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

180 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1997.Integrin function in muscle differentiation was addressed by ectopic expression of integrin alpha subunits in primary quail skeletal muscle. Ectopic expression of either the human or $\alpha$5 or the chicken $\alpha$6 subunit produced contrasting phenotypes. The $\alpha$5 transfected myoblasts remain in the proliferative phase and are differentiation inhibited. Myoblasts overexpressing the $\alpha$6 subunit exhibit inhibited proliferation and substantial differentiation. Antisense suppression of endogenous quail $\alpha$6 inhibits myoblast differentiation and promotes proliferation. These effects of ectopic $\alpha$ subunits are mediated by the cytoplasmic domains. Ectopic expression of chimeric alpha subunits, $\alpha$5ex/6cyto and $\alpha$6ex/5cyto, produced phenotypes opposite to those observed with ectopic $\alpha$5 or $\alpha$6 expression. Myoblasts that express $\alpha$5ex/6cyto show decreased proliferation and increased differentiation. The $\alpha$6ex/5cyto transfectants remain in the proliferative phase unless confluent. Expression of human $\alpha$5 subunit cytoplasmic domain truncations, localizes the active site to the conserved GFFKR motif. Ectopic $\alpha$5 and $\alpha$6 expression also results in contrasting responses to the mitogenic effects of serum growth factors. Myoblasts expressing the human $\alpha$5 subunit differentiate in the absence of serum while differentiation of untransfected and $\alpha$6 transfected myoblasts is insensitive to serum concentration. Addition of individual, exogenous growth factors to $\alpha$5 transfected myoblasts results in unique responses that differ from their effects on untransfected cells. bFGF or TGF$\beta$ inhibit the serum-free differentiation of $\alpha$5 transfected myoblasts. bFGF stimulates proliferation whereas TGF-$\beta$ inhibits it. Insulin or TGF-$\alpha$ promote proliferation and differentiation; insulin alters myotube morphology. TGF-$\alpha$ or PDGF-BB enhance muscle $\alpha$-actinin organization into myofibrils. bFGF and insulin promote survival of $\alpha$5 transfected myoblasts in serum-free medium. TGF-$\alpha$ and TGF-$\beta$ promote survival of untransfected myoblasts. Our observations demonstrate: (1) a specificity for integrin $\alpha$ 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 $\alpha$ subunit cytoplasmic domain in mediating proliferative and differentiative signals, (4) regulation of proliferation, differentiation, cytoskeletal assembly, and cell survival depends on the expression levels of different integrins and the growth factor environment in which the cells reside.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD