MMP-7 Knock-In Corneal Fibroblast Cell Lines Secrete MMP-7 with Proteolytic Activity towards Collagen XVIII

Author： Yeh Shu-I. Han Kyu-Yeon Sabri Abdellah Rosenblatt Mark I. Azar Dimitri T. Jain Sandeep Chang Jin-Hong

ISSN： 0271-3683

Source： Current Eye Research, Vol.35, Iss.9, 2010-09, pp. : 799-805

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Abstract

Purpose: To determine whether matrix metalloproteinase-7 (MMP-7) that is stably overexpressed by mouse corneal fibroblast cell lines exhibits proteolytic activity against the NC1 fragment of collagen XVIII.Methods: Corneal fibroblasts isolated from MMP-7 knockout (7ko) mice were subjected to SV40 T-antigen immortalization and stably transfected with a bicistronic retroviral vector encoding green fluorescence protein and active MMP-7. The resulting MMP-7 knock-in fibroblasts (7ko-MMP-7 cells) were isolated and enriched by fluorescence activated cell sorting (FACS). Culture media samples from 7ko and 7ko-MMP-7 cells were then incubated with the recombinant NC1 fragment of collagen XVIII, and NC1 degradation was monitored by immunoblotting.Results: Immunoblot analysis revealed that MMP-7 was present in lysates and culture media from 7ko-MMP-7 fibroblasts, but not media from immortalized 7ko fibroblasts. Importantly, lower amounts of the NC1 fragment were present in in vitro enzymatic reaction mixtures containing concentrated 7ko-MMP-7 media than in those containing concentrated 7ko media.Conclusion: Immortalized fibroblasts stably transfected with MMP-7 secrete active MMP-7 with proteolytic activity towards the NC1 fragment of collagen XVIII.