Identification of Overlapping AP-2/NF-
B-responsive
Elements on the Rat Cholecystokinin Gene Promoter*
Pavel L.
Katsel
§ and
Robert J.
Greenstein¶
From the
Department of Anesthesiology, Mount Sinai
Medical Center, New York, New York 10029 and ¶ Veterans
Affairs Medical Center, Bronx, New York 10468
Received for publication, August 18, 2000
 |
ABSTRACT |
In this study we evaluate both proximal and more
distal transcriptional regulation of the 5' flanking region of the rat
cholecystokinin gene in transfected GH3 (rat pituitary tumor) cells.
Transcriptional activity was measured on the intact (
400 to +73) 5'
flanking region of cholecystokinin (CCK), as well as with DNA
constructs, which were deleted in both the conventional 5' to 3', as
well as an unconventional 3' to 5' direction. Our in vivo
studies indicate complex phorbol ester and forskolin interactions in
the 10-base pair region between
130 and
140. We conclude, there are
at least two transcriptional factors involved in regulation of the rat CCK transcription in this region. In vitro studies
utilizing heterologous nuclear (HeLa) extract, as well as purified
transcription factors AP-2 and NF-
B, identify overlapped AP-2- and
NF-
B-responsive elements within the 17-base pair sequence between
149 and
134 of the distal 5' flanking region. In this region
complex transcriptional regulation occurs, which indicates inhibition
of AP-2 CCK promoter complexing by NF-
B. Six-point mutations
introduced into this sequence prevent AP-2 and NF-
B binding to CCK
promoter, as well as its transcriptional activation by phorbol ester
and forskolin in GH3 cells.
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INTRODUCTION |
Cholecystokinin (CCK),1
a prototypical brain-gut peptide (1-5), has hemacrine (3, 6) and
autocrine (7) action in the gut. In the brain CCK is a neurotransmitter
(8, 9). There is a commonly accepted gut, as well as brain, CCK
transcription initiation site in the rat (10), mouse (11), and man
(12). Alternatively, more distal 5' CCK transcription start sites have been identified in the brain of the rat (13). Transcriptional control
of CCK is regulated by multiple factors. These include phorbol ester
(14-16), cAMP (14, 17-20), and gonadal steroids (21), as well as
glucocorticoids (22). Several cis-elements, important
in transcriptional control, have been identified within the first 119 bp of the 5' flanking region of the rat CCK gene (10). This
119-bp fragment contains a sequence homologous with a
12-O-tetradecanoylphorbol 13-acetate (TPA)-responsive
element of the c-fos gene, as well as a cAMP-responsive
element identified in the proenkephalin gene (23). This sequence binds
several transcription factors including AP-1, jun, and
fos homodimers, as well as cAMP-responsive element-binding
protein (16, 19). Two other recognition sites on the human CCK
gene are Sp1 (at
39 to
34) and E-box or upstream stimulatory factor
(at
97 to
92) (16). Additionally, a negative interaction was
detected between TPA-responsive element and E-box or upstream
stimulatory factor-responsive element (24).
TPA and forskolin activate specific nuclear proteins, which regulate
gene transcription. These proteins include AP-2 (25) and NF-
B (26,
27). Transcription factor AP-2 is activated either directly or
indirectly by two signal transduction pathways (25). One route involves
cAMP-dependent protein kinase A. The other involves phorbol
ester and diacylglycerol-activated protein kinase C (28). AP-2
specifically recognizes and binds to a conserved DNA sequence motif,
found in promoters of a number of genes. These include the human growth
hormone, the oncogene c-myc, H-2Kb gene promoters
(25), as well as in the enhancer regions of the SV40 and hepatitis B
viruses (29, 30).
NF-
B activation occurs as a cellular response to multiple
extracellular signals. These include cytokines, phorbol esters, bacterial lipopolysaccharides, viral infection, and calcium ionophores, as well as physical stimuli (UV radiation and free radicals) (for review see Refs. 26, 31, and 32). NF-
B has multiple embodiments. It
belongs to the Rel family of transcriptional regulatory proteins (26,
31). The p50 subunit is specifically bound to an intron enhancer site
in a number of eukaryotic genes (33-36). NF-
B is involved in
cross-talk with other transcription regulators. The consequence is
either a synergistic activation with an AP-1 complex (36, 37) or mutual
inhibition with the glucocorticoid receptor (38-40).
