title: MMP-7 knock-in corneal fibroblast cell lines secrete MMP-7 with proteolytic activity towards collagen XVIII.
abstract: 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.
authors.affiliation: Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, USA.
authors.forename: Shu-I
authors.initials: SI
authors.lastname: Yeh
authors.forename: Kyu-Yeon
authors.initials: KY
authors.lastname: Han
authors.forename: Abdellah
authors.initials: A
authors.lastname: Sabri
authors.forename: Mark I
authors.initials: MI
authors.lastname: Rosenblatt
authors.forename: Dimitri T
authors.initials: DT
authors.lastname: Azar
authors.forename: Sandeep
authors.initials: S
authors.lastname: Jain
authors.forename: Jin-Hong
authors.initials: JH
authors.lastname: Chang
mesh_terms: 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
chemical_list: D000952:Antigens, Polyomavirus Transforming; D043170:Collagen Type XVIII; D020783:Matrix Metalloproteinase 7
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