title: IGF-II is present in bovine corneal stroma and activates keratocytes to proliferate in vitro.
abstract: Extracts of bovine corneal stroma have been shown to activate keratocytes in culture to proliferate. We fractionated stromal extract on a column of Sephacryl S-300 and tested the fractions for mitogenic activity using cell culture and for the presence of IGF-II and its binding protein IGFBP-2 by Western blot. We found that the mitogenic activity in the extract separated into major and minor peaks and that immunologically detectable IGF-II and IGFBP-2 co-eluted with the minor peak. We also compared the effects of 10 ng IGF-II/ml on keratocytes in culture to that of 2 ng TGF-beta/ml over a 7-day culture period. We found that IGF-II and TGF-beta, alone or combined, increased both (3)H-thymidine incorporation and DNA content of the cultures. The phenotype of the cells was determined by using antibodies to alpha-SM (smooth muscle) actin, fibronectin, SPARC, lumican and keratocan in Western blots of cell layers of media. Keratocytes cultured in IGF-II expressed no alpha-SM actin or fibronectin, low levels of SPARC and high levels of lumican and keratocan, indicating a native phenotype. Keratocytes in TGF-beta expressed alpha-SM actin, fibronectin, SPARC and lumican, and expressed no or low levels of keratocan, indicating a myofibroblast phenotype. Keratocytes cultured in IGF-II plus TGF-beta, however, expressed alpha-SM actin, fibronectin, SPARC, lumican, and keratocan by day 7 of culture. The results of this study show that IGF-II to be present in the corneal stroma, to stimulate keratocyte proliferation while maintaining native phenotype and to override the TGF-beta mediated down regulation of keratocan production. The IGF-II in the stroma may serve as a mechanism to immediately activate keratocytes upon wounding and to ameliorate the scarring effects of TGF-beta.
authors.affiliation: Department of Molecular Medicine, College of Medicine, University of South Florida, Tampa, FL 33612-4799, USA.
authors.forename: Kurt
authors.initials: K
authors.lastname: Musselmann
authors.forename: Bradley P
authors.initials: BP
authors.lastname: Kane
authors.forename: Bridgette
authors.initials: B
authors.lastname: Alexandrou
authors.forename: John R
authors.initials: JR
authors.lastname: Hassell
mesh_terms: D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002417:Cattle; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D003319:Corneal Stroma / Q000737:chemistry* / Q000166:cytology / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D018971:Insulin-Like Growth Factor Binding Protein 2 / Q000032:analysis; D007335:Insulin-Like Growth Factor II / Q000032:analysis* / Q000494:pharmacology; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology
chemical_list: D018971:Insulin-Like Growth Factor Binding Protein 2; D053773:Transforming Growth Factor beta1; D007335:Insulin-Like Growth Factor II