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 role in intracellular signaling, and this cascade may impinge on cAMP response elements (CREs) of target genes. Both the MAPK pathway and chromogranin A expression may be activated by cytosolic calcium influx, and calcium-dependent signals map onto the chromogranin A promoter proximal CRE. We therefore probed the role of the MAPK pathway in chromogranin A biosynthesis after secretory stimulation of PC12 pheochromocytoma cells by the nicotinic cholinergic pathway, the physiological secretory trigger. Chemical inhibition of either MAPK or MAPK kinase blocked 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. Activation of the MAP kinase cascade (Ras, Raf, MAPK, or CREB kinase) by cotransfection of pathway components stimulated the chromogranin A promoter. Cotransfection of MAPK pathway dominant negative mutants (for Raf, MAPK, or CREB kinase) blocked nicotinic or PMA activation of chromogranin A, although a dominant negative Ras 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 element was necessary in cis for stimulation by nicotine, PMA, or chemical activation of the MAPK pathway. Transfer of the CRE to a heterologous promoter conferred inducibility by not only nicotine or cAMP but also MAPK activation. Expression of the CREB antagonist KCREB blocked the response of the chromogranin A promoter to nicotine, cAMP, or MAPK pathway activation by either chemical stimulation or cotransfection of active cascade components. Chromogranin A mRNA responded to MAPK pathway manipulation in a fashion similar to the transfected chromogranin A promoter, in both direction and magnitude. We conclude that the MAPK pathway is a necessary intermediate in signaling from the nicotinic receptor to secretory protein transcription, although not to catecholamine secretion. In trans, this response seems to involve the following signal cascade: protein kinase C --> Raf --> MAPK kinase --> MAPK --> CREB kinase --> CREB. In cis, activation by the cascade maps onto the chromogranin A promoter proximal CRE, which is both necessary and sufficient to confer the response.
Tang
K
K
Department of Medicine and Center for Molecular Genetics, University of California, and Department of Veterans Affairs Medical Center, San Diego, California 92161, USA.
Wu
H
H
Mahata
S K
SK
O'Connor
D T
DT
D000310:Adrenal Gland Neoplasms / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D002273:Carcinogens / Q000494:pharmacology; D002395:Catecholamines / Q000378:metabolism; D053379:Chromogranin A; D002864:Chromogranins / Q000235:genetics / Q000378:metabolism*; D017362:Cyclic AMP Response Element-Binding Protein / Q000502:physiology; D009538:Nicotine / Q000494:pharmacology; D016716:PC12 Cells / Q000187:drug effects; D010673:Pheochromocytoma / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011978:Receptors, Nicotinic / Q000502:physiology; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection
D002273:Carcinogens; D002395:Catecholamines; D053379:Chromogranin A; D002864:Chromogranins; D017362:Cyclic AMP Response Element-Binding Protein; D011978:Receptors, Nicotinic; D009538:Nicotine; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium