Skeletal muscle cell differentiation is a highly ordered multistep process that involves the expression of myogenic transcription factors, followed by cyclin kinase inhibitor p21 protein induction, cell cycle arrest, muscle-specific protein expression, and cell fusion to form multinucleated myotubes (1-4). The commitment to differentiate into myotubes is influenced negatively by several factors. Treatment of myoblasts with fetal bovine serum, basic fibroblast growth factor 2, or transforming growth factor ␤1 is known to inhibit differentiation of myoblasts (5, 6). Myogenesis is also regulated negatively by oncogenes such as c-fos, Ha-ras, and E1a (7-9). The insulin-like growth factors (IGFs) 1 are the only known growth factors that are crucial to myogenesis (10, 11). IGF expression is increased during myoblast differentiation in response to serum withdrawal (12-15). The amount of IGF-II secreted correlates with the rate of spontaneous differentiation that, in the absence of exogenous IGF-II, can be inhibited by antisense oligonucleotides complementary to IGF-II mRNA (16). Because of their myogenic actions, IGFs have been postulated as potential therapeutic tools in conditions characterized by muscle myopathy, atrophy, or muscle injury. In this context, IGFs have been implicated in the regulation of satellite cell function during regeneration, a characteristic response of adult muscle to injury (17, 18). However, IGFs are pleiotropic growth factors, and they also affect the growth of several tissues other than skeletal muscle. This has led to the analysis of the intracellular myogenic process initiated by IGFs. It is known that IGF-I and IGF-II switch on the myogenic program by activating the IGF-I receptor (19). During the last 2 years, the phosphatidylinositol (PI) 3-kinase has emerged as an essential second messenger for skeletal muscle cell differentiation (20 -22). Moreover, by overexpressing a mutant p85 regulatory subunit of PI 3-kinase (⌬p85) lacking the ability to bind and activate the p110 catalytic subunit (L6E9-⌬p85), we showed that the heterodimeric p85-p110 is the PI 3-kinase isoform essential for IGF-induced myogenesis in L6E9 muscle cells (23). Currently, there is no information regarding the downstream signals activated by IGFs and PI 3-kinase or its PI 3-phosphate products during myogenesis, and we have recently shown that the serine/threonine p70 S6 kinase, a downstream element in several PI 3-kinase-dependent signaling cascades (24, 25), is not involved in the myogenic actions of IGFs in rat, mouse, or human cells (26). Among the signaling events involved in myogenesis, the induction of chick embryonic myoblast fusion in low serum conditions requires NO production and NF-B activity (27,28). However, the mechanisms that trigger NF-B and NOS activation in differentiating myoblasts and the involvement of these molecules in biochemical differentiation (i.e. expression of structural and functional muscle markers) remain to be defined. In the present study, we attempted to further characterize the...

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Phosphatidylinositol 3 (PI 3)-kinases are potently inhibited by two structurally unrelated membrane-permeant reagents: wortmannin and LY294002. By using these two inhibitors we first suggested the involvement of a PI 3-kinase activity in muscle cell differentiation. However, several reports have described that these compounds are not as selective for PI 3-kinase activity as assumed. Here we show that LY294002 blocks the myogenic pathway elicited by insulin-like growth factors (IGFs), and we confirm the specific involvement of PI 3-kinase in IGF-induced myogenesis by overexpressing in L6E9 myoblasts a dominant negative p85 PI 3-kinase-regulatory subunit (L6E9-delta p85). IGF-I, des(1-3)IGF-I, or IGF-II induced L6E9 skeletal muscle cell differentiation as measured by myotube formation, myogenin gene expression, and GLUT4 glucose carrier induction. The addition of LY294002 to the differentiation medium totally inhibited these IGF-induced myogenic events without altering the expression of a non-muscle-specific protein, beta1-integrin. Independent clones of L6E9 myoblasts expressing a dominant negative mutant of the p85-regulatory subunit (delta p85) showed markedly impaired glucose transport activity and formation of p85/p110 complexes in response to insulin, consistent with the inhibition of PI 3-kinase activity. IGF-induced myogenic parameters in L6E9-delta p85 cells, ie. cell fusion and myogenin gene and GLUT4 expression, were severely impaired compared with parental cells or L6E9 cells expressing wild-type p85. In all, data presented here indicate that PI 3-kinase is essential for IGF-induced muscle differentiation and that the specific PI 3-kinase subclass involved in myogenesis is the heterodimeric p85-p110 enzyme.

