signaling pathways might mediate IL-1β–induced uPA production by corneal fibroblasts cultured in a collagen gel. Immunoblot analysis revealed that IL-1β induced phosphorylation (activation) of the MAPKs ERK, p38, and JNK as well as that of the p65 subunit of NF-κB (Fig. 9), suggesting that these signaling molecules might contribute to the upregulation of uPA expression by IL-1β in this system. 
Figure 9
 
Interleukin-1β–induced activation of MAPK and NF-κB signaling in corneal fibroblasts cultured in a collagen gel. Cells were cultured in a collagen gel in the absence or presence of IL-1β (5 ng/mL) for 30 minutes, after which cell lysates were subjected to immunoblot analysis with antibodies to phosphorylated (p-) forms of ERK, p38 MAPK, JNK, or the p65 subunit of NF-κB. Data are representative of three independent experiments.
Figure 9
 
Interleukin-1β–induced activation of MAPK and NF-κB signaling in corneal fibroblasts cultured in a collagen gel. Cells were cultured in a collagen gel in the absence or presence of IL-1β (5 ng/mL) for 30 minutes, after which cell lysates were subjected to immunoblot analysis with antibodies to phosphorylated (p-) forms of ERK, p38 MAPK, JNK, or the p65 subunit of NF-κB. Data are representative of three independent experiments.
Functional Activity of uPA Produced by Corneal Fibroblasts
We next examined whether uPA produced by corneal fibroblasts in response to stimulation with IL-1β and collagen is able to catalyze the conversion of exogenous plasminogen to plasmin. Fibrin zymography revealed that, in the presence of IL-1β, plasmin was produced from plasminogen in the culture supernatants of cells embedded in collagen gels (Fig. 10). No such conversion was apparent in the absence of cell stimulation with IL-1β or in the presence of a selective uPA inhibitor (uPA-STOP). A similar pattern of plasmin production was apparent with cell lysates. 
Figure 10
 
Functional activity of uPA produced by corneal fibroblasts. Cells were cultured in collagen gels and in the absence or presence of IL-1β (5 ng/mL), plasminogen (50 μg/mL), or uPA-STOP (5 μg/mL) for 24 hours, after which culture supernatants and cell lysates were subjected to fibrin zymography to detect the conversion of plasminogen to plasmin. Lanes containing plasminogen (Plg) that had been incubated with or without uPA are included in the left panel to provide a marker for plasmin. Data are representative of three separate experiments.
Figure 10
 
Functional activity of uPA produced by corneal fibroblasts. Cells were cultured in collagen gels and in the absence or presence of IL-1β (5 ng/mL), plasminogen (50 μg/mL), or uPA-STOP (5 μg/mL) for 24 hours, after which culture supernatants and cell lysates were subjected to fibrin zymography to detect the conversion of plasminogen to plasmin. Lanes containing plasminogen (Plg) that had been incubated with or without uPA are included in the left panel to provide a marker for plasmin. Data are representative of three separate experiments.
Effects of IL-1β, Plasminogen, and a uPA Inhibitor on MMP Production and Activation in Collagen Gel Cultures of Corneal Fibroblasts
We examined the production and activation of collagenolytic MMPs in collagen gel cultures of corneal fibroblasts. Immunoblot analysis revealed that stimulation of the cells with IL-1β increased the abundance of the 57-kDa pro forms of both MMP-1 and MMP-3 in both culture supernatants and cell lysates (Fig. 11). In the additional presence of plasminogen, the intensity of the bands corresponding to proMMP-1 and proMMP-3 decreased and that of bands corresponding to the 41-kDa active form of MMP-1 and the 45-kDa active form of MMP-3 increased, consistent with previous observations.14,28 Further addition of a uPA inhibitor blocked the IL-1β–induced, plasminogen-dependent conversion of the pro forms of both MMPs to the active forms. 
Figure 11
 
Effects of IL-1β, plasminogen, and a uPA inhibitor on the production and activation of MMP-1 and MMP-3 in collagen gel cultures of corneal fibroblasts. Cells were cultured in collagen gels and in the absence or presence of IL-1β (5 ng/mL), plasminogen (50 μg/mL), or uPA-STOP (5 μg/mL) for 24 hours, after which culture supernatants and cell lysates were subjected to immunoblot analysis with antibodies to MMP-1 and to MMP-3. Data are representative of three separate experiments.
Figure 11
 
