Purchase this article with an account.
G.P. Kwon, C.M. Garcia, X. Wang, J. Guo, A. Murphy, G. McEnroe, S. Dugar, S. Chakravarty, D.C. Beebe; Regulation of Genes Expressed Early in the Formation of Myofibroblasts from Lens Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4504.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: To identify transcripts expressed early in the transdifferentiation of lens epithelial cells to myofibroblasts and to determine the effects of FGF and TGFß signaling on gene expression and myofibroblast formation. Methods: RNA from P3 or adult rat lens epithelia cultured for 0 or 20 hours in unsupplemented medium or in medium with added FGF2 (50 ng/ml) was used to synthesize probes for Affymetrix microarrays. Standard and quantitative PCR were used to measure the expression of selected mRNAs from cultured rat and bovine lens epithelia. Immunostaining and Western blotting were performed with an antibody to α-smooth muscle actin (α-SMA). An inhibitor of TGFß signaling, SD-208 (Scios Inc., Sunnyvale CA), a potent (IC50 < 100 nM) and selective small molecule inhibitor of the TGFß RI receptor kinase, was added to cultured bovine lens epithelial explants to test the role of endogenous TGFß signaling in myofibroblast formation. Results: Analysis of rat and bovine lens epithelial cells using microarrays, PCR and Western blotting showed that α-SMA mRNA and protein are normally expressed in untreated lens epithelial cells. In P3 explants the mRNAs for several genes that are characteristic of myofibroblasts increased in the first 20 hours of culture. These included α-SMA (>4X), transgelin (SM22, ~100X), and desmin (>15X). Treatment with FGF2 reduced (SM22) or prevented (α-SMA, desmin) the accumulation of these transcripts. In cultured bovine lens epithelial cells, α-SMA protein levels increased up to six days of culture, then declined. α-SMA localized to stress fibers after four days of culture, but was dispersed throughout the cytoplasm in small aggregates in older cultures. SD-208 reduced the accumulation of α-SMA in cultured bovine lens epithelial cells in a dose-dependent manner (100 – 1,000 nM). However, even at the highest concentration used, the drug did not prevent the expression of α-SMA. Conclusions: Lens epithelial cells are poised to transdifferentiate into myofibroblasts, as they express α-SMA constitutively. After explantation, lens epithelial cells rapidly increase the expression of genes characteristic of myofibroblasts. As in other cell types, FGF signaling antagonizes this process. TGFß signaling contributes to myofibroblast formation, but other factors are likely to be involved.
This PDF is available to Subscribers Only