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Hyperosmotic stress initiates adaptive responses, including phosphorylation of myosin light chain (MLC) and concomitant activation of [Na.sup.+] -[K.sup.+] -[Cl.sup.-] 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 (20 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.sup.+] and [K.sup.+] at normal total salinity failed to activate Rho, and [Cl.sup.-] 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; [Na.sup.+]-[K.sup.+]-[Cl.sup.-] cotransporter

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

Background: Transforming growth factor-beta (TGFbeta)-induced epithelial-myofibroblast transdifferentiation is a central mechanism contributing to the pathogenesis of progressive tubulo-interstitial fibrosis. We wanted to dissect the role of extracellular signal-regulated protein kinase (ERK1,2), p38 mitogen-activated protein kinase (p38 MAPK) and the receptor-regulated Smad proteins in the regulation of alpha-smooth muscle cell actin (alphaSMA) expression, a hallmark of myofibroblast formation, induced by TGFbeta in renal proximal tubular cells., Methods: Activation of signalling molecules was assessed by western blotting using phospho-specific antibodies. To specifically interfere with signalling cascades, porcine proximal tubular cells (LLC-PK/AT1) were infected with recombinant replication-deficient adenoviruses. In other experiments, specific kinase inhibitors were used. The alphaSMA synthesis was assessed by western blotting or immunofluorescent staining of cellular alphaSMA. To assess the regulation of the alphaSMA promoter, tubular cells were transiently transfected with a 785 bp alphaSMA promoter-luciferase reporter construct and vectors interfering with the Smad pathway., Results: Blocking ERK1,2 activation with PD98059 or p38 MAPK with SB 203580 potently inhibited the TGFbeta-induced alphaSMA synthesis in renal tubular cells. Adenoviral expression of dominant negative (DN) p38beta but not of p38alpha potently inhibited alphaSMA expression. Furthermore, adenoviral expression of DN MKK6b but not of DN MKK3b caused a substantial inhibition of the TGFbeta effect, confirming the role of p38beta in the regulation of TGFbeta-induced alphaSMA expression. Finally, inhibiting the Smad pathway with adenovirally delivered Smad7 and DN Smad3 also blocked TGFbeta-induced alphaSMA synthesis., Conclusion: 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.

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.