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Volume 17, Number 17,
Issue of September 1, 1997
pp. 6554-6564
Copyright ©1997 Society for Neuroscience
Differential Expression of 
-Bungarotoxin-Sensitive Neuronal
Nicotinic Receptors in Adrenergic Chromaffin Cells: A Role for
Transcription Factor Egr-1
Manuel Criado1, 4,
Eduardo Domínguez del Toro1, 4,
Carmen Carrasco-Serrano1, 4,
Frazer I. Smillie1, 4,
José M. Juíz2, 4,
Salvador Viniegra1, 4, and
Juan J. Ballesta3, 4
Departments of 1 Neurochemistry,
2 Histology, and 3 Pharmacology, and
4 Instituto de Neurociencias, Universidad Miguel Hernandez,
03550 San Juan, Alicante, Spain
ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
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 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 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.
Key words:
ACh receptors;
-bungarotoxin;
adrenergic;
chromaffin
cell;
7 subunit;
Egr-1
INTRODUCTION
Neuronal nicotinic acetylcholine
receptors (nAChR) sensitive to -bungarotoxin (-Bgt) are widely
expressed in the central and peripheral nervous systems (for review,
see Sargent, 1993
; McGehee and Role, 1995). The molecular cloning of
nAChR subunits that assemble into channels activated by cholinergic
agonists and are blocked by -Bgt (termed 7, 8, and 9;
Couturier et al., 1990; Schoepfer et al., 1990; Elgoyhen et al., 1994)
has allowed structural and functional studies of these nAChR subtypes. Although -Bgt-sensitive nAChRs on neurons function as ligand-gated ion channels (Alkondon and Albuquerque, 1993; Zhang et al., 1994), their physiological role in situ have not yet been
elucidated fully. It has been suggested that they could enhance fast
excitatory transmission via a presynaptic mechanism (McGehee et al.,
1995). In addition, they could have a growth or trophic role, because there is evidence that these receptors are involved in the guidance of
nerve fibers (Pugh and Berg, 1994), inhibition of neurite extension in
cultured cells (Chan and Quik, 1993), regulation of neurotrophic growth
factor expression in rat hippocampus (Freedman et al., 1993),
motoneuronal death (Hory-Lee and Frank, 1995), and modulation of
proliferative responses in lung tumor cells (Codignola et al., 1994;
Quik et al., 1994). Recently, postsynaptic responses for -Bgt-sensitive nAChRs on neurons have been detected (Zhang et al.,
1996).
We have shown previously that the -Bgt-sensitive nAChRs from the
chromaffin cells of the adrenal medulla contain an 7 subunit, homologous to those previously cloned from other species
(García-Guzmán et al., 1995). In the adrenal gland,
acetylcholine released on stimulation of the splanchnic nerve activates
nAChRs in chromaffin cells and triggers catecholamine secretion, but,
interestingly, this functional response is not blocked by -Bgt. This
suggests the involvement of other nAChRs in the activation of
exocytosis and would imply a segregation of functions for different
nAChRs subtypes in the same cell. Alternatively, different nAChRs with distinct functions might be expressed in specific chromaffin cell subsets. Given any of these cases, complex molecular mechanisms may
underlie nAChR subunit expression, which in turn may influence the
physiological characteristics of a cellular group or a neuronal circuit. To investigate these mechanisms, we studied the expression of
-Bgt-sensitive nAChRs in the adrenal gland of several mammals, consistently finding that they were present only in adrenergic cells.
This heterogenous distribution is attributable, at the very least, to
restricted transcription of the 7 subunit gene in this chromaffin
cell subset. Furthermore, we have identified transcription factor Egr-1
(Cao et al., 1990), which also is restricted to adrenergic cells, as
the candidate potentially involved in this differential expression.
MATERIALS AND METHODS
-Bgt autoradiography. The procedure followed for
[125I]-Bgt binding has been described
previously (Clarke et al., 1985). Cryostat sections (15-20 µm) were
incubated with 5 nM [125I]-Bgt for
2 hr at room temperature. After nonbound toxin was washed, sections
were dried and stored in cassettes for 48 hr at 4°C with Kodak X-OMAT
x-ray film.
Immunohistochemistry. Rat tissue was perfused and processed
as previously described (Domínguez del Toro et al., 1994). Cat and bovine adrenal glands were fixed by immersion in 4%
paraformaldehyde in 75 mM sodium phosphate buffer, pH 7.35, and then processed in the same manner as the rat tissue. Cryostat
sections (25-30 µm) were free-floated in continuously agitated
solutions at 4°C. Phenylethanolamine N-methyl transferase
(PNMT; EC2.1.1.28) was immunolabeled with a rabbit antiserum against
bovine PNMT (Eugene Tech, Ridgefield Park, NJ), diluted 1:1000. Egr-1
was localized with an affinity-purified polyclonal antibody (588)
raised in rabbit against a peptide that corresponds to the 14 C-terminal residues of mouse Egr-1 p82 (Santa Cruz Biotechnology, Santa
Cruz, CA). The antibody was used at a 1:1000 dilution.
