Induction of acetylcholine receptor gene expression by ARIA requires activation of mitogen-activated protein kinase

Transcription of genes encoding nicotinic acetylcholine receptor (AChR) subunits (alpha, beta, gamma or epsilon, and delta) is highest in nuclei localized to the synaptic region of the muscle, which contributes to maintain a high density of AChRs at the postjunctional membrane. ARIA (AChR inducing activity) is believed to be the trophic factor utilized by motor neurons to stimulate AChR synthesis in the subsynaptic area. To elucidate the signaling mechanism initiated by ARIA, we established stable C2C12 cell lines carrying the nuclear lacZ gene under the control of the mouse epsilon subunit promoter or chicken alpha subunit promoter. ARIA stimulated tyrosine phosphorylation of erbB proteins in these C2C12 cells within 15 s with a peak at 5 min. Immediately following tyrosine phosphorylation of erbB proteins, mitogen-activated protein (MAP) kinase was activated which occurred within 30 s and peaked at 8 min after ARIA stimulation. Concomitantly, expression of AChR genes was induced by ARIA. ARIA-induced AChR subunit transgene expression was observed only in differentiated myotubes and not in myoblasts, suggesting that downstream signaling component(s) are regulated in a manner dependent on the myogenic program. Inhibition of the MAP kinase activity by using a specific MAP kinase kinase inhibitor or by overexpressing dominant negative mutants of Raf or MAP kinase kinase attenuated or abolished the ARIA-induced activation of AChR alpha and epsilon subunit gene expression. These results indicate that regulation of AChR gene expression by ARIA in C2C12 cells requires activation of the MAP kinase signaling pathway.