Transcriptional Activation of the Rat Vesicular
Monoamine Transporter 2 Promoter in Gastric Epithelial Cells
REGULATION BY GASTRIN*
Fiona
Watson
,
Rachel S.
Kiernan,
Damian G.
Deavall,
Andrea
Varro, and
Rod
Dimaline
From the Physiological Laboratory, University of Liverpool,
Liverpool L69 3BX, United Kingdom
Received for publication, July 27, 2000, and in revised form, November 30, 2000
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ABSTRACT |
Vesicular monoamine transporter 2 is important
for the accumulation of monoamine neurotransmitters into synaptic
vesicles and histamine transport into secretory vesicles of the
enterochromaffin-like cell of the gastric corpus. In this study we have
investigated the mechanisms regulating the transcriptional activation
of the rat vesicular monoamine transporter 2 (VMAT2) promoter in
gastric epithelial cells. Maintenance of basal levels of transcription was dependent on the presence of SP1, cAMP-response element (CRE), and
overlapping AP2/SP1 consensus sequences within the region of promoter
from
86 to +1 base pairs (bp). Gastrin stimulation increased
transcriptional activity, and responsiveness was shown to be dependent
on the CRE (
33 to
26 bp) and AP2/SP1 (
61 to
48 bp) consensus
sites but independent of the SP1 site at
86 to
81 bp.
Gastrin-induced transcription was dependent on the cooperative
interaction of an uncharacterized nuclear factor of ~23.3 kDa that
bound to the putative AP2/SP1 site, CRE-binding protein (CREB), and
CREB-binding protein/p300. Gastrin stimulation resulted in the
increased binding of phosphorylated CREB to the promoter, but it did
not result in the increased binding of the AP2/SP1-binding protein. The
gastrin responsiveness of the promoter was shown to be dependent on
both the protein kinase C and mitogen-activated protein
kinase/extracellular signal-regulated kinase kinase-signaling pathways,
which may converge on the AP2/SP1-binding protein.
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INTRODUCTION |
The transport protein vesicular monoamine transporter 2 (VMAT2)1 plays a central role
in cellular physiology. It is responsible for the accumulation of
monoamine neurotransmitters into synaptic vesicles and has been
implicated in the synthesis and storage of histamine in the
interleukin-3-dependent cell line Ea3.123 (1-5). VMAT2 may
play a significant role in locomotor stimulation and/or the behavioral
reward produced by amphetamines, and malfunctions of monoamine
transport have been implicated in psychiatric disease (6, 7). Studies
on VMAT2 knockout mice support the idea that vesicular monoamine
transport is important in maintaining neuronal function with
heterozygote VMAT2+/
mice having dysfunctional monoamine storage and
release parameters (6, 8). VMAT2 is also likely to mediate histamine
transport into the secretory vesicles of the enterochromaffin-like cell
(ECL) of the gastric corpus (2, 4, 9- 11). In the rat stomach the ECL
cell is tightly regulated by the peptide hormone gastrin, with gastrin
stimulation leading to the release of histamine and the resultant
production of gastric acid from the parietal cell (12-14). Histamine
is synthesized within the cytosol of the ECL cell from
L-histidine by the action of the enzyme
L-histidine decarboxylase (HDC) (15) and is then sequestered by VMAT2 into secretory vesicles that are stabilized by
chromogranin A (CgA), a multi-functional acidic protein expressed both
in neuroendocrine cells and in ECL cells (16-21). Evidence suggests
that VMAT2 may be up-regulated to accommodate the increased histamine
biosynthesis and secretion that accompanies ECL stimulation. Hypergastrinaemia-induced degranulation of the ECL cell is accompanied by a parallel secretion of histamine and CgA followed by enhanced production of both molecules, and in the pre-B cell line Ea3.123, the
mRNA abundance for both VMAT2 and HDC is increased in a parallel fashion after mobilization of their intracellular calcium levels or by
activation of protein kinase C (4, 5, 20-23). In addition, increases
in the abundance of mRNA for HDC, VMAT2, and CgA were observed in
animal models of hypergastrinaemia, suggesting that the transcriptional
activity of these genes could be regulated by gastrin in
vivo (24-26).
Indeed, gastrin has been shown to transcriptionally activate both the
HDC and CgA promoters (27-29). Regulation of the human HDC promoter by
gastrin involves a protein kinase C-dependent, MAP
kinase/ERK-dependent, and AP1-dependent pathway
and involves the binding of distinct nuclear factors to two
cis-acting overlapping binding sites (GAS-RE1, +1 to +19;
and GAS-RE2 +11 to +27) (30-32). Gastrin transactivation of the mouse
CgA promoter was found to be dependent on the binding of Sp1 to an
Sp1/Egr motif located at
88 to
77 bp and a CRE-like element at
71
to
64 bp of the mCgA promoter (29). Gastrin stimulation resulted in
an increased binding of both SP1 and CREB to their consensus sequences
within the mCgA promoter, and overexpression of either SP1 or
phosphorylated CREB transactivated the promoter. Coexpression of both
transcription factors resulted in an additive mCgA promoter response,
suggesting that the effect of gastrin was brought about by their
cooperative action.
In the present study we have investigated the transcriptional
regulation of VMAT2 in the gastric epithelial cell line
AGS-GR. This cell line has been permanently transfected
with the CCKB-gastrin receptor (33). It has been shown to
express a functional CCK
receptor and utilizes signaling
pathways that are common to the ECL cell (33, 13). This has allowed us
to examine the controls of both basal and gastrin-stimulated VMAT2
transcriptional activity in these gastric epithelial cells. Maintenance
of the basal level of transcription was dependent on the presence of
SP1, CRE, and overlapping AP2/SP1 consensus sequences within the region
of promoter from
86 to +1 bp, whereas full gastrin responsiveness of
the promoter depended on the presence of the intact CRE and AP2/SP1 consensus sequences. Regulation of the rat VMAT2 promoter was found to
involve the protein kinase C-dependent and MAP
kinase/ERK-dependent pathway, binding of a distinct nuclear
factor of estimated molecular mass 23.3 kDa to the putative AP2/SP1
consensus site and the binding and phosphorylation of CREB to the CRE
site within the promoter. Thus, our study demonstrates that the rat
VMAT2 promoter can, like the HDC and mCgA promoters, be
transcriptionally regulated by gastrin, but the mechanism of this
regulation is distinct from that observed for these other
gastrin-sensitive promoters.
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EXPERIMENTAL PROCEDURES |
Materials--
Ham's F-12, fetal bovine serum, and
penicillin/streptomycin were obtained from Life Technologies, Inc.
Gastrin I (human) was obtained from Peninsula Laboratories Europe Ltd.,
and BIOTAQ DNA polymerase was obtained from Bioline, London,
UK. A gel shift assay system containing HeLa and AP2 nuclear
extracts and consensus oligos for AP1, OCT1, CREB, NF-K
, and TFIID
was obtained from Promega. Additional oligonucleotides were synthesized
by Sigma- Genosys, and all antibodies were purchased from Santa Cruz
Biotechnology, Inc. apart from the phospho-CREB antibody, which was
from Upstate Biotechnology and peroxidase-conjugated anti-goat IgG from
Sigma. Complete, mini-protease inhibitor mixture tablets were from
Roche Molecular Biochemicals. The inhibitors GF109303X and PD98059 were obtained from Calbiochem. All other reagents were obtained from Sigma.
DNA Constructs and Plasmids--
The promoter region of the rat
VMAT2 gene had been previously cloned, and a subclone pV2404-8 was
generated in the pGEM-TEasy vector (Promega, Southampton, UK) as
described previously (5). The subclone pV2404-8 was sequenced in both
directions by an automated dideoxy method and used to make a series of
VMAT2 promoter 5'-deletional constructs in the pGL3- Basic vector
(Promega) that contained 1632, 1289, 943, 609, 223, 86, and 36 nucleotides upstream of the start site together with 55 bp of exon 1. Polymerase chain reaction products from pV2404-8 were directionally
cloned between the SacI and XhoI sites of
pGL3-Basic, and the resultant constructs were sequenced in both
directions to confirm their integrity. In addition, polymerase chain
reaction products from pV2404-8 that contained 86 and 36 nucleotides
upstream of the transcriptional start site together with 55 bp of exon
1 were directionally cloned between the BamHI and
XhoI sites of the promoterless luciferase-reporter vector
PXP2. Again, the integrity of these constructs and the sequences of
mutated constructs generated by polymerase chain reaction were
confirmed before their use in experiments. The mutations generated in
these constructs are indicated in the appropriate figures and their legends.