In situ hybridization. Sense and antisense
digoxigenin-labeled riboprobes were synthesized with SP6 polymerase
from the corresponding linearized template (pSPT18) with a Boehringer
Mannheim (Barcelona, Spain) RNA labeling kit. Probes were directed to a
region of the M3-M4 cytoplasmic loop unique to the 7 subunit
[amino acids 315-382; see García-Guzmán et al. (1995)
for the bovine 7 sequence and Séguéla et al. (1993) for
its rat counterpart]. Frozen adrenal gland tissue was cut in 10 µm
sections, mounted on silanated slides, fixed in 4% formaldehyde (5 min
at room temperature), deproteinated (10 min in 0.2 M HCl at
room temperature), and dehydrated in ethanol. Hybridization was for 16 hr at 45°C in a buffer containing 50% formamide, 5× SSC, 1×
Denhardt's solution, 10% dextran sulfate, 0.2% SDS, 10 mM dithiothreitol, 250 µg/ml herring sperm DNA, and 250 µg/ml yeast tRNA. Digoxigenin-labeled probes were diluted (in the
range from 1:25 to 1:400) with hybridization buffer to obtain a
suitable and equivalent concentration of the different probes, as
verified by dot blot assays. After hybridization, sections were washed
for 2 × 30 min in 0.1× SSC at 60°C. Visualization of the probe
was performed with anti-digoxigenin antiserum, as indicated by the
manufacturer (Boehringer Mannheim).
Reverse transcription-PCR (RT-PCR) analysis. Total RNA was
extracted by guanidinium isothiocyanate and acid phenol extraction (Promega, Barcelona, Spain) from adrenomedullary tissue dissected from
areas near to or far from the adrenal cortex. 7 subunit transcripts
were detected by a combined RT-PCR assay (Singer-Sam et al., 1990).
Briefly, samples of total RNA (1.2 µg) in a final volume of 30 µl
were reverse-transcribed with 1.25 U avian myeloblastosis virus (AMV)
reverse transcriptase (10 min at 50°C); then PCR was performed for 30 cycles (1 min at 94°C, 2 min at 58°C, and 2.5 min at 72°C). The
following primers were chosen: sense, 5-CCAGGGCTGGTTTCCGAGA-3; antisense, 5TGTCCAAGTCATTTGTAGCCA-3. The PCR fragment had 281 base pairs (bp) from the 5 noncoding region to amino acid 80; the
amplified DNA fragment expanded over three introns to rule out the
possibility of amplifying a potential contamination of genomic DNA. In
fact, RT-PCR performed in the absence of AMV reverse transcriptase (see
Fig. 2B, inset) did not yield any fragment.
Fig. 2.
Localization of 7 transcripts in the adrenal
gland. In situ hybridization of 7 transcripts in the
bovine (A-C) and rat (D, E)
adrenal glands. A, Hybridization with bovine 7
antisense probe. B, Hybridization with bovine 7 sense
probe. C, Detail of A. B, Inset, Reverse transcription and further PCR amplification of 7 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 7 antisense probe; Co, adrenal cortex; M, adrenal medulla. E, Detail of
D. Scale bars: 300 µm in A,
B; 100 µm in D; 25 µm in
C, E.
[View Larger Version of this Image (189K GIF file)]
Isolation and analysis of the 5 flanking sequence of the 7
subunit. A cDNA probe containing 87 bp of the 3 end of exon 1 and
105 bp of the 5 end of exon 2 was used to screen a bovine genomic
library in EMBL-3 SP6/T7 (Clontech, Heidelberg, Germany), as previously
described (García-Guzmánet al., 1995). Several overlapping bacteriophage clones were purified and characterized. Clone
7-11, which contained ~17 kb of bovine genomic sequence, including exons 1 and 2, and ~7 kb of 5 flanking sequence, was characterized further.
5 RACE analysis of 5 mRNA ends. The rapid amplification of
cDNA ends (RACE) was performed as described
(García-Guzmán et al., 1995). The first-strand cDNA,
after reverse transcription of bovine adrenal medulla mRNA, was
polyadenylated and amplified by two PCR rounds. An oligo dT was the
sense primer, and the two antisense primers were
5-TGTCCAAGTCATTTGTAGCCA-3 (first round) and 5-ATGTTGGTGGTCAACAC
CTG-3 (second round), coding for amino acids 74-80 and 67-73,
respectively, of the 7 sequence. The resultant DNA was cloned into
pBluescript (Stratagene, Heidelberg, Germany), and the sequence of the
inserts was determined and compared with genomic DNA to establish the
5 end of 7 mRNA.
RNase protection. A 305 bp
SacII-BamHI fragment of the 7 gene that
included 126 bp 5 and 46 bp 3 to exon 1 was subcloned into
pBluescript. After linearization of the plasmid with EcoRI, a probe of 397 nucleotides was synthesized with T7 polymerase and
[-32P]CTP. For calibration, several other RNAs of
known size were synthesized also. RNase protection experiments were
performed with the RNase Protection Kit (Boehringer Mannheim).
Protected fragments were separated on a 6% acrylamide/urea gel.
Plasmid constructions. All 7promoter-LUC gene fusions
were made in the pGL2-Basic vector (Promega), introducing in its
polylinker upstream of the luciferase gene the suitable 7 promoter
fragments. These fragments were generated with restriction enzymes (as
indicated in Fig. 4) and cloned directly into pGL2-Basic or subcloned
first in pBluescript and then transferred to pGL2-Basic. The vector pGL2-Control, which express the luciferase gene under the regulation of
the SV40 promoter and enhancer sequences, was used to check luciferase
activity.
Fig. 4.
