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G. M. Gordon, D. R. Ledee, M. E. Fini; MMP-9 Regulation in Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3377.
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The critical role of MMP-9 in corneal wound healing is well established, therefore understanding the mechanisms regulating MMP-9 is important. Potential regulatory stimuli range from circulating salt, glucose or cytokine levels to cell-cell or cell-matrix interactions. Previously, our lab found that TGF-β and IL-1 families cooperate to strongly induce MMP-9 gene expression in corneal epithelial cells (CECs). In this study, we investigate the intracellular signaling pathways mediating this induction.
Primary cultures of Rabbit CECs, confirmed by immunostaining for cytokeratins 3/12, were used for all experiments. The CECs were co-transfected with an MMP-9 promoter-driven β-gal construct and an sv40 promoter-driven luciferase construct. The co-transfected cells were treated with TGF-β2 or IL-1β individually or in combination in the presence or absence of various pathway inhibitors (JSH-23, SB202190, PD98059, SIS3, and SP600125) for 24 hours. Cell lysates were harvested and β-Gal activity determined and normalized to luciferase activity.
Inhibition of the p38, NF-kB, or JNK pathways significantly reduced, but did not abrogate, basal MMP-9 promoter activity levels. Following TGF-β treatment, NF-kB, Smad3, or JNK inhibition significantly reduced MMP-9 promoter activity (p=0.002, 0.001, 0.009 respectively), whereas an effect on MMP-9 mediated reduction due to p38 inhibition was less significant (p=0.083). IL-1 mediated MMP-9 promoter activity was significantly reduced following NF-kB or p38 inhibition (p=0.014, 0.005 respectively), however, inhibition of Smad3 had a lower significant effect (p=0.051). Inhibition of the ERK pathway did not have an effect on MMP-9 mediated expression in either the treated or untreated co-transfected cells.
Many signaling pathways are involved in the regulation of MMP-9 in CECs, most notably the NF-kB, p38 and JNK pathways. Our data suggest TGF-β mediated induction of MMP-9 promoter activity is 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.
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