Integrin oL Subunit Ratios, Cytoplasmic Domains, and Growth Factor Synergy Regulate Muscle Proliferation and Differentiation

The role of integrins in muscle differentiation was addressed by ectopic expression of integrin o~ subunits in primary quail skeletal muscle, a culture system particularly amenable to efficient transfection and expression of exogenous genes. Ectopic expression of either the human a5 subunit or the chicken et6 subunit produced contrasting phenotypes. The ot5-transfected myoblasts remain in the proliferative phase and are differentiation inhibited even in confluent cultures. In contrast, myoblasts that overexpress the or6 subunit exhibit inhibited proliferation and substantial differentiation. Antisense suppression of endogenous quail et6 expression inhibits myoblast differentiation resulting in sustained proliferation. These effects of ectopic a subunit expression are mediated, to a large extent, by the cytoplasmic domains. Ectopic expression of chimeric a subunits, ct5ex/6cyto and a6ex/5¢yto, produced phenotypes opposite to those observed with ectopic or5 or or6 expression. Myoblasts that express a5ex/6mo show decreased proliferation while differentiation is partially restored. In contrast, the et6ex/5¢yto transfectants remain in the proliferative phase unless allowed to become confluent for at least 24 h. Furthermore, expression of human ct5 subunit cytoplasmic domain truncations, before and after the conserved GFFKR motif, shows that this sequence is important in a5 regulation of differentiation. Ectopic c~5 and o~6 expression also results in contrasting responses to the mitogenic effects of serum growth factors. Myoblasts expressing the human et5 subunit differentiate only in the absence of serum while differentiation of untransfected and ot6-transfected myoblasts is insensitive to serum concentration. Addition of individual, exogenous growth factors to a5transfected myoblasts results in unique responses that differ from their effects on untransfected ceils. Both bFGF or TGFI3 inhibit the serum-free differentiation of a5-transfected myoblasts, but differ in that bFGF stimulates proliferation whereas TGF-13 inhibits it. Insulin or TGF-ot promote proliferation and differentiation of ot5-transfected myoblasts; however, insulin alters myotube morphology. TGF-o~ or PDGF-BB enhance muscle ct-actinin organization into myofibrils, which is impaired in differentiated or5 cultures. With the exception of TGF-ot, these growth factor effects are not apparent in untransfected myoblasts. Finally, myoblast survival under serum-free conditions is enhanced by ectopic ct5 expression only in the presence of bFGF and insulin while TGF-et and TGF-~ promote survival of untransfected myoblasts. Our observations demonstrate (1) a specificity for integrin a subunits in regulating myoblast proliferation and differentiation; (2) that the ratio of integrin expression can affect the decision to proliferate or differentiate; (3) a role for the et subunit cytoplasmic domain in mediating proliferative and differentiative signals; and (4) the regulation of proliferation, differentiation, cytoskeletal assembly, and cell survival depend critically on the expression levels of different integrins and the growth factor environment in which the cells