Inhibition by a Selective IκB Kinase-2 Inhibitor of Interleukin-1–Induced Collagen Degradation by Corneal Fibroblasts in Three-Dimensional Culture (original) (raw)
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Investigative Opthalmology & Visual Science, 2004
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 threedimensional gels of type I collagen and in the absence or presence of IL-1 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-1-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-1-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-1-induced collagen degradation by these cells. Inhibition of the IL-1-induced activation of ERK and JNK may contribute to these effects of dexamethasone.
Investigative Ophthalmology & Visual Science, 2010
The authors previously showed that the expression of various junctional proteins in corneal epithelial cells or corneal fibroblasts (CFs) is regulated by the presence of the other cell type in a coculture system. In this study, the effect of corneal epithelial cells on the expression of matrix metalloproteinases (MMPs) in CFs was studied. Human CFs and simian virus 40-transformed human corneal epithelial (HCE) cells were cultured on opposite sides of a collagen vitrigel membrane. Expression of MMPs in CFs was examined by reverse transcription-polymerase chain reaction and immunoblot analyses. The amounts of MMP-2 mRNA and protein in CFs were decreased by the presence of HCE cells. HCE cells had no effect on the expression of MMP-1 in CFs. HCE cells released interleukin (IL)-1 receptor antagonist (IL-1RA) into the culture medium, and depletion of IL-1RA in HCE cells by RNA interference largely abolished the effect of these cells on MMP-2 expression in CFs. The downregulation of MMP-2 expression in CFs by HCE cells was blocked by an inhibitor of signaling by the mitogen-activated protein kinase (MAPK) ERK (PD98059) but was not affected by those of signaling by the MAPKs p38 (SB203580) or JNK (SP600125). Corneal epithelial cells downregulated expression of MMP-2 in CFs in a manner dependent at least in part on the release of IL-1RA from the former cells. This effect might contribute to the attenuation of corneal stromal remodeling by corneal epithelial cells.
Inhibition by Triptolide of IL-1–Induced Collagen Degradation by Corneal Fibroblasts
Investigative Opthalmology & Visual Science, 2003
PURPOSE. Extracts of the herb Tripterygium wilfordii hook f, the major component of which is triptolide, have been used in traditional Chinese medicine for the treatment of rheumatoid arthritis. Triptolide also exerts many other biological actions both in vitro and in vivo. The effect of this agent on collagen degradation by cultured corneal fibroblasts was examined. METHODS. Rabbit corneal fibroblasts were cultured in threedimensional gels of type I collagen and in the absence or presence of interleukin (IL)-1 or triptolide. The extent of collagen degradation was determined by measurement of the amount of hydroxyproline generated by acid-heat hydrolysis of the culture supernatants. The activities of matrix metalloproteinase (MMP)-1 and plasmin were measured with the specific substrates thiopeptolide and S-2251, respectively. The release of MMPs into the culture supernatant was assessed by immunoblot analysis and gelatin zymography, and the abundance of MMP mRNAs in the cells was determined by reverse transcription and real-time polymerase chain reaction. RESULTS. Triptolide inhibited the IL-1-induced degradation of collagen by corneal fibroblasts in a dose-and time-dependent manner. Neither the activity of purified recombinant MMP-1 nor that of plasmin in culture supernatants was affected by triptolide. The IL-1-induced expression of MMP-1,-2,-3, and-9 by corneal fibroblasts was inhibited by triptolide at the protein or mRNA level. CONCLUSIONS. Triptolide inhibits collagen degradation by corneal fibroblasts by inducing downregulation of the production of MMPs, without directly affecting the collagenolytic activity of these enzymes.
… 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.
Effects of Th2 Cytokines on Expression of Collagen, MMP-1, and TIMP-1 in Conjunctival Fibroblasts
Investigative Opthalmology & Visual Science, 2003
PURPOSE. To determine whether cytokines involved in chronic allergic conjunctival disorders may affect formation of giant papillae and tissue remodeling. METHODS. Conjunctival fibroblast cultures were challenged with different concentrations of human recombinant interleukin (IL)-4, IL-13, interferon (IFN)-␥ and tumor necrosis factor (TNF)-␣. Procollagens I (PIP) and III (PIIIP), matrix metalloproteinase (MMP)-1 and-9, and tissue inhibitor of metalloproteinase (TIMP)-1 were measured in supernatants, and their respective mRNAs were evaluated by RT-PCR. RESULTS. IL-4 and-13 (10 ng/mL) significantly increased production and expression of PIP compared with nonstimulated cells, whereas IFN-␥ elicited the opposite effect, at both the protein and mRNA levels. Both IL-4 and-13 significantly decreased production of MMP-1 and increased that of TIMP-1, whereas TNF-␣ increased production of MMP-1 and-9. Expression of MMP-1 was reduced by IL-4 and increased by the other tested cytokines, whereas expression of TIMP-1 was increased by all tested cytokines. CONCLUSIONS. IL-4 and-13 increased production of collagen and modified the equilibrium between MMP-1 and its inhibitor, TIMP-1. These effects were partially opposed by IFN-␥ and TNF-␣.
