1 Physiological Laboratory, University of Liverpool, Liverpool L69 3BX, United Kingdom; and 2 Massachusetts General Hospital, Boston, Massachusetts 02114
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ABSTRACT |
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The
mechanisms by which neuroendocrine stimulants regulate CCK gene
transcription are unclear. We examined promoter activation by pituitary
adenylate cyclase-activating polypeptide (PACAP), a known CCK
secretagogue, in the enteroendocrine cell line STC-1. The promoter
region from 70 to
87 bp, relative to the transcriptional start
site, contains a composite calcium/cyclic AMP response element (CRE)/activator protein 1 (AP1) site that may bind CRE binding protein
(CREB) and AP1. PACAP (with IBMX) stimulated expression of an 87-bp
construct 3.35 ± 0.36-fold but had no effect on a
70 construct.
The effect was blocked by the protein kinase A inhibitor H-89 and by a
dominant-negative CREB plasmid. Mutation of the CRE/AP1 site to a
canonical CRE site did not affect the response to PACAP, but mutation
to a canonical AP1 site prevented it. CREB phosphorylation was
increased after PACAP treatment. Electrophoretic mobility shift assay
and supershift analysis revealed that CREB and not AP1 bound to the
CRE/AP1 site and that PACAP increased the proportion of phosphorylated
CREB that was bound. We conclude that PACAP increases CCK gene
expression via a cAMP-mediated pathway involving CREB phosphorylation
by protein kinase A and activation of a composite CRE/AP1 site.
cholecystokinin; pituitary adenylate cyclase-activating polypeptide; calcium/cyclic AMP response element; calcium/3,5'-monophosphate response element binding protein; adenosine 3',5'-cyclic monophosphate
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INTRODUCTION |
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THE BRAIN-GUT HORMONE CHOLECYSTOKININ (CCK) is synthesized by central neurons and by enteroendocrine I cells of the proximal small intestine. The release of CCK from the latter is associated with stimulation of pancreatic enzyme secretion and gallbladder contraction and inhibition of gastric emptying and food intake (19, 35). Factors controlling CCK release have been relatively well studied both in vivo and in cell lines such as STC-1 (3, 11, 18, 21, 30). The STC-1 cell line is derived from an intestinal endocrine tumor that developed in a double-transgenic mouse expressing the rat insulin promoter linked to the SV40 large T antigen and the polyoma small t antigen. These cells express CCK mRNA and secrete the biologically active form of the peptide, CCK-8 (27), thus providing a useful model of the CCK-producing endocrine cell. In vivo, CCK release is stimulated by luminal nutrients such as fatty acids with a chain length greater than C12 (21), protein (18), neuropeptides including pituitary adenylate cyclase-activating polypeptide (PACAP) (14, 16), and luminal CCK-releasing peptides such as monitor peptide (13) and the luminal CCK-releasing factor LCRF (11, 31). In STC-1 cells, CCK is released by amino acids and fatty acids as well as by secretagogues such as bombesin and PACAP (3, 21, 24, 30). PACAP is a member of the secretin/glucagon/vasoactive intestinal polypeptide (VIP) regulatory peptide family and was originally identified in the hypothalamus as a stimulant of pituitary adenylate cyclase (2, 23). This neuropeptide is expressed in vivo by both central and peripheral neurons, including those of the submucosal plexus underlying the enteroendocrine cells. The secretory response to PACAP of the STC-1 cell is associated with rises in intracellular calcium and cAMP (3, 30).