Nuclear factor B (NF-B)-inducing kinase (NIK),The IGFs 1 are the only known growth factors that are crucial to myogenesis (1). IGF-I and IGF-II switch on the myogenic program through the IGF-I receptor (2), activating the expression of myogenic transcription factors, cell cycle arrest, musclespecific protein expression, and cell fusion to form multinucleated myotubes (3, 4). PI3K is an essential second messenger for myogenesis (5-9). We have recently described a myogenic signaling cascade initiated by IGF-II that leads to biochemical and morphological skeletal muscle cell differentiation and that involves PI3K activation, NF-B activation, and inducible nitricoxide synthase expression and activation (10). In this report, we further analyze the role of the NF-B-activating signaling cascade in myogenesis. NF-B transcription factors are key mediators of inflammatory responses, immune system functioning, transformation, oncogenesis, and anti-apoptotic signaling (11-13). NF-B exists in the cytoplasm in an inactive form by virtue of its association with inhibitory proteins termed IB (11-15). NF-B translocation to the nucleus and activation are most frequently achieved through the signal-induced proteolytic degradation of IB in the cytoplasm. Two kinases, IKK␣ and IKK␤, which are contained in a high-molecularweight multiprotein complex, show inducible IB kinase activity and play a key role in NF-B activation by a variety of stimuli (16 -19). Despite their high sequence similarity, IKK␣ and IKK␤ have different regulatory and functional roles. In mice lacking IKK␤, the activation of NF-B by cytokines is abolished, and mouse embryos die on days 12-13 of gestation due to massive liver apoptosis (20). In contrast, IKK␣ is dispensable for pro-inflammatory responses, but plays an essential role in embryonic development. Mice lacking IKK␣ exhibit defective proliferation and differentiation of epidermal keratinocytes and defective limb and skeletal patterning (21,22). IKK␣ and IKK␤ are themselves phosphorylated and activated by one or more upstream kinases, like NIK, which is a member of the mitogen-activating protein kinase kinase kinase family (23-25).We report here that IB␣ phosphorylation at Ser-32 and Ser-36 is required for both IGF-II-dependent NF-B activation and differentiation in L6E9 myoblasts. We show that IKK␣ is involved in IGF-II-dependent multinucleated myotube formation and muscle-specific gene expression, whereas IKK␤ is not essential for these processes. Our data suggest that NIK activation triggers myogenin expression and multinucleated myotube formation in the absence of IGF-II.

Insulin-like growth factors (IGFs) are potent stimulators of muscle differentiation, and phosphatidylinositol 3-kinase (PI 3-kinase) is an essential second messenger in this process. Little is known about the downstream effectors of the IGF/PI 3-kinase myogenic cascade, and contradictory observations have been reported concerning the involvement of p70 S6 kinase. In an attempt to clarify the role of p70 S6 kinase in myogenesis, here we have studied the effect of rapamycin on rat, mouse, and human skeletal muscle cell differentiation. Both insulin and IGF-II activated p70 S6 kinase in rat L6E9 and mouse Sol8 myoblasts, which was markedly inhibited at 1 ng/ml rapamycin concentrations. Consistent with previous observations in a variety of cell lines, rapamycin exerted a potent inhibitory effect on L6E9 and Sol8 serum-induced myoblast proliferation. In contrast, even at high concentrations (20 ng/ml), rapamycin had no effect on IGF-II-induced proliferation or differentiation. Indeed, neither the morphological differentiation, as assessed by myotube formation, nor the expression of muscle-specific markers such as myogenin, myosin heavy chain, or GLUT4 (glucose transporter-4) glucose carriers was altered by rapamycin. Moreover, here we extended our studies on IGF-II-induced myogenesis to human myoblasts derived from skeletal muscle biopsies. We show that, as observed for rat and mouse muscle cells, human myoblasts can be induced to form multinucleated myotubes in the presence of exogenous IGF-II. Moreover, IGF-II-induced human myotube formation was totally blocked by LY294002, a specific PI 3-kinase inhibitor, but remained unaffected in the presence of rapamycin.