Quantitative changes in integrin and focal adhesion signaling regulate myoblast cell cycle withdrawal

We previously demonstrated contrasting roles for integrin alpha subunits and their cytoplasmic domains in controlling cell cycle withdrawal and the onset of terminal differentiation (Sastry, S., M. Lakonishok, D. Thomas, J. Muschler, and A.F. Horwitz. 1996. J. Cell Biol. 133:169-184). Ectopic expression of the integrin alpha 5 or alpha 6A subunit in primary quail myoblasts either decreases or enhances the probability of cell cycle withdrawal, respectively. In this study, we addressed the mechanisms by which changes in integrin alpha subunit ratios regulate this decision. Ectopic expression of truncated alpha 5 or alpha 6A indicate that the alpha 5 cytoplasmic domain is permissive for the proliferative pathway whereas the COOH-terminal 11 amino acids of alpha 6A cytoplasmic domain inhibit proliferation and promote differentiation. The alpha 5 and alpha 6A cytoplasmic domains do not appear to initiate these signals directly, but instead regulate beta 1 signaling. Ectopically expressed IL2R-alpha 5 or IL2R-alpha 6A have no detectable effect on the myoblast phenotype. However, ectopic expression of the beta 1A integrin subunit or IL2R-beta 1A, autonomously inhibits differentiation and maintains a proliferative state. Perturbing alpha 5 or alpha 6A ratios also significantly affects activation of beta 1 integrin signaling pathways. Ectopic alpha 5 expression enhances expression and activation of paxillin as well as mitogen-activated protein (MAP) kinase with little effect on focal adhesion kinase (FAK). In contrast, ectopic alpha 6A expression suppresses FAK and MAP kinase activation with a lesser effect on paxillin. Ectopic expression of wild-type and mutant forms of FAK, paxillin, and MAP/erk kinase (MEK) confirm these correlations. These data demonstrate that (a) proliferative signaling (i.e., inhibition of cell cycle withdrawal and the onset of terminal differentiation) occurs through the beta 1A subunit and is modulated by the alpha subunit cytoplasmic domains; (b) perturbing a subunit ratios alters paxillin expression and phosphorylation and FAK and MAP kinase activation; (c) quantitative changes in the level of adhesive signaling through integrins and focal adhesion components regulate the decision of myoblasts to withdraw from the cell cycle, in part via MAP kinase.