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C-K Joo; A novel TGF-beta sigaling in EMT-related gene expression . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2986.
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Purpose: It has been reported that transdifferentiation such as epithelium-mesenchyme transition (EMT) is the multiple process in time-dependent regulation of various genes. To investigate an early event of EMT induced by TGF-beta, we focused on Crk-associated substrate (Cas), a adaptor protein localized at focal adhesions and stress fiber, which is known to have important functions in cell migration and the induction of immediate early gene expression. Methods: Analysis of Cas phosphorylation induced by TGF-beta was performed by immunoprecipitation using anti-Cas antibody and immunoblotting with anti-phosphotyrosine antibody. The activities of several kinases were analyzed by immunocomplex kinase assay using various substrates. In addition, to verify TGF-beta signal pathway, we also utilized other methods such as immunofluorescence staining, cell transfection, and the analysis for fibronectin promoter-responsive luciferase activity. Results: The tyrosine phosphorylation of Cas induced by TGF-beta is cell-type specific and related to src kinase signal pathway. Addition of TGF-beta to various epithelial cell lines, including human lens, kidney, breast, and skin, rapidly induced tyrosine phosphorylation of Cas and association between Cas and Crk. TGF-beta also stimulated the activity of Src kinase family and Src kinase specific inhibitors completely blocked the tyrosine phosphorylation of Cas by TGF-beta. Tyrosine phosphorylation of Cas was induced in an epithelial cell type specific manner. In addition, stable transfection of E-cadherin to L cells and E-cadherin blocking assay revealed that E-cadherin-mediated cell-cell interaction was required to Cas phosphorylation by TGF-beta. Especially, we found that TGF-beta-responsive fibronectin gene expression was subject to this Src-Cas mediated signaling pathway. Conclusion: Our data suggest that rapid Cas phosphorylation and Src kinase activation induced by TGF-beta may play a novel role in EMT-related gene expression.
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