Determination of the 7 subunit gene
transcription initiation site. A, Sequence of the 5 end
of the RACE product obtained by reverse transcription and PCR
amplification of 7 subunit mRNA. B, Mapping 5 ends
of 7 subunit mRNA by RNase protection by using an 7 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.
[View Larger Version of this Image (43K GIF file)]
Deletion analysis of the most promoter-proximal region was performed by
partially digesting an ApaI-HindIII fragment
(from 339 to +41 in the 7 sequence) with BstUI, which
leaves blunt ends at GCGC sequences, and subcloning the partial digests
into pBluescript vector cut with HincII-HindIII.
Sequence analysis allowed selection of the appropriate fragments, which
were cloned into pGL2-Basic and transfected further.
For site-directed mutagenesis of the putative Egr-1 sites at the 38
to 15 region of the 7 promoter (see Fig. 6), the following mutagenic primers were synthesized: mut1,
5-CaaaaaCGGTCGGGGCGTGGGCG-3; mut2,
5-CGGGGGCGGTaaaaaCGTGGGCGCGCGCTGGG-3; mut3,
5-CGGGGGCGGTCGGGGaaaaaGCGCGCGCTGGGCTTTTTA-3 (the introduced
mutations are indicated in lower case letters). PCR (25 cycles of
94°C for 10 sec, 62°C for 30 sec, and 68°C for 45 sec, using
the Expand kit from Boehringer Mannheim) was performed with these
oligonucleotides and an antisense primer (5-CTTTATGTTTTTGGCGTCTTCC-3) that anneals to the pGL2-Basic vector downstream of the site of transcription initiation. PCR products were cloned into pBluescript, sequenced, and transferred further to pGL2-Basic.
Fig. 6.
Egr-1 induction of luciferase expression from the
7 subunit promoter. Neuro-2a and bovine chromaffin cells were
cotransfected with p7LUC 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.
[View Larger Version of this Image (28K GIF file)]
Cell culture and reporter assays. Neuro-2a mouse
neuroblastoma cells were cultured in 90% Eagle's minimal essential
medium (EMEM), 10% fetal calf serum (FCS), and 2 mM
glutamine. SH-SY5Y human neuroblastoma cells were grown in a 1:1
mixture of Ham's F12 medium and EMEM containing 1% nonessential amino
acids and 10% FCS. Chromaffin cells were isolated from bovine adrenal
glands as described (Gandía et al., 1991) and cultured in 90%
DMEM, 10% FCS, and 10 µM cytosine arabinoside to prevent
fibroblast proliferation.
Plasmids were purified by Wizard maxipreps (Promega) and then banded in
CsCl. All cell types were transfected by the calcium phosphate
procedure (Graham and van der Eb, 1973). Cells (1-3 × 105) on 3.5 cm plates were incubated with 3 µg of
pGL2 vector or an equivalent amount (in molar terms) of the different
constructs derived from this vector and with 3 µg of
-galactosidase expression vector pCH110 (Pharmacia, Uppsala, Sweden)
as a control of transfection efficiency. Two micrograms of Egr-1
expression plasmid (pCMVEgr-1) (Gupta et al., 1991) or of its mutated
inactive form (pCMVEgr-1
331-374) also were present in the
transfection mixture when the effect of this transcription factor was
studied. Cells were harvested after 48 hr and lysed with reporter lysis
buffer (Promega). Then -galactosidase and luciferase were determined
in the lysates with the corresponding assay systems (Promega).
Luciferase activity was normalized to values obtained with the
pGL2-Control vector in the same cell type. Relative light units
obtained with pGL2-Control in the different cell types are as follows
(mean of three individual experiments and corrected for transfection
efficiency): chromaffin, 273; neuro-2a, 371; SHSY-5Y, 801.
Electrophoretic mobility shift assay. Crude nuclear extracts
were prepared from chromaffin cells, as described by Schreiber et al.
(1989). DNA fragments corresponding to the region 38 to +41, mutated
or not at the putative Egr-1 sites, were obtained by digesting
pBluescript subclones with EcoRI-HindIII and
end-labeled by Klenow filling with [-32P]dATP. The
DNA-protein binding reaction volumes were 20 µl containing (in
mM): 10 Tris, pH 7.5, 50 NaCl, 1 EDTA, and 1 dithiotreitol with 10% glycerol, 5 µg of bovine serum albumin, 2 µg of
poly(dA-dT)·(dA-dT) (Pharmacia), 5 µg of nuclear extract protein,
and 20,000 cpm of 32P-labeled probe. Reactions were
incubated for 10 min at room temperature, labeled probe was added, and
the incubation was continued for an additional 20 min. For competition
studies the nuclear extract was incubated with the competing probe
before the labeled probe during 20 min. Competition was performed
either with the same purified fragments or with double-stranded
oligonucleotides containing consensus sites for Egr-1 (Santa Cruz
Biotechnology), Sp1, or cAMP response element binding proteins (CREB;
Promega). Supershift assays were performed by incubating with 2 µl of
anti-Egr-1 antibody (588X from Santa Cruz Biotechnology) or rabbit IgG
(Sigma, Madrid, Spain) after probe addition, and the incubation
continued for an additional 30 min at room temperature.
In some experiments purified Egr-1 protein fused to glutathione
S-transferase (GST) was used. The expression vector for the Egr-1/GST fusion protein was kindly provided by Dr. X. Cao (National University of Singapore), and the protein was expressed in bacteria and
purified as described by Jain et al. (1996). The mutated inactive form
of Egr-1 (Egr-1331-374) fused to GST also was cloned in the same
expression vector (pGEX-KG; Guan and Dixon, 1991) and used as a
negative control.
