The development of fibrosis promotes the differentiation of myofibroblasts, pro-fibrotic cells, which contribute to tissue dysfunction. Myofibroblast differentiation is dependent on actin assembly, which in response to force, is mediated by various actin-binding proteins including the mammalian Diaphanous-related formins (mDia). We examined the role of mDia in the mechano-sensing pathway that leads to force-induced expression of ␣-smooth muscle actin (SMA), a marker and critical determinant of myofibroblast differentiation. In cells treated with siRNA to knockdown mDia and then subjected to tensile force using collagen-coated magnetite beads attached to ␤1 integrins, actin assembly was inhibited at bead contact sites. Force-induced nuclear translocation of MRTF-A, a transcriptional co-activator of SMA, was reduced 50% by mDia knockdown. The expression of the transcriptional co-activator of SMA, serum response factor, was reduced by 50% after siRNA knockdown of mDia or by 100% in cells transfected with catalytically inactive mDia. Force-induced activation of the SMA promoter and SMA expression were blocked by knockdown of siRNA of mDia. In anchored collagen gel assays to measure myofibroblast-mediated contraction, knockdown of mDia reduced contraction by 50%. We conclude that mDia plays an important role in the development of force-induced transcriptional activation of SMA and myofibroblast differentiation.During the development of fibrosis in heart, skin, liver, and kidney, fibroblasts are activated to become myofibroblasts (1). These cells strongly express ␣-smooth muscle actin (SMA) 3 and secrete abundant, disorganized collagen (2, 3), and other extracellular matrix molecules (4). Fibroblasts adhere to extracellular matrices through integrins (5, 6), cell surface receptors that provide sites for force transfer to the actin cytoskeleton and that are required for force-induced stimulation of SMA expression and myofibroblast differentiation (7). At sites of force transfer, focal adhesion proteins act as proximal sensors (8, 9) for translating mechanical forces into the signals that activate the Rho pathway (10). As a result, actin assembly is triggered (8), and there is enhanced nuclear translocation of the SMA transcriptional co-activator, myocardin-related transcription factor-A (MRTF-A) (10). Previous studies have shown that Rho signaling regulates the nuclear translocation of MRTF-A (11). Currently, the mechanisms by which force application regulates actin assembly that leads to myofibroblast differentiation are not well-defined.Actin assembly is regulated by formin proteins, which are involved in many cytoskeletal processes such as cytokinesis, actin stress fiber formation, neurite outgrowth, and intracellular trafficking (12). Members of a subfamily of formins, the mammalian Diaphanous-related formins (mDia), interact with activated, GTP-bound Rho family small GTPases. Interactions between Rho and mDia disrupt the binding of the Rho-binding domain to the diaphanous autoregulatory domain in formins, thereby ...

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