Purchase this article with an account.
JL Funderburgh, MM Mann, ML Funderburgh; Cell-Cycle Dependent Pleiotropic Action of Transforming Growth Factor Beta on Keratocytes . Invest. Ophthalmol. Vis. Sci. 2002;43(13):859.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose:In corneal wounds, keratocytes of the stroma develop smooth muscle alpha actin cytoskeletal elements. Formation of these myofibroblasts results from transforming growth factor beta (TGFß) action both in vivo and in vitro. We recently showed (J Biol Chem 276, 44173) that myofibroblasts derived from primary bovine keratocytes secrete of components of fibrotic extracellular matrix typical of corneal scar tissue. This fibrotic matrix secretion is elicited by TGFß, in the presence of serum but not in serum-free media. The purpose of this study was to investigate the role of serum and other mitogens in the action of TGFß on induction of transdifferentiation of keratocytes to fibrogenic myofibroblasts. Methods:Primary keratocytes were obtained by collagenase digestion of bovine stromas and maintained as quiescent cultures in serum-free media. Myofibroblast transdifferentiation was induced by recombinant TGFß-1 in the presence of of sera or purified mitogens. Transdifferentiation was monitored using western blotting, immunohistology, and metabolic labeling methods for identification of collagen, proteoglycans, cytoskeletal components, and fibronectin. Cell cycle entry was determined by incorporation of tritiated thymidine, bromodeoxyuridine, or by immunodetection of Ki67 antigen. Results:Fibrotic matrix components and smooth muscle actin accumulation in keratocyte cultures in response to TGFß was minimal in serum-free media but was markedly stimulated by a variety of mitogens including: sera, platelet-derived growth factor, insulin-like growth factor, and fibroblast growth factor-2. Biotinylation of cell-surface proteins and immune-precipitation with antibodies to TGFß receptors I and II showed the presence of these proteins on quiescent keratocytes. TGFß treatment of keratocytes in serum-free conditions led to rapid phosphorylation of SMAD 2 and nuclear translocation of SMADs 2 and 4. TGFß stimulated thymidine and bromodeoxyuridine incorporation into keratocytes that were exposed to mitogens for more than 24 hr, but inhibited DNA synthesis and Ki67 antigen expression when TGFß was present during the first 6 hr of mitogen exposure. Conclusion:TGFß appears to act on keratocytes with two distinct modes. In quiescent cells TGFß stimulates signal transduction and retards entry into the cell cycle but has no fibrogenic action. In actively dividing (fibroblastic) stromal cells, TGFß is both mitogenic and fibrogenic. This pleiotropic response pattern may be important in determining the response of keratocytes to inflammation and wounding in vivo.
This PDF is available to Subscribers Only