The insulin-like growth factors (IGFs) are unique in that they are the only known mitogens that, when free of other serum components, stimulate both the proliferation and differentiation of skeletal muscle cells. The mechanisms by which IGFs can be both mitogenic and differentiation- promoting in skeletal muscle, however, are unclear since these events are believed to be mutually exclusive in this tissue. In this proposal we will examine the mechanisms by which IGF-I initially inhibits and subsequently stimulates myogenin gene transcription, and we will evaluate the potential role of the cdk4/cdk6 inhibitor p19 in mediating the switch in IGF-I response from proliferation to differentiation. We will test three hypotheses: 1) The early inhibitory and subsequent stimulatory effects of IGF-I on myogenin gene transcription are mediated by an IGF response element (or elements) contained in the 5' flanking region of the myogenin gene; 2) The early inhibitory effect of IGF-I on myogenin transcription is mediated by the Ras/MAP kinase pathway, while the subsequent stimulatory effect of IGF-I on myogenin transcription is mediated by the phosphatidylinositol (PI)-3 kinase pathway; and 3) During the initial mitogenic response to IGF-I, p19 expression increases in S phase myoblasts, limiting further cell cycle entry and causing IGF- I to switch from stimulating proliferation to promoting cell cycle exit and stimulating differentiation. We propose the following specific aims, to be carried out in murine skeletal muscle cells: 1) Identify the region(s) in the regulatory portion of the myogenin gene which confer the early inhibitory and late stimulatory effects of IGF-I on myogenin transcription, and examine the interaction of nuclear proteins with this region or regions; 2) Identify the IGF-I receptor signaling pathways which mediate the early inhibition and subsequent stimulation of myogenin gene transcription; and 3) Assess the potential role of the cdk4/cdk6 inhibitor p19 in mediating the switch in IGF-I response from proliferation to differentiation in skeletal myoblasts. These studies should further our understanding of the mechanisms by which IGFs influence the decision of skeletal myoblasts to proliferate or differentiate. In addition, these studies of IGF response elements, signaling pathways, and cell cycle regulatory components may have more general implications for understanding the mechanisms by which IGFs regulate proliferation and differentiation in a variety of tissues.
