INTEGRIN ALPHA-SUBUNIT RATIOS, CYTOPLASMIC DOMAINS, AND GROWTH-FACTORSYNERGY REGULATE MUSCLE PROLIFERATION AND DIFFERENTIATION

The role of integrins in muscle differentiation was addressed by ectop ic expression of integrin alpha subunits in primary quail skeletal mus cle, a culture system particularly amenable to efficient transfection and expression of exogenous genes. Ectopic expression of either the hu man alpha 5 subunit or the chicken alpha 6 subunit produced contrastin g phenotypes. The alpha 5-transfected myoblasts remain in the prolifer ative phase and are differentiation inhibited even in confluent cultur es. In contrast, myoblasts that overexpress the alpha 6 subunit exhibi t inhibited proliferation and substantial differentiation. Antisense s uppression of endogenous quail alpha 6 expression inhibits myoblast di fferentiation resulting in sustained proliferation. These effects of e ctopic alpha subunit expression are mediated, to a large extent, by th e cytoplasmic domains. Ectopic expression of chimeric a subunits, (alp ha 5(ex)/6(cyto) and alpha 6(ex)/5(cyto), produced phenotypes opposite to those observed with ectopic alpha 5 or alpha 6 expression. Myoblas ts that express alpha 5(ex)/6(cyto) show decreased proliferation while differentiation is partially restored. In contrast, the alpha 6(ex)/5 (cyto) transfectants remain in the proliferative phase unless allowed to become confluent for at least 24 h. Furthermore, expression of huma n alpha 5 subunit cytoplasmic domain truncations, before and after the conserved GFFKR motif, shows that this sequence is important in alpha 5 regulation of differentiation. Ectopic alpha 5 and alpha 6 expressi on also results in contrasting responses to the mitogenic effects of s erum growth factors. Myoblasts expressing the human alpha 5 subunit di fferentiate only in the absence of serum while differentiation of untr ansfected and alpha 6-transfected myoblasts is insensitive to serum co ncentration. Addition of individual, exogenous growth factors to alpha 5-transfected myoblasts results in unique responses that differ from their effects on untransfected cells. Both bFGF or TGF beta inhibit th e serum-free differentiation of alpha 5-transfected myoblasts, but dif fer in that bFGF stimulates proliferation whereas TGF-beta inhibits it . Insulin or TGF-alpha promote proliferation and differentiation of al pha 5-transfected myoblasts; however, insulin alters myotube morpholog y. TGF-alpha or PDGF-BB enhance muscle alpha-actinin organization into myofibrils, which is impaired in differentiated alpha 5 cultures. Wit h the exception of TGF-alpha, these growth factor effects are not appa rent in untransfected myoblasts. Finally, myoblast survival under seru m-free conditions is enhanced by ectopic alpha 5 expression only in th e presence of bFGF and insulin while TGF-alpha and TGF-beta promote su rvival of untransfected myoblasts. Our observations demonstrate (1) a specificity for integrin alpha subunits in regulating myoblast prolife ration and differentiation; (2) that the ratio of integrin expression can affect the decision to proliferate or differentiate; (3) a role fo r the alpha subunit cytoplasmic domain in mediating proliferative and differentiative signals; and (4) the regulation of proliferation, diff erentiation, cytoskeletal assembly, and cell survival depend criticall y on the expression levels of different integrins and the growth facto r environment in which the cells reside.