Cell Culture and Transfection Studies--
AGS-GR
cells, which were permanently transfected with the human
CCKB-gastrin receptor (33) driven by the EF1-
promoter under puromycin selection, were grown in Ham's F-12 medium
supplemented with 10% (fetal bovine serum) and penicillin/streptomycin
(100 IU/ml) at 37 °C in 5% CO2. Transient transfections
were carried out using TransfastTM transfection reagent
from Promega. Cells were plated out at a density of 5 × 105 cells/65-mm well 24 h before transfection. During
transfection, cells were incubated for 1.5 h in 2 ml of
transfection mix in serum-free media. The transfection mix consisted
typically of 3.4 µl of TransfastTM,1.5 µg of firefly
luciferase reporter construct, and 0.1 µg of Renilla
luciferase (pRL-TK) control vector (Promega) per well. The quantity of
firefly luciferase reporter was reduced to 1 µg/ml when additional
expression constructs were added. These were typically added at 0.5 µg/well. Cells were stimulated 24 h post-transfection as
described in the figure legends and harvested at appropriate time
points after stimulation. Luciferase activity was determined using the
dual luciferase reporter system (Promega), and each individual
transfection was assayed in duplicate.
Electrophoretic Mobility Shift Assays--
Nuclear extracts from
AGS-GR cells and rat gastric corpus epithelium were
prepared using the methodology described in Schreiber et al.
(34). Double-stranded oligonucleotides were radiolabeled with
[
-32P]dATP, and EMSAs were performed with 10 µg of
nuclear extracts in a final volume of 20 µl of binding buffer
containing 10 mM Tris-HCl (pH 7.5), 50 mM NaCl,
1 mM dithiothreitol, 1 mM EDTA, 1 µg of
poly(dA-dT), 10% glycerol, and 10 µM Zn2+.
Mixtures were incubated with 10 fmol of double-stranded oligonucleotide probes for 20 min at room temperature (29, 32). For supershift assays,
nuclear extracts were incubated with 1 µl of anti-phospho-CREB, anti-ATF, and anti-AP2 antibodies but 2 µl of anti-Sp family
antibodies. In addition, the Sp supershifts required the addition of 4 µg of bovine serum albumin to the binding reactions. DNA-protein complexes were electrophoresed on a 6% nondenaturing polyacrylamide gel. The dried gel was exposed to a phosphor storage screen, and the
image was revealed with a PhosphorImager (Molecular Dynamics, Sevenoaks, Kent, UK).
UV Cross-linking--
20-µg aliquots of AGS-GR
nuclear extract were incubated with 10 fmol of Klenow
[
-32P]dATP end-labeled probes (A,
B, and E, as described in the legend to Fig. 6)
in EMSA binding buffer for 30 min on ice. The samples were exposed to
UV light for 60 min before the addition of an equal volume of 2× SDS
sample buffer and heating to 99 °C for 5 min. 10 µg of each sample
and prestained molecular weight markers were electrophoresed on a 12%
SDS-polyacrylamide gel in Tris/glycine electrophoresis buffer. The gels
were dried, and radioactivity associated with the proteins was
visualized using a PhosphorImager.
Western Blot Analysis--
5 × 105
AGS-GR cells were plated in 65-mm culture dishes containing
5 ml of complete medium. 18 h later, the medium was removed and
replaced with 5 ml of serum-free medium, and the cells were incubated
in the absence or presence of gastrin (5 × 10
8 M) for 3, 6, 12, and 24 h before preparation of a total cell lysate. To prepare the cellular
lysate AGS-GR, cells were washed twice with ice-cold
phosphate-buffered saline and resuspended in 250 µl of lysis buffer
containing 20 mM Tris (pH 7.8), 150 mM NaCl, 2 mM EDTA, 0.5% Nonidet P-40, 10 mM NaF, 15 mg/ml benzamide, 8.8 mg/ml sodium orthovanadate, 0.5 mM
dithiothreitol, 10 mg/ml phenylmethylsulfonyl fluoride, and one
protease inhibitor mixture tablet/10 ml of lysis buffer. 40 µg of the
lysates were electrophoresed on a 12% SDS-polyacrylamide gel. After
electrophoresis, the proteins were blotted on nitrocellulose membranes,
and immunodetection of the proteins was performed. The primary antibody
used was obtained from Santa Cruz Biotechnology and is specific for
mouse, rat, and human VMAT2 but is noncross-reactive with VMAT1. The
secondary antibody was a peroxidase-conjugated anti-goat IgG. Enhanced
chemiluminescence (SuperSignal® West Pico chemiluminescent
substrate; Pierce) was used to identify the VMAT2 protein.
Statistical Analysis--
Results were analyzed for statistical
significance using the Student's t test for independent samples.
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RESULTS |
Basal Transcriptional Activity of the Rat VMAT2 Promoter in the
AGS-GR Cell Line--
The basal transcriptional activity
of the rat VMAT2 promoter in the gastric environment was characterized
by ligating polymerase chain reaction-generated segments of the
5'-flanking region of the VMAT2 gene upstream of the gene for firefly
luciferase in the reporter vector pGL3-Basic and transfection of the
resultant constructs together with a Renilla luciferase
vector into AGS-GR cells. The AGS-GR cell line
has been permanently transfected with the CCKB-gastrin
receptor and has been demonstrated to show specific activation by
gastrin (33). As shown in Fig. 1,
transfection of all the constructs resulted in significant luciferase
activity with the largest construct v1632pg (encompassing nucleotides
1632 to +55 bp) showing an approximate 10.3-fold increase in
luciferase activity over that seen in the promoterless vector. Activity
was maximal in the construct v223pg (
223 to +55 bp), which showed an
average 44.7-fold increase in luciferase activity over promoterless vector. This level of expression was maintained in the construct v86pg
(
86 to +55 bp). The increase in luciferase activity observed in the
v223pg construct as compared with v609pg (
609 to +55 bp) suggests the
presence of at least one negative regulatory element in the region from
609 to
223 bp. The schematic illustrated in Fig.
2a shows the sequence of the
5'-flanking region of the rat VMAT2 gene from
86 to +55 bp, with
bases numbered relative to the transcriptional start site. Within this
region there is a SP1 consensus sequence spanning from
86 to
81 bp,
a putative AP2/SP1 site spanning from
61 to
48 bp, and a CRE
consensus sequence spanning from
33 to
26 bp. To assess the
contribution of the cis-regulatory elements contained in this region to
maintenance of basal transcriptional activity, constructs were made in
which the SP1, AP2/SP1, or CRE consensus sequences were mutated. A
schematic representation of these and other constructs used in this
study is shown in Fig. 2b. As Fig.
3 shows, when the intact SP1 site starting at
86 bp was mutated, basal activity of the v86pg construct was reduced to 36.61 ± 5.91% (mean ± S.E.,
n = 17) wild type. When the AP2 region of the AP2/SP1
composite site was mutated (Fig. 3), there was no effect on basal
activity of the construct v86px. However mutation of the SP1 part of
the site reduced basal activity to 37.4 ± 0.04% (mean ± S.E., n = 12) of the control level (Fig. 3). Mutation
of the CRE also reduced the basal transcriptional activity of the
construct v36pg to 39.9 ± 4.8% (mean ± S.E.,
n = 18) of the control level (Fig. 3). Thus the SP1,
putative AP2/SP1, and CRE consensus sequences are all important
determinants of basal transcriptional activation in the
AGS-GR cell line.

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Fig. 1.