The mechanisms that control expression of the CCK gene are less clear,
although there is evidence from studies in vivo that dietary and
hormonal factors influence intestinal CCK mRNA abundance (17). Cloning and characterization of the CCK promoter
region has identified several recognized regulatory DNA sequences,
including consensus binding sites for a number of transcription
factors, e.g., SP1 and AP4, within 100 bp of the transcriptional start site (10). In SK-N-MC cells, studies of CCK
promoter-reporter constructs confirmed the functionality of a number of
cis-regulatory domains (25). Of particular
interest is a putative composite calcium response element
(CRE)/activator protein 1 (AP1) site that may bind both the
calcium/3,5'-monophosphate response element binding protein (CREB) and
AP1 (10). Composite CRE/AP-1 sites of a similar type occur
in a number of genes involved in neurotransmitter synthesis, including
dopamine -hydroxylase (29), prodynorphin (4,
22), and proenkephalin (5). CRE/AP1 sites are
potentially able to bind members of both the CREB/activating
transcription factor (ATF) and AP1 transcription factor
families (15), thereby facilitating transcriptional
integration by a wide range of stimuli acting through CREB or AP1,
representing convergence of separate signal transduction pathways. In
PC12 cells, cAMP-mediated transcriptional responses of the dopamine
-hydroxylase promoter are mediated by AP1 proteins acting through a
composite CRE/AP1 site (33). In SK-N-MC cells, basic
fibroblast growth factor increases CCK gene transcription via binding
of CREB to the composite CRE/AP1 site (8). The CRE/AP1
site in the CCK promoter lies downstream of an E box binding domain for
the basic helix-loop-helix family of transcription factors, and
interaction between these two sites in cAMP-stimulated activity has
also recently been described (28). We hypothesized that
the CCK secretagogue PACAP may also increase CCK gene transcription,
and in the present study we describe cis-regulatory elements
within the promoter and trans-activating STC-1 cell nuclear proteins mediating the response. Our data suggest that PACAP-induced CCK gene expression is achieved by CREB family transcription factors acting at the CRE/AP1 site.
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MATERIALS AND METHODS |
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Materials. Ovine PACAP-38 was purchased from Calbiochem-Novabiochem (Nottingham, UK). Forskolin and IBMX were obtained from Sigma (Poole, UK). Tissue culture media, supplements, and plasticware were obtained from Life Technologies (Paisley, UK). Luciferase assay reagents were obtained from Promega (Southampton, UK).
Tissue culture. STC-1 cells were maintained in DMEM supplemented with 10% horse serum, 5% fetal bovine serum, 100 µg/ml streptomycin, 100 µU/ml penicillin, and 5 µg/ml ascorbate in a humidified incubator at 37°C under 5% CO2-95% O2.
CCK-luciferase plasmids.
The promoter fragment of the C-1089 construct was created from rat
genomic DNA by PCR. The forward primer was directed between 1089 and
1061 bases upstream of the published major transcriptional start site
(10), and a reverse primer was directed between +38 and
+55 of exon 1 (which was common to all constructs). C-87, C-125, and
C-70 were created by a similar strategy using C-1089 as a template.
C-87CRE, C-87AP, C-87m1, C-87m2,
C-87m3, and C-87m4 were generated using C-87 as a
template. The primers used to generate constructs are shown in Table
1. PCR products were purified from agarose gels and cloned into pCRII (Invitrogen, Groningen, The Netherlands) before insertion as Hind III/Xho I
fragments into pGL3-Basic luciferase reporter vector (Promega).
Integrity of the constructs was confirmed by automated sequencing.
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Transient transfection.
In initial experiments, cells were transfected by electroporation, and
5 × 106 cells were incubated on ice for 5 min in DMEM
with 20 µg CCK-luciferase (CCK-LUC) and 2.5 µg pRL-TK
(Promega) as an internal control. The pRL-TK vector contains a gene
encoding sea pansy luciferase driven by the thymidine kinase promoter.
