| Abstract |
Regulation of actin reorganization and contractility allows cells to control their shape, movement, division and secretion, vital processes known to be coordinated by the action of several signal transduction pathways. Rapid reorganization of the actin cytoskeleton is one of the earliest cellular responses to many extracellular signals. Transforming growth factor β (TGF-β) regulates cell growth and differentiation and induces motility of various cell types. The basic TGF-β signaling apparatus consists of a plasma membrane complex of type I and type II receptors and downstream Smad signaling effectors that regulate expression of target genes. Other signaling pathways are also activated by TGF-β. The existence of such pathways raises several important questions, such as if they are tryly independent of Smads. Reorganization of the actin cytoskeleton in response to growth factor signaling, such as transforming growth factor β (TGF-β), controls cell adhesion, motility and growth of diverse cell types. In Swiss3T3 and mouse embryonic fibroblasts (MEFs), treatment with TGF-β1 resulted in cell flattening, which was supported by changes in the organization of the actin cytoskeleton. Newly formed stress fibers were prominent in the treated cells and their abundance and length clearly increased with prolonged TGF-β1 treatment. Concomitantly, TGF-β1 induced activation of RhoA and RhoB small GTPases. Cells expressing dominant-negative RhoA o dominant-negative RhoB could not form new actin stress fibers in response to TGF-β1 whereas the surrounding non-transfected cells showed robust actin reorganization. As Rho GTPases are known to regulate the activity of LIM-kinases (LIMK), we found that TGF-β1 induced LIMK2 phosphorylation with similar kinetics to Rho activation. Cofilin and LIMK2 co-precipitated and cofilin became phosphorylated in response to TGF-β1, while RNA interference against LIMK2 blocked formation of new stress fibers by TGF-β1. No significant phosphorylation of LIMK1 was detected after short or long term incubation of Swiss3T3 cells with TGF-β1. Since the kinase ROCK1 links Rho GTPases to LIMK2, we found that inhibiting ROCK1 activity blocked completely TGF-β1-induced LIMK2/cofilin phosphorylation and downstream stress fiber formation. A wounding assay revealed that TGF-β1 induces increased cell motility of Swiss3T3 fibroblasts, which is blocked by the ROCK1 specific inhibitor Y27632. We then tested whether the canonical TGF-β receptor/Smad pathway mediates regulation of the above effectors and actin reorganization. Adenoviruses expressing constitutively activated TGF-β type I receptor, led to robust actin reorganization and RhoA activation, while the constitutively activated TGF-β type I receptor with mutated Smad docking sites (L45 loop) did not affect neither actin organization nor Rho activity. In line with this, ectopic expression of the inhibitory Smad7 inhibited TGF-β1-induced Rho activation and cytoskeletal reorganization. Adenoviral expression of the cytoplasmic Smads, Smad2 and Smad3 induced actin reorganization and RhoA activation in the absence of TGF-β1. Finally, TGF-β1 induces α-smooth muscle actin expression, which is not blocked by the ROCK1 inhibitor Y27632. Ectopic expression of Smad3 (and to a lesser degree of Smad2), induced α-SMA expression in the absence of TGF-β1. In conclusion, our study reveals a new non-genomic signaling cascade downstream of the TGF-β type I receptor that involves well-known effectors such as Rho GTPases and downstream kinases ROCK1 and LIMK2, transmitting a signal to cofilin and modulating its ability to shift the actin polymerization equilibrium in a positive manner. Furthermore, we have identified novel structural determinants of the type I receptor that may link to the activators of the Rho GTPases and a possible point of convergence of the non-genomic Rho pathway with the cytoplasmic Smads. Thus, our study offers a deeper insight into the alternative signaling pathways initiated by the multifunctional polypeptide factor TGF-β aiming at the regulation of critical physiological responses such as actin cytoskeleton polymerization.
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