Injury of the tubular epithelium and TGF-beta1-induced conversion of epithelial cells to alpha-smooth muscle actin (SMA)-expressing myofibroblasts are key features of kidney fibrosis. Since injury damages intercellular junctions and promotes fibrosis, we hypothesized that cell contacts are critical regulators of TGF-beta 1-triggered epithelial-to-mesenchymal transition (EMT). Here we show that TGF-beta 1 was unable to induce EMT in intact confluent monolayers, but three different models of injury-induced loss of epithelial integrity (subconfluence, wounding, and contact disassembly by Ca(2+)-removal) restored its EMT-inducing effect. This manifested in loss of E-cadherin, increased fibronectin production and SMA expression. TGF-beta 1 or contact disassembly alone only modestly stimulated the SMA promoter in confluent layers, but together exhibited strong synergy. Since beta-catenin is a component of intact adherens junctions, but when liberated from destabilized contacts may act as a transcriptional co-activator, we investigated its role in TGF-beta 1-provoked EMT. Contact disassembly alone induced degradation of E-cadherin and beta-catenin, but TGF-beta1 selectively rescued beta-catenin and stimulated the beta-catenin-driven reporter TopFLASH. Moreover, chelation of free beta-catenin with the N-cadherin cytoplasmic tail suppressed the TGF-beta1 plus contact disassembly-induced SMA promoter activation and protein expression. These results suggest a beta-catenin-dependent two-hit mechanism in which both an initial epithelial injury and TGF-beta 1 are required for EMT.

Epithelial-mesenchymal-myofibroblast transition (EMT),

New research suggests that, during tubulointerstitial fibrosis, α-smooth muscle actin (SMA)-expressing mesenchymal cells might derive from the tubular epithelium via epithelial-mesenchymal transition (EMT). Although transforming growth factor-β1(TGF-β1) plays a key role in EMT, the underlying cellular mechanisms are not well understood. Here we characterized TGF-β1-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-β1 treatment caused delocalization and downregulation of cell contact proteins (ZO-1, E-cadherin, β-catenin), cytoskeleton reorganization (stress fiber assembly, myosin light chain phosphorylation), and robust SMA synthesis. TGF-β1induced 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-β1-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-β1-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.

Smad3 inhibits activation of the smooth muscle actin promoter and functions as a timer for myogenic programming in the epithelium.

Two novel mechanisms are shown by which injury of intercellular junctions via β-catenin promotes epithelial–myofibroblast transition. β-Catenin interacts with Smad3, thereby preventing the inhibitory effect of the latter on myocardin-related transcription factor (MRTF), and maintains MRTF stability by inhibiting Smad3-mediated, GSK-3β–dependent degradation of MRTF.

Hyperosmotic stress initiates adaptive responses, including phosphorylation of myosin light chain (MLC) and concomitant activation of Na ϩ -K ϩ -Cl Ϫ cotransporter (NKCC). Because the small GTPase Rho is a key regulator of MLC phosphorylation, we investigated 1) whether Rho is activated by hyperosmotic stress, and if so, what the triggering factors are, and 2) whether the Rho/Rho kinase (ROK) pathway is involved in MLC phosphorylation and NKCC activation. Rho activity was measured in tubular epithelial cells by affinity pulldown assay. Hyperosmolarity induced rapid (Ͻ1 min) and sustained (Ͼ20 min) Rho activation that was proportional to the osmotic concentration and reversed within minutes upon restoration of isotonicity. Both decreased cell volume at constant ionic strength and elevated total ionic strength at constant cell volume were capable of activating Rho. Changes in [Na ϩ ] and [K ϩ ] at normal total salinity failed to activate Rho, and Cl Ϫ depletion did not affect the hyperosmotic response. Thus alterations in cellular volume and ionic strength but not individual ion concentrations seem to be the critical triggering factors. Hyperosmolarity induced mono-and diphosphorylation of MLC, which was abrogated by the Rho-family blocker Clostridium toxin B. ROK inhibitor Y-27632 suppressed MLC phosphorylation under isotonic conditions and prevented its rise over isotonic levels in hypertonically stimulated cells. ML-7 had a smaller inhibitory effect. In contrast, it abolished the hypertonic activation of NKCC, whereas Y-27632 failed to inhibit this response. Thus hyperosmolarity activates Rho, and Rho/ROK pathway contributes to basal and hyperosmotic MLC phosphorylation. However, the hypertonic activation of NKCC is ROK independent, implying that the ROK-dependent component of MLC phosphorylation can be uncoupled from NKCC activation. cell volume; ionic strength; small GTPases; Y-27632; ML-7; myosin light chain; NaOSMOTIC STRESS induces a variety of compensatory responses that can be classified into three major categories: activation of ion transport systems; changes in the transcription of osmosensitive genes and, consequently, in the expression of osmolyte-generating enzymes or solute carriers; and reorganization of the cytoskeleton (for reviews, see Refs. 28,35,38,and 53). Whereas considerable knowledge has accumulated about these vital adaptive responses, comparatively little is known about the signaling processes that link them to the initial osmotic insult. Regarding the cytoskeletal effects, our previous studies (8, 29) have shown that Rac and Cdc42, two members of the Rho family small GTPases, are stimulated by hyperosmotic stress and that their activation contributes to the osmotically provoked cytoskeleton remodeling. Specifically, these small GTPases appear to play a role in the reinforcement of the cortical cytoskeleton by facilitating peripheral de novo F-actin assembly (8, 29) and by inducing translocation of cortactin (8), a protein that potentiates actin polymerization and stabilizes newly gener...

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TGFbeta-induced alphaSMA expression is regulated by the coordinated activation of a complex system of parallel MAPK and Smad signalling pathways in renal proximal tubular cells during epithelial-mesenchymal transdifferentiation.