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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.