Differential Inhibition of Collagenase and Interleukin la Gene Expression in Cultured Corneal Fibroblasts by TGF-/3, Dexamethasone, and Retinoic Acid (original) (raw)
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… ophthalmology & visual …, 1999
PURPOSE. Expression of the genes for collagenase and interleukin-la (IL-la) are induced as stromal cells become activated to the repair fibroblast phenotype after injury to the cornea. This investigation examines the mechanisms whereby expression of these genes is inhibited by transforming growth factor-j3 (TGF-/3), dexamethasone (DEX), or retinoic acid (RET A). METHODS. A model of freshly isolated cultures of corneal stromal cells and early passage cultures of corneal fibroblasts was used in these studies. This model reproduces the events of stromal cell activation in the corneal wound. RESULTS. In early passage cultures of corneal fibroblasts, expression of collagenase is under obligatory control by autocrine IL-la. IL-la controls its own expression through an autocrine feedback loop that is dependent on transcription factor NF-KB. TGF-0, DEX, and RET A were each effective inhibitors of collagenase gene expression in these cells. Furthermore, these agents have the capacity to inhibit expression of IL-la and this was correlated with their ability to affect DNA-binding activity of NF-KB. However, TGF-/3, DEX, and RET A were also effective inhibitors of the low level of collagenase expressed by freshly isolated corneal stromal cells that cannot express IL-la. CONCLUSIONS. In cells with an active IL-la autocrine loop there are at least two distinct signaling pathways by which collagenase gene expression can be modulated. The results of this study demonstrate that TGF-/3, DEX, and RET A differentially inhibit collagenase and IL-la gene expression. This information will be useful in the design of therapeutic modalities for fibrotic disease in the cornea and other parts of the eye.
PURPOSE. Connective tissue growth factor (CTGF) has been linked to fibrosis in several tissues. In this study, the interactions between CTGF and transforming growth factor (TGF)- were assessed in human corneal fibroblasts, and the levels and location of CTGF protein and mRNA were measured during healing of excimer laser ablation wounds in rat corneas. METHODS. Human corneal fibroblasts were incubated with TGF-1, -2, and -3 isoforms, and CTGF mRNA and protein were measured. CTGF was immunolocalized in the cultured fibroblasts by using a specific antibody. Regulation of collagen synthesis by TGF- and CTGF was assessed in human corneal fibroblasts with a neutralizing antibody and an antisense oligonucleotide to CTGF. CTGF mRNA and protein were measured in rat corneas up to day 21 after excimer ablation of the cornea. CTGF protein was immunolocalized in rat corneas after photorefractive keratectomy (PRK), and the presence of CTGF mRNA and protein in ex vivo rat corneal scrapings was established. RESULTS. All three TGF- isoforms stimulated expression of CTGF in human corneal fibroblasts, and CTGF was immunolocalized in the cells. Both TGF- and CTGF increased collagen synthesis in corneal fibroblasts. Furthermore, CTGF antibody or antisense oligonucleotide blocked TGF--stimulated collagen synthesis. CTGF protein and mRNA increased in rat corneas through day 21 after PRK. CTGF expression was also detected in ex vivo scrapings of rat corneas. CONCLUSIONS. These data demonstrate that CTGF is expressed by corneal cells after stimulation by TGF-, that CTGF expression increases significantly during corneal wound healing, and that CTGF mediates the effects of TGF- induction of collagen synthesis by corneal fibroblasts. These data support the hypothesis that CTGF promotes corneal scar formation and imply that regulating CTGF synthesis and action may be an important goal for reducing corneal scarring. (Invest Ophthalmol Vis Sci.
Transforming growth factor-?1 expression in cultured corneal fibroblasts in response to injury
Journal of Cellular Biochemistry, 2000
Aims: To evaluate the expression of transforming growth factor β1 (TGF-β1) and fibroblast growth factor 2 (FGF-2) mRNA in stromal cells in response to injury in the presence of either TGF-β1 or FGF-2. It has been shown previously that heparan sulfate proteoglycans and FGF-2 are present transiently during wound repair in vivo and that an increase in TGF-β1 mRNA is detected rapidly after injury. Methods: Primary corneal fibroblasts were cultured to confluency, serum starved, and linear wound(s) were made in medium containing TGF-β1 or FGF-2. TGF-β1 and FGF-2 mRNA expression were evaluated using both northern blot analysis and in situ hybridisation. Both dose dependent and time course experiments were performed. Whole eye organ culture experiments were also carried out and growth factor expression was assessed.
