α-Smooth Muscle Actin Is Crucial for Focal Adhesion Maturation in Myofibroblasts

Abstract

Cultured myofibroblasts are characterized by stress fibers, containing alpha-smooth muscle actin (alpha-SMA) and by supermature focal adhesions (FAs), which are larger than FAs of alpha-SMA-negative fibroblasts. We have investigated the role of alpha-SMA for myofibroblast adhesion and FA maturation. Inverted centrifugation reveals two phases of initial myofibroblast attachment: during the first 2 h of plating microfilament bundles contain essentially cytoplasmic actin and myofibroblast adhesion is similar to that of alpha-SMA-negative fibroblasts. Then, myofibroblasts incorporate alpha-SMA in stress fibers, develop mature FAs and their adhesion capacity is significantly increased. When alpha-SMA expression is induced in 5 d culture by TGFbeta or low serum levels, fibroblast adhesion is further increased correlating with a "supermaturation" of FAs. Treatment of myofibroblasts with alpha-SMA fusion peptide (SMA-FP), which inhibits alpha-SMA-mediated contractile activity, reduces their adhesion to the level of alpha-SMA negative fibroblasts. With the use of flexible micropatterned substrates and EGFP-constructs we show that SMA-FP application leads to a decrease of myofibroblast contraction, shortly followed by disassembly of paxillin- and beta3 integrin-containing FAs; alpha5 integrin distribution is not affected. FRAP of beta3 integrin-EGFP demonstrates an increase of FA protein turnover following SMA-FP treatment. We conclude that the formation and stability of supermature FAs depends on a high alpha-SMA-mediated contractile activity of myofibroblast stress fibers.

Vascular disease-causing mutation, smooth muscle α-actin R258C, dominantly suppresses functions of α-actin in human patient fibroblasts

“…These observations are consistent with findings by others, where FA number correlates with cell size, and it is thought that global shape of cells plays an important role in control of FA assembly and organization through mechanical tension in the cytoskeleton (36,37). SM α-actin promotes high actin-myosin contractile activity leading to formation of thick stress fibers that are implicated in the time-dependent maturation of vinculin-containing FAs in myofibroblasts (38)(39)(40)(41). FA categories and size ranges have been described for human Dupuytren's fibroblasts as immature (<2 μm 2 ), mature (2-6 μm 2 ), and supermature (>6 μm 2 ), the latter categories associated with the presence of SM α-actin (41).…”

Insights into the Role of Focal Adhesion Modulation in Myogenic Differentiation of Human Mesenchymal Stem Cells

“…A major function of FA was to transmit the extracellular signal into the nucleus, resulting in a proper cellular response to the microenvironment. Elongated FA, which was found in mature human myofibroblasts, was able to increase phosphorylation of FA kinase and regulate stress fiber development in myogenic differentiation [47]. Under optimal conditions, recruited FA components such as ITB3 and vinculin formed elongated clusters along the micropatterns as the response to the modulated ECM environment.…”

Smooth Muscle Actin Determines Mechanical Force-induced p38 Activation

“…SMA is required for focal adhesion maturation and for the initial formation of cortical filament bundles in spreading rat lung myofibroblasts (29). SMA comprises up to 14% total actin content (14) in well differentiated fibroblasts and up to 60 -70% of total actin in smooth muscle cells (45,46).…”