The purpose of this study is to further define transcriptional
regulation mechanisms of the distal 5' flanking region of the rat CCK
promoter. We hypothesized that AP-2 and NF-
B would have regulatory
effects on CCK transcription. We have evaluated the effect of the
transcription inducers forskolin and TPA in transient transfection
studies in GH3 rat pituitary tumor cells (41). Additionally, we map DNA
binding in vitro using DNase I protection studies, as well
as mobility shift assays. For these in vitro studies
purified nuclear proteins, as well as heterologous nuclear protein
extracts, were used.
Our results demonstrate that TPA and forskolin regulate transcription
of the rat CCK gene in the distal 5' flanking region. In this region we
identified AP-2/NF-
B overlapping recognition sites. We additionally
demonstrated a direct interaction between NF-
B and AP-2, which
results in inhibition of AP-2 DNA binding.
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MATERIALS AND METHODS |
Cloning CCK 5' Flanking Region and Plasmid Constructs--
The
cloning of the rat CCK 5' flanking regulatory region (pCCKUER-luc) is
as described (13). The 5' to 3' direction nested deletions (see Fig. 1)
were generated using PCR (upstream oligos were as follows:
244,
5'AGGTGGGGAGGAAGCTGTTTAG 3';
210, 5' ACTCATACAAAGTACCCCGCCTG 3';
170, 5' TCACTGGGCCGTTTCCCTTC 3'; and
84, 5' TGCGTCAGCACTGGGTAAACAG 3', and the downstream oligo was 5' GGATCTGCCAGCCACTTACC 3'). Linearized pCCKUER (2 ng) was mixed with 1× PCR buffer, 1 unit of
AmpliTaq DNA polymerase (Applied Biosystems, Foster City,
CA). There were twenty five amplification cycles (denaturation at
94 °C for 30 s, primer annealing at 58 °C for 30 s, and
extension at 72 °C for 2 min). PCR-generated fragments were cloned
into the polylinker of pBL-luc, which has a Photinus pyralis
luciferase reporter gene as described (13). Both strands of all PCR
fragments used in this study were sequenced to confirm CCK authenticity and orientation (ABI Prism ABI50; Biotechnology Center, Utah
State University, Logan, UT). Sequence analysis was performed using MacVector (Version 5.0; Kodak Scientific Imaging Systems). A
series of 3' to 5' direction nested deletions were generated using
exonuclease III digestion (Erase A Base kit; Promega, Madison, WI)
(13). Six constructs were subsequently used in the transient
transfection experiments (see Fig. 2).
GH3 Cell Culture and Cationic Lipid-mediated
Transfection--
GH3 rat pituitary tumor cells (ATCC, Manassas,
VA) were cultured and transfected using Lipofectin® (Life
Technologies, Inc.) as described (13). Two transcriptional
enhancers were evaluated, forskolin and TPA (both from Sigma). Samples
were reconstituted in dimethyl sulfoxide and further diluted in sterile
water (molecular biology grade; Whittaker, Walkersville, MD) to a final
concentration of 10 µM. Fresh solutions of these
transcriptional enhancers were prepared for each transfection. Aqueous
solutions (5 µl) of 10 µM solutions of phorbol ester
and/or forskolin were added to the medium following 6 h of
transfection. In preliminary studies we determined that 100 nM forskolin, as well as TPA, was the optimal concentration
(data not presented). 12 h later cells were harvested, lysed, and
assayed for luciferase activity as described (13). To compare data from
different experiments, values were normalized to intact CCKUER,
arbitrarily assigned the value of 100. Data is presented as mean ± S.E.
