STIMULUS-TRANSCRIPTION COUPLING IN PHEOCHROMOCYTOMA CELLS

To explore stimulus-transcription coupling in pheochromocytoma cells, we studied the biosynthetic response of chromogranin A, the major solu ble protein co-stored and co-released with catecholamines, to chromaff in cells' physiologic nicotinic cholinergic secretory stimulation. Chr omogranin A mRNA showed a time-dependent 3.87-fold response to nicotin ic stimulation, and a nuclear run-off experiment indicated that the re sponse occurred at a transcriptional level, Transfected chromogranin A promoter/luciferase reporter constructs were activated by nicotinic s timulation, in time- and dose-dependent fashions, in both rat PC12 phe ochromocytoma cells and bovine chromaffin cells. Cholinergic subtype a gents indicated that nicotinic stimulation was required, Promoter dele tions established both positive and negative nicotinic response domain s. Transfer of candidate promoter domains to a heterologous (thymidine kinase) promoter conferred region-specific nicotinic responses onto t hat promoter. A proximal promoter domain (from -93 to -62 base pairs) was activated in copy number- and distance-dependent fashion, and thus displayed features of a promoter element, Its activation was sufficie nt to account for the overall positive response to nicotine, Within th is proximal region, a cAMP response element (CRE) was implicated as a major nicotinic response element, since a CRE point-gap mutation decre ased nicotinic induction, transfer of CRE to a thymidine kinase promot er augmented the promoter's response to nicotine, and nicotine activat ed the CRE-binding protein CREB through phosphorylation at serine 133. We conclude that secretory stimulation of pheochromocytoma cells also activates the biosynthesis of the major secreted protein (chromograni n A), that the activation is transcriptional, and that a small proxima l domain, including the CRE box, is, at least in part, both necessary and sufficient to account for the positive response to nicotine.