Effects of IL-1β, plasminogen, and a uPA inhibitor on the production and activation of MMP-1 and MMP-3 in collagen gel cultures of corneal fibroblasts. Cells were cultured in collagen gels and in the absence or presence of IL-1β (5 ng/mL), plasminogen (50 μg/mL), or uPA-STOP (5 μg/mL) for 24 hours, after which culture supernatants and cell lysates were subjected to immunoblot analysis with antibodies to MMP-1 and to MMP-3. Data are representative of three separate experiments.
Effects of IL-1β, Plasminogen, and a uPA Inhibitor on Collagen Degradation by Corneal Fibroblasts
Finally, we examined the effects of IL-1β, plasminogen, and a uPA inhibitor on the collagenolytic activity of corneal fibroblasts. Whereas the addition of IL-1β or plasminogen alone had no effect on collagen degradation, the presence of both agents induced a marked increase in collagenolytic activity (Fig. 12). Furthermore, this stimulatory effect of IL-1β and plasminogen was abolished in the additional presence of a uPA inhibitor. These results thus indicated that the stimulation of collagen degradation by IL-1β and plasminogen in this system is dependent on the production of uPA by corneal fibroblasts. 
Figure 12
 
Effects of IL-1β, plasminogen, and a uPA inhibitor on collagen degradation by corneal fibroblasts. Cells were cultured in collagen gels and in the absence or presence of IL-1β (5 ng/mL), plasminogen (50 μg/mL), or uPA-STOP (5 μg/mL) for 24 hours, after which culture supernatants were assayed for products of collagen degradation. Data are expressed as micrograms of hydroxyproline (HYP) per well and are means ± SEM from three separate experiments. *P < 0.001 versus the value for cells incubated without addition (unpaired Student's t-test).
Figure 12
 