Basal transcriptional activity of the rat
VMAT2 promoter in AGS-GR cells. AGS-GR
cells were transfected with 5'-deletional VMAT2 promoter/luciferase
constructs containing from 1632 down to 36 bp of promoter together with
55 bp of exon 1 in the promoterless vector pGL3b (constructs v1632pg,
v1289pg, v943pg, v609pg, v223pg, v86pg, and v36pg are shown
schematically on the left). Transcriptional activity is expressed as
luciferase activity, and the results are representative of nine
independent experiments assayed in duplicate.
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Fig. 2.
a, sequence of the 5'-flanking
region of the rat VMAT2 gene. Bases are numbered relative to the
transcriptional start site (+1), which was determined previously by
primer extension analysis (5). Putative cis-regulatory elements (SP1,
AP2/SP1, and CRE) are underlined; the 55 bp of exon 1 included in the VMAT2 promoter/luciferase constructs are
double-underlined. The schematic in b illustrates
the deletional and mutated VMAT2 promoter/luciferase constructs used in
this study. v, VMAT2; 86, 48, or
36 is the number of bases of promoter in the construct;
px, PXP2; pg, pGL3b. The sites mutated are
indicated both in the construct name and in their associated line
diagrams and are as follows: v86sp1pg, mutation of authentic SP1 site
GGGCGG to GAGCTC ( 86 to 81 bp); v86ap2sp1px, mutation throughout
putative AP2/SP1 site CCCCTCCGCCC to AACGTCAGAAC ( 61 to 51 bp);
v86ap2px, mutation of AP2 part of AP2/SP1 site CCCCT to AACGG ( 61 to
57 bp); v86sp1/2px, mutation of SP1 part of AP2/SP1 site CCGCCC to
CCGAAT ( 56 to 51 bp); v86crepx, mutation of CRE site TGACGT to
TGTAGA ( 33 to 28 bp); v36mpx/pg, mutation of CRE site TGACGT to
TGTAGA ( 33 to 28 bp).
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Fig. 3.
Contribution of cis-regulatory elements SP1,
AP2/SP1, and CRE to maintenance of basal transcriptional activity of
the rat VMAT2 promoter. AGS-GR cells were transiently
transfected with mutated VMAT2 promoter/luciferase constructs
(v86sp1pg, v86sp1/2px, v86ap2px, and v36 mpg), and their luciferase
activity was compared with that found for their parental controls. The
luciferase activity of each mutant construct is expressed as the
percentage of its parental wild type construct (=100%). Results are
the mean ± S.E. and are representative of a minimum of six
separate experiments.
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Gastrin Responsiveness of the Rat VMAT2 Promoter in the
AGS-GR Cell Line--
AGS-GR cells were
transiently transfected with the VMAT2-promoter luciferase constructs
before stimulation with gastrin. Constructs that contained from 1632 to
86 bp of VMAT2 promoter in the vector pGL3b all showed significant
increases in luciferase activity after stimulation with gastrin (Fig.
4a) compared with their basal levels of activity. The luciferase activity of the largest construct, v1632pg, was increased 8.2 ± 0.9-fold (mean ± S.E.,
n = 9) over basal activity after stimulation with
gastrin (5 × 10
8 M) for a
24-h period. Truncation of the promoter region of this construct down
to 86 bp did not lead to significant reductions in response. Gastrin
stimulation led to a 7.1 ± 1.5-fold (mean ± S.E.,
n = 9) increase in the luciferase activity of the
construct v609pg, which was not significantly different from that of
construct v86pg, which was on average increased 5.2 ± 0.4-fold
(mean ± S.E., n = 9). Stimulation of the v86px
construct with 5 × 10
8 M
gastrin increased the transcriptional activity of the promoter within
4 h of stimulation and was maximal at 24 h post-stimulation. Maximal responses were seen with 5 × 10
8 M gastrin, but significant
stimulation was seen with 5 × 10
9
M gastrin (data not shown). Gastrin responsiveness was
inhibited by the gastrin antagonist L740093, and Gly-extended gastrin
was unable to increase the transcription of the v86px construct (data not shown). Western blot analysis demonstrated VMAT2 protein in the
AGS-GR cell line, but its abundance was not significantly elevated after gastrin stimulation of the cells (data not shown). The
luciferase activity of the promoterless pGL3b-empty vector was modestly
increased after stimulation with 5 × 10
8 M gastrin (2.67 ± 0.6-fold over basal levels, mean ± S.E., n = 9;
Fig. 4a). Although this increase was not comparable with those observed with constructs containing the rat VMAT2 promoter segments, new constructs were made in the PXP2 luciferase vector to
confirm and extend the data on gastrin responsiveness of the promoter.
Stimulation with gastrin did not increase the intrinsic luciferase
activity of the promoterless PXP2 vector (Fig. 4b). However
the construct v86px showed a 4.44 ± 0.8 (mean ± S.E., n = 11) fold increase in luciferase activity over basal
levels after gastrin stimulation (Fig. 4b), confirming our
findings originally made using the pGL3b vector that the region of rat
VMAT2 promoter from
86 to +55 bp was capable both of sustaining basal
transcription and of increasing its transcriptional activity in
response to gastrin. The gastrin responsiveness of constructs v48px (48 to +55 bp) and v36px was significantly reduced when compared with that of v86px (p < 0.05) but was significantly greater
than that of the promoterless PXP2 empty vector (Fig. 4b,
p < 0.005). The region of promoter (from
86 to 49 bp) contains both the SP1 and AP2/SP1 composite sites, and gastrin
responsiveness was reduced on its deletion. As shown by the similar
responsiveness of the v48px and v36px constructs, the region from
48
to
37 bp did not appear to contribute to the gastrin responsiveness
of the promoter. However, a significant level of gastrin responsiveness was maintained in the smallest construct v36px, which contains the CRE.
These data therefore suggested the possibility that all three consensus
sites, SP1, AP2/SP1, and CRE, might contribute to the gastrin
responsiveness of the promoter. To dissect the gastrin responsiveness
further, comprehensive mutagenesis of the constructs v36px and v86px
was performed.

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Fig. 4.
Gastrin increases the transcriptional
activity of the rat VMAT2 promoter in AGS-GR cells.
a, AGS-GR cells were transiently transfected
with 5'-deletional VMAT2 promoter/luciferase constructs containing from
1632 down to 86 bp of promoter together with 55 bp of exon 1 in the
promoterless vector pGL3b (constructs v1632pg, v1289pg, v943pg, v609pg,
v223pg, and v86pg are shown schematically on the left). b,
AGS-GR cells were transiently transfected with the
5'-deletional VMAT2 promoter/luciferase constructs v86px, v48px, and
v36px (constructs shown schematically on the left). In both
a and b, cells were stimulated with 5 × 10 8 M gastrin as described under
"Experimental Procedures" before measurement of luciferase
activity. Luciferase activity is expressed as fold increase over
unstimulated controls, and results are representative of nine
independent experiments assayed in duplicate for a and 6 for
b.
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Mutagenesis of the Constructs v36px and v86px--
When the intact
SP1 site at position
86 to
81 bp was mutated in construct v86sp1px,
the gastrin responsiveness of this construct was not different from its
parental construct, v86px (101.9 ± 17.03% gastrin response;
mean ± S.E., n = 9). This indicated that this
site was not contributing to the gastrin responsiveness of the promoter
(Fig. 5a), and experiments
performed with the construct v86pg and the mutated construct v86sp1pg
confirmed this finding (data not shown). In contrast, when the CRE site
was mutated in the construct v36mpx, the gastrin responsiveness of this
construct was reduced to 51 ± 10% (mean ± S.E.,
n = 7) of its parental construct, v36px, indicating the
importance of the CRE consensus sequence. The importance of this CRE
consensus sequence for gastrin responsiveness of the rat VMAT2 promoter
was confirmed through its mutation from within the construct v86px
(Fig. 5b). Gastrin increased the luciferase activity of the
resultant vcrepx construct by only 1.3 ± 0.2-fold (mean ± S.E., n = 14) over basal levels, which was
significantly less than the responsiveness of the parental v86px
construct (4.4 ± 0.8-fold over basal levels; mean ± S.E.,
n = 11). However the gastrin-stimulated luciferase
activity of the construct v86ap2sp1px, in which the complete AP2/SP1
site was mutated, also showed a significantly reduced gastrin
responsiveness (2.26 ± 0.44-fold over basal levels; mean ± S.E., n = 6) as compared with v86px, indicating that
this site also might regulate the gastrin response (Fig.
