Central role for Rho in TGF-β1-induced α-smooth muscle actin expression during epithelial-mesenchymal transition

New research suggests that, during tubulointerstitial fibrosis, alpha- smooth muscle actin ( SMA)- expressing mesenchymal cells might derive from the tubular epithelium via epithelial- mesenchymal transition ( EMT). Although transforming growth factor-beta(1) ( TGF-beta(1)) plays a key role in EMT, the underlying cellular mechanisms are not well understood. Here we characterized TGF-beta(1)- induced EMT in LLC- PK1 cells and examined the role of the small GTPase Rho and its effector, Rho kinase, ( ROK) in the ensuing cytoskeletal remodeling and SMA expression. TGF-beta(1) treatment caused delocalization and downregulation of cell contact proteins ( ZO- 1, E- cadherin, beta- catenin), cytoskeleton reorganization ( stress fiber assembly, myosin light chain phosphorylation), and robust SMA synthesis. TGF-beta(1) induced a biphasic Rho activation. Stress fiber assembly was prevented by the Rho- inhibiting C3 transferase and by dominant negative ( DN) ROK. The SMA promoter was activated strongly by constitutively active Rho but not ROK. Accordingly, TGF-beta(1)- induced SMA promoter activation was potently abrogated by two Rho- inhibiting constructs, C3 transferase and p190RhoGAP, but not by DN- ROK. Truncation analysis showed that the first CC( A/ T) richGG ( CArG B) serum response factor- binding cis element is essential for the Rho responsiveness of the SMA promoter. Thus Rho plays a dual role in TGF-beta(1)- induced EMT of renal epithelial cells. It is indispensable both for cytoskeleton remodeling and for the activation of the SMA promoter. The cytoskeletal effects are mediated via the Rho/ ROK pathway, whereas the transcriptional effects are partially ROK independent.