Effects of IL-1β, plasminogen, and a uPA inhibitor on collagen degradation by corneal fibroblasts. Cells were cultured in collagen gels and in the absence or presence of IL-1β (5 ng/mL), plasminogen (50 μg/mL), or uPA-STOP (5 μg/mL) for 24 hours, after which culture supernatants were assayed for products of collagen degradation. Data are expressed as micrograms of hydroxyproline (HYP) per well and are means ± SEM from three separate experiments. *P < 0.001 versus the value for cells incubated without addition (unpaired Student's t-test).
Discussion
We showed here that collagen enhances the stimulatory effect of IL-1β on uPA production by corneal fibroblasts. This effect of collagen may be mediated in part by upregulation of IL-1R expression in the cells. The uPA released from corneal fibroblasts was able to catalyze the conversion of plasminogen to plasmin and thereby to promote the plasmin-dependent activation of the pro forms of MMP-1 and MMP-3 produced by the cells in response to IL-1β stimulation. 
We have previously shown that culture of corneal fibroblasts in a collagen gel inhibits their proliferation.4 In addition, our previous4 and present results show that the morphology of corneal fibroblasts embedded in such a gel resembles that of corneal fibroblasts in vivo, suggesting that three-dimensional culture of the cells in a collagen matrix more closely reflects the in vivo situation than does their two-dimensional culture on plastic. 
The ECM modulates a variety of cellular functions. For example, the function of neutrophils in the inflammatory response is modulated by contact with collagen, which inhibits the secretion of IL-8 from these cells.29 Consistent with the regulation of the morphology and function of various cell types by the ECM,30,31 we found that collagen and laminin each enhanced the expression of uPA in corneal fibroblasts. The effect of collagen on uPA expression was IL-1β dependent, whereas that of laminin was not. Moreover, the stimulatory effect of collagen on uPA expression was more pronounced in three-dimensional culture, whereas that of laminin was similar in both two- and three-dimensional environments. These results suggest that the interaction of corneal fibroblasts with collagen in three dimensions is important for optimal stimulation of uPA expression by IL-1β. 
We showed that culture in a collagen gel increases the sensitivity of corneal fibroblasts to the stimulatory effect of IL-1β on uPA production, likely at least in part by upregulating the expression of IL-1R in these cells. In addition, RNA interference–mediated depletion of IL-1R greatly attenuated the IL-1β–induced upregulation of uPA in corneal fibroblasts cultured in a collagen gel, suggesting that this effect of IL-1β is indeed mediated by IL-1R. 
The precise mechanism of the combined effect of IL-1β and collagen on uPA expression in corneal fibroblasts remains to be elucidated. The NF-κB signaling pathway has been implicated in collagen degradation by corneal fibroblasts.11 Furthermore, NF-κB is a key mediator of IL-1β effects and the uPA gene promoter contains binding sites for this transcription factor.19,3234 Interleukin-1β also activates MAPK signaling pathways including those mediated by ERK, JNK, and p38 MAPK.35,36 We confirmed that IL-1β activates these MAPKs and NF-κB in corneal fibroblasts cultured in a collagen gel. It is therefore possible that IL-1β induces uPA expression in corneal fibroblasts by activation of MAPK and NF-κB signaling pathways. 
The actions of uPA are modulated by its binding with high affinity to the uPA receptor (uPAR), which restricts the activation of plasminogen and other proteolytic reactions to the vicinity of the cell surface.3739 Corneal fibroblasts have previously been shown to express uPAR.40 Binding of uPA to uPAR not only promotes proteolytic activity at the cell surface but also triggers intracellular signaling that is dependent on the presence of coreceptors including integrins and growth factor receptors.41,42 The interaction of uPA with uPAR on corneal fibroblasts may thus enhance the conversion of plasminogen to plasmin as well as MMP activation and consequent collagen degradation. 
Given that uPA-STOP is a reversible competitive inhibitor of trypsin-like serine proteases that has a relatively high specificity for uPA and inhibits the activity of uPA in the free or uPAR-bound state,43 our finding that uPA-STOP completely inhibited IL-1β–induced, plasminogen-dependent collagen degradation by corneal fibroblasts implicates uPA secreted from the cells in this process. 
Activated MMPs mediate collagen degradation, and plasminogen triggers collagen degradation via MMP activation.12,44 Interleukin-1 has been shown to increase MMP expression in fibroblasts in an IL-1R–dependent manner,45 and the production of MMP-1 and MMP-3 by corneal fibroblasts is upregulated by IL-1β in vitro.13 We showed that uPA production is required for the plasmin-mediated activation of MMP-1 and MMP-3 produced in collagen gel cultures of corneal fibroblasts stimulated with IL-1β, consistent with the notion that uPA contributes to the activation of MMPs in other systems.4648 
In summary, we showed that a three-dimensional collagen matrix enhances IL-1β–induced uPA expression in corneal fibroblasts, and we identified a pathway for IL-1β–induced collagen degradation by these cells that is strictly dependent on uPA production and is mediated by the conversion of plasminogen to plasmin by uPA and the activation of MMPs by plasmin. Our results thus provide potential insight into the pathogenesis of corneal ulcer. 
Acknowledgments
The authors thank Mihoko Iwata for technical support. 
Supported in part by grants from Osaka Eye Bank and a Grant-in-Aid (15k20294) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. 
Disclosure: K. Sugioka, None; A. Kodama-Takahashi, None; K. Yoshida, None; K. Aomatsu, None; K. Okada, None; T. Nishida, None; Y. Shimomura, None 
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Figure 1
 
Effect of culture conditions on the morphology of human corneal fibroblasts. Cells cultured for 24 hours either in a collagen matrix (A) or on plastic (B) were stained for F-actin with fluorescently labeled phalloidin (green) and examined with a fluorescence microscope. Data are representative of three separate experiments. Scale bars: 50 μm.
Figure 1
 
Effect of culture conditions on the morphology of human corneal fibroblasts. Cells cultured for 24 hours either in a collagen matrix (A) or on plastic (B) were stained for F-actin with fluorescently labeled phalloidin (green) and examined with a fluorescence microscope. Data are representative of three separate experiments. Scale bars: 50 μm.
Figure 2
 
Fibrin zymographic analysis of the effects of IL-1β and ECM proteins on cell-associated uPA abundance in corneal fibroblasts in two-dimensional culture. Cells were cultured for 24 hours on plates coated with BSA either alone or together with fibronectin (FN), laminin (LN), or collagen and in the absence or presence of IL-1β (5 ng/mL), after which the amount of uPA in cell lysates was examined by fibrin zymography. Representative results (left) and quantitative data (means ± SEM) from three independent experiments (right) are shown. *P < 0.05 (Dunnett's test). NS, not significant.
Figure 2
 