Cells were pulsed at 200 mV/1,070 µF and plated into 60-mm
poly-L-lysine-coated dishes containing Ultraculture media
(BioWhittaker, Wokingham, UK). Cells were maintained for 18 h
before stimulation and harvested at 24 h after transfection. For
all subsequent experiments, lipofection was used. Cells (1 × 106 cells per well) were plated into six-well plates
24 h before transfection. Cells were transfected with Trans FAST
reagent (Promega) using 0.7 µg CCK-LUC, 0.2 µg of either
dominant-negative plasmid, empty vector, or pBluescript II SK
(Stratagene, LaJolla, CA) and 0.1 µg pRL-TK, according to the
manufacturer's protocol. Cells were then maintained in full media for
18 h before stimulation and harvested after 24 h. Luciferase
activity was determined by luminometry (Turner Designs TD20-20)
using the dual luciferase assay system (Promega). Transfection
efficiency was assessed following transfection of cells (1 × 106 cells per well), with 1 µg of the green fluorescent
protein (GFP) expression vector pEGFP-C1 (Clontech,
Basingstoke, UK). GFP-expressing cells were visualized 24 h later
by fluorescence microscopy, and the number present in four visual
fields for each of five wells was counted. Transfection efficiency was
determined to be 5.58 ± 0.28% (mean ± SE;
n = 5).
Northern blot analysis.
Cells were plated (1 × 106 cells per well) in
six-well plates 18 h before use and then treated with PACAP/IBMX
(107 M/0.5 mM) or vehicle for 6, 24, or 48 h. Total
RNA was extracted with TRIzol (Life Technologies), and Northern blot
analysis was performed as previously described (7) using a
cRNA probe to rat CCK.
Western blot analysis. Immunoblotting of STC-1 cell proteins was performed using a PhosphoPlus CREB (Ser133) antibody kit (New England Biolabs, Beverley, MA) according to the manufacturer's protocol. Briefly, STC-1 cells were plated at 106 cells per well on six-well plates and harvested into SDS-sample buffer 48 h later. Whole cell extracts were run on 10% SDS-PAGE and immunoblotted with primary antibodies for phosphorylated and total CREB. After incubation with horseradish peroxidase-conjugated secondary antibody and the chemiluminescence reaction, the signal was detected by exposure to HyperFilm (Amersham Biotech, Little Chalfont, UK).
Electrophoretic mobility shift assay.
Crude nuclear extracts from STC-1 cells were made as previously
described (26). Double-stranded oligonucleotides were
radiolabeled with [-32P]dCTP and incubated with 4 µg of nuclear extract in a reaction containing 10 mM
Tris · HCl (pH 7.5), 50 mM NaCl, 5 mM
MgCl2, 1 mM dithiothreitol, 1 mM EDTA, 1 µg poly(dA-dT),
and 10% glycerol. Reactions containing 10 fmol of double-stranded
oligonucleotide probe were incubated at room temperature for 20 min
before electrophoresis at 30 mA on 6% nondenaturing polyacrylamide
gels containing 0.25× Tris borate/EDTA. Gels were then dried and
exposed to a Phosphor screen before image analysis using ImageQuant
(Molecular Dynamics), or gels were exposed to HyperFilm (Amersham
Biotech) at room temperature for up to 72 h. For competition
experiments, extracts were incubated with 200-fold excess of competitor
oligonucleotide for 10 min at room temperature before addition of the
labeled probe. For supershift band analysis, extracts were incubated
with antibody for 10 min at room temperature followed by 20 min on ice
before addition of the probe for 10 min at room temperature. The
anti-P-CREB antibody used in supershift analyses recognizes
p43-phosphorylated CREB (Upstate Biotechnology, Lake Placid, NY), the
anti-CREB ATF-1 antibody recognizes CREB-1 p43, ATF-1 p35, and cyclic
AMP response element modulator (CREM)-1 (sc-270 X, Santa Cruz
Biotechnology, Santa Cruz, CA), and the anti-Jun/AP1 antibody
recognizes c-Jun, Jun B, and Jun D p39 proteins (sc-44-G X, Santa Cruz Biotechnology).
Statistical analysis. All results are expressed as means ± SE. Statistical difference was determined by Student's t-test or one-way ANOVA
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RESULTS |
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Basal activity.
