MMP-7 knock-in corneal fibroblast cell lines secrete MMP-7 with proteolytic activity towards collagen XVIII.
OBJECTIVE
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.


CONCLUSIONS
Immortalized fibroblasts stably transfected with MMP-7 secrete active MMP-7 with proteolytic activity towards the NC1 fragment of collagen XVIII.
Yeh
Shu-I
SI
Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, USA.
Han
Kyu-Yeon
KY
Sabri
Abdellah
A
Rosenblatt
Mark I
MI
Azar
Dimitri T
DT
Jain
Sandeep
S
Chang
Jin-Hong
JH
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D002460:Cell Line; D043170:Collagen Type XVIII / Q000378:metabolism*; D003315:Cornea / Q000201:enzymology*; D005347:Fibroblasts / Q000201:enzymology*; D005434:Flow Cytometry; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D055879:Gene Knock-In Techniques; D005822:Genetic Vectors; D020783:Matrix Metalloproteinase 7 / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D016133:Polymerase Chain Reaction; D012190:Retroviridae / Q000235:genetics; D014162:Transfection
D000952:Antigens, Polyomavirus Transforming; D043170:Collagen Type XVIII; D020783:Matrix Metalloproteinase 7
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