Anchorage-dependent control of muscle-specific gene expression in C2C12 mouse myoblasts

Our previous studies have demonstrated that expression of growth-associated genes is regulated by the adhesive state of the cell. To understand the role of cell adhesion in regulating the switch from growth to differentiation, we are studying the differentiation of mouse myoblasts into multinucleated contractile myotubes. In this report, we describe 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 asynchronous, proliferating population of myoblasts in a viscous gel of methylcellulose dissolved in medium containing 20% serum induces growth arrest in G0 phase of the cell cycle without a concomitant induction of muscle-specific genes. Reattachment to a solid substratum in 20% serum, 0.5 nM bFGF, or 10 nM IGF-1 rapidly activates entry of the quiescent cells into G1 followed by a synchronous progression of the cell population through into S phase. bFGF or IGF-1 added separately facilitate only one passage through the cell cycle, whereas 20% serum or the two growth factors added together support multiple cell divisions. Adhesion of suspended cells in DMEM alone or with 3 nM IGF-1 induces myogenesis as evidenced by the synthesis of myogenin and myosin heavy chain (MHC) proteins followed by fusion into myotubes. bFGF completely inhibits this differentiation process even in the presence of myogenic doses of IGF-1. Addition of 3 nM IGF-1 to quiescent myoblasts maintained in suspension culture in serum-free conditions does not induce myogenin or MHC expression. Thus, adhesion is a requirement for the induction of muscle gene expression in mouse myoblasts. The development of a muscle cell culture environment in which proliferating myoblasts can be growth arrested in G0 without activating muscle-specific gene expression provides a means of analyzing the synchronous activation of either the myogenic or growth programs and how adhesion affects each process, respectively.