5b). When only the AP2 part of the putative AP2/SP1 site was
mutated in the construct v86ap2px, the gastrin response was not
significantly different from that of the v86px construct. In contrast,
when the SP1 part of the AP2/SP1 site was mutated in the construct
v86sp1/2px, the gastrin responsiveness was significantly reduced
compared with that of v86px (2.23 ± 0.25-fold increase over
basal; mean ± S.E., n = 12). Thus, both the CRE
and part of the composite AP2/SP1 site may be important in achieving
full transcriptional activation after gastrin stimulation.

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Fig. 5.
Contribution of the cis-regulatory elements
SP1 and CRE to gastrin responsiveness of the rat VMAT2 promoter.
a, AGS-GR cells were transiently transfected
with the mutated VMAT2 promoter/luciferase constructs v86sp1px and
v36mpx or parental constructs before stimulation with 5 × 10 8 M gastrin as described under
"Experimental Procedures." The gastrin-induced luciferase activity
of the mutated constructs was expressed as a percentage of that
observed for the wild type (=100%). Results are the mean ± S.E.
and are representative of a minimum of six separate experiments. The
n value for each construct pair is given in the text.
b, AGS-GR cells were transiently transfected
with the mutated VMAT2 promoter/luciferase constructs v86ap2/sp1px,
v86ap2px, v86sp1/2px, and vcrepx or the parental construct v86px before
gastrin stimulation, as described under "Experimental Procedures."
Luciferase activity is expressed as a fold increase relative to
unstimulated controls (=1.0), and results are representative of a
minimum of six independent experiments assayed in duplicate.
n values are included in the text.
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Binding of Nuclear Proteins from the AGS-GR Cell Line
and Rat Gastric Corpus to the AP2/SP1 Consensus Sequence--
EMSA
analysis showed that a nuclear protein from the AGS-GR cell
line could bind to the putative AP2/SP1 site in the rat VMAT2 promoter
(Fig. 6a). Binding of this
protein could be inhibited by inclusion in the binding reaction of a
100-fold excess of unlabeled wild type probe. However a 100-fold excess
of competitor in which the AP2/SP1 site is completely mutated could not
inhibit binding, indicating that the binding of this protein is
specific to the AP2/SP1 site (Fig. 6a). Competitors in which
only the AP2 part or the SP1 part of the site had been mutated could
only partially inhibit the binding reaction, indicating that the
protein bound to a site that was not the intrinsic AP2 or SP1 site. In
support of this we found that a 100-fold excess of authentic AP2 or SP1 double-stranded oligonucleotides could not inhibit the binding of this
protein; neither could double-stranded oligonucleotides containing the
binding sites AP1, OCT1, CREB, NFkB, and TFIID (data not shown),
raising the possibility that the protein binding to the putative
AP2/SP1 site is an uncharacterized nuclear factor. A nuclear protein
isolated from rat gastric corpus also specifically bound to the
putative AP2/SP1 site, as shown in Fig. 6e. The similar mobilities of the protein/AP2/SP1 site complexes that are observed for
both the AGS-GR cell line and for the rat corpus suggest
that the proteins are probably the same. A cellular extract enriched for AP2-binding protein was used to demonstrate the ability of AP2
protein to bind specifically to a canonical AP2 binding site in our
assay system (Fig. 6b). A supershift of the bands binding to
the AP2 oligonucleotide was observed in the presence of AP2
antibody
but not by AP2
or -
antibodies (Fig. 6c). None of
these antibodies could supershift the band produced by nuclear extracts from the AGS-GR cells binding to the AP2/SP1 site (Fig.
6d). In addition, we were unable to detect a specific AP2
protein in the AGS-GR nuclear extracts that recognized our
AP2 probe (data not shown), suggesting that this protein may not be
abundant in this cell line. Specific binding was observed of
AGS-GR nuclear proteins to a radiolabeled authentic SP1
probe (Fig. 7). In two cases this binding
was shown to be specific. One of the proteins was supershifted by a SP1
antibody, whereas the binding of another protein was inhibited in the
presence of a SP3 antibody. The AGS-GR protein, which bound
to the AP2/SP1 site, was, however, not supershifted by any of the Sp
family antibodies (Fig. 7). In addition the mobility of the
AP2/SP1-binding protein as observed in the gel-shift assay suggests
that the protein is smaller than SP family proteins.

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Fig. 6.
EMSA analysis of the putative AP2/SP1 site
found in the rat VMAT2 promoter. The double-stranded
oligonucleotides used are the wild type AP2/SP1 site (A),
complete mutation of AP2/SP1 site (B), mutation of AP2 part
of AP2/SP1 site (C), and mutation of SP1 part of AP2/SP1
site (D). The AP2/SP1 site in A and mutated
sequence in B, C, and D are
underlined. E, F, and G are
canonical SP1, AP2, and AP1 consensus sequences, respectively.
Panel a, binding of AGS-GR nuclear extracts to
radiolabeled probe A (wild type) (lane 1).
Binding of AGS-GR nuclear extracts to radiolabeled probe
A in the presence of a 100-fold excess of unlabeled
competitor A-F (lanes 2-7 as indicated).
Panel b, lane 1, binding of AP2-enriched extract
to radiolabeled AP2 probe (F). Lanes 2-5,
binding of AP2-enriched extract to radiolabeled AP2 probe
(F) in the presence of a 100-fold excess of unlabeled
competitors F (AP2), E (SP1), A (wild
type AP2/SP1), and G (AP1). Panel c, lane
1, binding of AP2-enriched extract to radiolabeled AP2 probe
(F). Lane 2, binding of AP2-enriched extract to
radiolabeled AP2 probe (F) in the presence of a 100-fold
excess of unlabeled AP2 competitor (F). Lanes
3-5, binding of AP2-enriched extract to radiolabeled AP2 probe
(F) in the presence of anti-AP2 , and antibodies
as indicated. Panel d, lane 1, binding of
AGS-GR nuclear extracts to radiolabeled probe A. Lane 2, binding of AGS-GR nuclear extracts to
radiolabeled probe A in the presence of a 100-fold excess of
unlabeled competitor A. Lanes 3-5, binding of
AGS-GR nuclear extracts to radiolabeled probe A
in the presence of anti-AP2 , , and antibodies as indicated.
Panel e, lane 1, binding of
AGS-GR nuclear extracts to radiolabeled probe A. Lane 2, binding of AGS-GR nuclear extracts to
radiolabeled probe A in the presence of a 100-fold excess of
unlabeled competitor A. Lane 3, binding of rat
gastric corpus nuclear extracts to radiolabeled probe A. Lane 4, binding of rat corpus nuclear extracts to
radiolabeled probe A in the presence of a 100-fold excess of
unlabeled competitor A.
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Fig. 7.
The protein that recognizes the AP2/SP1
putative site is not SP 1. See Fig. 6 for sequences of
double-stranded oligonucleotides used. Lane 1, binding of
AGS-GR nuclear extracts to radiolabeled SP1 probe
(E). Lane 2, binding of AGS-GR
nuclear extracts to radiolabeled probe (E) in the presence
of a 100-fold excess of unlabeled wild type competitor (E).
Lanes 3-6, binding of AGS-GR nuclear extracts
to radiolabeled probe (E) in the presence of anti-SP family
antibodies as indicated. Lane 7, binding of
AGS-GR nuclear extracts to radiolabeled wild type AP2/SP1
probe (A). Lane 8, binding of AGS-GR
nuclear extracts to radiolabeled AP2/SP1 probe (A) in the
presence of a 100-fold excess of unlabeled wild type competitor
A. Lanes 9-12, binding of AGS-GR
nuclear extracts to radiolabeled AP2/SP1 probe (A) in the
presence of anti-SP family antibodies as indicated.
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|
Binding of Nuclear Proteins from the AGS-GR Cell Line
to Regulatory Elements Present in the HDC and mCgA Promoters--
Fig.
