Differential expression of alpha-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 alpha-bungarotoxin. One subtype is involved in the activation step of the catecholamine secretion process and is not blocked by the toxin. The other is alpha-bungarotoxin-sensitive, and its functional role has not yet been defined. The alpha7 subunit is a component of this subtype. Autoradiography of bovine adrenal gland slices with alpha-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 alpha7 subunit also are localized exclusively to adrenergic cells. To identify possible transcriptional regulatory elements of the alpha7 subunit gene involved in the restricted expression of nicotinic receptors, we isolated and characterized its 5' 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 alpha7 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 alpha-bungarotoxin-sensitive nicotinic receptors in the adrenal gland.

Fig. 2.

Localization of α7 transcripts in the adrenal gland. In situ hybridization of α7 transcripts in the bovine (A–C) and rat (D, E) adrenal glands. A, Hybridization with bovine α7 antisense probe. B, Hybridization with bovine α7 sense probe. C, Detail of A. B, Inset, Reverse transcription and further PCR amplification of α7 subunit mRNA isolated from sites close (Me) or far (Mi) from the adrenal cortex (Co); + and − indicate reaction performed in the presence or absence of reverse transcriptase, respectively. D, Hybridization with rat α7 antisense probe; Co, adrenal cortex;M, adrenal medulla. E, Detail ofD. Scale bars: 300 μm in A,B; 100 μm in D; 25 μm inC, E.

Fig. 4.

Determination of the α7 subunit gene transcription initiation site. A, Sequence of the 5′ end of the RACE product obtained by reverse transcription and PCR amplification of α7 subunit mRNA. B, Mapping 5′ ends of α7 subunit mRNA by RNase protection by using an α7 probe that included exon 1 and its 5′ and 3′ flanking regions. T, Undigested probe, 397 bases; N, probe digested in the presence of 5 μg of yeast tRNA; P, protected fragments using 5 μg of bovine adrenal medulla poly(A⁺) mRNA. The size of several RNA fragments used for calibration of the gel is indicated at the left.

Fig. 6.

Egr-1 induction of luciferase expression from the α7 subunit promoter. Neuro-2a and bovine chromaffin cells were cotransfected with pα7LUC plasmids and Egr-1 expression vectors encoding a nonfunctional (+Mut. Egr-1) or a functional (+Egr-1) Egr-1 protein. Numbers above the columns indicate the fold induction by Egr-1. Data are expressed as in Figure 5.

Fig. 1.

Localization of α-Bgt binding sites and PNMT in the adrenal gland. A–F, Autoradiography of [¹²⁵I]α-Bgt binding to adrenal gland slices.A, B, Bovine; C,D, rat; E, F, cat. Total binding is observed in A, C, andE, whereas nonspecific binding, determined by incubating in the presence of 0.1 mm nicotine, is observed inB, D, and F.G–I, Immunolocalization of PNMT in the bovine (G), rat (H), and cat (I) adrenal glands. Scale bars: 1000 μm inA–G; 500 μm inH–I. Note that sizes are not comparable between species, because sections were not obtained from analogous locations in each gland.

Fig. 3.

The 5′ region of the bovine α7 subunit gene. A 2 kb EcoRI–PstI fragment of genomic clone λα7–11 carrying exons 1 and 2 (with the protein sequence indicatedunderneath in italics), the intron in between (within arrowheads, ▾), and 1500 bp of 5′ flanking sequence, was analyzed. The translation start codon isunderlined, and the major transcription initiation site (+1) is denoted by the arrow. Perfect matches to transcription factors Sp1 (solid lines rectangle), Egr-1 (long dashed lines rectangle), Myc-Max (short dashed lines rectangle), and E-box (hatched lines rectangle) are indicated.

Fig. 5.

Analysis of α7 gene promoter activity. The indicated cell types were transfected with each of the plasmids (namedpα7LUC, with the number of promoter base pairs included in the construct) containing the luciferase reporter under the control of the different fragments of the α7 subunit promoter and pCH110/β-galactosidase as a transfection efficiency control. All experimental points were run in triplicate. The mean ± SE (error bars) are given for three individual experiments. The restriction enzymes used to clone the different fragments also are shown in the scheme.

Fig. 7.

Two sites in the proximal promoter region of the α7 gene are involved in Egr-1 activation of transcription initiation.A, Deletion constructs (from p77α7LUCto p+7α7LUC) were obtained, using BstUI restriction enzyme, as described in Materials and Methods. The α7 promoter sequences contained between the constructs p38α7LUC and p15α7LUC were mutated by PCR (mut1–mut3). Each construct was cotransfected with Egr-1 expression vectors into chromaffin cells, and its activity was measured. Numbers above the columns indicate the fold induction by Egr-1. Luciferase activity was normalized to values obtained with the p77α7LUC construct. Data are expressed as in Figure 5.B, The region between −38 and −15 contains several putative binding sites for Egr-1 (boxed inp38). Five nucleotides of each potential element were mutated as indicated in mut1, mut2, andmut3 to yield constructs analyzed in transfection (p38α7LUCmut1–3 in A) and bandshift (Figs. 8, 9) experiments.

Fig. 8.

Gel mobility shift assays. A, Labeled wild-type (WT-38/+41) and mutated (Mut1, Mut2, Mut3; see Fig. 7) DNA fragments were used as gel mobility shift probes in the presence of 0.3 μg of purified Egr-1/GST fusion protein (E) or its mutated, inactive counterpart (Δ). B, The same DNA fragments were used with 5 μg of crude chromaffin cell nuclear extracts (N). The results of a supershift assay with Egr-1 specific antibody (E) or control IgG (I) are shown in lanes 3 and 4, respectively, of this panel.Dot, Egr-1 bound probe; arrowhead, supershifted Egr-1 bound probe; −, probe run in the absence of protein extracts. C, D, The gel mobility assay was run, using the WT-38/+41 DNA fragment as the labeled probe and nuclear extracts from chromaffin cells. The first lane in each series (−) is always without added competitor. C, Competitor DNA fragments WT-38/+41, Mut1, and Mut3were added in 15-, 45-, 150-, or 300-fold excess. D, Competitor oligonucleotides with consensus sites for Egr-1 (E), Sp1 (Sp1), or CREB (CREB) were added in 400-fold molar excess.Dot, Egr-1 bound probe.

Fig. 9.

Purified Egr-1 binds two nonequivalent sites within the most promoter-proximal region of the α7 gene. Labeled wild-type (WT-38/+41) and mutated (Mut1,Mut3) DNA fragments were used as gel mobility shift probes in the presence of 0.7 and 4 μg of purified Egr-1/GST fusion protein. Dot, Egr-1 bound to a single site;double dot, Egr-1 bound to two sites; −, probe run in the absence of protein.

Fig. 10.

Egr-1 immunolocalization in bovine and rat adrenal gland sections. A, A general view of the bovine gland shows that immunoreaction is present in the areas previously demonstrated to express α-Bgt-sensitive nAChRs and PNMT.B, Detail of the bovine gland showing nuclear labeling.C, High magnification of immunostaining in the rat adrenal medulla also shows nuclear labeling of Egr-1. Scale bars: 1000 μm in A; 50 μm in B; 25 μm inC.