Fibrin zymographic analysis of the effects of IL-1β and ECM proteins on cell-associated uPA abundance in corneal fibroblasts in two-dimensional culture. Cells were cultured for 24 hours on plates coated with BSA either alone or together with fibronectin (FN), laminin (LN), or collagen and in the absence or presence of IL-1β (5 ng/mL), after which the amount of uPA in cell lysates was examined by fibrin zymography. Representative results (left) and quantitative data (means ± SEM) from three independent experiments (right) are shown. *P < 0.05 (Dunnett's test). NS, not significant.
Figure 3
 
Fibrin zymographic analysis of the effects of IL-1β and collagen on secreted and cell-associated uPA abundance in corneal fibroblast cultures. Cells were cultured for 24 or 48 hours in a collagen matrix or on plastic and in the absence or presence of IL-1β (5 ng/mL), after which the amounts of uPA in culture supernatants (A) and in cell lysates (B) were examined by fibrin zymography. Markers for tissue plasminogen activator (tPA) and uPA were included in the analysis. Representative results (left) and quantitative data (means ± SEM) from three independent experiments (right) are shown. *P < 0.05 (unpaired Student's t-test).
Figure 3
 
Fibrin zymographic analysis of the effects of IL-1β and collagen on secreted and cell-associated uPA abundance in corneal fibroblast cultures. Cells were cultured for 24 or 48 hours in a collagen matrix or on plastic and in the absence or presence of IL-1β (5 ng/mL), after which the amounts of uPA in culture supernatants (A) and in cell lysates (B) were examined by fibrin zymography. Markers for tissue plasminogen activator (tPA) and uPA were included in the analysis. Representative results (left) and quantitative data (means ± SEM) from three independent experiments (right) are shown. *P < 0.05 (unpaired Student's t-test).
Figure 4
 
Concentration dependence of the effects of IL-1β on uPA secretion and cell-associated uPA abundance in corneal fibroblasts. Cells were cultured in collagen gels for 24 hours in the presence of the indicated concentrations of IL-1β, after which the amounts of uPA in culture supernatants (A) and in cell lysates (B) were examined by fibrin zymography. Representative results (left) and quantitative data (means ± SEM) from three independent experiments (right) are shown. *P < 0.05 versus the corresponding value for cells incubated in the absence of IL-1β (Dunnett's test).
Figure 4
 
Concentration dependence of the effects of IL-1β on uPA secretion and cell-associated uPA abundance in corneal fibroblasts. Cells were cultured in collagen gels for 24 hours in the presence of the indicated concentrations of IL-1β, after which the amounts of uPA in culture supernatants (A) and in cell lysates (B) were examined by fibrin zymography. Representative results (left) and quantitative data (means ± SEM) from three independent experiments (right) are shown. *P < 0.05 versus the corresponding value for cells incubated in the absence of IL-1β (Dunnett's test).
Figure 5
 
Concentration dependence of the ability of collagen to enhance the effects of IL-1β on uPA secretion and cell-associated uPA abundance in corneal fibroblasts. Cells were cultured for 24 hours in gels formed by the indicated concentrations of collagen and in the presence of IL-1β (5 ng/mL), after which the amounts of uPA in culture supernatants (A) and in cell lysates (B) were examined by fibrin zymography. Representative results (left) and quantitative data (means ± SEM) from three independent experiments (right) are shown. *P < 0.05 versus the corresponding value for cells incubated in a gel formed by collagen at 0.02 mg/mL (Dunnett's test).
Figure 5
 
Concentration dependence of the ability of collagen to enhance the effects of IL-1β on uPA secretion and cell-associated uPA abundance in corneal fibroblasts. Cells were cultured for 24 hours in gels formed by the indicated concentrations of collagen and in the presence of IL-1β (5 ng/mL), after which the amounts of uPA in culture supernatants (A) and in cell lysates (B) were examined by fibrin zymography. Representative results (left) and quantitative data (means ± SEM) from three independent experiments (right) are shown. *P < 0.05 versus the corresponding value for cells incubated in a gel formed by collagen at 0.02 mg/mL (Dunnett's test).
Figure 6
 
Fibrin zymographic analysis of the effects of laminin and collagen on cell-associated uPA abundance in corneal fibroblasts in two- or three-dimensional cultures. Cells were cultured for 24 hours in the presence of IL-1β (5 ng/mL) and either on plates coated with collagen-BSA or laminin-BSA or in collagen or laminin gels, after which the amount of uPA in cell lysates was examined by fibrin zymography. Representative results (left) and quantitative data (means ± SEM) from three independent experiments (right) are shown. *P < 0.05 (Dunnett's test).
Figure 6
 