RESULTS
-Bgt binding sites and 7 subunit mRNA colocalize in
chromaffin adrenergic cells
Autoradiography of adrenal gland slices labeled with
[125I]-Bgt was performed to localize
-Bgt-sensitive nAChRs in chromaffin cells (Fig.
1). Three mammalian species were tested:
cow, cat, and rat. The bovine adrenal medulla showed a characteristic
labeling: the highest -Bgt levels were found in the areas close to
the adrenal cortex, at the periphery, and around the portal vein at the
center (Fig. 1A). Labeling of the rat adrenal medulla
was almost complete (Fig. 1C), whereas the cat showed a
patchy distribution of binding sites, which was difficult to categorize
(Fig. 1E). In all cases labeling was abolished by
preincubation with nicotine (Fig. 1B,D,F). The
fact that the adrenergic cell-rich rat adrenal medulla was labeled
almost totally by -Bgt, whereas its bovine counterpart, in which
only ~50% of cells are adrenergic, was partially labeled, suggested
that -Bgt-sensitive nAChRs were differentially expressed in
adrenergic cells. To verify this, we performed immunolocalization of
PNMT, the enzyme converting norepinephrine to epinephrine and, therefore, conferring the adrenergic phenotype. PNMT was present only
in the bovine chromaffin cells close to the cortex or the portal vein
(Fig. 1G), whereas most rat chromaffin cells were labeled
(Fig. 1H). PNMT immunostaining in the cat gland had a patchy distribution (Fig. 1I). Hence, PNMT
localization for all three mammals was shown to be very similar to the
one observed for -Bgt binding sites.
Fig. 1.
Localization of -Bgt binding sites and PNMT in
the adrenal gland. A-F, Autoradiography
of [125I]-Bgt binding to adrenal gland slices.
A, B, Bovine; C,
D, rat; E, F, cat.
Total binding is observed in A, C, and
E, whereas nonspecific binding, determined by incubating
in the presence of 0.1 mM nicotine, is observed in
B, D, and F.
G-I, Immunolocalization of PNMT in the bovine
(G), rat (H), and
cat (I) adrenal glands. Scale bars: 1000 µm in
A-G; 500 µm in
H-I. Note that sizes are not comparable between species, because sections were not obtained from analogous locations in each gland.
[View Larger Version of this Image (136K GIF file)]
Because the 7 subunit present in bovine chromaffin cells is probably
a major component of -Bgt-sensitive nAChRs
(García-Guzmán et al., 1995), it seemed possible that the
restricted expression of -Bgt-sensitive nAChRs was regulated by the
transcriptional activity of the 7 subunit gene. This hypothesis was
supported by a qualitative assay, the RT-PCR analysis of 7 mRNA
levels in bovine adrenomedullary tissue (Fig.
2B, inset) dissected
from places near to (Me) or far (Mi) from the adrenal cortex, i.e., where -Bgt-sensitive nAChRs were in high or low abundance,
respectively. This experiment demonstrates that 7 mRNA was more
abundant in the region of the medulla close to the cortex. This
evidence was confirmed by in situ hybridization of bovine
7 transcripts: as shown by an antisense probe, they were present in
the area adjacent to the adrenal cortex (Fig. 2A,
Me; 2C), where adrenergic cells had been
localized previously. The same probe in the sense orientation gave no
labeling at all (Fig. 2B). On the other hand, most of the rat adrenal medulla was labeled with an antisense rat 7 probe (Fig. 2D,E), as in the case of PNMT immunolabeling.
Thus transcriptional regulation of the 7 subunit gene seems to be
involved in the differential expression of -Bgt-sensitive nAChRs in
adrenergic cells.
Functional analysis of the 7 subunit promoter
To examine the requirements for 7 subunit transcription, we
isolated and analyzed its promoter region. A bovine genomic library was
screened, and several overlapping clones were isolated. Their mapping
showed the presence of the 7 5 untranslated region (first exon) and
further upstream sequences, as well as the second and third exons; the
latter were separated by an intron of ~30 kb. Subcloning and
sequencing of an isolated EcoRI-PstI fragment
from one of the clones (Fig. 3) and
further comparison of this sequence to a database of binding sequences
of known transcription factors revealed the main features of the
promoter/regulatory region of the 7 gene: lack of a TATA box and the
presence of perfect matches to three potential Sp1 sites, one Egr-1
site, one Myc-Max site, and one E-box (Fig. 3), all of them
concentrated into ~360 bases located 5 to transcription initiation
site. Additional sites with one mismatch, regarding perfect consensus
sequences, also were observed.
Fig. 3.
The 5 region of the bovine 7 subunit gene. A 2 kb EcoRI-PstI fragment of genomic clone
7-11 carrying exons 1 and 2 (with the protein sequence indicated
underneath in italics), the intron in
between (within arrowheads, 
), and 1500 bp of 5
flanking sequence, was analyzed. The translation start codon is
underlined, 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.