Investigative Opthalmology & Visual Science, 2008
PURPOSE. Corneal ulcer results from excessive collagen degradation in the corneal stroma. Interleukin (IL)-1 promotes this process by activating signaling molecules that include nuclear factor (NF)-B and stimulating the synthesis of matrix metalloproteinases (MMPs) in corneal fibroblasts. NF-B activation is mediated by phosphorylation of the inhibitor IB by IB kinase (IKK)-2 and consequent IB degradation. The authors investigated the effects of the IKK-2 inhibitor [5-(p-fluorophenyl)-2ureido]thiophene-3-carboxamide (TPCA-1) on collagen degradation by corneal fibroblasts. METHODS. Rabbit corneal fibroblasts were cultured in threedimensional collagen gels. Collagen degradation was evaluated by spectrophotometric quantitation of hydroxyproline in culture supernatants subjected to acid-heat hydrolysis. Expression of MMPs was evaluated by immunoblot analysis, gelatin zymography, and real-time reverse transcription polymerase chain reaction analysis. The phosphorylation and degradation of IB␣ and the subcellular localization of NF-B were examined by immunoblot and immunofluorescence analyses, respectively. RESULTS. IL-1-induced collagen degradation by corneal fibroblasts was inhibited by TPCA-1 in a concentration-and timedependent manner. TPCA-1 inhibited the IL-1-induced expression of MMP-1,-3, and-9 in these cells at both the mRNA and protein levels and the IL-1-induced activation of pro-MMP-2. In contrast to dexamethasone, TPCA-1 inhibited the phosphorylation and degradation of IB␣ and the nuclear translocation of NF-B induced by IL-1. CONCLUSIONS. An IKK-2 inhibitor blocked IL-1-induced collagen degradation by corneal fibroblasts by inhibiting the activation of the NF-B signaling pathway and the upregulation of MMPs. IKK-2 inhibitors are thus potential alternatives to dexamethasone for the treatment of corneal ulcer.
Investigative ophthalmology & visual science, 1993
We have previously reported that corneal endothelial modulation takes place when rabbit corneal endothelial (CE) cells are exposed to corneal endothelium modulation factor (CEMF) released by polymorphonuclear leukocytes (PMN) (Kay, E. P., L. Rivela, and Y. G. He, 1990. Invest Ophthalmol Vis Sci. 31:313-322). The modulation was involved in phenotypic switches from polygonal cell shape to fibroblastic morphology and from basement membrane collagen (type IV-rich) synthesis to fibrillar collagen (type I-rich) synthesis. In the current study, we tested the effect of several growth-modulating factors on corneal endothelial modulation. The effect of basic fibroblast growth factor (bFGF) on cell proliferation was measured by [3H]thymidine incorporation into DNA and cell numbers. Collagen expression was determined by SDS-polyacrylamide gel electrophoresis and by Northern blot analysis. Transcription rate was determined by nuclear run-off assay. Basic fibroblast growth factor synthesis was an...
TGF-β3 Stimulates Stromal Matrix Assembly by Human Corneal Keratocyte-Like Cells
Investigative Opthalmology & Visual Science, 2013
PURPOSE. We have previously shown that TGF-b3 (T3) stimulates extracellular matrix (ECM) assembly while maintaining antifibrotic characteristics in a model using human corneal fibroblasts (HCFs). This model, however, requires non-physiological levels of serum. In the current study, we tested whether T3 could stimulate human corneal keratocytes (HCKs) in vitro to assemble a functional ECM, while maintaining their characteristics. METHODS. Human corneal keratocytes and HCFs were isolated and cultured using 1% or 10% serum, respectively 6T3. The constructs were processed for indirect immunofluorescence (IF), transmission electron microscopy (TEM), and qRT-PCR, analyzing for keratocyte marker, keratocan, and ECM components, collagen (col) types I, III, and V. RESULTS. Quantitative reverse transcriptase PCR data showed that keratocan, col I, and V were all upregulated in HCKs compared with HCFs, whereas col III was expressed at low levels in HCKs. Transforming growth factor beta 3 stimulation further enhanced the level of change. Without T3, HCK constructs were very thin, approximately 5 lm; however, as with HCFs, upon stimulation with T3, HCK constructs increased in thickness by approximately 5-fold. Cell counts and ECM production revealed that HCKs assembled more ECM per unit area compared with HCFs, and IF revealed downregulation of fibrotic markers, col III, and thrombospondin-1, with T3 stimulation. Transmission electron microscopy data revealed aligned ECM with long fibrils for all conditions except HCK Controls. Human corneal keratocytesþT3 also showed denser collagen fibrils with more consistent fibril diameter. CONCLUSIONS. Overall, the data suggests that it is possible to stimulate matrix secretion and assembly by HCKs in vitro by using a single growth factor, T3.