The pattern of basal activity of the CCK promoter in STC-1 cells was
established by performing 5' deletional analysis. Deletion from C-1089
to C-125 resulted in significantly elevated activity, but further
deletion to C-87 reduced basal activity to a level similar to the
C-1089 construct. Further deletion to C-70 decreased basal activity
14.02 ± 0.02-fold compared with C-125 (Fig.
1), indicating the importance of the
region between 125 and
70.
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The region between 87 and
70 of the rat CCK promoter confers
PACAP responsiveness.
Because the region between
87 and
70 includes the CRE/AP1 site, we
asked whether this sequence conferred responsiveness to extracellular
stimuli by administration of PACAP and the adenylyl cyclase activator
forskolin. The latter stimulated activity of C-1089, C-125, and C-87 by
1.65 ± 0.24-, 2.34 ± 0.18-, and 2.39 ± 0.19-fold,
respectively (P < 0.05) but had no effect on C-70 (Fig. 1). The C-1089 and C-87 constructs, but not C-70, were also stimulated by PACAP (Fig. 2). Stimulation
was both dose (Fig. 2) and time dependent (not shown), being maximal at
6 h with 10
7 M PACAP in the presence of 0.5 mM IBMX.
We therefore established that PACAP responsiveness lay between
87 and
70 bp upstream of the reported transcriptional start site. Compatible
with the idea that PACAP stimulates CCK gene transcription, we found
using Northern blot that endogenous CCK mRNA abundance was also
elevated when STC-1 cells were treated for 48 h with PACAP
(10
7 M) in the presence of 0.5 mM IBMX (Fig. 2).
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Inhibition of protein kinase A and overexpression of a dominant
negative for CREB inhibits the response to PACAP.
PACAP receptors may be linked exclusively to adenylyl cyclase or to
adenylyl cyclase and phospholipase C (3). To examine the
role of protein kinase A (PKA)-mediated phosphorylation in our system,
we used the specific PKA inhibitor H-89. At a concentration of 20 µM,
H-89 completely inhibited PACAP stimulation of C-87 (Fig.
3). We considered the possibility that
PKA activated CREB, and to investigate this we cotransfected a plasmid
encoding a dominant negative for CREB (A-CREB). This plasmid possesses
an acidic extension of the CREB leucine zipper domain that binds with
the basic region of wild-type CREB to prevent the latter from
interacting with the CRE (1). PACAP-induced activation of
C-87 was decreased from 3.14 ± 0.09-fold to 1.36 ± 0.10-fold in the presence of A-CREB (Fig. 3), indicating a role for
CREB in PACAP-stimulation of the CCK promoter.
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PACAP and forskolin increase CREB phosphorylation.
The data presented above are compatible with PACAP activation of CREB,
and to determine whether the abundance of phosphorylated CREB was
altered, we performed Western blot analysis on cells stimulated with
PACAP or forskolin. The total CREB abundance (i.e., phosphorylated and
unphosphorylated) did not change (Fig.
4). However, the abundance of both
phosphorylated CREB and phosphorylated ATF was increased in response to
both forskolin and PACAP (Fig. 4).
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Electrophoretic mobility shift assay reveals complex formation with
STC-1 nuclear extracts and a CRE/AP1 probe.
To establish the nature of the protein/DNA interactions at the
composite site, in vitro binding assays were performed using electrophoretic mobility shift assay (EMSA). Nuclear protein extracts from STC-1 cells formed a complex with a 24mer oligonucleotide probe
that included the wild-type CCK promoter CRE/AP1 sequence (Fig.
