PAF-induced furin and MT1-MMP expression is independent of MMP-2 activation in corneal myofibroblasts

Abstract

Purpose: Corneal stromal myofibroblasts express the platelet-activating factor (PAF) receptor, but its role is unclear. In the present study, the effect of PAF on induction of metalloproteinases (MMPs) was investigated.

Methods: Rabbit corneal myofibroblasts were identified by immunodetection of alpha-smooth muscle (alpha-SM)-actin. MT1-MMP, MMP-2, MMP-9, and tissue inhibitor of matrix metalloproteinase (TIMP)-2 were detected by immunofluorescence. Cells were treated with 100 nM cPAF, with or without the PAF antagonist BN 50730 or the furin inhibitor nona-D-arg-NH(2). Gene-expression levels for furin, urokinase plasminogen activator, MMP-2, MMP-9, MT1-MMP, and TIMP-2 were determined by real-time PCR. Protein expression was assessed by Western blot. MMP-2 and -9 activity was determined by gelatin zymography. Active MT1-MMP levels were measured by ELISA.

Results: cPAF triggered significantly increased MT1-MMP, MMP-2, MMP-9, and TIMP-2 mRNA expression, followed by increased active MT1-MMP protein expression at 12 hours, whereas TIMP-2 protein increased at 24 hours. PAF also induced furin gene expression, followed by increased protein expression. Nona-D-arg-NH(2) blocked cPAF induction of MT1-MMP activity. PAF-treated myofibroblasts showed increased active MMP-9 protein, but unchanged MMP-2 activity. Pretreatment with BN 50730 blocked PAF-induced transcription and translation of these proteins.

Conclusions: PAF, through a receptor-mediated mechanism, induces a specific pattern of furin, MMP, and TIMP-2 expression in corneal myofibroblasts. MMP-2 activity was unchanged by PAF treatment. These results suggest that in response to the inflammatory mediator PAF, induction of MT1-MMP is independent of MMP-2 activity in corneal myofibroblasts. Thus, PAF-mediated changes in extracellular matrix composition surrounding the myofibroblasts could be important in regulating the corneal scarring process. Moreover, PAF antagonists could be useful in maintaining corneal transparency.