LIM-kinase 2 and cofilin phosphorylation mediate actin cytoskeleton reorganization induced by transforming growth factor-beta

Reorganization of the actin cytoskeleton in response to growth factor signaling, such as transforming growth factor beta (TGF-beta), controls cell adhesion, motility, and growth of diverse cell types. In Swiss3T3 fibroblasts, a widely used model for studies of actin reorganization, TGF-beta1 induced rapid actin polymerization into stress fibers and concomitantly activated RhoA and RhoB small GTPases. Consequently, dominant-negative RhoA and RhoB mutants blocked TGF-beta1-induced actin reorganization. Because Rho GTPases are known to regulate the activity of LIM-kinases (LIMK), we found that TGF-beta1 induced LIMK2 phosphorylation with similar kinetics to Rho activation. Cofilin and LIMK2 co-precipitated and cofilin became phosphorylated in response to TGF-beta1, whereas RNA interference against LIMK2 blocked formation of new stress fibers by TGF-beta1. Because the kinase ROCK1 links Rho GTPases to LIMK2, we found that inhibiting ROCK1 activity blocked completely TGF-beta1-induced LIMK2/cofilin phosphorylation and downstream stress fiber formation. We then tested whether the canonical TGF-beta receptor/Smad pathway mediates regulation of the above effectors and actin reorganization. Adenoviruses expressing constitutively activated TGF-beta type I receptor led to robust actin reorganization and Rho activation, whereas the constitutively activated TGF-beta type I receptor with mutated Smad docking sites (L45 loop) did not affect either actin organization or Rho activity. In line with this, ectopic expression of the inhibitory Smad7 inhibited TGF-beta1-induced Rho activation and cytoskeletal reorganization. Our data define a novel pathway emanating from the TGF-beta type I receptor and leading to regulation of actin assembly, via the kinase LIMK2.     

LIM-domain proteins in transforming growth factor β-induced epithelial-to-mesenchymal transition and myofibroblast differentiation

Epithelial to mesenchymal transition (EMT) is a process during which junctions of the cell-cell contacts are dissolved, actin cytoskeleton is deformed, apical-basolateral cell polarity is lost and cell motility is increased. EMT is needed during normal embryonal development and wound healing, but may also lead to pathogenic transformation and formation of myofibroblasts. Transforming growth factor β (TGFβ) is a multifunctional cytokine promoting EMT and myofibroblast differentiation, and its dysregulation is involved in pathological disorders like cancer and fibrosis. Lin11, Isl-1 and Mec-3 (LIM) domain proteins are associated with actin cytoskeleton and linked to regulation of cell growth, damage signaling, cell fate determination and signal transduction. LIM-domain proteins generally do not bind DNA, but are more likely to function via protein-protein interactions. Despite being a disparate group of proteins, similarities in their functions are observed. In this review we will discuss the role of LIM-domain proteins in TGFβ-signaling pathway and in EMT-driven processes. LIM-domain proteins regulate TGFβ-induced actin cytoskeleton reorganization, motility and adhesion, but also dissolution of cell-cell junctions during EMT. Finally, the role of LIM-domain proteins in myofibroblasts found in fibrotic foci and tumor stroma will be discussed.     

TGF-beta1-mediated fibroblast-myofibroblast terminal differentiation-the role of Smad proteins

