Central role for Rho in TGF-[[beta].sub.1]-induced [alpha]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].sub.1] (TGF-[beta].sub.1]) plays a key role in EMT, the underlying cellular mechanisms are not well understood. Here we characterized TGF-[beta].sub.1]-induced EMT in LLC-[PK.sub.1] 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].sub.1] treatment caused delocalization and downregulation of cell contact proteins (ZO-1, E-cadherin, [beta].sub.1]-catenin), cytoskeleton reorganization (stress fiber assembly, myosin light chain phosphorylation), and robust SMA synthesis. TGF-[beta].sub.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].sub.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].sub.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. Rho kinase; epithelial-mesenchymal transdifferentiation; transforming growth factor-[beta].sub.1]; kidney proximal tubule cells