DNase I Protection Assay--
DNase protection assays were
performed as described (13). The only variation was the final binding
buffer for the NF-
B experiments (10 mM HEPES (pH 7.9),
0.2 mM EDTA, 50 mM KCl, 2.5 mM
dithiothreitol, 10% glycerol, and 0.05% Nonidet P-40). The transcription factors studied were AP-2, as well as NF-
B (p50 subunit) (all from Promega, Madison, WI). The digestion products were
analyzed on denaturing polyacrylamide gels. The markers are sequenced
single-stranded M13mp18 bacteriophage (T7 Sequenase, Version 2.0, DNA
sequence kit; Amersham Pharmacia Biotech).
PCR-Site-directed Mutagenesis--
PCR-site-directed mutagenesis
was performed as described (42). This particular method of mutagenesis
requires two sequential PCR reactions. The upstream primer is mutagenic
for the AP-2 recognition site (5'
CCTTCTATCCTCGGATCCACTTCGATCGG 3'). The
downstream primer recognizes a sequence from the luciferase reporter
(5' ATGTTTTTGGCGTCTTCCA 3'). First step PCR with Deep Vent
(exo
) DNA polymerase (New England Biolabs, Inc., Beverly, MA)
has thirty amplification cycles. Denaturation was at 94 °C for 1 min, annealing was at 65 °C for 1 min, and extension was at 72 °C
for 1 min. PCR products were mixed with SYBRTM Green I
(Molecular Probes, Inc., Eugene, OR) and loaded into 1% agarose gel.
The resulting band (327 bp) was purified using a gel extraction kit
(Qiagen, Chatsworth, CA). Isolated fragment was then used as the
mutagenic primer in the second PCR reaction, where it was mixed with
the 20-mer forward primer (20 pmol, 5' TCACTGCATTCTAGTTGTGG 3'). The
conditions in the second PCR reaction were identical to those used in
the first. The resulting PCR product was religated into pBL-luc.
Mutated plasmids contained an additional BamHI restriction
site, which had been introduced by the mutagenesis. Successful
mutagenesis and the correct orientation were confirmed by restriction
digest mapping, as well as by DNA sequencing (Biotechnology Center,
Utah State University, Logan, UT).
Mobility Shift Assay--
The fragment to be radiolabeled was
PCR-generated using linearized pCCKUER as a template with primers (5'
TCACTGGGCCGTTTCCCTTC 3' and 5' AGTCATCTGTTTACCCAGTGCTGAC 3'). Obtained
fragment (113 bp) was than dephosphorylated using calf intestine
phosphatase (Promega, Madison, WI). The termini of this fragment were
labeled with [
-32P]ATP using T4 polynucleotide
kinase (Promega, Madison, WI). Labeled, double-stranded DNA (50,000 cpm, 0.01 pmol) was incubated for 30 min at room temperature.
Titrations were performed with increasing amounts of HeLa cell nuclear
extract (see Fig. 5, panel A), AP-2 protein extract (see
Fig. 5, panel B), or NF-
B (p50) protein (see Fig. 6).
Following incubation, samples were mixed with loading buffer and
analyzed on a 4% (80:1 acrylamide:bisacrylamide) non-denaturing polyacrylamide gel in 0.5 × Tris borate, EDTA. The
resulting gel was dried prior to autoradiography. Following exposure,
the bands of interest were excised and quantified in a scintillation
counter (Beckman LS 7000).
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RESULTS |
The effect of TPA and forskolin were evaluated both independently
and in combination. We used the full-length CCKUER (473 bp), as well as
four 5' to 3' direction deletions of the 5' flanking region of the rat
CCK gene (Fig. 1, left panel).
These constructs were then stimulated with TPA and forskolin (Fig. 1,
right panel). Both TPA and forskolin enhance CCKUER-luc
expression. Deletion to
170 results in loss of forskolin-induced
expression. By contrast, TPA-induced expression is maintained until
deletion proceeds to
84. Thus, these data identify a TPA-activated
transcription factor-responsive element between
84 and
170 bp.

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Fig. 1.
