A CRUCIAL ROLE FOR THE MITOGEN-ACTIVATED PROTEIN-KINASE PATHWAY IN NICOTINIC CHOLINERGIC SIGNALING TO SECRETORY PROTEIN TRANSCRIPTION IN PHEOCHROMOCYTOMA CELLS

The mitogen-activated protein kinase (MAPK) pathway plays a pivotal ro le in intracellular signaling, and this cascade may impinge on cAMP re sponse elements (CREs) of target genes. Both the MAPK pathway and chro mogranin A expression may be activated by cytosolic calcium influx, an d calcium-dependent signals map onto the chromogranin A promoter proxi mal CRE, We therefore probed the role of the MAPK pathway in chromogra nin A biosynthesis after secretory stimulation of PC12 pheochromocytom a. cells by the nicotinic cholinergic pathway, the physiological secre tory trigger. Chemical inhibition of either MAPK or MAPK kinase blocke d the response of a transfected chromogranin A promoter to nicotine or protein kinase C activation [by phorbol-12-myristate-13-acetate (PMA) ], although nicotine-evoked catecholamine secretion was unaffected. Ac tivation of the MAP kinase cascade (Ras, Raf, MAPK, or CREB kinase) by cotransfection of pathway components stimulated the chromogranin A pr omoter. Cotransfection of MAPK pathway dominant negative mutants (for Raf, MAPK, or CREB kinase) blocked nicotinic or PMA activation of chro mogranin A, although a dominant negative Pas mutant was without effect . MAPK pathway enzymatic activity was stimulated by both nicotine and PMA. Point mutations of the chromogranin A CRE suggested that this ele ment was necessary in cia for stimulation by nicotine, PMA, or chemica l activation of the MAPK pathway. Transfer of the CRE to a heterologou s promoter conferred inducibility by not only nicotine or cAMP but als o MAPK activation. Expression of the CREB antagonist KCREB blocked the response of the chromogranin A promoter to nicotine, cAMP, or MAPK pa thway activation by either chemical stimulation or cotransfection of a ctive cascade components. Chromogranin A mRNA responded to MAPK pathwa y manipulation in a fashion similar to the transfected chromogranin A promoter, in both direction and magnitude. We conclude that the MAPK p athway is a necessary intermediate in signaling from the nicotinic rec eptor to secretory protein transcription, although not to catecholamin e secretion. In trans, this response seems to involve the following si gnal cascade: protein kinase C --> Raf --> MAPK kinase --> MAPK --> CR EB kinase --> CREB. In cis, activation by the cascade maps onto the ch romogranin A promoter proximal CRE, which is both necessary and suffic ient to confer the response.