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68 (2) DESARROLLO MUSCULAR

de la línea celular C2C12, la activación de las rutas de la PI3 quinasa y de p38 MAP
quinasa es necesaria para la miogénesis de la línea celular transformada por v-ras, como
hemos observado mediante el uso de inhibidores específicos de ambas rutas. Finalmente,
una construcción constitutivamente activa de Akt (siempre en presencia de PD) es capaz
de imitar a la insulina induciendo la miogénesis de la línea celular transformada por v-
ras. Además en estas condiciones Akt constitutivamente activo induce las rutas p70S6
quinasa y p38 MAP quinasa.

Palabras clave: desarrollo muscular-transformación por Ras-C2C12-insulina-NFkB

Differential insulin signaling involved in skeletal muscle development

SUMMARY

v-H-ras transformed C2C12 (C2Ras) myoblasts, overexpressing p21-Ras pro-
tein 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 (pre-
cluding the expression of PCNA, cyclin-D1 and retinoblastoma at the hyperphosphory-
lated state and inducing the expression of the cell cycle inhibitor p21Cip) and myogenesis
(multinucleated myotubes formation and induction of creatine kinase, caveolin-3 and -
actin). Both events were accompanied by down-regulation of AP-1 and up-regulation of
NF- B transcriptional activities. Furthermore, inhibition of NF B transcriptional activ-
ity by the use of the proteasome inhibitor MG132 totally precluded differentiation by
insulin+PD98059, demonstrating a direct role for NF B 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.

Key words: muscle differentiation-Ras transformation-C2C12-Insulin-NFkappaB

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