Journal of Cellular Physiology, 2009
Matrix metalloproteinase-9 (MMP-9) is a well known regulator and effecter of many cellular processes including wound healing. In the cornea, either too much or too little MMP-9 can be detrimental to overall wound repair. We investigated the secreted factors as well as the intracellular signaling pathways and the promoter sequences that mediate this regulation. Primary culture rabbit corneal epithelial cells were treated with various cytokines alone or in different combinations and MMP-9 induction was assessed by gel zymography. Pharmacological inhibitors were used to determine the intracellular signaling pathways induced by the cytokines tested and deletion promoter constructs were created to determine the regions of the MMP-9 promoter involved in the cytokine regulation, thereby assessing the exact transcription factors binding the MMP-9 promoter. We found that two cytokine families, TGF-β and IL-1, act additively in an isoform non-specific manner to induce MMP-9 in this cell type. Our data suggest TGF-β mediated MMP-9 induction may be regulated by the NF-kB, Smad3, and JNK pathways, whereas the IL-1β mediated induction may be regulated by the NF-kB and p38 pathways. Inhibition of the p38, NF-kB, or JNK pathways significantly reduced, but did not abrogate, basal MMP-9 levels. Inhibition of the ERK pathway did not have an effect on MMP-9 mediated expression in either the treated or untreated co-transfected cells.
Journal of Cellular Biochemistry, 2007
Collagenase-1 is a protease expressed by active fibroblasts that is involved in remodeling of the extracellular matrix (ECM). In this study, we characterize the intracellular signaling mechanism of collagenase-1 production by IL-1a in subcultured normal fibroblasts (NF) from uninjured normal corneas, compared to that in repair wound fibroblasts (WF). In NF, collagenase-1 was induced specifically after the exogenous addition of IL-1a via activation of ERK and p38MAPK. Collagenase-1 expression was strongly suppressed upon treatment with either a MEK or p38MAPK inhibitor. In contrast, repair WF constitutively synthesized both IL-1a and collagenase-1. Combined treatment with both mitogen-activated protein kinase (MAPK) inhibitors dramatically reduced collagenase-1 synthesis, while individual MEK1 or p38 inhibitors weakly modulated the collagenase-1 level. The results indicate that both pathways are crucial in the regulation of collagenase-1 synthesis. Furthermore, an IL-1a receptor antagonist (IL-1ra) could not abolish constitutive collagenase-1 synthesis, even at high doses, suggesting that other cytokines/factors are additionally involved in this process. We propose that induction of collagenase-1 by IL-1a in both WF and NF depends on a unique combination of cell type-specific signaling pathways.
Matrix metalloproteinase inhibition in corneal ulceration
Veterinary Clinics of North America: Small Animal Practice, 2004
The cornea and precorneal tear film combine to function as a strong refractive lens. To produce such an optically powerful structure, the corneal microanatomy consists of an epithelium and thin epithelial basement membrane, a thick relatively acellular stroma, Descemet's membrane, and a monolayered endothelium.
Investigative Ophthalmology & Visual Science, 2005
PURPOSE. Substance P (SP) is present in the sensory nerve fibers of the corneal epithelium. Various biological agents, including epidermal growth factor, fibronectin, interleukin-6, and the combination of SP and insulin-like growth factor (IGF)-1, promote the healing of corneal epithelial wounds. The role of SP in corneal epithelial migration was examined. METHODS. The effects of various agents on corneal epithelial migration were investigated with the rabbit cornea in an organ culture system. RESULTS. An SP-derived tetrapeptide, FGLM-amide, shifted the dose-response relations for the induction of corneal epithelial migration not only by an IGF-1-derived peptide (C-domain peptide) but also by fibronectin or interleukin-6 to lower concentrations. This action of SP was prevented by inhibitors of phospholipase C, of the inositol 1,4,5-trisphosphate receptormediated release of Ca 2ϩ from intracellular stores, and of Ca 2ϩand calmodulin-dependent protein kinase II (CaM-PK II).