Myofibroblasts are contractile, smooth muscle-like cells that are characterized by the de novo expression of smooth muscle α-actin (SMαA) and normally function to assist in wound closure, but have been implicated in pathological contractures. Transforming growth factor beta-1 (TGF-β1) helps facilitate the differentiation of fibroblasts into myofibroblasts, but the exact mechanism by which this differentiation occurs, in response to TGF-β1, remains unclear. Myocardin-related transcription factors-A and -B (MRTFs, MRTF-A/B) are transcriptional co-activators that regulate the expression of smooth muscle-specific cytoskeletal proteins, including SMαA, in smooth muscle cells and fibroblasts. In this study, we demonstrate that TGF-β1 mediates myofibroblast differentiation and the expression of a contractile gene program through the actions of the MRTFs. Transient transfection of a constitutively-active MRTF-A induced an increase in the expression of SMαA and other smooth muscle-specific cytoskeletal proteins, and an increase in myofibroblast contractility, even in the absence of TGF-β1. MRTF-A/B knockdown, in TGF-β1 differentiated myofibroblasts, resulted in decreased smooth muscle-specific cytoskeletal protein expression levels and reduced contractile force generation, as well as a decrease in focal adhesion size and number. These results provide direct evidence that the MRTFs are mediators of myofibroblast differentiation in response to TGF-β1.

During wound healing, fibroblasts transition from quiescence to a migratory state, then to a contractile myofibroblast state associated with wound closure. We found that the myofibroblast phenotype, characterized by the expression of high levels of contractile proteins, suppresses the expression of the pro-migratory gene, MMP-2. Fibroblasts cultured in a 3-D collagen lattice and allowed to develop tension showed increased contractile protein expression and decreased MMP-2 levels in comparison to a stress-released lattice. In 2-D cultures, factors that promote fibroblast contractility, including serum or TGF-β, down-regulated MMP-2. Pharmacologically inducing F-actin disassembly or reduced contractility increased MMP-2 expression, while conditions that promote F-actin assembly suppressed MMP-2 expression. In all cases, changes in MMP-2 levels were inversely related to changes in the contractile marker, smooth muscle α-actin. To determine if the mechanisms involved in contractile protein gene expression play a direct role in MMP-2 regulation, we used RNAi-mediated knock-down of the myocardin-like factors, MRTF-A and MRTF-B, which induced the down-regulation of contractile protein genes by fibroblasts under both serum-containing and serum-free conditions. In the presence of serum or TGF-β, MRTF-A/B knock-down resulted in the up-regulation of MMP-2; serum-free conditions prevented this increased expression. Together, these results indicate that, while MMP-2 expression is suppressed by F-actin formation, its up-regulation is not simply a consequence of contractile protein down-regulation.

Myofibroblasts (MFs) are specialized contractile cells that arise from fibroblasts and facilitate wound closure and can promote pathological contractures. MFs have “super‐mature” focal adhesions and express α‐smooth muscle actin (ASMA) and other smooth muscle specific cytoskeletal proteins (SMCPs). Myocardin‐related transcription factor A (MRTF‐A/MAL/MKL‐1) is a putative mechanical stress‐induced co‐activator of serum response factor that activates expression of contractile proteins. We hypothesize that MRTF‐A is a key regulator of these contractile genes which allows for the formation and function of MFs. Rat embryonic fibroblasts (REF‐52) transfected with a constitutively active MRTF‐A acquired the MF phenotype as determined by increased expression of ASMA, SM22α, and smooth muscle γ‐actin (SMGA), as well as, an increased ability to contract a deformable silicone substrate. REF‐52 cells were treated with transforming growth factor‐β1 to promote MF formation. Small interfering RNA specific to MRTF‐A was used to knockdown expression of MRTF‐A in MFs, and resulted in decreased expression of ASMA, SM22α, and SMGA and decreased ability of MFs to contract a wrinkling substrate. These results suggest MRTF‐A functions as a regulator of MF formation by playing a critical role in regulating the expression of SMCPs and the resultant contractile activity. Funded by NIH grant R01 GM60651.

Myofibroblasts (MFs) differentiate from fibroblasts and function to facilitate wound closure. Under pathological conditions, MFs fail to undergo apoptosis and continue to remodel the extracellular matrix, leading to increased contracture of connective tissue. MFs are characterized as having "super‐mature" focal adhesions and expression of smooth muscle α‐actin (SMAA). Myocardin‐related transcription factors A and B (MRTF‐A/MAL/MKL‐1 and MRTF‐B/MKL‐2) are putative mechanical stress‐induced co‐activators that activate expression of contractile proteins such as SMAA. Small interfering RNA (siRNA) has been used to study the effects of MRTF‐A and MRTF‐B reduction on the MF contractile phenotype in rat embryonic fibroblasts. Decreased SMAA levels, as determined by immunoblot analyses, and reduced contractile force generation, as determined by a wrinkling assay, were observed in cells transfected with MRTF‐A and ‐B‐targeted siRNA. Immunocytochemistry has shown a decrease in the size and number of focal adhesions in these cells when compared to control cells. Finally, transiently transfected myocardin, which acts similarly to constitutively‐active MRTF‐A, increased SMAA promoter activity and increased the contractile ability of cells plated on a deformable wrinkling substrate. These results suggest MRTFs play a critical role in regulating MF formation and function. (Funded by NIH 2R01GM60651)Grant Funding SourceNIH