TPA and forskolin transcriptional induction
in the 5' flanking region of the rat CCK gene, using conventional 5' to
3' deletion. Left panel, a map of the 5' to 3' deletion
constructs of the fragments generated by PCR and subcloned in the
luciferase reporter construct. The number to the
left of each fragment indicates the number of base pairs
deleted 5' to the cap site. CAP, conventionally accepted
transcription initiation site (23). ATIS, alternative
transcription initiation site (located between 210 and 170 bp)
(13). Right panel, pCCKUER-luc and each of the four nested
deletions were transiently transfected in GH3 cells. Values were
normalized to CCKUER, which has been arbitrarily assigned the value of
100. Error bars represent S.E.; n = 4 from
each experiment.
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Alternatively, we evaluated the effect of TPA and forskolin treatment
of transfected GH3 cells for a series of 3' to 5' nested deletions
(Fig. 2, left panel). In our
preliminary studies we have determined that by removing the dominant
signal generated at the conventional transcription initiation site as a
result of 3' to 5' direction of deletion, the rate of basal
transcription would be significantly declined resulting in decrease of
luciferase signal (13). This enabled us to isolate the effect of more
distal 5' regulatory elements associated with upstream alternative
start sites of the rat CCK gene. Both TPA and forskolin induced
expression until CCKUER was deleted to
130 (Fig. 2, right
panel, lanes
50 and
130). When the next
10 bp were deleted TPA induction was lost (Fig. 2, right
panel, lane
140). In contrast, the same deletion construct (
140) still exhibits forskolin-induced expression. Thus,
these data identify a TPA-activated transcription factor-responsive element between
130 and
140 bp of the 5' flanking region of the rat
CCK gene.

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Fig. 2.
TPA and forskolin transcriptional induction
in the 5' flanking region of the rat CCK gene, using
"unconventional" 3' to 5' deletion. Left panel, a
map of the deletion constructs of the fragments used in this study
(13). The conventionally accepted cap site has been deleted by 3' to 5'
exonuclease III digestion. The number on the
right of each fragment indicates the number of base pairs
deleted 5' to the cap site. CAP, conventionally accepted
transcription initiation site (4). ATIS, alternative
transcription initiation site (located between 210 and 170 bp)
(13). Right panel, pCCKUER-luc and each of the four nested
deletions were transiently transfected in GH3 cells. Values were
normalized to CCKUER, which has been arbitrarily assigned the value of
100. Error bars represent S.E.; n = 7 from
each experiment.
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We next evaluated, in combination, the effect of TPA and forskolin on
the series of 3' to 5' nested deletions (Fig.
3). Individually both TPA and forskolin
induce CCKUER. By contrast, combination of TPA and forskolin reduces
CCKUER expression to a baseline level. This combined TPA/forskolin
inhibition is maintained for the first two deletion constructs studied
(CCK
50 and CCK
130). However, when deletion proceeds an additional
10 bp, to CCK
140, the inhibition was lost, and signal increased up
to 3-fold. Forskolin, but not TPA, remains active at CCK
140 when
deletion proceeds to
140 (Fig. 2). These data corroborate our initial
observation that there is a major site of transcriptional regulation in
the
130 to
140 region of the 5' flanking region of the rat CCK
gene.

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Fig. 3.
TPA and forskolin inhibit each other within
the sequence between 140 and 130 bp of the 5' flanking region of
the rat CCK gene. Four fragments of the 5' flanking region of the
rat CCK gene were used in this experiment. Used were the intact
full-length upstream enhancer region, as well as three nested deletions
(see Fig. 2, left panel). Distances (5' to the cap site) are
indicated on abscissa.
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Sequence analysis suggests AP-2, as well as NF-
B, binding sites in
this region of
130 to
140 (see Fig. 9). Accordingly, DNase
protection assays with purified AP-2 and NF-
B transcription factors
were performed (Fig. 4). Purified AP-2
protects the coding strand in two regions, between
37 and
48 and
between
139 and
149. Purified NF-
B (p50) protects the coding
strand between
134 and
145. Thus, both NF-
B and AP-2 bind in the
sequence between
130 and
140 of the 5' flanking region of the rat
CCK gene, which is regulated by TPA and forskolin stimulation (Figs. 2
and 3).

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Fig. 4.