Investigative Ophthalmology & Visual Science, 2010
PURPOSE. The purpose of this study was to elucidate the pathophysiological process in primary cultured corneal fibroblasts (PCFs) from normal subjects and granular corneal dystrophy (GCD) II patients, by using cDNA microarrays. METHODS. PCFs were isolated from the corneas of normal subjects and GCD II patients who were heterozygous and homozygous for the TGFBI R124H mutation. RNA was isolated from each sample, and gene expression profiles were analyzed with a cDNA microarray consisting of approximately 29,000 genes. Cell adhesion assays were performed to confirm the functionality of the detected gene expression profiles. RESULTS. Twofold differences were detected in the expression of 555 genes between wild-type and homozygous GCD II PCFs. Of these, 319 genes were upregulated, and 236 genes were downregulated in the homozygous GCD II PCFs. The most abundant and consistent changes were observed in gene families encoding signal transduction pathways involving the TGF- receptor-and integrin-mediated signaling, cell differentiation and proliferation, immune responses, cell adhesion, extracellular matrix (ECM) proteolytic enzymes, cell cycle, cytoskeletal organization, mitochondrial energy metabolism, collagen catabolism, response to wounding, response to oxidative stress, and the ubiquitin-mediated proteasomal degradation pathway. Cell adhesion assays demonstrated that heterozygous and homozygous GCD II PCFs strongly attached to collagen-I, collagen-IV, fibronectin, and laminin, compared with wild-type cells. CONCLUSIONS. Alterations in the TGF- receptor-and integrinmediated signaling pathway may play a key role in GCD II pathophysiology. If the novel factors identified in this study are involved in GCD II pathogenesis, they could assist in designing further studies to elucidate specific mechanisms of this disease.
Investigative Opthalmology & Visual Science, 2009
PURPOSE. To determine the relationship between signaling by different growth factors and the phases of corneal stromal wound repair. The authors hypothesize that the process involves sequential signaling, resulting first in proliferation and then in extracellular matrix (ECM) synthesis. METHODS. The effects of IGF-I, TGF-1, FGF-2, and PDGF on proliferation and ECM production by primary cultured bovine keratocytes were evaluated. DNA synthesis was determined by 3 H-thymidine incorporation, and maximal cell density was determined by measurement of DNA content. Relative levels of ECM components synthesized by keratocytes and secreted into the media were evaluated by 3 H-glycine incorporation into total ECM protein and collagen, by 3 H-glucosamine incorporation into chondroitin sulfate, keratan sulfate, and hyaluronan, and by Western blotting with antibodies specific to procollagen types ⌱ and ⌱⌱⌱. RESULTS. FGF-2 stimulated the highest level of proliferation and the lowest level of glycosaminoglycan synthesis and inhibited the synthesis of collagen types ⌱ and ⌱⌱⌱. IGF-I, in contrast, stimulated the lowest level of proliferation and the highest levels of collagen synthesis. PDGF and TGF-1 had intermediate effects on proliferation and collagen synthesis. Although FGF-2 inhibited collagen production, it could be restored by subsequent treatment with IGF-I, TGF-1, and PDGF. CONCLUSIONS. The results of this study showed that the level of proliferation induced by the growth factors was inversely related to the levels of collagen production. The authors suggest that FGF-2 initiates the hypercellular phase of corneal wound healing and that IGF-I and PDGF are involved in the restoration of a normal ECM.