Changes in expression and organization of smooth-muscle-specific α-actin during fibronectin-mediated modulation of arterial smooth muscle cell phenotype

Abstract

Abstract The spreading of freshly isolated arterial smooth muscle cells on a substrate of fibronectin is mediated by an integrin receptor on the cell surface. It is associated with organization of actin filaments in stress fibers and marked changes in cell morphology and function, collectively referred to as a transition from a contractile to a synthetic phenotype. To study further how extracellular matrix components affect smooth muscle phenotype, we have analyzed the expression and organization of smooth-muscle-specific α-actin in freshly isolated rat aortic smooth muscle cells cultured on a substrate of fibronectin under serum-free conditions. Northern-blot analysis showed that the expression of mRNA for smooth muscle α-actin, but not for nonmus-cle actin, was strongly repressed during primary culture. On the other hand, the cellular content of α-actin was only moderately changed during the same period. Indirect immunofluorescence staining revealed that non-muscle actin was rapidly organized in stress fibers, which did not stain with a monoclonal antibody against smooth muscle α-actin. Filament bundles containing α-actin were most prominent in the central parts of the cytoplasm and gradually disappeared as the spreading of the cells progressed. In contrast to the situation with nonmuscle actin, there was no apparent overlap in the staining for α-actin and the fibronectin receptor (α 5β 1), indicating that this receptor interacted with nonmuscle actin during the initial spreading process. Taken together, the results show that the expression and organization of smooth muscle α-actin are changed during interaction of the cells with fibronectin early in primary culture. They support the notion that integrin-mediated interactions between extracellular matrix components and arterial smooth muscle cells take part in the control of smooth muscle phenotype.