8 shows the pattern of protein/DNA
binding that is obtained using EMSA when nuclear extracts from
AGS-GR cells are incubated with radiolabeled probes
corresponding to the AP2/SP1 consensus sequence found in the rat VMAT2
promoter, an AP2/SP1 consensus site, which is found in the mCgA
promoter (
90 to
77 bp) and where it has been reported that SP1
protein binds to regulate transcription (29), and the GAS-RE (+1 to +27
bp) of the HDC promoter, which has been shown to regulate gastrin
responsiveness through the binding of two as yet uncharacterized
nuclear proteins (32). Binding of proteins to the (
90 to
77 bp)
fragment of the mCgA promoter (lane 3) was inhibited by a
100-fold excess of unlabeled wild type probe (lane 4) but
not by a 100-fold excess of the AP2/SP1 sequence (lane 5).
In addition we used EMSA to investigated the binding of
AGS-GR nuclear extracts to the
100 bp to
43 bp region
of the mCgA promoter and found no evidence for binding of the
uncharacterized AP2/SP1 protein to this region (data not shown). The
pattern of AGS-GR protein/DNA binding observed with the
GAS-RE (+1 to +27 bp, lane 6) was also inhibited with a
100-fold excess of its unlabeled wild type probe but not by a 100-fold
excess of the AP2/SP1 probe. These results indicate that the
uncharacterized AP2/SP1-binding protein, which is important in the
gastrin responsiveness of the VMAT2 promoter, is unlikely to be
involved in regulation of the mCgA and human HDC promoters.

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Fig. 8.
Binding of AGS-GR nuclear
proteins to the mCgA and HDC promoters. The double-stranded
oligonucleotides used are wild type (VMAT2) AP2/SP1 site
(A); mCgA sequence ( 90 to 77 bp) as in Hocker et
al. (29) (B), and GAS-RE (+1 to +27 bp) of HDC as in
Raychowdhury et al. (32) (C). Lane 1,
binding of AGS-GR nuclear extracts to radiolabeled probe
A (wild type). Lane 2, binding of
AGS-GR nuclear extracts to radiolabeled probe A
in the presence of a 100-fold excess of unlabeled competitor
A. Lane 3, binding of AGS-GR nuclear
extracts to radiolabeled probe B (wild type). Lanes
4 and 5, binding of AGS-GR nuclear extracts
to radiolabeled probe B (wild type) in the presence of a
100-fold excess of unlabeled competitor B and A
as indicated. Lane 6, binding of AGS-GR nuclear
extracts to radiolabeled probe C (wild type). Lanes
7 and 8, binding of AGS-GR nuclear extracts
to radiolabeled probe C (wild type) in the presence of a
100-fold excess of unlabeled competitor C and A
as indicated.
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|
Estimation of the Molecular Weight of AP2/SP1-binding Protein from
AGS-GR Cells--
To investigate the size of the
AGS-GR nuclear protein that bound to the putative AP2/SP1
consensus sequence, UV cross-linking studies were performed as
described under "Experimental Procedures." The molecular weight of
the oligonucleotide probes was subtracted from the molecular weight of
the protein. As shown in Fig. 9 the wild-type probe (A), which contained the AP2/SP1 consensus
sequence, bound to a protein of 23.3 kDa that did not bind to the
mutant probe (B) or the SP1 probe (E). In
addition, two proteins with molecular weights compatible with them
being members of the SP family bound to the SP1 probe
(E).

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Fig. 9.
Estimated molecular weight of AP2/SP1 binding
protein. Probes A, B, and E are
as described in Fig. 6. AGS-GR nuclear extracts were
incubated with the 32P-radiolabeled probes before
UV-cross-linking and analysis by SDS-polyacrylamide electrophoresis as
described under "Experimental Procedures." The positions of the
UV-cross-linked protein bands are indicated by arrows, and
the positions of the prestained molecular mass markers (kilodaltons)
are shown. mw, molecular mass.
|
|
Binding of Nuclear Proteins from the AGS-GR Cell Line
to the CRE Consensus Sequence in the Rat VMAT2 Promoter--
We
investigated the binding of nuclear proteins from the
AGS-GR cells to a radiolabeled probe that contained from
36 to +16 bp of rat VMAT2 promoter. The radiolabeled probe was bound
by a complex of proteins as shown in Fig.
10 (lane 1), and binding was
inhibited in the presence of a 100-fold excess of unlabeled probe (Fig.
10, lane 2). Binding specificity was shown by the failure of
a 100-fold excess of unlabeled competitor in which the CRE was mutated
to inhibit binding (Fig. 10, lane 3). This was confirmed by
use of an ATF family antibody to supershift the complex (Fig. 10,
lane 4). Stimulation of the AGS-GR cells with
gastrin (5 × 10
8 M) for 15 min before the extraction of nuclear proteins led to an increase in the
binding of phosphorylated CREB to the promoter as determined using a
phospho-CREB antibody (Fig. 10, lanes 5 and 7).
However, gastrin stimulation did not lead to a detectable increase in
the quantity of CREB bound to the promoter and neither did it increase
the binding of the AP2/SP1 protein to its consensus sequence (data not
shown).

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Fig. 10.
Electrophoretic mobility shift assay of the
rat VMAT2 36 to +16-bp region. Upper panel, the
double-stranded oligonucleotide (CRE) containing the CRE consensus
sequence and representing the region 36 to +16 bp and its mutant.
Lower panel, EMSA with 32P-end-labeled CRE
probe. Electrophoretic mobility shift assays were as described under
"Experimental Procedures." Lane 1, binding of
AGS-GR nuclear extracts to radiolabeled CRE probe.
Lane 2, binding of AGS-GR nuclear extracts in
the presence of a 100-fold excess of unlabeled CRE competitor.
Lane 3, binding of AGS-GR nuclear extracts in
the presence of a 100-fold excess of mutated CRE competitor (TGACGT
mutated to TGTAGA). Lanes 4, 6, and
8, binding of AGS-GR nuclear extracts to
radiolabeled CRE probe in the presence of anti-ATF antibody.
Lanes 5, 7, and 9, binding of
AGS-GR nuclear extracts to radiolabeled CRE probe in the
presence of anti-phospho-CREB (PC) antibody. In some
instances the AGS-GR cell nuclear extracts had been
stimulated with 5 × 10 8 M
gastrin for 0 min (lanes 4 and 5), 15 min
(lanes 6 and 7), and 30 min (lanes 8 and 9) before protein extraction.
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Effect of Protein Kinase C Inhibitor GF109303X and MAPK Inhibitor
PD98059 on the Gastrin Responsiveness of the Rat VMAT2
Promoter--
The increase in transcriptional activity of the
construct v86px, which was observed after gastrin stimulation (4.2 ± 0.39-fold over basal; mean ± S.E., n = 15),
was reduced (2.61 ± 0.5-fold over basal; mean ± S.E.,
n = 11) to a level comparable with that observed for
the construct v36px in the presence of the protein kinase C inhibitor
GF109303X (Fig. 11a).
However, the response of the v36px construct was not significantly
affected by the presence of this inhibitor and neither was the gastrin
responsiveness of the construct v86sp1/2px, in which the SP1 part of
the AP2/SP1 site has been mutated (Fig. 11, b and
c). The addition of the MAPK inhibitor PD98059 also reduced
the gastrin responsiveness of the v86px construct (2.00 ± 0.15-fold over basal; mean ± S.E., n = 15; Fig.
11a) to a value comparable with that found for the v36px construct while having no significant effect on the responses observed
for both the v36px and v86sp1/2px constructs. These data suggest that
the AP2/SP1-binding protein may be regulated by a protein kinase
C/MAPK-dependent signaling pathway.

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Fig. 11.
Effect of the MAPK
inhibitor PD98059 and the protein kinase C (PKC)
inhibitor GF109303X on the gastrin responsiveness of the rat VMAT2
promoter. AGS-GR cells were transfected with the VMAT2
promoter/luciferase constructs v86px (a), v86sp1/2px
(b), and v36px (c) (see Fig. 2b).