Fibrin zymographic analysis of the effects of laminin and collagen on cell-associated uPA abundance in corneal fibroblasts in two- or three-dimensional cultures. Cells were cultured for 24 hours in the presence of IL-1β (5 ng/mL) and either on plates coated with collagen-BSA or laminin-BSA or in collagen or laminin gels, after which the amount of uPA in cell lysates was examined by fibrin zymography. Representative results (left) and quantitative data (means ± SEM) from three independent experiments (right) are shown. *P < 0.05 (Dunnett's test).
Figure 7
 
Effect of collagen on IL-1R expression in corneal fibroblasts. (A) Cells were cultured for 24 hours in gels formed by the indicated concentrations of collagen, after which cell lysates were subjected to immunoblot analysis with antibodies to IL-1R and to β-actin (loading control). Representative results (left) and quantitative data for the IL-1R/β-actin ratio (means ± SEM) from three independent experiments (right) are shown. *P < 0.05 versus the value for collagen at 0.02 mg/mL (Dunnett's test). (B) Cells were cultured for 24 hours either on plates coated with collagen (2 mg/mL) and BSA or in collagen gels (2 mg/mL), after which cell lysates were subjected to immunoblot analysis as in (A). Representative results (top) and quantitative data (means ± SEM) from three independent experiments (bottom) are shown. *P < 0.05 versus collagen-BSA (unpaired Student's t-test). (C) Cells were cultured in collagen gels in the absence or presence of α2-antiplasmin (50 μg/mL) for 24 hours, after which cell lysates were analyzed as in (A). Representative results (top) and quantitative data (means ± SEM) from three independent experiments (bottom) are shown.
Figure 7
 
Effect of collagen on IL-1R expression in corneal fibroblasts. (A) Cells were cultured for 24 hours in gels formed by the indicated concentrations of collagen, after which cell lysates were subjected to immunoblot analysis with antibodies to IL-1R and to β-actin (loading control). Representative results (left) and quantitative data for the IL-1R/β-actin ratio (means ± SEM) from three independent experiments (right) are shown. *P < 0.05 versus the value for collagen at 0.02 mg/mL (Dunnett's test). (B) Cells were cultured for 24 hours either on plates coated with collagen (2 mg/mL) and BSA or in collagen gels (2 mg/mL), after which cell lysates were subjected to immunoblot analysis as in (A). Representative results (top) and quantitative data (means ± SEM) from three independent experiments (bottom) are shown. *P < 0.05 versus collagen-BSA (unpaired Student's t-test). (C) Cells were cultured in collagen gels in the absence or presence of α2-antiplasmin (50 μg/mL) for 24 hours, after which cell lysates were analyzed as in (A). Representative results (top) and quantitative data (means ± SEM) from three independent experiments (bottom) are shown.
Figure 8
 
Inhibition of the stimulatory effect of IL-1β on uPA expression in corneal fibroblasts by knockdown of IL-1R. (A) Corneal fibroblasts were transfected with an siRNA specific for IL-1R mRNA or were subjected to mock transfection, after which the cells were cultured for 24 hours and then subjected to immunoblot analysis for quantitation of IL-1R abundance normalized by that of β-actin. Data are means ± SEM from three independent experiments. *P < 0.05 versus mock transfection (unpaired Student's t-test). (B) Cells transfected as in (A) were cultured in collagen gels for 24 hours in the absence or presence of IL-1β (5 ng/mL), after which cell lysates were subjected to immunoblot analysis with antibodies to IL-1R and to β-actin as well as to fibrin zymography for detection of uPA. Representative results (left) and quantitative data (means ± SEM) from three independent experiments (right) are shown. *P < 0.05 (unpaired Student's t-test).
Figure 8
 
Inhibition of the stimulatory effect of IL-1β on uPA expression in corneal fibroblasts by knockdown of IL-1R. (A) Corneal fibroblasts were transfected with an siRNA specific for IL-1R mRNA or were subjected to mock transfection, after which the cells were cultured for 24 hours and then subjected to immunoblot analysis for quantitation of IL-1R abundance normalized by that of β-actin. Data are means ± SEM from three independent experiments. *P < 0.05 versus mock transfection (unpaired Student's t-test). (B) Cells transfected as in (A) were cultured in collagen gels for 24 hours in the absence or presence of IL-1β (5 ng/mL), after which cell lysates were subjected to immunoblot analysis with antibodies to IL-1R and to β-actin as well as to fibrin zymography for detection of uPA. Representative results (left) and quantitative data (means ± SEM) from three independent experiments (right) are shown. *P < 0.05 (unpaired Student's t-test).
Figure 9
 
