Regulation of MMP-9 production by human corneal epithelial cells

The matrix metalloproteinases, MMP-2 and MMP-9. are known to be critical ex tracellular-remodeling enzymes in wound healing and other diseases of the o cular surface. This study investigated the regulation of MMP-2 and MMP-9 in human corneal epithelial cells by growth factors and proinflammatory cytok ines (IL-1 beta and TNF-alpha) they are exposed to, and by doxycycline, a m edication used to treat ocular surface disease. Primary human corneal epith elial cell cultures were treated with one of the following cytokines (IL-1 alpha, IL-1 beta, IL-6, IL-8, TNF-alpha) or growth factors (EGF, HGF, KGF. PDGF-BB. TGF-alpha, TGF-beta), with or without their corresponding inhibito rs. The conditioned media were collected after 24 hr for gelatin zymography and MMP-9 activity assay. Total RNA was extracted from the cells treated f or 6 hr and was subjected to RT-PCR and Northern hybridization. Between the two gelatinises. MMP-2 and MMP-9, detected by zymography. the 92 kDa MMP-9 in the conditioned medium was markedly up-regulated by the pro-inflammator y cytokines, IL-1 beta and TNF-alpha. The MMP-9 protein and activity were d ose-dependently stimulated by IL-1 beta or TNF-alpha at 0.1, 1.0 and 10 ng ml(-1). This upregulation was nearly abolished by neutralizing antibodies ( IL-1 beta and TNF-alpha) and by IL-1 receptor antagonist. Semi-quantitative RT-PCR and Northern hybridization disclosed that the MMP-9 transcript was also markedly up-regulated in a dose-dependent manner by IL-1 beta and TNF- alpha. Doxycycline (10 mug ml(-1)) suppressed MMP-9 protein level and activ ity. but not its mRNA, that was stimulated by IL-1 beta and TNF-alpha (1 ng ml(-1)). In contrast. the 72 kDa MMP-2 was not significantly modulated by any of these cytokines. In conclusion. production of MMP-9 is stimulated by the pro-inflammatory cytokines. IL-1 beta and TNF-alpha. These factors may play a role in the pathogenesis of MMP-9 mediated corneal matrix degradati on. The efficacy of doxycycline in treating ocular surface diseases may be related to its ability to suppress MMP-9 production in the corneal epitheli um. (C) 2001 Academic Press.