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 nu clei localized to the synaptic region of the muscle, which contributes to maintain a high density of AChRs at the postjunctional membrane. A RIA (AChR inducing activity) is believed to be the trophic factor util ized by motor neurons to stimulate AChR synthesis in the subsynaptic a rea. To elucidate the signaling mechanism initiated by ARIA, we establ ished stable C2C12 cell lines carrying the nuclear lacZ gene under the control of the mouse epsilon subunit promoter or chicken ru subunit p romoter. ARIA stimulated tyrosine phosphorylation of erbB proteins in these C2C12 cells within 15 s with a peak at 5 min. Immediately follow ing tyrosine phosphorylation of erbB proteins, mitogen-activated prote in (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 w as observed only in differentiated myotubes and not in myoblasts, sugg esting that downstream signaling component(s) are regulated in a manne r dependent on the myogenic program. Inhibition of the MAP kinase acti vity by using a specific MAP kinase kinase inhibitor or by overexpress ing dominant negative mutants of Raf or MAP kinase kinase attenuated o r abolished the ARIA-induced activation of AChR alpha and epsilon subu nit gene expression. These results indicate that regulation of AChR ge ne expression by ARIA ill C2C12 cells requires activation of the MAP k inase signaling pathway.