Binding AP2 and NF- B
to the 5' flanking region of the rat CCK gene. Single end-labeled
DNA fragments from the coding strand of the 400-bp 5' flanking region
of the rat CCK gene were combined with purified AP2 or NF- B (p50)
proteins either alone as described (0, 1, or 2 footprint units as
indicated) or in combination (MIX = 1:1 footprint units
as indicated). Limited DNase I digestion was followed by polyacrylamide
gel electrophoresis and autoradiography.
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Binding to this
130 to
140 region was then evaluated in a mobility
shift assay. HeLa cell nuclear extract, which contains AP-2, was used.
As the HeLa cell nuclear extract amount is increased there is linear
increase in signal (Fig. 5, panel
A, lanes 5, 10, and 20). Next a
competitive inhibition study was performed using 1- and 10-fold molar
excess of "cold" wild type CCK fragment between
57 and
170
(Fig. 5, panel A, lanes 5 and 6 from the left). As the amount of cold CCK is increased, the
radiolabeled signal band (cpm) decreases by 70% (data not presented).
This indicates competition by the wild type CCK for the fraction of the
HeLa cell nuclear extract, which binds to the radiolabeled fragment of
CCK. The same experiment was then repeated using the unlabeled 113-bp
mutated (
) CCK fragment between
57 and
170 (Fig. 5, panel
A, lanes 7 and 8 from the left). There were
six bp mutations introduced in the AP-2 consensus recognition sequence located between
130 and
140 (see "Materials and Methods").
These mutations result in only 7% decrease in binding (cpm data not presented). This competitive inhibition by the wild type cold CCK
demonstrates the specificity of the interaction between HeLa cell
nuclear extract and AP-2 cis-element on the CCK gene. Thus, these data
identify the binding of the HeLa cell nuclear extract to the 5'
flanking region of the rat CCK gene, which contains both the AP-2
cis-element located between
138 and
149 and the NF-
B cis-element
between
134 and
145. Purified AP-2 and NF-
B (p50) bind, with a
linear titration, to the same CCK region (Fig. 5, panel B
and Fig. 6, lanes 2 and
3 from the left).

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Fig. 5.
HeLa cell nuclear extract and AP-2 protein
binding to the 5' flanking region of the rat CCK gene. Panel
A, the 113-bp fragment between 170 and 57 was labeled with
[ -32P]ATP. The control is the labeled fragment
incubated in buffer without any nuclear protein extract. In the
next three lanes, the labeled fragment was incubated with
increasing amounts (5, 10, and 20 µg) of HeLa cell nuclear extract.
In the next two lanes, the labeled 113-bp fragment was
incubated with 10 µg of HeLa nuclear extract in the presence of
either 1- or 10-fold molar excess of a 113-bp cold fragment of the rat
CCK gene ( 170 to 57). In the last two lanes, the labeled
113-bp fragment was incubated with 10 µg of HeLa nuclear extract in
the presence of either 1- or 10-fold molar excess of a 113-bp cold
mutated fragment CCK ( 170 to 57). This competitive inhibition by
wild type cold CCK demonstrates the specificity of the interaction
between HeLa cell nuclear extract with wild type AP-2 cis-element on
the CCK gene. Panel B, a DNA-protein binding study performed
with increasing amounts (1.4, 2.8, and 4.2 µg) of purified AP-2
protein and the [ -32P]ATP-labeled fragment of CCK
( 170 to 57).
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Fig. 6.
Mutations in the
AP-2/NF- B consensus recognition sequence
reduces binding by NF- B (p50). The 113-bp
fragments CCK, 170 to 57 (three lanes on the
left), and the mutated CCK, 170 to 57 (three
lanes on the right), were labeled with
[ -32P]ATP and incubated with increasing amounts (0.13 and 0.26 µg) of NF- B (p50) protein.
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In contrast, there is negligible NF-
B (p50) binding of the mutated
CCK
170 to
57 (Fig. 6, lanes 4 and 5 from the
left). These data indicate that six-point mutation in the sequence
between
140 and
149 of the 5' flanking region of the rat CCK gene
prevents binding of NF-
B protein.
A mobility shift assay was performed using both AP-2 and NF-
B
individually, as well as in combination (Fig.