It is now clear that resident myofibroblasts play a central role in the mediation of tissue fibrosis. The aim of the work outlined in this study is to increase our understanding of the mechanisms which drive the phenotypic and functional changes associated with the differentiation process. We have used an in vitro model of transforming growth factor-beta1 (TGF-beta1)-induced pulmonary fibroblast-myofibroblast differentiation to examine the role of the TGF-beta1 Smad protein signaling intermediates, in alterations of fibroblast phenotype and function associated with terminal differentiation. TGF-beta1 induced marked alteration in cell phenotype, such that cells resembled "epithelioid-postmitotic fibroblasts." This was associated with marked reorganization of the actin cytoskeleton and upregulation of alphaSMA gene expression. TGF-beta1 stimulation also induced alphaSMA protein expression with increased incorporation of alphaSMA into stress fibers. Following stimulation with TGF-beta1, subsequent addition of serum-free medium did not reverse TGF-beta1-induced morphological change, suggesting that TGF-beta1 induced a relatively stable alteration in fibroblast cell phenotype. Functionally, these phenotypic changes were associated with induction of type I, type III, and type IV collagen gene expression and an increase in the concentrations of the respective collagens in the cell culture supernatant. The role of Smad proteins in terminal differentiation of fibroblasts was examined by transfection of cells, with expression vectors for the TGFbeta1 receptor-regulated Smads (R-Smads) or the co-Smad, Smad 4. Transfection with Smad2 but not Smad3 resulted in TGF-beta1 independent alteration in fibroblast cell phenotype, up-regulation of alphaSMA mRNA and reorganization of the actin cytoskeleton. Transfection with Smad4 also induced alteration in cell phenotype, although this was not as pronounced as the effect of overexpression of Smad2. Overexpression of the Smad2, Smad3, or Smad4 proteins was associated with increased production of all collagen types. The study suggests that the phenotypic and functional changes associated with TGF-beta1-induced fibroblast terminal differentiation are differentially regulated by Smad proteins.     

Cofilin-1 signaling mediates epithelial-mesenchymal transition by promoting actin cytoskeleton reorganization and cell-cell adhesion regulation in colorectal cancer cells

Colorectal cancer (CRC) is frequently a lethal disease because of metastasis. Actin cytoskeletal rearrangement is an essential step in cell migration during activation of the epithelial-mesenchymal transition (EMT) program, which is associated with metastatic properties of cancer cells. Cofilin-1 protein modulates actin dynamics by promoting actin treadmilling, thereby driving membrane protrusion and cell migration and invasion. However, the role of cofilin-1 during EMT in CRC is unknown. Here, we show that cofilin-1 and p-cofilin-1 have distinct subcellular distribution in EMT cells, as determined by super-resolution microscopy images, indicating distinct roles in different areas of cells. Silenced cofilin-1 cells treated with TGF-β (siCofilin-1/TGF-β) evaded p-LIMK2-p-cofilin-1 status, leading to recovery of E-cadherin and claudin-3 at the cell-cell contact and their respective protein levels, actin reorganization, and decreased mesenchymal protein level. Furthermore, siCofilin-1/TGF-β cells exhibited decreased migration and invasion rates as well as MMP-2 and -9 activity and augmented focal adhesion size. The expression of an inactive phospho-cofilin-1 mimetic (S3E) reduced E-cadherin and claudin-3 in cell-cell contacts, reduced their protein levels, and increased vimentin protein. Based on our findings, we suggest that cofilin-1 is crucial to switching from epithelial to mesenchymal-like morphology and cell migration and invasion by regulating actin cytoskeleton organization through activation of RhoA-LIMK2-cofilin-1 signaling, impacting the cell-cell adhesion organization of colon cancer cells in EMT.     

Inflammatory cytokines augments TGF-beta1-induced epithelial-mesenchymal transition in A549 cells by up-regulating TbetaR-I

Epithelial-mesenchymal transition (EMT) is believed to play an important role in fibrosis and tumor invasion. EMT can be induced in vitro cell culture by various stimuli including growth factors and matrix metalloproteinases. In this study, we report that cytomix (a mixture of IL-1beta, TNF-alpha and IFN-gamma) significantly enhances TGF-beta1-induced EMT in A549 cells as evidenced by acquisition of fibroblast-like cell shape, loss of E-cadherin, and reorganization of F-actin. IL-1beta or TNF-alpha alone can also augment TGF-beta1-induced EMT. However, a combination of IL-1beta and TNF-alpha or the cytomix is more potent to induce EMT. Cytomix, but not individual cytokine of IL-1beta, TNF-alpha or IFN-gamma, significantly up-regulates expression of TGF-beta receptor type I (TbetaR-I). Suppression of TbetaR-I, Smad2 or Smad3 by siRNA partially blocks EMT induction by cytomix plus TGF-beta1, indicating cytomix augments TGF-beta1-induced EMT through enhancing TbetaR-I and Smad signaling. These results indicate that inflammatory cytokines together with TGF-beta1 may play an important role in the development of fibrosis and tumor progress via the mechanism of epithelial-mesenchymal transition.