[View Larger Version of this Image (71K GIF file)]
The 5 end of 7 mRNA was mapped by 5 RACE and RNase protection
analyses (Fig. 4), because primer
extension experiments produced inconsistent results. In 5 RACE only
one DNA fragment was detected, the 5 end sequence of which is shown in
Figure 4A. This would map transcription initiation to
a stretch of 5Ts and 3As (see black arrow in Fig. 3). RNase
protection analyses with a 397 residue antisense riboprobe
complementary to +180 to 125 and poly(A+) RNA from
bovine adrenal medulla yielded two main protected fragments of ~139
and 137 bases (Fig. 4B). The major one mapped to the
first adenosine detected in the 5 RACE fragment and was located
downstream of a GC-rich region. Therefore, this tentatively was
considered the main transcription initiation site (position +1,
black arrow in Fig. 3), although other initiation sites may
exist, because this is a typical feature of promoters without TATA
boxes.
The transcriptional activity of the 7 promoter was examined with a
series of constructs made by fusing the 7 5 flanking region to a
luciferase reporter gene. The nested set of 7 expression plasmids,
containing 5900-128 bp of 7 promoter sequence plus 41 bp of 5
noncoding region was transfected into a variety of cell types. Two of
them, bovine chromaffin cells and the human neuroblastoma SH-SY5Y
(which is of sympathetic adrenergic ganglion origin; see Biedler et
al., 1973), endogenously express -Bgt-sensitive nAChRs (Lukas et
al., 1993), whereas the other, mouse neuroblastoma neuro-2a, does not
(our unpublished results). All reporter constructs were found to be
active in chromaffin and SH-SY5Y cells, showing much reduced activity
in neuro-2a cells (Fig. 5). The smallest 7 promoter fragment tested, 128 bp, was sufficient to drive
transcription, although larger fragments increased activity, especially
in chromaffin cells. No luciferase expression was detected in any of
the cell types tested when no promoter segments were present upstream
of the luciferase gene (pGL2-Basic vector; data not shown).
Fig. 5.
Analysis of 7 gene promoter activity. The
indicated cell types were transfected with each of the plasmids (named
p7LUC, with the number of promoter base pairs
included in the construct) containing the luciferase reporter under the
control of the different fragments of the 7 subunit promoter and
pCH110/-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.
[View Larger Version of this Image (42K GIF file)]
The similar pattern of expression in the bovine adrenal gland of the
enzyme PNMT and the 7 subunit suggested that they share common
transcriptional regulatory elements. This idea was reinforced when the
promoter regions were compared: both were markedly G/C rich and
contained several consensus sequences for transcription factors Sp1 and
Egr-1. Moreover, it is known that these factors regulate PNMT
expression in PC12 cells (Ebert et al., 1994; Ebert and Wong, 1995). To
determine the possible regulation of 7 expression by Egr-1, we
cotransfected different 7-luciferase constructs into neuro-2a and
chromaffin cells with either a construct allowing strong expression of
Egr-1 (pCMVEgr-1) or a control yielding a truncated, nonfunctional
Egr-1 protein (pCMVEgr-1331-374) (Fig. 6). In neuro-2a cells the functional
Egr-1 increased the luciferase activity up to fourfold with respect to
that observed with the truncated Egr-1. In chromaffin cells activation
was between three- and fivefold, depending on the construct used.
In both cell types activation already was observed with the smallest
construct, which contains 128 bp of 7 promoter region. Several
potential binding sites for Egr-1 are found in this region, although
only one, located 31 bp 5 to the transcription initiation site (+1) of
the 7 gene, comprises a perfect match to the Egr-1 consensus binding
element (GCGGGGGCG) (Gashler and Sukhatme, 1995). To demonstrate a
possible direct interaction of this transcription factor with the 7
gene, we performed several deletions, studying in each case the
activation produced by Egr-1 expression on transfection into chromaffin
cells (Fig. 7A). Thus, removal
of nucleotides (nt) 128 to 78 did not affect Egr-1 activation
(p777LUC, Fig. 7A), which was even
larger than with previous constructs. The promoter basal activity was
similar to that supported by p1287LUC. Further removal of nt 77 to
39 (p387LUC, Fig. 7A) resulted in
~30% decrease in promoter activity (relative to p777LUC). However, Egr-1 was able to activate this shortened construct to a
similar extent. Additional removal of nt 38 to 16
(p157LUC, Fig. 7A) drastically reduced
promoter activity (~20% of p777LUC), which was not modified by
the presence of Egr-1. A construct lacking the site of transcription
initiation (p+77LUC, Fig. 7) was used to
demonstrate further that downstream sequences as well as vector sequences were not involved in the observed effects. Therefore, these
experiments indicate that the region between nt 38 to 16, although
probably lacking important elements for basal activity, can support
Egr-1 activation. This region contains several potential sites for
Egr-1 (boxed and termed 1, 2, and 3 in
Fig. 7B), which were mutated in an attempt to identify the
area of interaction between this factor and the 7 gene. When site 1, starting at the 5 end, was abolished
(p387LUCmut1, Fig. 7A), the activation produced by Egr-1 decreased with respect to that observed with the wild
construct but was still significant. This site also was modified in
p1287LUC by mutating the area at 37 to 39 from GCG to CAT,
yielding a similar degree of Egr-1 activation to the one observed for
p387LUCmut1 (data not shown). Mutation of site 2 (p387LUCmut2, Fig. 7A) did not produce
any decline on Egr-1 activation. Finally, mutation of site 3 (p387LUCmut3, Fig. 7A) totally
abolished the effect of Egr-1. Therefore, two areas (at 38 to 31
and 24 to 16) seem to be responsible for the effect of Egr-1,
emerging as more important the most proximal to the initiation start
site (site 3, Fig. 7B). In addition, it is
important to note that the three mutant constructs showed reduced basal promoter activity, indicating that the whole region is involved in
providing essential elements for transcriptional activity.
