Functional receptors for transforming growth factor-β are retained by biochemically differentiated C2 myocytes in growth factor-deficient medium containing EGTA but down-regulated during terminal differentiation

Transforming growth factor-β (TGF-β) has been shown to block the morphological and molecular events associated with myoblast differentiation. During fusion of C2 myoblasts, TGF-β receptors are down-regulated, and muscle-specific genes become refractory to the inhibitory effects of TGF-β. To define further the mechanisms that modulate TGF-β receptor expression during myogenesis, we have developed culture conditions that support the differentiation of C2 cells in the absence of fusion and have examined the expression of functional TGF-β receptors in biochemically differentiated mononucleated myocytes. Exposure of C2 myoblasts to growth factor-deficient medium containing 1.4 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) leads to withdrawal from the cell cycle and high level expression of muscle-specific mRNAs and proteins. Under these conditions, TGF-β receptors fail to be down-regulated, and the differentiation program remains sensitive to repression by TGF-β. These studies demonstrate that EGTA uncouples muscle-specific gene expression from fusion in C2 cells and that in the absence of fusion, C2 myocytes retain a functional TGF-β signaling system.