Central role for Rho in TGF-beta1-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(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-PK(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(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.
Masszi
Andras
A
Department of Surgery, The Toronto General Hospital, Toronto, Ontario M5G 1L7, Canada.
Di Ciano
Caterina
C
Sirokmány
Gábor
G
Arthur
William T
WT
Rotstein
Ori D
OD
Wang
Jiaxu
J
McCulloch
Christopher A G
CA
Rosivall
László
L
Mucsi
István
I
Kapus
András
A
D000199:Actins / Q000378:metabolism*; D000209:Acute-Phase Proteins / Q000502:physiology*; D000818:Animals; D002454:Cell Differentiation / Q000502:physiology; D003599:Cytoskeleton / Q000502:physiology; D004847:Epithelial Cells / Q000166:cytology; D018374:LLC-PK1 Cells; D008648:Mesoderm / Q000166:cytology; D009130:Muscle, Smooth / Q000378:metabolism*; D013552:Swine; D016212:Transforming Growth Factor beta / Q000502:physiology*; D053773:Transforming Growth Factor beta1
D000199:Actins; D000209:Acute-Phase Proteins; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; C010781:acute-phase protein rho