Fig. 7.
Two sites in the proximal promoter region of the
7 gene are involved in Egr-1 activation of transcription initiation.
A, Deletion constructs (from p777LUC
to p+77LUC) were obtained, using BstUI
restriction enzyme, as described in Materials and Methods. The 7
promoter sequences contained between the constructs p387LUC and
p157LUC 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
p777LUC construct. Data are expressed as in Figure 5.
B, The region between 38 and 15 contains several
putative binding sites for Egr-1 (boxed in
p38). Five nucleotides of each potential element were
mutated as indicated in mut1, mut2, and
mut3 to yield constructs analyzed in transfection (p387LUCmut1-3 in A) and
bandshift (Figs. 8, 9) experiments.
[View Larger Version of this Image (39K GIF file)]
To examine further the binding sites of Egr-1 detected in the 38 to
16 region of the 7 promoter region, we performed gel mobility
shift assays. DNA fragments carrying the wild-type 38/+41 promoter
region and the corresponding mutants (mut1, mut2, and mut3, Fig. 7B) were labeled and used either with
nuclear extracts from chromaffin cells or with purified Egr-1 protein.
A prominent retarded band was observed when Egr-1 was used either with
the wild-type DNA fragment or with mut1 or mut2 (Fig.
8A, lanes 2, 5, and
8, respectively, filled dot). The same band also
was obtained with mut3 but with much less intensity (Fig.
8A, lane 11). Because a mutant, inactive Egr-1
protein was unable to bind any of the DNA fragments (Fig.
8A, lanes 3, 6, 9, and 12), we deduced
that Egr-1 was binding specifically to the DNA probes. Moreover, the fact that mut3 showed very reduced capacity to bind Egr-1 confirms the
significance, with respect to Egr-1 activation, of the Egr-1 binding
site most proximal to the transcription initiation site (site
3). When nuclear extracts were used, several retarded bands were observed (Fig. 8B). One of them (indicated by a
dot) was retarded further by an anti-Egr-1 antibody
(arrowhead Fig. 8B, lane 3), but not by an
unrelated IgG. The same band also was observed with mut1, mut2, and
mut3, but, again, the latter fragment showed reduced binding capacity.
Interestingly, mut3 revealed the presence of a new band, only weakly
observed with the other constructs, that suggests the existence of
another protein, the binding of which is possible only when Egr-1 is
not bound. Alternatively, the introduced mutation would create a new
recognition site not present in the other fragments.
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
(). 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 Mut3
were 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.
[View Larger Version of this Image (95K GIF file)]
To evaluate further the specificity of Egr-1 binding to the proximal
promoter region of the 7 gene, we performed competition experiments
with oligonucleotides containing Egr-1, Sp1, or CREB consensus
sequences or the same DNA fragments used in the bandshift assays (Fig.
8C,D). Preincubation of the nuclear extracts with increasing
amounts (ranging from a 15- to a 300-fold excess) of the WT-38/41
fragment gradually decreased the intensity of the different complexes
(Fig. 8C, lanes 2-5). Competition with mut1 produced a
similar decrease (Fig. 8C, lanes 6-9), although the band
corresponding to Egr-1 appeared slightly more intense (compare, for
instance, the corresponding lanes 2 and 6 in Fig.
8C), suggesting that the absence of the Egr-1 site 1 in this
fragment caused a decrease in its competition ability. The mut3
fragment was the less effective competitor, although it also induced a
decrease in band intensity (Fig. 8C, lanes 10-13),
suggesting that (1) the proximal Egr-1 site 3, absent in this fragment,
has a higher affinity for the factor, and (2) the distant Egr-1 site,
although weaker, can bind Egr-1 and induce competition. Finally, when
competition was performed with a 400-fold molar excess of synthetic
oligonucleotides (Fig. 8D, lanes 1-4), only
the Egr-1 consensus fragment abolished the formation of the Egr-1
complex. Therefore, Egr-1 specifically interacts with the most
promoter-proximal region of the 7 gene.
The above data also suggest that Egr-1 categorizes two nonequivalent
sites in this area. Experiments with purified Egr-1 confirmed this
assertion (Fig. 9). Thus, by raising the
amount of protein used to shift the nonmutated DNA fragment WT
38/+41, it was possible to observe the formation of a second, larger
complex (Fig. 9, lanes 2 and 3, double dot). This
complex was not observed when any of the two Egr-1 sites (sites
1 and 3) were mutated (Fig. 9, lanes
5-6 and lanes 8-9), suggesting that it arises from
the binding of Egr-1 polypeptides to both sites. The different
intensity of the bands obtained with mut1 and mut3 DNA fragments
(compare lanes 5 and 6 vs lanes 8 and
9) indicated, again, that the Egr-1 affinity of site 1, which is kept intact in mut3, is much lower than the one of site 3. If
this is the case, one could assume that Egr-1 binds first to site 3, and, subsequently, a second Egr-1 binds to site 1. Moreover, the
binding of the second Egr-1 molecule might be facilitated when the
first one is already bound, because the band intensity of the larger
complex, which would result on binding of Egr-1 to site 1 when site 3 is already occupied (Fig. 9, lane 3, double dot), is clearly
stronger than the one produced on binding of Egr-1 to site 1 when site
3 is not available for binding (Fig. 8, lane 9).
