Regulation of MMP-9 production by human corneal epithelial cells.
The matrix metalloproteinases, MMP-2 and MMP-9, are known to be critical extracellular-remodeling enzymes in wound healing and other diseases of the ocular surface. This study investigated the regulation of MMP-2 and MMP-9 in human corneal epithelial cells by growth factors and pro-inflammatory cytokines (IL-1beta and TNF-alpha) they are exposed to, and by doxycycline, a medication used to treat ocular surface disease. Primary human corneal epithelial cell cultures were treated with one of the following cytokines (IL-1alpha, IL-1beta, IL-6, IL-8, TNF-alpha) or growth factors (EGF, HGF, KGF, PDGF-BB, TGF-alpha, TGF-beta), with or without their corresponding inhibitors. The conditioned media were collected after 24 hr for gelatin zymography and MMP-9 activity assay. Total RNA was extracted from the cells treated for 6 hr and was subjected to RT-PCR and Northern hybridization. Between the two gelatinases, MMP-2 and MMP-9, detected by zymography, the 92 kDa MMP-9 in the conditioned medium was markedly up-regulated by the pro-inflammatory cytokines, IL-1beta and TNF-alpha. The MMP-9 protein and activity were dose-dependently stimulated by IL-1beta or TNF-alpha at 0.1, 1.0 and 10 ng ml(-1). This up-regulation was nearly abolished by neutralizing antibodies (IL-1beta 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-1beta and TNF-alpha. Doxycycline (10 microg ml(-1)) suppressed MMP-9 protein level and activity, but not its mRNA, that was stimulated by IL-1beta 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-1beta and TNF-alpha. These factors may play a role in the pathogenesis of MMP-9 mediated corneal matrix degradation. The efficacy of doxycycline in treating ocular surface diseases may be related to its ability to suppress MMP-9 production in the corneal epithelium.
Li
D Q
DQ
Ocular Surface Center, Cullen Eye Institute, Baylor College of Medicine, Houston, TX 77030, USA.
Lokeshwar
B L
BL
Solomon
A
A
Monroy
D
D
Ji
Z
Z
Pflugfelder
S C
SC
D018929:Cell Culture Techniques; D017077:Culture Media, Conditioned / Q000378:metabolism; D016207:Cytokines / Q000494:pharmacology; D019573:Epithelium, Corneal / Q000187:drug effects / Q000201:enzymology*; D005786:Gene Expression Regulation; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D020778:Matrix Metalloproteinase 2 / Q000096:biosynthesis; D020780:Matrix Metalloproteinase 9 / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D017077:Culture Media, Conditioned; D016207:Cytokines; D007375:Interleukin-1; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9