Sequential expression of smooth muscle and sarcomeric alpha-actin isoforms during BC3H1 cell differentiation.

Abstract

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.