Transfected cells were incubated in the absence or presence of either 2 µM GF109303X or 20 µM PD98059 during
stimulation with 5 × 10 8 M gastrin.
Luciferase activity is expressed as a fold increase relative to
unstimulated controls, and results are representative of a minimum of
six independent experiments assayed in duplicate. n values
are given in the text.
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Effect of Dominant-negative ERKs and Acidic CREB on the Gastrin
Responsiveness of the Rat VMAT2 Promoter--
Kinase-deficient ERK
mutants, which were previously shown to behave like dominant-negative
constructs and inhibit endogenous ERK function (30), were used to
determine whether ERK function was necessary for gastrin-stimulated
VMAT2 promoter function. Transfection of either ERK1 or ERK2 reduced
the gastrin response of the construct v86px to ~50% of its original
value (Fig. 12a), but
transfection of ERK1 failed to inhibit the response of the construct
v36px (Fig. 12b). Transfection of the ERK2 construct did
however lead to a small reduction in the gastrin responsiveness of the
v36px construct (66% ± 3% of original response; mean ± S.E., n = 6; Fig. 12b). These data thus
in part support the concept of MAPK-dependent signaling
through the AP2/SP1-binding protein. The overexpression of acidic CREB
was used to confirm CREB transactivation of the VMAT2 promoter (Fig.
12c). An acidic extension of A-CREB interacts with the basic
region of CREB, forming a coiled-coil extension of the leucine zipper
and, thus, preventing the basic region of wild type CREB from binding
to DNA (36). Overexpression of A-CREB led to a 56 ± 2%
(mean ± S.E., n = 6) reduction in the gastrin
responsiveness of the construct v86px (Fig. 12c).

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Fig. 12.
Inhibition of the gastrin responsiveness of
the rat VMAT2 promoter by overexpression of dominant-negative ERKS and
acidic CREB. AGS-GR cells were cotransfected with
either v86px (panel a) or v36px (panel b) in the
presence of either vehicle control, dominant-negative ERK1, or
dominant-negative (dn) ERK2 before stimulation with 5 × 10 8 M gastrin. Panel
c, AGS-GR cells were transfected with v86px together
with either vehicle control or acidic CREB before gastrin stimulation.
Luciferase responses to gastrin are expressed as a percentage of the
unstimulated activity (=100%) of each construct. Results are
representative of at least six independent experiments assayed in
duplicate. Individual n values are given in the text.
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The Adenoviral Oncoprotein E1A Inhibits the Gastrin Responsiveness
of the Rat VMAT2 Promoter--
E1A is an adenoviral oncoprotein that
binds to and inactivates p300/CBP (35). The gastrin-induced
transcription of the construct v86px was inhibited by coexpression of
wild-type E1A (44.5% ± 5.6% reduction of stimulated value; mean ± S.E., n = 6) but not by a mutant of E1A (
2-36E1A)
that is unable to bind CBP (Fig. 13).
Thus, the effect of gastrin on transcription requires the involvement
of p300/CBP.

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Fig. 13.
Gastrin-induced transcriptional increases
are inhibited by cotransfection of adenoviral oncoprotein E1A.
AGS-GR cells were cotransfected with v86px together with
either mutant E1A, 2-36 E1A, or oncoprotein E1A before stimulation
with 5 × 10 8 M gastrin.
Luciferase responses to gastrin are expressed as the percentage of
unstimulated activity (= 100%). Results are representative of six
independent experiments assayed in duplicate.
|
|
 |
DISCUSSION |
The key physiological regulator of gastric acid secretion is
histamine, whose synthesis, storage, and secretion from the ECL cell is
regulated by the antral hormone gastrin (14). In the gastric mucosa of
humans and rodents, the ECL cell is the major source of the histamine
that is generated in the cytosol by HDC and sequestered into the ECL
cell secretory vesicles by VMAT2 (2-4, 9-13). The abundance of
mRNAs encoding VMAT2, HDC, and CgA are increased to accommodate the
increased histamine biosynthesis and secretion that accompanies
ECL-cell stimulation by gastrin (4, 5, 20-26). Elegant studies have
clearly demonstrated that gastrin can regulate the human and rat HDC
promoters together with the mouse CgA promoter (27-29), and in this
study, we have investigated the possibility that gastrin can also
regulate transcription of VMAT2. We had previously cloned and
characterized the 5'-flanking region of the rat VMAT2 gene (5) and
found it to contain no TATA or TATA-like sequence upstream of the
transcriptional start site, but we identified multiple potential
cis-regulatory elements. Of particular interest is the region of
promoter from
86 to +1 bp, which contains SP1, CRE, and putative
overlapping AP2/SP1 consensus sequences.
To investigate both basal and gastrin-stimulated transcriptional
regulation of the VMAT2 promoter, we generated a series of 5'-flanking
deletional constructs of the VMAT2 gene in the luciferase reporter
vectors pGL3b and PXP2. All constructs generated were capable of
driving the transcription of the VMAT2 gene in the AGS-GR
cell line, and responses were maintained in the construct v86pg, which
contained the SP1, AP2/SP1, and CRE regulatory elements. Mutagenesis of
either the SP1, AP2/SP1, or CRE consensus sequence significantly
reduced basal activity of the parental construct, suggesting that basal
transcription is maintained through interaction of a combination of
transcription factors with these cis-regulatory elements. Gastrin
stimulation resulted in the transactivation of all rat VMAT2-promoter
luciferase constructs with significant responsiveness (~4-5-fold
over basal) again being maintained in constructs that contained from
86 to +55 bp of the gene. Mutagenesis of the intact SP1 site at
position
86 to
81 bp from within construct v86px did not affect the
gastrin responsiveness of the promoter; however, mutation of the CRE
from within the construct v36px did significantly reduce the gastrin
response. The importance of the CRE in gastrin-stimulated
transactivation of the VMAT2 promoter was confirmed by the significant
reduction in responsiveness of the v86crepx construct when the CRE site
was also mutated and the inhibition of the gastrin responsiveness of
the v86px construct in the presence of acidic CREB. Moreover, using
EMSA supershift analysis it was demonstrated that CREB bound to the CRE
consensus sequence and that it was phosphorylated after gastrin
stimulation. Because stimulation of CREB-dependent
transcriptional activity is generally achieved by phosphorylation of
the transcription factor, this was an important observation (37). We
did not detect, however, any increase in the amount of CREB binding to
the CRE site. Although our data indicate that CREB is necessary for
gastrin responsiveness of the VMAT2 promoter, it also suggests that an additional factor that might act cooperatively with CREB is necessary for full gastrin responsiveness. Total mutagenesis of the putative AP2/SP1 site significantly reduced the gastrin responsiveness of the
construct v86px as did partial disruption of this site through mutation
of the SP1 section of this AP2/SP1 sequence. Mutation of the AP2
portion of the AP2/SP1 site did not inhibit the response of the v86px
construct. We showed using EMSA supershift analysis that the protein
that bound specifically to this site was unlikely to be a member of
either the AP2 or SP family. Additionally the finding that the binding
of the protein was not totally dependent on the presence of zinc and
that the mobility of the protein-DNA complex was much greater than the
SP family-DNA complexes suggests that it is smaller than an SP protein.
This was confirmed by UV-cross-linking experiments, which estimated the
molecular mass of the protein to be 23.3 kDa. A protein from rat
gastric corpus nuclear extracts also bound to the AP2/SP1 consensus
sequence, and the mobility of the corresponding protein-DNA complex as
observed in EMSA suggested that the protein found in the corpus is the
same as that found in the AGS-GR cells. The
AGS-GR cells utilize similar signaling pathways to the ECL
cell (13, 30, 33), and the finding that they contain transcription
factors common to cells in the corpus make them an appropriate cell
type to use in this study. The overexpression of the adenoviral
oncoprotein E1A inhibited the gastrin responsiveness of the v86px
construct, whereas overexpression of a mutant form that is unable to
bind p300/CBP failed to do so. These data imply that gastrin might
transcriptionally activate the rat VMAT2 promoter through a mechanism
involving the cooperative interaction of CREB, the AP2/SP1-binding
protein, and p300/CBP.
