ANCHORAGE-DEPENDENT CONTROL OF MUSCLE-SPECIFIC GENE-EXPRESSION IN C2C12 MOUSE MYOBLASTS

Our previous studies have demonstrated that expression of growth-assoc iated genes is regulated by the adhesive state of the cell. To underst and the role of cell adhesion in regulating the switch from growth to differentiation, we are studying the differentiation of mouse myoblast s into multinucleated contractile myotubes. In this report, we describ e a novel means of culturing C2C12 myoblasts that permits an analysis of the role of cell adhesion in regulating the sequential induction of muscle-specific genes that control myogenesis. Suspension of an async hronous, proliferating population of myoblasts in a viscous gel of met hylcellulose dissolved in medium containing 20% serum induces growth a rrest in G(0) phase of the cell cycle without a concomitant induction of muscle-specific genes. Reattachment to a solid substratum in 20% se rum, 0.5 nM bFGF, or 10 nM IGF-1 rapidly activates entry of the quiesc ent cells into G(1) followed by a synchronous progression of the cell population through into S phase. bFGF or IGF-1 added separately facili tate only one passage through the cell cycle, whereas 20% serum or the two growth factors added together support multiple cell divisions. Ad hesion of suspended cells in DMEM alone or with 3 nM IGF-1 induces myo genesis as evidenced by the synthesis of myogenin and myosin heavy cha in (MHC) proteins followed by fusion into myotubes. bFGF completely in hibits this differentiation process even in the presence of myogenic d oses of IGF-1. Addition of 3 nM IGF-1 to quiescent myoblasts maintaine d in suspension culture in serum-free conditions does not induce myoge nin or MHC expression. Thus, adhesion is a requirement for the inducti on of muscle gene expression in mouse myoblasts. The development of a muscle cell culture environment in which proliferating myoblasts can b e growth arrested in G(0) without activating muscle-specific gene expr ession provides a means of analyzing the synchronous activation of eit her the myogenic or growth programs and how adhesion affects each proc ess, respectively.