Accordingly, the enhancing activity of Egr-1 may take place via a
cooperative interaction with two close elements at the 7 gene
promoter region.
Fig. 9.
Purified Egr-1 binds two nonequivalent sites
within the most promoter-proximal region of the 7 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.
[View Larger Version of this Image (87K GIF file)]
Egr-1 transcription factor is expressed only in
adrenergic cells
If Egr-1 activates 7 and PNMT transcription, it should be
present in the cells that express these two proteins.
Immunolocalization of Egr-1 in the bovine adrenal gland (Fig.
10A) with a
polyclonal antibody against this transcription factor shows its
expression in only the areas previously demonstrated to express PNMT,
-Bgt-sensitive nAChRs, and 7 transcripts, i.e., the domains close
to the cortex and the portal vein. In these cells Egr-1 immunostaining
was concentrated in the cell nuclei (Fig. 10B),
corresponding to a transcriptional factor. In contrast to this
restricted localization, Egr-1 labeling of the rat adrenal medulla was
almost complete in accordance with PNMT and -Bgt binding
localization. In this case, Egr-1 also was found in the cell nuclei
(Fig. 10C).
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 -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 in
C.
[View Larger Version of this Image (133K GIF file)]
DISCUSSION
At least two classes of nAChRs have been identified on chromaffin
cells. One class, probably formed by 3 (Criado et al., 1992), 5,
and 4 subunits, is present in all chromaffin cells of the adrenal
medulla (Campos-Caro et al., 1997) and seems to be responsible for
mediating catecholamine secretion. The other binds -Bgt and contains
7 subunits (García-Guzmán et al., 1995). It was
demonstrated only recently that these receptor subtype functions as a
nAChR (Alkondon and Albuquerque, 1993; Zhang et al., 1994, 1996). The
first conclusion that can be drawn from the present study is that, in
contrast with the other nAChR subtype, -Bgt-sensitive nAChRs are
localized heterogeneously in the adrenal medulla at sites where the
enzyme PNMT also is present. Moreover, 7 subunit transcripts also
are detected in the same place. Cell type-specific gene expression
frequently depends on cis-acting DNA sequences that are
primarily located within the 5 flanking region of the gene. Activatory
or inhibitory trans-acting proteins can bind to these
sequence elements and regulate transcription. Thus, the 5 flanking
region of the 7 subunit was isolated and characterized. The 128 bp
sequence immediately upstream from the transcription initiation site
was able to drive transcription of reporter genes in cells that express
endogenous levels of the 7 gene, such as chromaffin cells
(García-Guzmán et al., 1995) and SH-SY5Y neuroblastoma
cells (Peng et al., 1994). Thus it was deduced that this region seems
to contain the essential sequence elements required to establish a core
promoter, although other elements further upstream also may be
important, as indicated by the fact that the larger 7 promoter
constructs yielded increased activity. The core promoter region does
not contain TATA and CAAT boxes, but it does coincide with a G+C-rich
domain, characteristic of many promoters, including the chick 7
promoter (Matter-Sadzinski et al., 1992). When this domain was
inspected for motifs that might be relevant for the regulation of
transcription, several putative binding sites for transcription factors
Sp1 and Egr-1 were detected. Because Sp1 generally is expressed
constitutively, our interest was directed to Egr-1 (also termed NGF1-A,
krox-24, and zif268), an immediate early gene transcription factor
known to activate PNMT gene expression in PC12 cells (Ebert et al., 1994; Ebert and Wong, 1995). Egr-1 activated the different 7 gene
constructs tested in chromaffin and neuro-2a cells, indicating the
requirement of this transcription factor for 7 gene expression.
The minimal promoter region of the 7 gene, which shows activity
enhanced by Egr-1 (38 to 16), contains several sequences that are
similar to Egr-1 binding sites. Egr-1 binds to this region at two
locations. Interestingly, the site located 24 to 16, which contains
a 1 bp mismatch regarding the Egr-1 consensus sequence, seems to have
the highest affinity in bandshift experiments; consequently, its
modification abolishes Egr-1 activation in transfection experiments. By
contrast, the more distal site, which comprises a perfect Egr-1 consensus sequence, seems to play a secondary role, because Egr-1 activation is not eliminated totally when this site is modified. Accordingly, bandshift experiments showed that the site is recognized by Egr-1, although with lower affinity. These facts suggest that other
elements, perhaps additional factors and/or DNA sequences outside the
proximal Egr-1 binding site, may favor the binding of Egr-1 to this
site. On the other hand, it may worth mentioning that the sequence of
the proximal site (GCGTGGGCG), although containing a 1 bp mismatch, has
been shown previously to be able to bind with high affinity an Egr-1
zinc finger peptide with which it was cocrystalized (Pavletich and
Pabo, 1991). In any case, we propose that Egr-1 exerts its enhancing
action via a mechanism that involves direct binding to two very close
nonequivalent sites. However, this fact does not preclude the
involvement of additional factors on 7 gene transcription. Consider,
for instance, that the maximal activity obtained with Egr-1 in neuro-2a
cells is always lower than the one exhibited by chromaffin cells,
suggesting that, in the latter case, additional factors, probably not
available in neuro-2a cells, are needed for enhanced transcription.
