Abstract
Insulin-like growth factors (IGFs) are well known to play essential roles in enhancement of myogenic differentiation. In this report we showed that initial IGF-I signal activation but long-term IGF-1 signal termination are required for myogenic differentiation. L6 myoblast stably transfected with myc-epitope tagged insulin receptor substrate-1, myc-IRS-1 (L6-mIRS1) was unable to differentiate into myotubes, indicating that IRS-1 constitutive expression inhibited myogenesis. To elucidate the molecular mechanisms underlying myogenic inhibition, IGF-I signaling was examined. IGF-I treatment of control L6 cells for 18 h resulted in a marked suppression of IGF-I stimulated IRS-1 association with the p85 PI 3-kinase and suppression of activation of Akt that correlated with a down regulation of IRS-1 protein. L6-mIRS1 cells, in contrast, had sustained high levels of IRS-1 protein following 18 h of IGF-I treatment with persistent p85 PI 3-kinase association with IRS-1, Akt phosphorylation and phosphorylation of the downstream Akt substrate, Foxo1. Consistent with Foxo1 phosphorylation, Foxo1 protein was excluded from the nuclei in L6-mIRS1 cells, whereas Foxo1 was localized in the nuclei in control L6 cells during induction of differentiation. In addition, L6 cells stably expressing a dominant-interfering form of Foxo1, Δ256Foxo1 (L6-Δ256Foxo1) were unable to differentiate into myotubes. Together, these data demonstrate that IGF-I regulation of Foxo1 nuclear localization is essential for the myogenic program in L6 cells but that persistent activation of IGF-1 signaling pathways results in a negative feedback to prevent myogenesis.
Highlights
Myogenic differentiation is a tightly regulated complex process in which mononucleated myoblasts first proliferate, withdraw from the cell cycle, differentiate, and fuse to form multinucleated myotubes
Protein expression of myogenin or myosin heavy chain (MyHC) was significantly suppressed in L6-mIRS1 cells (Fig. 1C, S1A). mRNA expression of myogenin or MyHC was suppressed in L6-mIRS1 cells (Fig. S1B)
We have shown that constitutive expression of Insulin Receptor Substrate (IRS)-1 inhibited Foxo1 nuclear localization, resulting in inhibition of myogenesis in L6 myoblast cells
Summary
Myogenic differentiation is a tightly regulated complex process in which mononucleated myoblasts first proliferate, withdraw from the cell cycle, differentiate, and fuse to form multinucleated myotubes. IGFs can induce either differentiation or proliferation [7], suggesting that other factors influence myoblast response. Both responses are elicited through binding to the same type 1 IGF tyrosine protein kinase receptor [7]. How a single receptor can elicit two opposite responses is not clear To address this issue, the IGF-I signal transduction pathways in L6 myogenic cells have been extensively dissected. The activated receptor phosphorylates several substrates, including insulin receptor substrates (IRSs) [13,14] Phosphotyrosine residues of these substrates are recognized by several SH2 domain containing signaling molecules, including the p85 PI 3-kinase regulatory subunit and Grb2 [13,15]. Our data demonstrated that prolonged activation of IGF-I signaling did not enhance but inhibited myogenesis
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