Differential Expression of α-Bungarotoxin-Sensitive Neuronal Nicotinic Receptors in Adrenergic Chromaffin Cells: A Role for Transcription Factor Egr-1

Abstract

Adrenomedullary chromaffin cells express at least two subtypes of acetylcholine nicotinic receptors, which differ in their sensitivity to the snake toxin ␣-bungarotoxin. One subtype is involved in the activation step of the catecholamine secretion process and is not blocked by the toxin. The other is ␣-bungarotoxin-sensitive, and its functional role has not yet been defined. The ␣7 subunit is a component of this subtype. Autoradiography of bovine adrenal gland slices with ␣-bungarotoxin indicates that these receptors are restricted to medullary areas adjacent to the adrenal cortex and colocalize with the enzyme phenylethanolamine N-methyl transferase (PNMT), which confers the adrenergic phenotype to chromaffin cells. Transcripts corresponding to the ␣7 subunit also are localized exclusively to adrenergic cells. To identify possible transcriptional regulatory elements of the ␣7 subunit gene involved in the restricted expression of nicotinic receptors, we isolated and characterized its 5Ј flanking region, revealing putative binding sites for the immediate early gene transcription factor Egr-1, which is known to activate PNMT expression. In reporter gene transfection experiments, Egr-1 increased ␣7 promoter activity by up to sevenfold. Activation was abolished when the most promoter-proximal of the Egr-1 sites was mutated, whereas modification of a close upstream site produced a partial decrease of the Egr-1 response. Because Egr-1 was found to be expressed exclusively in adrenergic cells, we suggest that this transcription factor may be part of a common mechanism involved in the induction of the adrenergic phenotype and the differential expression of ␣-bungarotoxin-sensitive nicotinic receptors in the adrenal gland.

Glucocorticoid activation of the neuronal nicotinic acetylcholine receptor $alpha;7 subunit gene: involvement of transcription factor Egr-1

“…In order to examine the possibility that glucocorticoids were activating the transcription of the a7 subunit gene, bovine chromaffin cells were cotransfected with a plasmid coding for the human glucocorticoid receptor (pRS-hGRa kindly provided by Dr. R. Evans, San Diego, USA) and the a7 promoter-luciferase gene construct p2700a7LUC, which contains about 2700 bp of a7 promoter sequence plus 43 bp of 5 0 noncoding region linked to the luciferase gene and yields the maximal promoter activity in chromaffin cells [4]. The effect of the synthetic glucocorticoid dexamethasone on a7 promoter activity was examined by measuring luciferase expression in cells lysed at different times of dexamethasone treatment.…”

“…For instance, phosphorylation [15], the redox state of Egr-1 [16] or the interaction with corepressors [17] might modify its DNA binding capacity. Whatever the mechanism, in the bovine adrenal gland a specific stimulation of Egr-1 function by glucocorticoids appears very plausible, since localization of adrenergic cells expressing Egr-1 and also the enzyme phenylethanolamine N-methyl transferase (PNMT) and a7 nAChRs is so peculiar: they are only in the region of the adrenal medulla close to the adrenal cortex and to the portal vein [4]. In this scenario, glucocorticoids arising from the cortex would diffuse into the nearby area of the adrenal medulla and then act directly on adrenergic chromaffin cells, especially because the glucocorticoid receptor has been localized only to the cytoplasm of adrenergic cells [18].…”

“…The isolation and characterization of the 5 0 flanking sequence of the bovine a7 subunit gene has been previously described [4]. All a7 promoter-LUC gene fusions were made in the pGL2-Basic vector (Promega, Spain), introducing in its polylinker, upstream of the luciferase gene, the suitable a7 promoter fragments.…”

“…Considerable effort has been dedicated to understanding how the regional and developmental expression of neuronal nAChRs is controlled at the transcriptional level (reviewed in [3]). Previously, we have characterized the promoters of the bovine a7 [4,5], a5 [6], and b4 [7] subunits which are components of nAChRs present in chromaffin cells of the bovine adrenal medulla. One of the subtypes, sensitive to a-bungarotoxin, is formed by a7 subunits and is differentially expressed in adrenergic cells adjacent to the adrenal cortex [4].…”

“…Previously, we have characterized the promoters of the bovine a7 [4,5], a5 [6], and b4 [7] subunits which are components of nAChRs present in chromaffin cells of the bovine adrenal medulla. One of the subtypes, sensitive to a-bungarotoxin, is formed by a7 subunits and is differentially expressed in adrenergic cells adjacent to the adrenal cortex [4]. Since the latter may provide high levels of glucocorticoids to the contiguous adrenergic cells, we asked whether the a7 promoter would be regulated by glucocorticoids.…”