Stimulus-transcription Coupling in Pheochromocytoma Cells

Abstract

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 timeand 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 pointgap 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.When chromaffin cells and sympathetic axons discharge their vesicular stores of peptides and catecholamines after secretory stimuli, how do they resynthesize the released components? Does the same signal that causes secretion also trigger transcriptional activation of secretory peptide genes?Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, is transcriptionally activated (1-3) in chromaffin cells or noradrenergic neurons by the usual secretory stimulus, a nicotinic cholinergic agonist. Other catecholamine biosynthetic enzymes which respond to such stimuli include phenylethanolamine-N-methyltransferase and dopamine-␤-hydroxylase (4, 5). Chromaffin granule soluble components, such as proenkephalin A and carboxypeptidase H, also respond to secretory stimuli (4, 5).Since the quantitatively major soluble protein co-stored and co-released by exocytosis with catecholamines is chromogranin A (6, 7), we studied the response of the chromogranin A gene to activation of PC12 pheochromocytoma cell secretion by nicotinic cholinergic stimulation (4, 5, 8, 9). Our results suggest that chr...

Catecholamine Secretory Vesicle Stimulus-Transcription Coupling in Vivo

“…Nicotine also acts centrally to stimulate dopamine release from nigrostriatal and mesocorticolimbic neurons and norepinephrine release from hippocampus, cerebellum, and locus coeruleus neurons (61)(62)(63). In agreement with these findings as well as our previous in vitro studies (1,7), here we documented acute nicotinic stimulation of catecholamine release as reflected by ϳ2.7-fold increments in plasma catecholamine levels (Fig. 5).…”

“…In cultured chromaffin cells in vitro (7)(8)(9), exocytotic stimuli also program the resynthesis of just released catecholamine storage vesicle contents, a process known as "stimulus-secretion-synthesis coupling" or "stimulus-transcription coupling." Does this phenomenon occur in vivo?…”

“…Such nicotinic stimulation in cultured chromaffin cells in vitro (7)(8)(9) triggers the resynthesis of chromogranin A at the transcriptional level. This process of "stimulus-secretion-synthesis coupling" or "stimulus-transcription coupling" may serve to replete storage vesicles of just released transmitters (7)(8)(9).…”

“…This process of "stimulus-secretion-synthesis coupling" or "stimulus-transcription coupling" may serve to replete storage vesicles of just released transmitters (7)(8)(9).…”

“…We have characterized this process (sometimes called "stimulus-secretion-synthesis coupling" or "stimulustranscription coupling") in chromaffin cells in vitro (7)(8)(9) and established transcriptional activation of chromogranin A by nicotinic cholinergic (physiologic pathway) stimulation. The process occurs at the level of transcript initiation (7), requires particular elements in cis in the proximal promoter (7), and has well defined signal transduction pathways in trans (8,9). However, whether this nicotinic cholinergic stimulation of chromogranin A occurs in vivo is uncertain.…”