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

Oncogene volume 21, pages 3739–3753 (2002)Cite this article

Abstract

v-H-ras transformed C2C12 (C2Ras) myoblasts, overexpressing p21-Ras protein in the Ras-GTP active form, showed a differentiation-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 differentiation. 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 p21Cip) and myogenesis (multinucleated myotubes formation and induction of creatine kinase, caveolin-3 and α-actin). Both events were accompanied by down-regulation of AP-1 and up-regulation of NF-κB transcriptional activities. Furthermore, inhibition of NF-κB transcriptional activity by the use of the proteasome inhibitor MG132 totally precluded differentiation by insulin+PD98059, demonstrating a direct role for NF-κB on C2Ras myogenesis. C2Ras myoblasts failed to restore differentiation 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 differentiated 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 differentiation.

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Acknowledgements

R Conejo and C De Alvaro were recipients of fellowships from the Ministerio de Educacion y Cultura, Spain. We thank A Alvarez (Universidad Complutense, Madrid) for expert technical assistance with the flow cytometer and L Bosca (Consejo Superior de Investigaciones Cientificas, Madrid) for expert advice on the proteasome inhibitor. This work was supported by grants from the Direccion General de Enseñanza Superior e Investigacion Cientifica; Contract grant number: PM98-0082 and from Comunidad de Madrid; Contract grant number: 08.6/0043/2000.1.

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  1. Departamento de Bioquimica y Biologia Molecular, Facultad de Farmacia, Universidad Complutense, Madrid, 28040, Spain

    Ruben Conejo, Cristina de Alvaro, Manuel Benito & Margarita Lorenzo

  2. Departamento de Bioquimica, Facultad de Medicina, Universidad Autonoma de Madrid, Madrid, 28029, Spain

    Antonio Cuadrado

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Correspondence to Margarita Lorenzo.

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Conejo, R., de Alvaro, C., Benito, M. et al. Insulin restores differentiation of Ras-transformed C2C12 myoblasts by inducing NF-κB through an AKT/P70S6K/p38-MAPK pathway. Oncogene 21, 3739–3753 (2002). https://doi.org/10.1038/sj.onc.1205469

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