IGFBP-5 regulates muscle cell differentiation by binding to IGF-II and switching on the IGF-II auto-regulation loop
Skeletal muscle differentiation, maturation, and regeneration are regulated by interactions between signaling pathways activated by hormones and growth factors, and intrinsic genetic programs controlled by myogenic transcription factors, including members of the MyoD and myocyte enhancer factor 2 (MEF2) families. Insulin-like growth factors (IGFs) play key roles in muscle development in the embryo, and in the maintenance and hypertrophy of mature muscle in the adult, but the precise signaling pathways responsible for these effects remain incompletely defined. To study mechanisms of IGF action in muscle, we have developed a mouse myoblast cell line termed C2BP5 that is dependent on activation of the IGF-I receptor and the phosphatidyl inositol 3-kinase (PI3-kinase)-Akt pathway for initiation of differentiation. Here, we show that differentiation of C2BP5 myoblasts could be induced in the absence of IGF action by recombinant adenoviruses expressing MyoD or myogenin, but it was reversibly impaired by the PI3-kinase inhibitor LY294002. Similar results were observed using a dominant-negative version of Akt, a key downstream component of PI3-kinase signaling, and also were seen in C3H 10T1/2 fibroblasts. Inhibition of PI3-kinase did not prevent accumulation of muscle differentiation-specific proteins (myogenin, troponin T, or myosin heavy chain), did not block transcriptional activation of E-box containing muscle reporter genes by MyoD or myogenin, and did not inhibit the expression or function of endogenous MEF2C or MEF2D. An adenovirus encoding active Akt could partially restore terminal differentiation of MyoD-expressing and LY294002-treated myoblasts, but the resultant myofibers contained fewer nuclei and were smaller and thinner than normal, indicating that another PI3-kinase-stimulated pathway in addition to Akt is required for full myocyte maturation. Our results support the idea that an IGF-regulated PI3-kinase pathway functions downstream of or in parallel with MyoD, myogenin, and MEF2 in muscle development to govern the late steps of differentiation that lead to multinucleated myotubes.