| The cytoskeleton of eukaryotic cells is a network structure composed of many kinds of structural and contractile proteins and plays an important role in maintaining cell shape and contraction. There are three types that include microfilaments, microtubles and intermediate filaments. The microfilaments (filamentous actin, F-actin) are a major kind of cytoskeleton in vascular smooth muscle cells (VSMC), which is assembled by actin and its binding proteins. The stress fibers (SF) are the contractile apparatus of VSMC, which is composed of microfilament bundles and myosin filaments (thick myofilaments). The microfilaments are a complex, plastic, dynamic network structure, which is associated with the shape and function changes of VSMC. Many proteins binding to actin play a regulatory role in microfilament remodeling progress.Smooth muscle 22 alpha (SM22α) is a 22-kD protein of smooth muscle-specific expression. Purified SM22α protein binds directly to actin filaments in a ratio of 1:6 monomers in vitro. SM22α and SM α-actin colocalize to stress fibers in cultured VSMC. Whether SM22α is a functional actin-associated protein in VSMC is controversial. In order to determine the role of SM22α in cytoskeleton remodeling, contraction and migration of VSMC, the model of VSMC redifferentiation induced by serum starvation was used in present study. We observed the changes of SM22α expression and its relation with cell contraction and migration during phenotype reversion of VSMC. After VSMC was transfected by recombinant SM22α antisense or sense plasmids, changes of the cytoskeleton structure and the ability of contraction and migration of VSMC were observed. It was investigated whether the expression of SM22α in VSMC could affect the cytoskeleton remodeling.1 Effect of SM22α on VSMC cytoskeleton and contraction SM22α is a marker of contractile VSMC. Here we observed the effect of SM22α on VSMC phenotype and function. During reversion of synthetic VSMC to contractile phenotype induced by serum starvation, the expression of SM22α gene in different phenotypes was detected by RT-PCR. To investigate the relationship between SM22α expression and cytoskeleton remodeling, VSMC was transfected by antisense SM22α plasmids, and then contraction of VSMC was evaluated. The results were as follows:1.1 As analyzed by RT-PCR, the mRNA of SM22α was almost undetected in VSMC stimulated by serum. The expression of SM22α was quickly upregulated following serum starvation and maintained at higher levels. In serum-restimulated VSMC after serum starvation for 72 hours, the expression of SM22α was down-regulated quickly again.1.2 Western analysis showed that the expression change of SM α-actin was consistent with SM22α. The level of SM α-actin protein increased about one and a half of folds in serum-deprived VSMC, and declined to the base level following serum restimulated.1.3 The VSMC cytoskeleton was shown by modified coomassie brilliant blue stainning. The cytoskeletal structure of VSMC was remodulated from discrete network to well arranged and dense bundles after serum starvation. This processe was blocked by tranfection of antisense plasmids.1.4 Acetylcholine (ACh) didn't induce contraction of synthetic VSMC. When the expressions of SM22α and SM α-actin were upregulated, contraction of VSMC induced by ACh was found, suggesting that VSMC is capable of reversion from synthetic to contractile phenotype. However, the contraction induced by ACh was sustained in VSMC transfected by antisense SM22α recombinant. These data suggested that SM22α is only expressed in contractile VSMC. Compared with other VSMC markers, the expression specificity of SM22α is higher, suggesting that SM22α may play a role in regulation of VSMC cytoskeleton remodeling and contraction.2 SM22α colocalization with SM α-actin during microfilament remodelingIn order to identify the localization association between SM22α and SM α-actin during microfilament remodeling, VSMC was treated with the inducers, inc...
|
PDF Full Text Request