5). Competition shift experiments
revealed that complex formation was dependent on an intact CRE/AP1 site
since the wild-type oligonucleotide, but not an oligonucleotide
containing a mutated CRE/AP1 site, effectively inhibited the binding
reaction. In extracts of unstimulated cells, the consensus
CRE-containing sequence, but not a consensus AP1 oligonucleotide, also
successfully inhibited the binding reaction. The data indicate that
CRE/ATF family proteins, but not AP1 family transcription factors, bind
to the CRE/AP1 site. This was further confirmed by supershift band
analysis, which showed that an antibody to CREB shifted the bound
complex in both unstimulated and forskolin/PACAP stimulated cells (Fig. 5). However, supershifts were not seen using an antibody to the AP1
transcription factor (anti-Jun/AP1; Fig. 5). In the extracts from
forskolin and PACAP-stimulated cells, but not unstimulated cells, there
was an increase in the binding of phosphorylated CREB to the CRE/AP1
probe because anti-P-CREB produced a supershift of a proportion of the
bound material (Fig. 5).
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Mutagenesis of the region between 87 and
70 reveals the
importance of an intact CRE in cis for PACAP responsiveness.
We hypothesized that CREB activated CRE, and to establish the
importance of an intact CRE in cis we performed mutagenesis of the region between
87 and
70 of the CCK promoter (Fig.
6). The unstimulated transcriptional
activity of C-87 was maintained by C-87mAP (89.9 ± 2.5% of C-87; n = 4), whereas C-87mCRE
showed a reduced basal activity compared with the wild type (62.2 ± 1.5%; n = 4). Analysis of the mutants in response
to PACAP revealed the importance of an intact CRE for maximal activity.
Thus when the composite CRE/AP1 site was mutated randomly or to a pure
AP1 site, there was significantly decreased stimulation in response to
PACAP compared with wild-type C-87. Mutations outside the CRE site or
to a pure CRE consensus did not significantly diminish PACAP
responsiveness (Fig. 6).
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DISCUSSION |
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The main finding of the present study is that PACAP increased CCK gene transcription in STC-1 cells. Our data indicate that PACAP acts via PKA-mediated phosphorylation of CREB/ATF transcription factors at Ser133 to activate the CCK promoter. Increased phosphorylation of CREB-1 and ATF-1 was confirmed by Western blot analysis. CREB/ATF protein was shown to bind to a CRE/AP1-containing oligonucleotide probe in EMSAs, and the bound material was supershifted with a CREB/ATF antibody. Supershift band analysis using anti-P-CREB antibody delayed the migration of the binding complex in the forskolin- and PACAP-treated cells but not in control cells. An intact CRE site in cis was shown to be essential for increased transcription, because deletion or mutation of the composite CRE/AP1 site to a non-CRE-like sequence in the promoter-reporter constructs prevented PACAP-induced activation. The importance of CREB in extracts from unstimulated cells was further verified by competition EMSAs in which CREB- and not AP1-family transcription factors bound to the CRE/AP1 site.
The composite CRE/AP1 site potentially facilitates convergence of signal transduction pathways impinging on both the AP1 and CREB transcription factors. Previously, it has been demonstrated that the CCK promoter exhibited increased activation in response to overexpression of CREB and the AP1 dimerization partners, Fos and Jun, in SK-N-MC cells (25). More recently, promoter activation with basic fibroblast growth factor, via activation of CREB by mitogen-activated protein kinase and PKA, has been demonstrated in the SK-N-MC cell line. This has been proposed as a mechanism by which growth and neurotrophic factors, in concert with neuropeptides, may regulate expression of the CCK gene (10).
