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.

The myocardium responds to chronic pressure or volume overload by activation and proliferation of cardiac fibroblasts and their differentiation into myofibroblasts. Because alpha-smooth muscle actin (SMA) expression is the classical marker for myofibroblast differentiation, we examined force-induced SMA expression and regulation by specific MAPK pathways. Rat cardiac fibroblasts were separated from myocytes and smooth muscle cells, cultured, and phenotyped by using the presence of SMA, vimentin, and ED-A fibronectin and the absence of desmin as myofibroblast markers. Static tensile forces (0.65 pN/microm2) were applied to fibroblasts via collagen-coated magnetite beads. In neonatal cardiac fibroblasts cultured for 1 day, immunostaining and Western and Northern blotting showed very low basal levels of SMA. After the application of force, there were 1.5- to 2-fold increases of SMA protein and mRNA within 4 h. Force-induced SMA expression was dependent on ERK phosphorylation and on intact actin filaments. In contrast to cells cultured for 1 day, cells grown for 3 days on rigid substrates showed prominent stress fibers and high basal levels of SMA, which were reduced by 32% within 4 h after force application. ERK was not activated by force, but p38 phosphorylation was required for force-induced inhibition of SMA expression. These results indicate that mechanical force-induced regulation of SMA content is dependent on myofibroblast differentiation and by selective activation of MAPKs.

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We examined mechanotranscriptional regulation of the contractile gene, ␣-smooth muscle actin (SMA), in osteoblastic cells. Tensile forces were applied through collagen-coated magnetite beads to ROS17/2.8 cells. These cells were desmin؊, vimentin؉ and expressed low levels of SMA. After force application (480 piconewton/ cell), SMA protein and mRNA were increased but ␤-actin was unchanged. Beads coated with bovine serum albumin or poly-L-lysine produced no change of SMA. In cells transiently transfected with plasmids containing the SMA promoter fused to ␤-galactosidase or green fluorescent protein coding sequences, SMA promoter activity was increased by ϳ60% after 4 h of force, whereas control (Rous sarcoma virus) promoter activity was unaffected. Transfections with ␤-galactosidase or green fluorescent protein reporter constructs showed that forceloaded cells exhibited higher ␤-galactosidase activity than cells without force. Cytochalasin D and latrunculin B inhibited force-induced increases of SMA promoter activity. Deletion analyses showed that SMA promoter activity was increased ϳ70% after force with a minimal construct containing 155 bp upstream of the translation start site. The force effect on the SMA promoter was abrogated in cells transfected with CArG-B box mutants. Gel mobility shift analyses of nuclear extracts showed strong binding to the CArG-B motif after force. We conclude that the CArG-B box is a force-responsive element in the SMA promoter.

The “Stribeck curve” is a well-known concept, describing the frictional behavior of a lubricated interface during the transition from boundary and mixed lubrication up to full-film hydrodynamic/elastohydrodynamic lubrication. It can be found in nearly every tribology textbook/handbook and many articles and technical papers. However, the majority of the published Stribeck curves are only conceptual without real data from either experiments or numerical solutions. The limited number of published ones with real data is often incomplete, covering only a portion of the entire transition. This is because generating a complete Stribeck curve requires experimental or numerical results in an extremely wide range of operating conditions, which has been a great challenge. Also, numerically calculating a Stribeck curve requires a unified model with robust algorithms that is capable of handling the entire spectrum of lubrication status. In the present study, numerical solutions in counterformal contacts of rough surfaces are obtained by using the unified deterministic mixed elastohydrodynamic lubrication (EHL) model recently developed. Stribeck curves are plotted in a wide range of speed and lubricant film thickness based on the simulation results with various types of contact geometry using machined rough surfaces of different orientations. Surface flash temperature is also analyzed during the friction calculation considering the mutual dependence between friction and interfacial temperature. Obtained results show that in lubricated concentrated contacts, friction continuously decreases as speed and film thickness increase even in the full-film regime until extremely high speeds are reached. This is mainly due to the reduction of lubricant limiting shear stress caused by flash temperature rise. The results also reveal that contact ellipticity and roughness orientation have limited influence on frictional behaviors, especially in the full-film and boundary lubrication regimes.

The mitogen-activated protein kinase p38 is activated by mechanical force, but the cellular elements that mediate force-induced p38 phosphorylation are not defined. As ␣-smooth muscle actin (SMA) is an actin isoform associated with force generation in fibroblasts, we asked if SMA participates in the activation of p38 by force. Tensile forces (0.65 pN/m 2 ) generated by magnetic fields were applied to collagen-coated magnetite beads bound to Rat-2 cells. Immunoblotting showed that p38␣ was the predominant p38 isoform. Analysis of bead-associated proteins demonstrated that SMA enrichment of collagen receptor complexes required the ␣2␤1 integrin. SMA was present almost entirely as filaments.