Maturation of the myogenic program is induced by postmitotic expression of insulin-like growth factor I.

During mammalian embryogenesis, skeletal muscle undergoes a series of developmental changes that are reflected in a progression of structural gene expression patterns, such that the specific contractile protein composition of a myocyte is considered a measure of its maturity. The orchestration of this progression presumably involves a complex interplay between intrinsic programs of lineage specification and extrinsic controls such as innervation, extracellular milieu, and humoral factors in the embryonic environment (46). In the adult, muscle injury and subsequent regeneration result in a partial recapitulation of these embryonic programs of maturation, underscoring the enduring plasticity of the muscle phenotype in the face of changing external conditions. The insulin-like growth factors (IGFs) have been implicated in the control of skeletal muscle growth and differentiation in both embryonic development and in the muscle regeneration process (20, 22, 29, 36). Recent studies of the effects of IGFs on the myogenic program in muscle cell culture have focused specifically on IGF-I, since it is the predominant form in mature muscle tissue. In cultured myoblasts, as in other cell types, IGF-I induces cell proliferation (14, 37). However, unlike other growth factors, IGF-I also stimulates myogenic differentiation and generates pronounced myocyte hypertrophy (14, 18, 37), suggesting that this growth factor can regulate both proliferative and differentiative responses in muscle cells. In previous reports, we and others have established that proliferation precedes differentiation in IGF-I-stimulated myogenesis in culture (14, 41). From these studies, it is clear that the effects of IGF-I on proliferation and differentiation are temporally separated. IGF-I plays its roles step by step, first acting upon myoblast replication, increasing the expression pattern of factors involved in the cell cycle progression (14, 41), and subsequently promoting myogenic differentiation by induction of myogenic regulatory factors (MRFs). The effects of IGF-I are also modulated by a family of six IGF binding proteins (IGFBPs) (25), which can either inhibit or potentiate the IGF-stimulated actions in vivo and in vitro. Recent experimental evidence suggests that IGF-I exerts its functions by activating two different intracellular signal transduction pathways, conveying either proliferative or differentiative signals (10, 26). The proliferative response is mediated by the mitogen-activated protein (MAP) kinase pathway, whereas the pathway leading to differentiation involves the activation of phosphatidylinositol (PI) 3-kinase. The phosphorylated products of this lipid kinase, PI 3,4-bisphosphate and PI 3,4,5-trisphosphate activate downstream effectors, ultimately leading to induction of muscle gene expression. The alternate activation of these two pathways presumably underlies the observed pleiotropic effects of IGF-I on muscle cells. To determine whether IGF-I can stimulate myogenesis in the absence of proliferation, we established an experimental cell culture system where the action of IGF-I on proliferation and on differentiation could be uncoupled. The neonatal rat L6E9 myogenic line has been used extensively to study the role of IGF-I in myogenesis (33, 47), since it does not express this growth factor itself and can respond to IGF-I stimulation through presentation of IGF-I receptors (39). Although the L6 cells fail to express a number of characteristic myogenic regulators such as MyoD (5, 38) or myocyte enhancer factor (MEF) 2C (30), they are still able to undergo fusion and to activate a subset of muscle structural genes in response to exogenous stimuli (19). Treatment of L6E9 myoblasts with exogenous IGF-I in defined serum-free medium induces a transient increase in cell cycle markers and cell proliferation (14, 41). This response is rapidly followed by withdrawal from the cell cycle and activation of myogenic factors, resulting in a net increase in structural gene expression and larger myotubes (14). To bypass the proliferative effects of IGF-I, we stably transfected L6E9 cultures with a muscle-specific IGF-I expression vector, myosin light chain (MLC)–IGF-I, which is activated only after myoblasts have withdrawn from the cell cycle and have committed to differentiation. In this way, the influence of IGF-I on myogenic differentiation could be dissected and manipulated independently of its role in myoblast proliferation. Studies of the MLC–IGF-I transfectants suggest that postmitotic expression of IGF-I potentiates and accelerates the myogenic program, induces dramatic myotube hypertrophy, activates a new battery of genes, and initiates a shift towards a more mature fiber type. Moreover, disruption of specific signal transduction pathways in these cells reveals a potential third PI 3-kinase-independent signal transduction pathway that presumably participates in later stages of myogenic differentiation. These studies in cell culture support a role for IGF-I in the establishment and maintenance of the mature muscle phenotype in normal and regenerating muscle tissue.