Insulin restores differentiation of Ras-transformed C2C12 myoblasts by inducing NF-κB through an AKT/P70S6K/p38-MAPK pathway

Abstract

v-H-ras transformed C2C12 (C2Ras) myoblasts, overexpressing p21-Ras protein in the Ras-GTP active form, showed a di erentiation-defective phenotype when cultured in low serum as compared with C2C12 myoblasts. Accordingly, the purpose of the present study was to delineate the signaling pathways that restore C2Ras myoblasts di erentiation. Inhibition of p42/p44-MAPK with the chemical inhibitor PD98059, and activation of AKT/P70S6K and p38-MAPK with insulin, produced growth arrest (precluding the expression of PCNA, cyclin-D1 and retinoblastoma at the hyperphosphorylated state and inducing the expression of the cell cycle inhibitor p21 Cip ) and myogenesis (multinucleated myotubes formation and induction of creatine kinase, caveolin-3 and a-actin). Both events were accompanied by down-regulation of AP-1 and up-regulation of NF-kB transcriptional activities. Furthermore, inhibition of NFkB transcriptional activity by the use of the proteasome inhibitor MG132 totally precluded di erentiation by insulin+PD98059, demonstrating a direct role for NFkB on C2Ras myogenesis. C2Ras myoblasts failed to restore di erentiation when rapamycin or PD169316 were added in the presence of insulin+PD98059, indicating that the activation of both P70S6K and p38-MAPK was necessary to reach a fully di erentiated phenotype. Finally, transient transfection of a constitutively active Myr-EGFP-AKT-HA construct (in the presence of PD98059) restored C2Ras myogenesis by its ability to activate P70S6K and p38-MAPK. A crosstalk between P70S6K and p38-MAPK was observed under rapamycin treatment in both insulin or active AKT induced myogenesis. Our results are delineating an AKT/ P70S6K/p38-MAPK pathway involved in skeletal muscle di erentiation.

Ambient glucose levels qualify the potency of insulin myogenic actions by regulating SIRT1 and FoxO3a in C2C12 myocytes

“…2 and 3) both of which have recently been implicated as negative regulators of myogenesis (14) (21). Consistent with many previous reports studying myogenesis by using mostly IGFs (6,7,12), insulin also exerts a myogenic action in a manner dependent on PI 3-kinase and mTOR activities, as assessed by MHC expression (Fig. 6); however, we cannot rule out the possibility that insulin activates IGF-I receptors since a relatively high concentration of insulin was required to stimulate MHC expression within 24 h (Fig.…”

“…However, sirtinol failed to restore MHC expression (Fig. 6E, panel 3, lanes [5][6][7][8] in the presence of insulin, a condition in which FoxO3a is highly expressed (Fig. 6E, panel 2, lanes 5-8).…”

Tumor Necrosis Factor α Produces Insulin Resistance in Skeletal Muscle by Activation of Inhibitor κB Kinase in a p38 MAPK-dependent Manner

“…In this work, we show that TNF-␣ produced a sustained phosphorylation of JNK, p38 MAPK, and p42/p44 MAPK during the first 6 h of treatment. Acute insulin stimulation (5 min) produces a transient phosphorylation of MAPKs, as we reported previously (38,41), but insulin activates p38␣ MAPK, whereas TNF-␣ activates the ␤ isoform, as we demonstrate in this work. To evaluate the contribution of sustained activation of these kinases to insulin resistance, we used the chemical inhibitors SP, PD*/SB, and PD, which specifically prohibited activation of these pathways by TNF-␣ in primary muscle cells.…”

NF-κB Regulation of YY1 Inhibits Skeletal Myogenesis through Transcriptional Silencing of Myofibrillar Genes

“…In our own studies, we described the low constitutive NF-B activity contained by proliferating myoblasts, which functions to inhibit myogenesis by stimulating cell cycle progression (26) and by suppressing the synthesis of MyoD, especially in response to proinflammatory mediators (27,36). However, other reports suggest that NF-B signaling may also possess promyogenic activity (5,19,34), which highlights the complexity of this signaling pathway in relation to muscle differentiation and argues that additional investigation into the role of this transcription factor is warranted. The current study was thus undertaken to gain insight into the mechanisms underlying NF-B regulation of myogenesis.…”