In the STC-1 cell line, both CCK mRNA abundance and activity of an 800-bp CCK promoter construct were increased by peptone, which represents a luminal stimulus, although the transcriptional activation pathway was not reported (6). In addition, however, gene expression in enteroendocrine cells may be modulated by neurohormonal agents. Recently, PACAP was shown to be a potentially important modulator of the gastric enterocromaffin-like cell and the chromaffin cell (34, 36). Furthermore, PACAP has been shown to stimulate CCK secretion from the STC-1 cell line and from a mucosal I cell-enriched preparation from rat small intestine (3). Given its localization in central neurons and within the submucosal plexus of the small intestine, PACAP has itself been proposed as a candidate neuromodulator of CCK release in vivo (16). The mechanisms behind such secretagogue effects are unclear but are known to be associated with elevation of cAMP levels (3). PACAP is known to act through at least three receptor subtypes (9). The type II receptor VPAC1 is activated by both PACAP and VIP and is coupled exclusively to adenylyl cyclase (12), whereas the type I PAC1 receptor is not responsive to VIP and utilizes both cAMP and inositol 1,4,5-trisphosphate as second messengers (32). It has previously been reported that the effects of PACAP on STC-1 cells are mediated by the VPAC1 receptor and thus involve only cAMP as the second messenger (3). The present data also suggest that cAMP is the second messenger involved in the PACAP signaling pathway, because responsiveness is lost in the presence of the PKA inhibitor H-89. Therefore, as well as being a candidate neuromodulator of secretion, PACAP may serve to regulate expression of the CCK gene via cAMP-dependent mechanisms.
It has been proposed that the CCK promoter CRE/AP1 site and the
proenkephalin CRE-2 site preferentially bind CREB over AP1 because of
the nonpalindromic nature of the site and its core C residue
(TGCGTCA) (28). However, it has also been
reported that increases in AP1 proteins may directly mediate
cAMP-induced activation of the composite site in the dopamine
-hydroxylase promoter, which is also of the type TGCGTCA
(33), implicating both families of transcription factors
in cAMP-induced modulation of this site. Moreover, AP1-related proteins
have been shown to represent the majority of protein binding to the
proenkephalin promoter CRE-2 site in the adrenal chromaffin cell, which
contrasts with the predominance of CREB binding to this site in the
central nervous system (20). We did not observe
an AP1-mediated element to the PACAP-induced activation of the CCK
CRE/AP1 site within the STC-1 cell line. The dominant-negative A-CREB
vector has previously been shown not to interfere with dimerization of
members of the AP1 complex (1), and yet PACAP-induced
activation is inhibited in the presence of A-CREB. Furthermore,
mutation of the CRE/AP site to a consensus AP1 site in the
promoter-reporter gene greatly decreased the PACAP effect. The lack of
a supershift with a Jun/AP1 antibody in the EMSA and the lack of
competition for the binding reaction with the consensus AP1-containing
oligonucleotide suggest that AP1 binding to the composite site is less
important than CREB binding in the PACAP-stimulated or unstimulated
STC-1 cells. We demonstrated, however, that the unstimulated activity
of C-87 is fully maintained by C-87mAP and is slightly
reduced by C-87mCRE. Thus AP1 proteins may direct activity
of C-87mAP in a manner not observed with the composite site,
whereas CREB binding to the composite site may be of a higher affinity
than binding to the C-87mCRE. In PACAP-stimulated cells, AP1
does not appear to play a role in increasing transcriptional activation
of the CCK promoter. Thus the signaling pathway used by PACAP to
activate the CCK promoter in this cell type appears to be the classic
pattern of CREB/ATF activation by PKA, whereby phosphorylation at
Ser133 stimulates association of the CREB binding protein
required for trans-activation of the target gene.
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ACKNOWLEDGEMENTS |
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A-CREB dominant-negative plasmid was a kind gift from Dr. D. D. Ginty (The Johns Hopkins University School of Medicine, Baltimore, MD). We are grateful to Dr. Tim Wang for helpful advice.
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FOOTNOTES |
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This work was funded by the Wellcome Trust and the Medical Research Council.
Address for reprint requests and other correspondence: R. Dimaline, Physiological Laboratory, Univ. of Liverpool, Crown St., Liverpool L69 3BX, UK (E-mail: r.dimaline{at}liverpool.ac.uk).
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. §1734 solely to indicate this fact.
Received 4 January 2000; accepted in final form 2 April 2000.
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