Strauch A R, Reeser J C
Department of Anatomy, College of Medicine, Ohio State University, Columbus 43210-1239.
J Biol Chem. 1989 May 15;264(14):8345-55.
High cell density and cell cycle withdrawal stimulate the differentiation of BC3H1 smooth muscle-like cells. The differentiation process is accompanied by extensive changes in cell shape and the increased expression of a variety of muscle-specific proteins including the vascular smooth muscle-specific isoform of the contractile protein, alpha-actin. Results of actin peptide map analyses described in this report now indicate that a second, sarcomeric muscle-specific alpha-actin isoform is expressed in serum-deprived BC3H1 myocytes and that the induction of this actin isoform occurs late in differentiation well after the observed upregulation of vascular alpha-actin synthesis. The sarcomeric alpha-actin was identified in myocytes on the basis of the unique electrophoretic mobility of its NH2-terminal tryptic peptide, the distribution of cleavage products that were obtained when the NH2-terminal tryptic peptide was subjected to secondary proteolytic cleavage with thermolysin and Staphylococcus aureus V8 protease, and the presence of an additional cysteine residue at the NH2 terminus of the biosynthetic precursor of this novel alpha-actin. While expression of vascular alpha-actin was stimulated when myoblasts reached confluence, a 6-day post-confluent treatment with serum-free medium was required to induce maximal expression of the sarcomeric alpha-actin. Blot hybridization analysis of total BC3H1 myocyte RNA using actin gene-specific cDNA probes indicated that the sarcomeric alpha-actin corresponds to the skeletal muscle-specific isoform. This is the first report describing dual expression of smooth muscle and sarcomeric muscle alpha-actins in a clonal myogenic cell line. The results indicate the potential usefulness of the BC3H1 cell line for studying relationships between divergent muscle alpha-actin gene sequences and transcriptional and translational controls during myogenesis.
高细胞密度和细胞周期停滞刺激BC3H1平滑肌样细胞的分化。分化过程伴随着细胞形态的广泛变化以及多种肌肉特异性蛋白表达的增加，包括收缩蛋白的血管平滑肌特异性同工型α-肌动蛋白。本报告中描述的肌动蛋白肽图谱分析结果现在表明，第二种肌节肌特异性α-肌动蛋白同工型在血清剥夺的BC3H1肌细胞中表达，并且这种肌动蛋白同工型的诱导发生在分化后期，远远晚于观察到的血管α-肌动蛋白合成上调之后。肌节α-肌动蛋白是根据其NH2末端胰蛋白酶肽的独特电泳迁移率、当NH2末端胰蛋白酶肽用嗜热菌蛋白酶和金黄色葡萄球菌V8蛋白酶进行二级蛋白水解切割时获得的切割产物分布以及这种新型α-肌动蛋白生物合成前体的NH2末端存在一个额外的半胱氨酸残基而在肌细胞中鉴定出来的。当成肌细胞达到汇合时，血管α-肌动蛋白的表达受到刺激，但需要在汇合后用无血清培养基处理6天才能诱导肌节α-肌动蛋白的最大表达。使用肌动蛋白基因特异性cDNA探针对总BC3H1肌细胞RNA进行印迹杂交分析表明，肌节α-肌动蛋白对应于骨骼肌特异性同工型。这是第一份描述在克隆性成肌细胞系中平滑肌和肌节肌α-肌动蛋白双重表达的报告。结果表明BC3H1细胞系在研究不同肌肉α-肌动蛋白基因序列与成肌过程中转录和翻译控制之间关系方面的潜在用途。
