Abstract
Abstract: :
Purpose: Studies in our laboratory have shown that platelet activating factor (PAF) stimulates the gene expression of the inhibitors of metalloproteinases TIMP-1 and TIMP-2, in corneal epithelium. Furthermore, PAF induced an over-expression of MMP-9 mRNA relative to that of TIMP-1 and -2 (Ottino P. et al, Exp. Eye. Res., 2001, in press). In this study we investigated whether the increase in TIMP-1 and TIMP-2 mRNA expression in corneal epithelial cells is a direct result of PAF treatment or secondary to an increase in MMP-9 expression. Methods: Rabbit corneas were incubated in MEM medium containing 100 nM cPAF (carbamyl PAF, a non-hydrolyzable analog) for a total of 48 hours. At timed intervals (4, 8, 12, 24, 36, and 48 hours), supernatant was collected and TIMP-1 and -2 levels were determined by Western blot analysis. For mRNA studies, two sets of corneas were treated with cPAF for 4, 8 and 12 hours. From one set of corneas mRNA was extracted from the epithelium and the levels of gene expression for MMP-9, TIMP-1 and TIMP-2 were determined by real-time PCR. In the second set of corneas, the medium was replaced at 4, 8, and 12 hours with a medium containing PAF antagonist BN-50730 (10 µM) and further incubated for a total of 24 and 36 hours. This was followed by extraction of mRNA from corneal epithelium and quantitation of TIMP-1 and -2 gene expression by real-time PCR. Changes in the gene expression were reported as fold increase relative to those of untreated controls. Results: Treatment of rabbit corneas with cPAF for 4, 8, and 12 hours produced a significant increase in MMP-9 gene expression, while protein levels were increased as early as 8 hours. When such treatments were followed by inhibition of cPAF effect by BN 50730 for up to 36 hours, TIMP-1 mRNA expression was increased to levels similar to that of corneas treated only with cPAF for 36 hours. However, TIMP-2 mRNA expression correlated directly with the duration of cPAF treatment. Western blot analysis showed marked increases in TIMP-1 expression at 36, 48, and 72 hours in cPAF-treated corneas as compared to untreated controls, while TIMP-2 expression was increased as early as 24 hours. Conclusion: In summary, the PAF-induced increase in TIMP-1 gene expression is due to an increase in MMP-9 levels, while the induction of TIMP-2 gene is independent of MMP-9 expression. Induction of TIMP-1 could, therefore, be a mechanism by which the corneal epithelium attempts to counteract the strong induction of MMP-9 by cPAF during an injury. [Supported by NIH NEI EY04298]
Keywords: 372 cornea: epithelium • 417 gene/expression • 399 enzymes/enzyme inhibitors