Stimulus-transcription coupling in pheochromocytoma cells. Promoter region-specific activation of chromogranin a biosynthesis.
To explore stimulus-transcription coupling in pheochromocytoma cells, we studied the biosynthetic response of chromogranin A, the major soluble protein co-stored and co-released with catecholamines, to chromaffin cells' physiologic nicotinic cholinergic secretory stimulation. Chromogranin A mRNA showed a time-dependent 3.87-fold response to nicotinic stimulation, and a nuclear run-off experiment indicated that the response occurred at a transcriptional level. Transfected chromogranin A promoter/luciferase reporter constructs were activated by nicotinic stimulation, in time- and dose-dependent fashions, in both rat PC12 pheochromocytoma cells and bovine chromaffin cells. Cholinergic subtype agents indicated that nicotinic stimulation was required. Promoter deletions established both positive and negative nicotinic response domains. Transfer of candidate promoter domains to a heterologous (thymidine kinase) promoter conferred region-specific nicotinic responses onto that 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 sufficient to account for the overall positive response to nicotine. Within this proximal region, a cAMP response element (CRE) was implicated as a major nicotinic response element, since a CRE point-gap mutation decreased nicotinic induction, transfer of CRE to a thymidine kinase promoter augmented the promoter's response to nicotine, and nicotine activated 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 (chromogranin A), that the activation is transcriptional, and that a small proximal domain, including the CRE box, is, at least in part, both necessary and sufficient to account for the positive response to nicotine.
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. doconnor@ucsd.edu
Wu
H
H
Mahata
S K
SK
Taupenot
L
L
Rozansky
D J
DJ
Parmer
R J
RJ
O'Connor
D T
DT
D000818:Animals; D015152:Blotting, Northern; D015220:Calcium Channels / Q000378:metabolism; D002417:Cattle; D053379:Chromogranin A; D002864:Chromogranins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D051379:Mice; D009538:Nicotine / Q000494:pharmacology; D009638:Norepinephrine / Q000378:metabolism; D016716:PC12 Cells; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D014158:Transcription, Genetic; D014162:Transfection; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism
D015220:Calcium Channels; D053379:Chromogranin A; D002864:Chromogranins; D017362:Cyclic AMP Response Element-Binding Protein; D012333:RNA, Messenger; C506155:chromogranin A, mouse; C506153:chromogranin A, rat; D009538:Nicotine; D014446:Tyrosine 3-Monooxygenase; D009638:Norepinephrine