title: Insulin restores differentiation of Ras-transformed C2C12 myoblasts by inducing NF-kappaB 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 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 p21(Cip)) and myogenesis (multinucleated myotubes formation and induction of creatine kinase, caveolin-3 and alpha-actin). Both events were accompanied by down-regulation of AP-1 and up-regulation of NF-kappaB transcriptional activities. Furthermore, inhibition of NF-kappaB transcriptional activity by the use of the proteasome inhibitor MG132 totally precluded differentiation by insulin+PD98059, demonstrating a direct role for NF-kappaB 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.
authors.affiliation: Departamento de Bioquimica y Biologia Molecular, Facultad de Farmacia, Universidad Complutense, 28040-Madrid, Spain.
authors.forename: Ruben
authors.initials: R
authors.lastname: Conejo
authors.forename: Cristina
authors.initials: C
authors.lastname: de Alvaro
authors.forename: Manuel
authors.initials: M
authors.lastname: Benito
authors.forename: Antonio
authors.initials: A
authors.lastname: Cuadrado
authors.forename: Margarita
authors.initials: M
authors.lastname: Lorenzo
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D002471:Cell Transformation, Neoplastic / Q000378:metabolism / Q000473:pathology*; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology*; D051379:Mice; D048308:Mitogen-Activated Protein Kinase 14; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D016328:NF-kappa B / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D019893:Ribosomal Protein S6 Kinases / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection; D014407:Tumor Cells, Cultured; D018631:ras Proteins / Q000502:physiology*
chemical_list: D016895:Culture Media, Serum-Free; D007328:Insulin; D016328:NF-kappa B; D011518:Proto-Oncogene Proteins; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D019893:Ribosomal Protein S6 Kinases; D048308:Mitogen-Activated Protein Kinase 14; D020928:Mitogen-Activated Protein Kinases; D018631:ras Proteins