Interleukin-1β–induced activation of MAPK and NF-κB signaling in corneal fibroblasts cultured in a collagen gel. Cells were cultured in a collagen gel in the absence or presence of IL-1β (5 ng/mL) for 30 minutes, after which cell lysates were subjected to immunoblot analysis with antibodies to phosphorylated (p-) forms of ERK, p38 MAPK, JNK, or the p65 subunit of NF-κB. Data are representative of three independent experiments.
Figure 9
 
Interleukin-1β–induced activation of MAPK and NF-κB signaling in corneal fibroblasts cultured in a collagen gel. Cells were cultured in a collagen gel in the absence or presence of IL-1β (5 ng/mL) for 30 minutes, after which cell lysates were subjected to immunoblot analysis with antibodies to phosphorylated (p-) forms of ERK, p38 MAPK, JNK, or the p65 subunit of NF-κB. Data are representative of three independent experiments.
Figure 10
 
Functional activity of uPA produced by corneal fibroblasts. Cells were cultured in collagen gels and in the absence or presence of IL-1β (5 ng/mL), plasminogen (50 μg/mL), or uPA-STOP (5 μg/mL) for 24 hours, after which culture supernatants and cell lysates were subjected to fibrin zymography to detect the conversion of plasminogen to plasmin. Lanes containing plasminogen (Plg) that had been incubated with or without uPA are included in the left panel to provide a marker for plasmin. Data are representative of three separate experiments.
Figure 10
 
Functional activity of uPA produced by corneal fibroblasts. Cells were cultured in collagen gels and in the absence or presence of IL-1β (5 ng/mL), plasminogen (50 μg/mL), or uPA-STOP (5 μg/mL) for 24 hours, after which culture supernatants and cell lysates were subjected to fibrin zymography to detect the conversion of plasminogen to plasmin. Lanes containing plasminogen (Plg) that had been incubated with or without uPA are included in the left panel to provide a marker for plasmin. Data are representative of three separate experiments.
Figure 11
 
Effects of IL-1β, plasminogen, and a uPA inhibitor on the production and activation of MMP-1 and MMP-3 in collagen gel cultures of corneal fibroblasts. Cells were cultured in collagen gels and in the absence or presence of IL-1β (5 ng/mL), plasminogen (50 μg/mL), or uPA-STOP (5 μg/mL) for 24 hours, after which culture supernatants and cell lysates were subjected to immunoblot analysis with antibodies to MMP-1 and to MMP-3. Data are representative of three separate experiments.
Figure 11
 
Effects of IL-1β, plasminogen, and a uPA inhibitor on the production and activation of MMP-1 and MMP-3 in collagen gel cultures of corneal fibroblasts. Cells were cultured in collagen gels and in the absence or presence of IL-1β (5 ng/mL), plasminogen (50 μg/mL), or uPA-STOP (5 μg/mL) for 24 hours, after which culture supernatants and cell lysates were subjected to immunoblot analysis with antibodies to MMP-1 and to MMP-3. Data are representative of three separate experiments.
Figure 12
 
Effects of IL-1β, plasminogen, and a uPA inhibitor on collagen degradation by corneal fibroblasts. Cells were cultured in collagen gels and in the absence or presence of IL-1β (5 ng/mL), plasminogen (50 μg/mL), or uPA-STOP (5 μg/mL) for 24 hours, after which culture supernatants were assayed for products of collagen degradation. Data are expressed as micrograms of hydroxyproline (HYP) per well and are means ± SEM from three separate experiments. *P < 0.001 versus the value for cells incubated without addition (unpaired Student's t-test).
Figure 12
 
Effects of IL-1β, plasminogen, and a uPA inhibitor on collagen degradation by corneal fibroblasts. Cells were cultured in collagen gels and in the absence or presence of IL-1β (5 ng/mL), plasminogen (50 μg/mL), or uPA-STOP (5 μg/mL) for 24 hours, after which culture supernatants were assayed for products of collagen degradation. Data are expressed as micrograms of hydroxyproline (HYP) per well and are means ± SEM from three separate experiments. *P < 0.001 versus the value for cells incubated without addition (unpaired Student's t-test).
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