A growing number of studies demonstrate a role for p300/CBP in
connecting CREB to different transcription factors and the MAP
kinase/ERK intracellular signaling pathways (41-44). The use of
inhibitors and dominant-negative ERKs demonstrated that the gastrin
response of the rat VMAT2 promoter was potentially regulated by both
the protein kinase C and MAPK signaling pathways, and the putative
target of these signaling systems is the protein that binds to the
AP2/SP1 site. Activation of our constructs in which only the CRE
consensus sequence remained intact (truncated and mutated constructs
v36px and v86sp1px) was less dependent on these signaling pathways. Our
previous studies showed that mobilization of intracellular calcium can
increase the transcriptional activity of the VMAT2 promoter in a manner
dependent on the CRE consensus sequence, and since activation of the
CCKB-gastrin receptor is known to lead to an increase in
free cytosolic calcium, it is possible that in the absence of the
AP2/SP1 consensus site, activation of the rat VMAT2 promoter may be
largely through this pathway. The finding that the gastrin response of
the construct v36px was slightly inhibited by the overexpression of the
dominant-negative ERK-2 is not inconsistent with the finding that it is
largely the activity of the AP2/SP1 protein, which is regulated by the protein kinase C/MAPK pathways, as it is possible that there is some
interaction of the AP2/SP1-binding protein and p300/CBP in the absence
of DNA binding. Indeed, c-Jun can stimulate CBP-mediated transcription
in a manner that is independent of its ability to bind DNA (35).
However it is recognized that CREB phosphorylation through activation
of Ca2+-calmodulin or MAP kinase-dependent
pathways can occur (38-40). Gastrin- dependent regulation of the mCgA
promoter has been shown to involve the cooperative interaction of both
SP1 and CREB transcription factors (29), but the signal transduction
pathways regulating these proteins in this system remain to be elucidated.
The gastrin-stimulated transcription of the rat VMAT2 promoter is
similar to that of the mCgA promoter in that it involves the
phosphorylation of CREB, but unlike the mCgA promoter, the gastrin
responsiveness of the VMAT2 promoter appears to be independent of the
SP1 transcription factor. The
100 to
43-bp region of the mCgA
promoter is transcriptionally responsive to gastrin, and within this
region, the SP1 protein binds to a consensus sequence contained within
a larger AP2 consensus site (Fig. 8). This sequence is different from
the AP2/SP1 consensus sequence, which we have described for the rat
VMAT2 promoter, and the AP2/SP1-binding protein, which is described in
this study did not bind to the CgA consensus sequence or to any part of
the
100 to
43-bp region. The gastrin-dependent
regulation of the HDC promoter is dependent on the protein kinase
C/MAPK pathways (28, 30, 31) and is mediated by two distinct nuclear
factors of 52- and 35-kDa, which remain to be characterized (32). The
gastrin responsiveness of the rat VMAT2 promoter shares with the HDC
promoter its dependence on the protein kinase C/MAPK-signaling
pathways, but we have shown that the AP2/SP1 binding protein does not
recognize the consensus sequence through which gastrin responsiveness
of the HDC promoter is mediated (GAS-RE; +1 to +27 bp). The present
study demonstrates that the rat VMAT2 promoter can, like the HDC and
mCgA promoters, be transcriptionally regulated by gastrin, but although
the mechanism of this gastrin regulation shares some similarities with
that observed for each of these promoters, there are important
differences. Thus, gastrin-dependent regulation of the rat
VMAT2 promoter involves the cooperative interaction of the CREB
transcription factor not with the SP1 transcription factor, as seen for
the mCgA promoter, but with an unidentified factor of 23.3 kDa, which
binds to a putative AP2/SP1 site and possibly interacts with CREB
utilizing p300/CBP. The amount of protein binding to the AP2/SP1 site
was not increased after gastrin stimulation, but the observation that gastrin responsiveness of the rat VMAT2 promoter is regulated by the
protein kinase C/MAPK pathways suggests that the activity of the
protein may be regulated by phosphorylation. Further work will
therefore be necessary both to identify the AP2/SP1-binding protein and
to test this hypothesis. The cluster of cis-regulatory elements and
transactivating factors identified here to be important in
gastrin-stimulated VMAT2 gene transcription is likely to have significant roles in the regulation of VMAT2 gene expression in a
number of systems. The widespread importance of VMAT2 means that it is
pertinent to investigate the controls of VMAT2 transcriptional regulation in diverse physiological environments and to determine whether the uncharacterized AP2/SP1-binding protein is of universal importance.
 |
ACKNOWLEDGEMENTS |
AGS-GR gastric cancer cells were
kindly provided by Dr. A. Varro, Dept. of Physiology, University of
Liverpool, UK. These AGS cells were permanently transfected with the
human CCKB-gastrin receptor driven by the EF1-
promoter
under puromycin selection, which was a kind gift of Dr. Ramnik Xavier
(Massachusetts General Hospital, Boston). The promoterless-luciferase
vector PXP2 and the plasmids encoding kinase-deficient ERKs were kind
gifts from Prof. T. C. Wang (Massachusetts General Hospital,
Boston). The expression plasmids for the adenoviral oncoprotein E1A and
its mutant were kind gifts from Dr. Joseph R. Nevins, Duke University Medical Center, Durham, NC, whereas the expression plasmid encoding the
dominant-negative inhibitor acidic CREB was a kind gift from Dr. David
D. Ginty, The Johns Hopkins University School of Medicine, Baltimore.
The assistance of H. Davies and C. McLean is gratefully acknowledged.
 |
FOOTNOTES |
*
This work was supported by Wellcome Trust Grant 050830.The costs of publication of this
article were defrayed in part by the payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: The Physiological
Laboratory, University of Liverpool, Crown St., Liverpool L69 3BX, UK.
E-mail: watso@liv.ac.uk.
Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.M006697200
 |
ABBREVIATIONS |
The abbreviations used are:
VMAT2, vesicular
monoamine transporter 2;
ECL, enterochromaffin-like cell;
HDC, L-histidine decarboxylase;
CgA, chromogranin A;
mCgA, mouse
CgA;
MAP, mitogen-activated protein;
MAPK, MAP kinase;
ERK, extracellular signal-regulated kinase;
bp, base pair(s);
CRE, cAMP-response element;
CREB, CRE-binding protein;
CBP, CREB-binding
protein;
EMSA, electrophoretic mobility shift assay;
CCK, cholecystokinin.
 |
REFERENCES |
1.
|
Liu, Y.,
and Edwards, R. H.
(1997)
Annu. Rev. Neurosci.
20,
125-156[CrossRef][Medline]
[Order article via Infotrieve]
|
2.
|
Merickel, A.,
and Edwards, R. H.
(1995)
Neuropharmacology
34,
1534-1547
|
3.
|
Takahashi, N.,
and Uhl, G.
(1997)
Mol. Brain Res.
49,
7-14[CrossRef][Medline]
[Order article via Infotrieve]
|
4.
|
Dimaline, R.,
and Struthers, J.
(1996)
J. Physiol.
490,
249-256[Abstract]
|
5.
|
Watson, F.,
Deavall, D. G.,
Macro, J. A.,
Kiernan, R.,
and Dimaline, R.
(1999)
Biochem. J.
337,
193-199[CrossRef][Medline]
[Order article via Infotrieve]
|
6.
|
Takahashi, N.,
Miner, L. L.,
Sora, I.,
Ujike, H.,
Revay, R. R.,
Kostic, V.,
Jackson,
Lewis, V.,
Przedborski, S.,
and Uhl, G. R.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
9938-9943[Abstract/Free Full Text]
|
7.
|
Liu, Y.,
Peter, D.,
Roghani, A.,
Schuldiner, S.,
Prive, G. G.,
Eisenberg, D.,
Brecha, N.,
and Edwards, R. H.
(1992)
Cell
70,
539-551[Medline]
[Order article via Infotrieve]
|
8.
|
Gainetdinov, R. R.,
Fumagalli, F.,
Wang, Y-M.,
Jones, S. R.,
Levey, A. I.,
Miller, G. W.,
and Caron, M. G.
