This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 149/2/793    most recent Author Manuscript (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via Google Scholar Google Scholar Articles by De Alvaro, C. Articles by Lorenzo, M. Search for Related Content PubMed PubMed Citation Articles by De Alvaro, C. Articles by Lorenzo, M. Pubmed/NCBI databases Gene GEO Profiles HomoloGene Nucleotide Protein UniGene

Nuclear Exclusion of Forkhead Box O and Elk1 and Activation of Nuclear Factor-B Are Required for C2C12-RasV12C40 Myoblast Differentiation

Departamento de Bioquimica y Biologia Molecular II (C.D.A., I.N.-V., M.L.), Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain; and Unidad de Biologia Celular (J.M.R.), Centro Nacional de Microbiologia, Instituto de Salud Carlos III, 28220 Majadahonda, Madrid, Spain

Address all correspondence and requests for reprints to: Margarita Lorenzo, Departamento de Bioquimica y Biologia Molecular II, Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain. E-mail: mlorenzo{at}farm.ucm.es.

Activating ras point mutations are frequently found in skeletal muscle tumors such as rhabdomyosarcomas. In this study we investigated the impact of two different H-ras mutants in skeletal muscle differentiation: RasV12, a constitutively active form, and RasV12C40, a mutant deficient in Raf1 activation. Stably transfected C2C12-RasV12 myoblasts actively proliferated as indicated by the sustained expression of proliferating cell nuclear antigen and retinoblastoma at the hyperphosphorylated state and failed to express differentiation markers. This differentiation-defective phenotype was a consequence of the chronic p44/p42MAPK phosphorylation and the inability of the cells to activate AKT. Moreover, we observed that p44/p42MAPK activation in C2C12-RasV12 myoblasts phosphorylated the ETS-like transcription factor (ELK) 1, which translocates to the nuclei and seemed to be involved in maintaining myoblast proliferation. C2C12-RasV12C40 myoblasts cultured in low serum repressed phosphorylation of p44/p42MAPK and ELK1, resulting in cell cycle arrest and myogenic differentiation. Under this condition, activation of AKT, p70S6K, and p38MAPK was produced, leading to formation of myotubes in 3 d, 1 d earlier than in control C2C12-AU5 cells. Moreover, the expression of muscle-specific proteins, mainly the terminal differentiation markers caveolin-3 and myosin heavy chain, also occurred 1 d earlier than in control cells. Furthermore, AKT activation produced phosphorylation of Forkhead box O that led to nuclear exclusion and inactivation, allowing myogenesis. In addition, we found an induction of nuclear factor-B activity in the nucleus in C2C12-RasV12C40 myotubes attributed to p38MAPK activation. Accordingly, muscle differentiation is associated with a pattern of transcription factors that involves nuclear exclusion ELK1 and Forkhead box O and the increase in nuclear factor-B DNA binding.