7). There is a linear increase in binding
for both transcription factors when studied individually (Fig. 7,
lanes 2, 3, 5, and 6 from the
left). However, when both proteins are studied in combination, the
AP-2·DNA complex, previously identified, is completely lost
(Fig. 7, center lane, see arrow on the
right, indicating AP-2·DNA complex migration). These data
show an AP-2/NF-
B interaction, which interferes with AP-2·CCK
promoter complexing.

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Fig. 7.
Combination of NF- B
and AP-2 prevents AP-2/wild type CCK complexing. The fragment
between 170 and 57 labeled with [ -32P]ATP was
incubated with increasing amounts (0.13 and 0.26 µg) of purified
NF- B and 1.4 and 2.8 µg of purified AP-2 both individually and
subsequently in combination (center lane). When both
proteins are studied in combination, the AP-2·DNA complex, previously
identified, is completely lost (center lane, see
arrow on the right, indicating AP-2·DNA complex
migration).
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Cell culture was then used to investigate the functional significance
of the region between
134 and
149. When this region is mutated, the
CCK promoter loses its capacity to be induced by either TPA or
forskolin (Fig. 8). These data confirm
the functional significance of this region in the binding of nuclear
proteins activated by both TPA and forskolin.

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Fig. 8.
Mutation in
AP-2/NF- B consensus recognition sequence
eliminates transcriptional activation of CCK by both TPA and
forskolin. Open column, intact pCCKUER. Hatched
column, mutated p CCKUER. The mutations are identified by *. The
mutations straddle the AP-2, as well as NF- B, binding sites (see
"Materials and Methods"). The evaluated transcription enhancers are
indicated on the abscissa. Unst, unstimulated,
TPA, phorbol ester. Results are normalized to pCCKUER, which
is arbitrarily assigned the value of 100 (indicated on the
ordinate).
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Thus, the inhibition of combined TPA and forskolin is mediated within
the region between
130 and
140 bp 5' to the cap site. This
region contains, in part, AP-2 and NF-
B (p50) consensus recognition
sequences (see Fig. 4).
 |
DISCUSSION |
In this study we identify hitherto undescribed transcription
regulatory sites in the 5' flanking region of the rat CCK gene. The
region of most interest in this study was a 10-bp sequence located
between
130 and
140. In the course of this study we demonstrate
that this region is responsive to both TPA and forskolin. In addition,
we determine the complex relationship between these two transcription
enhancers in the same region of CCK promoter.
Four from six previously described 3'to 5' direction deletion
constructs (13) were utilized in this study (Fig. 2). We additionally generated four constructs, which were deleted in the more conventional 5' to 3' direction (Fig. 1). Mutational analysis was
performed using six bp mutations, which were inserted
between
130 and
140. These constructs permitted a more detailed
evaluation of the TPA- and forskolin-responsive regions, which had been
identified during the course of the study. Deleted, as well as mutated,
constructs were transiently transfected into GH3 cells. We had
previously used GH3 (rat pituitary tumor) cells in our studies with CCK
(13). GH3 cells produce neither endogenous CCK mRNA nor any CCK
peptides. However, GH3 cells self-evidently have the transcriptional
mechanisms that induce transcription of the 5' flanking region of the
rat CCK gene in the transient transfection studies that we performed. The data from cell culture show complex transcriptional regulation of
the 5' flanking region of the rat CCK gene as a result of TPA and
forskolin treatment (Figs. 1-3 and Fig. 8). However, pathways of TPA
and forskolin action remain to be fully defined in GH3 cells.
In this study, in addition to the role of TPA and forskolin, we show
that two transcription factors have overlapping recognition sites in
the 17-bp region located between
134 and
149. These two factors are
AP-2 and NF-
B. We speculate that in GH3 cells forskolin induction of
AP-2 may well occur. Preliminary data determine AP-2 expression of in
GH3 cells (data not presented).