This fact also is suggested by previous studies on the expression of the chicken 7 subunit, which described the ubiquitous activity of
its gene promoter in undifferentiated neural cells and mesoderm stem
cells (Matter-Sadzinski et al., 1992), as well as the presence of 7
transcript and protein in embryonic muscle (Corriveau et al., 1995).
Thus 7 regulatory elements would acquire their cell specificity in
the course of development (Matter-Sadzinski et al., 1992), depending on
the availability of determined factors.
The Egr-1 polypeptide was localized exclusively to the same chromaffin
cell subset as PNMT and -Bgt-sensitive nAChRs; thus it seems likely
that Egr-1 regulation is a common enhancing mechanism of transcription
for these proteins. The transcription factors Egr-1-Egr-4 constitute a
family of DNA binding proteins termed "early growth response
genes," which are induced rapidly and transiently in a variety of
cells by mitogenic stimulation and by signals involved in cell growth
and differentiation (for review, see Gashler and Sukhatme, 1995). In
the brain Egr-1 displays manifest basal expression (Mack et al., 1990),
which is highly responsive to neuronal stimulation. The level of Egr-1
transcript expression increases when high-frequency electric
stimulation is associated with the induction in vivo of
long-term potentiation. This increase is inhibited by NMDA receptor
antagonists (Cole et al., 1989; Wisden et al., 1990) and suggests the
potential involvement of Egr-1 in synaptic plasticity. In the bovine
adrenal gland a specific stimulation of Egr-1 expression seems very
plausible, because localization of adrenergic cells expressing Egr-1
(and also PNMT and 7 nAChRs) is so peculiar: they are only in the
region of the adrenal medulla close to the adrenal cortex and to the
portal vein. This might indicate that factors arising from the cortex diffuse into the nearby area of the adrenal medulla and induce, directly or indirectly, the expression of Egr-1, which, in turn, regulates the expression of PNMT and nAChRs. If this scenario is
correct, glucocorticoids are strong candidates for substances that are
released from the cortex and then act directly on nearby chromaffin
cells, especially because the glucocorticoid receptor has been
localized only to the cytoplasm of adrenergic cells (Ceccatelli et al.,
1989). Interestingly, a functional glucocorticoid response element in
the 5 flanking region of the enzyme PNMT has been described (Ross et
al., 1990). Moreover, by using a PC12 cell line that was stably
expressing a glucocorticoid receptor, Ebert et al. (1994) subsequently
demonstrated a concerted activation of Egr-1 and glucocorticoids.
Preliminary experiments indicate that 7 promoter activity increases
substantially when 7 constructs and a glucocorticoid receptor vector
are cotransfected into chromaffin cell cultures (M. Criado, C. Carrasco-Serrano, S. Viniegra, unpublished observations), reinforcing
our hypothesis that a direct or indirect interaction via the
glucocorticoid receptor regulates 7 expression.
A final important question highlighted by this study is the functional
significance of -Bgt-sensitive nAChRs in adrenergic cells. A
potential role is indicated by the high Ca2+
permeability of this receptor subtype (Séguéla et al.,
1993), allowing the capacity for substantial control of intracellular Ca2+ levels, which may modulate exocytosis.
Alternatively, the expression of 7 nAChRs in adrenergic cells may
provide them with an additional mechanism, perhaps faster and
specifically directed to trigger epinephrine release. This hypothetical
mechanism might have been obscured until now if we consider that (1)
-Bgt-sensitive receptors have very rapid rates of desensitization,
and the method of agonist application when testing catecholamine
release would have not been fast enough to detect responses caused by
-Bgt-nAChR (see Zhang et al., 1994), and (2) the existence of other
nAChR subtypes that desensitize more slowly than does -Bgt-nAChR
(Campos-Caro et al., 1997) would have darkened responses from the
latter. Whatever the case, many studies point out significant
differences in the secretory process of adrenergic and noradrenergic
cells (Choi et al., 1993; Langley and Grant, 1995; Lomax et al., 1997),
and the restricted expression of -Bgt-sensitive nAChRs may
contribute to this heterogeneity.
FOOTNOTES
Received Feb. 18, 1997; revised June 6, 1997; accepted June 13, 1997.
This work was supported by Grants from the Ministries of Education
(Dirección General de Investigación Cientifica y
Ténica: PB92-0346, PB95-0690, and PB93-0931) and Health (Fis
95/1672) of Spain, the Commission of the European Economic Community
(SC1*CT91-0666), Generalitat Valenciana (GV-D-VS-20-158-96), and the
Wellcome Trust (039284). E.D. del T. was the recipient of a predoctoral
fellowship from the Ministry of Education of Spain. F.I.S. was a
postdoctoral fellow of the Wellcome Trust. C.C.-S. was the recipient of
a fellowship from Generalitat Valenciana. We thank Y. Wang, V. P. Sukhatme, X. Cao, and R. Bravo for Egr-1 plasmids; J. Strotmann for
communicating his in situ hybridization protocol to us;
and A. García for the suggestion of using PNMT antisera to
label adrenergic cells. The excellent technical assistance of Eva
Martínez is also appreciated.
Correspondence should be addressed to Dr. M. Criado, Department of
Neurochemistry and Instituto de Neurociencias, Universidad Miguel
Hernandez, Campus de San Juan, 03550 San Juan, Alicante, Spain.
Dr. Smillie's present address: Wythenshawe Hospital Research Centre,
Manchester M23 9LT, England.
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