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.
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.
Authors: Bridgette L Berryhill; Ronald Kader; Bradley Kane; David E Birk; Jessie Feng; John R Hassell Journal: Invest Ophthalmol Vis Sci Date: 2002-11 Impact factor: 4.799
Authors: James V Jester; Jiying Huang; Stephen Fisher; Jennifer Spiekerman; Jin Ho Chang; Woodring E Wright; Jerry W Shay Journal: Invest Ophthalmol Vis Sci Date: 2003-05 Impact factor: 4.799
Authors: Julie T Daniels; Alison D Cambrey; Nicholas L Occleston; Qian Garrett; Roy W Tarnuzzer; Gregory S Schultz; Peng T Khaw Journal: Invest Ophthalmol Vis Sci Date: 2003-03 Impact factor: 4.799
Authors: Susanne Strand; Petra Vollmer; Lothar van den Abeelen; Daniela Gottfried; Vijay Alla; Hans Heid; Jürgen Kuball; Matthias Theobald; Peter R Galle; Dennis Strand Journal: Oncogene Date: 2004-04-29 Impact factor: 9.867