The unconventional 3' to 5' direction of deletion, which we use, shows
that when deletion proceeds to
140, forskolin, but not TPA, is still
stimulatory (Fig. 2). In contrast, the conventional 5' to 3' deletion
constructs support that TPA is still stimulatory in the fragment that
contains this
134 to
149 region, and forskolin induction is lost
between
170 and
210 (Fig. 1). The deletion construct (
170)
contains the AP-2/NF-
B binding sites identified in this study. A
possible explanation for this paradox may lie in the presence of other
forskolin-responsive elements associated with alternative initiation
sites (13) in the distal region of CCK promoter. These elements are
deleted as a result of 5'to 3' deletion but are still present in the 3'
to 5' deletion constructs. Our preliminary data indicate, for example,
previously unreported AP-1 binding sequences, which we identify between
354 and
387 (data not presented). As a consequence of our data, we
hypothesize that forskolin may activate binding to the identified AP-2,
as well as some other distal responsive elements, possibly, including the distal AP-1 site in GH3 cells. This hypothesis will be determined in subsequent study.
There is inter-species sequence conservation in the proximal 5'
flanking region of CCK genes. The first 100 bp 5' to the conventionally accepted transcription initiation site in rat (10, 23), mouse (11), and
man (16) have 80% homology. Sp1 consensus recognition sequence has
been described in the human CCK promoter between
37 and
48 (16). In
the rat gene, we were unable to demonstrate SP1 binding to this
sequence (data not presented). However, we have demonstrated that AP-2
binds in a homologous sequence (
37 to
48) in the 5' flanking region
of rat CCK gene (Fig. 4). In the more distal 5' flanking regulatory
region of rat, mouse, and man CCK DNA sequence homology falls to
~40%. In this study we identify an AP-2/NF-
B binding site in the
rat (
134 to
149). By contrast, we find no analogous AP-2/NF-
B
binding region in a sequence analysis of the 5' flanking region of man
or mouse. A similar 17-bp sequence in mouse CCK gene (11) contains 3-bp mismatches and a 4-bp mismatches in the human CCK gene (16). These data imply that the proximal 5' flanking region is of more significance in basal CCK transcription. The distal upstream 5' flanking regulatory region may be of greater relevance in
species-specific regulation of CCK transcription.
Both the human, as well as the rat, CCK promoter have a highly
conserved cAMP-responsive element/TPA-responsive element binding region (
79 and
87) (16, 23). A three-point mutation within this
site eliminates transcriptional activation by both TPA and forskolin
(16). We confirm a homologous region in the rat gene (
79 and
87)
(Fig. 9) and an additional (
42 to
46), which is protected from
DNase I digestion by AP-1 (data
not presented).

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Fig. 9.
The sequence of 5' flanking region of the rat
CCK gene. The consensus transcription initiation site is indicated
by an arrow and is assigned the number 0. Brain
ATIS* is the alternative initiation site (located at 41)
identified in the cerebral cortex in the rat (13). cAMP-responsive
element/TPA-responsive element (AP-1, between 87 and 79) and
upstream stimulatory factor cis-elements were previously identified in
both human and rat CCK DNA sequences (16, 23). NF- B and AP-2
cis-elements are identified in this study. Sp1 cis-elements
were determined by DNase I protection
assay.2 These are
boxed and marked.
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Our data indicate a complex inhibition of transcription by TPA and
forskolin (Figs. 1-3 and Fig. 8). We additionally show that in this
region of inhibition are overlapped responsive elements of AP-2 and
NF-
B, characterized by complex negative interaction of each other
(Fig. 7). NF-
B and a glucocorticoid receptor mutually inhibit
transcriptional activation (38-40). However, to our knowledge, these
data are the first that demonstrate transcriptional inhibition involving NF-
B and AP-2. Studies of the mechanism involved in the
interactions by which inhibition of these nuclear factors is mediated
should be addressed in the future.
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FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
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Published, JBC Papers in Press, October 5, 2000, DOI 10.1074/jbc.M007553200
2
Manuscript in preparation.
 |
ABBREVIATIONS |
The abbreviations used are:
CCK, cholecystokinin;
CCKUER, 5' flanking region of the rat CCK gene;
pCCKUER-luc, plasmid of CCKUER luciferase reporter fusion construct;
bp, base pair(s);
FRSK, forskolin;
TPA, 12-O-tetradecanoylphorbol 13-acetate;
PCR, polymerase chain
reaction.
 |
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