(1998)
J. Neurochem.
70,
1973-1978[Medline]
[Order article via Infotrieve]
|
9.
|
Hakanson, R.
(1970)
Acta Physiol. Scand.
340 (suppl.),
1-134
|
10.
|
Peter, D.,
Liu, Y.,
Sternini, C.,
De, Giorgio, R.,
Brecha, N.,
and Edwards, R. H.
(1995)
J. Neurosci.
15,
6179-6188[Abstract]
|
11.
|
Weihe, E.,
Schafer, M. K. H.,
Erickson, J. D.,
and Eiden, L. E.
(1994)
J. Mol. Neurosci.
5,
149-164[Medline]
[Order article via Infotrieve]
|
12.
|
Prinz, C.,
Kajimura, M.,
Scott, D. R.,
Mercier, F.,
Helander, H. F.,
and Sachs, G.
(1993)
Gastroenterology
105,
449-461[Medline]
[Order article via Infotrieve]
|
13.
|
Prinz, C.,
Zanner, R.,
Gerhard, M.,
Mahir, S.,
Neumayer, N.,
Hohne-Zell, B.,
and Gratzl, M.
(1999)
Am. J. Physiol.
277,
C845-C855[Medline]
[Order article via Infotrieve]
|
14.
|
Black, J. W.,
and Shankley, N. P.
(1987)
Trends Pharmacol. Sci.
8,
486-490
|
15.
|
Beaven, M. A.
(1982)
in
Pharmacology of Histamine Receptors
(Ganellin, C. R.
, and Parsons, M. E., eds)
, pp. 101-105, Wright, Bristol, UK
|
16.
|
O'Connor, D. T.,
Wu, H.,
Gill, B. M.,
Rozansky, D. J.,
Tang, K.,
Mahata, S. K.,
Mahata, M.,
Eskeland, N. L.,
Videen, J. S.,
Zhang, X.,
Takiyuddin, M. A.,
and Parmer, R. J.
(1993)
Ann. N. Y. Acad. Sci.
729,
36-45[Medline]
[Order article via Infotrieve]
|
17.
|
Bauerfeind, R.,
Ohashi, M.,
and Huttner, W. B.
(1994)
Ann. N. Y. Acad. Sci.
733,
385-390
|
18.
|
Iacangelo, A.,
and Eiden, L.
(1995)
Regul. Pept.
58,
65-88[CrossRef][Medline]
[Order article via Infotrieve]
|
19.
|
Stabile, B. E.,
Howard, T. J.,
Passaro, E.,
and O'Connor, D. T.
(1990)
Arch. Surg.
125,
451-453[Abstract]
|
20.
|
Cetin, Y.,
and Grube, D.
(1991)
Cell Tissue Res.
264,
231-241[Medline]
[Order article via Infotrieve]
|
21.
|
Syversen, U.,
Mignon, M.,
Bonfils, S.,
Kristensen, A.,
and Waldum, H. L.
(1993)
Acta Oncol. (Madr.)
32,
161-165
|
22.
|
Watkinson, A.,
and Dockray, G. J.
(1992)
Regul. Pept.
40,
51-61[CrossRef][Medline]
[Order article via Infotrieve]
|
23.
|
Hakanson, R.,
Ding, X-Q.,
Norlen, P.,
and Chen, D.
(1995)
Gastroenterology
108,
1445-1452[Medline]
[Order article via Infotrieve]
|
24.
|
Dimaline, R.,
and Sandvik, A. K.
(1991)
FEBS Lett.
281,
20-22[CrossRef][Medline]
[Order article via Infotrieve]
|
25.
|
Dimaline, R.,
Evans, D.,
Forster, E. R.,
and Sandvik, A. K.
(1993)
Am. J. Physiol.
264,
G583-G588[Abstract/Free Full Text]
|
26.
|
Dimaline, R.,
Sandvik, A. K.,
Evans, D.,
Forster, E. R.,
and Dockray, G. J.
(1993)
J. Physiol.
465,
449-458[Abstract]
|
27.
|
Hocker, M.,
Zhang, Z.,
Fenstermacher, D. A.,
Tagerud, S.,
Chulak, M.,
Joseph, D.,
and Wang, T. C.
(1996)
Am. J. Physiol.
270,
G619-G633[Abstract/Free Full Text]
|
28.
|
Zhang, Z.,
Hocker, M.,
Koh, T. J.,
and Wang, T. C.
(1996)
J. Biol. Chem.
271,
14188-14197[Abstract/Free Full Text]
|
29.
|
Hocker, M.,
Raychowdhury, R.,
Plath, T.,
Wu, H.,
O'Conner, D. T.,
Wiedenmann, B.,
Rosewicz, S.,
and Wang, T. C.
(1998)
J. Biol. Chem.
273,
34000-34007[Abstract/Free Full Text]
|
30.
|
Hocker, M.,
Henihan, R. J.,
Rosewicz, S.,
Riecken, E.,
Zhang, Z.,
Koh, T. J.,
and Wang, T. C.
(1997)
J. Biol. Chem.
272,
27015-27024[Abstract/Free Full Text]
|
31.
|
Hocker, M.,
Zhang, Z.,
Merchant, J. L.,
and Wang, T. C.
(1997)
Am. J. Physiol.
272,
G822-G830[Abstract/Free Full Text]
|
32.
|
Raychowdhury, R.,
Zhang, Z.,
Hocker, M.,
and Wang, T. C.
(1999)
J. Biol. Chem.
274,
20961-20969[Abstract/Free Full Text]
|
33.
|
Varro, A.,
Wroblewski, L.,
Noble, P. J.,
Bishop, L.,
Ashcroft, F.,
Varro, J.,
Thompson, C.,
Dimaline, R.,
and Dockray, G. J.
(2000)
Gut
46,
56[CrossRef] (abstr.)
|
34.
|
Schreiber, E.,
Matthias, P.,
Muller, M. M.,
and Schaffner, W.
(1989)
Nucleic Acids Res.
17,
6419[Medline]
[Order article via Infotrieve]
|
35.
|
Hu, P. P.,
Harvat, B. L.,
Hook, S. S.,
Shen, X.,
Wang, X.,
and Means, A. R.
(1999)
Mol. Endocrinol.
13,
2039-2048[Abstract/Free Full Text]
|
36.
|
Ahn, S.,
Olive, M.,
Aggarwal, S.,
Krylov, D.,
Ginty, D. D.,
and Vinson, C.
(1998)
Mol. Cell. Biol.
18,
967-977[Abstract/Free Full Text]
|
37.
|
Montminy, M. R.
(1993)
in
Gene Expression: General and Cell Type-specific
(Karin, M., ed)
, pp. 72-92, Birkhaeuser Boston, Cambridge, MA
|
38.
|
Sheng, M.,
Thompson, M. A.,
and Greenberg, M. E.
(1991)
Science
252,
1427-1430[Medline]
[Order article via Infotrieve]
|
39.
|
Ginty, D. D.,
Bonni, A.,
and Greenberg, M. E.
(1994)
Cell
77,
713-725[Medline]
[Order article via Infotrieve]
|
40.
|
Xing, J.,
Ginty, D. D.,
and Greenberg, M. E.
(1996)
Science
273,
959-963[Abstract]
|
41.
|
Janknecht, R.,
and Hunter, T.
(1996)
Nature
383,
22-23[CrossRef][Medline]
[Order article via Infotrieve]
|
42.
|
Arias, J.,
Alberts, A. S.,
Brindle, P.,
Claret, F. X.,
Smeal, T.,
Karin, M.,
Feramisco, J.,
and Montminy, M.
(1994)
Nature
370,
226-229[CrossRef][Medline]
[Order article via Infotrieve]
|
43.
|
Nakajima, T.,
Fukamizu, A.,
Takahashi, J.,
Gage, F. H.,
Fisher, T.,
Blenis, J.,
and Montminy, M. R.
(1996)
Cell
86,
465-474[Medline]
[Order article via Infotrieve]
|
44.
|
Cobb, M. H.,
and Goldsmith, E. J.
(1995)
J. Biol. Chem.
270,
14843-14846[Free Full Text]
|
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