Retrovirally mediated overexpression of insulin-like growth factor binding protein 4: evidence that insulin-like growth factor is required for skeletal muscle differentiation

Recent studies have indicated that the insulin-like growth factors (IGFs) stimulate skeletal myoblast proliferation and differentiation. However, the question of whether IGFs are required for myoblast differentiation has not been resolved. To address this issue directly, we used a retroviral vector (LBP4SN) to develop a subline of mouse C2 myoblasts (C2-BP4) that constitutively overexpress IGF binding protein-4 (IGFBP-4). A control C2 myoblast subline (C2-LNL6) was also developed by using the LNL6 control retroviral vector. C2-BP4 myoblasts expressed sixfold higher levels of IGFBP-4 protein than C2-LNL6 myoblasts. 125I-IGF-I cross linking indicated that IGFBP-4 overexpression reduced IGF access to the type-1 IGF receptor tenfold. At low plating densities, myoblast proliferation was inhibited, and myoblast differentiation was abolished in C2-BP4 cultures compared with C2-LNL6 cultures. At high plating densities in which nuclear numbers were equal in the two sets of cultures, C2-BP4 myoblast differentiation was inhibited completely. Differentiation was restored in C2-BP4 cells by treatment with high levels of exogenous IGF-I or with des(1-3)IGF-I, an analog of IGF-I with reduced affinity for IGFBPs. These findings confirm the hypothesis that positive differentiation signals from the IGFs are necessary for C2 myoblast differentiation, and they suggest that the present model of myogenic differentiation, which involves only negative external control of differentiation by mitogens, may be incomplete.