Mitogen-Activated Protein Kinase and Protein Kinase A Signaling Pathways Stimulate Cholecystokinin Transcription via Activation of Cyclic Adenosine 3',5'-Monophosphate Response Element-Binding Protein
Thomas v. O. Hansen,
Jens F. Rehfeld and
Finn C. Nielsen
Department of Clinical Biochemistry Rigshospitalet DK-2100
Copenhagen Ø, Denmark
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ABSTRACT
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Cholecystokinin (CCK) is a potent neuropeptide
expressed in the small intestine and in the central nervous system. We
have examined the effect of basic fibroblast factor (bFGF) and
forskolin on CCK gene transcription and depicted the signaling pathways
that lead to promoter activation. bFGF and forskolin stimulated
promoter activity via a cAMP response element
(CRE)/12-O-tetradecanoylphorbol-13-acetate response element
(TRE) located 80 bp upstream from the transcription initiation site. In
nuclear extracts from unstimulated as well as stimulated cells, only
CRE-binding protein (CREB) and activating transcription factor-1
(ATF-1) bound to the CRE/TRE, and activation was associated with
phosphorylation of CREB serine-133 and ATF-1 serine-63. In murine F9
cells, CREB stimulated promoter activity 10-fold in the presence of
protein kinase A (PKA), and in SK-N-MC cells activation was inhibited
6070% by a dominant negative CREB mutant. In contrast, ATF-1 had no
effect in F9 cells and exhibited a dominant negative effect in SK-N-MC
cells. bFGF stimulation led to phosphorylation of the p38
mitogen-activated protein kinase (MAPK), and the extracellular
signal-regulated kinase (ERK) MAPK and promoter activation,
phosphorylation of CREB, and GAL4-CREB-dependent transcription were
selectively prevented by a dominant negative Ras-mutant, the p38
MAPK-specific inhibitor SB203580, and the MAP/ERK kinase 1 (MEK1)
inhibitor PD098059. Forskolin stimulation proceeded via the PKA
pathway, and to a minor extent via the p38 and ERK MAPK pathways. We
conclude that bFGF and forskolin stimulate the CCK gene promoter via
the CRE/TRE(-80) in the proximal promoter region. Signaling proceeds
through the p38 MAPK, the ERK MAPK, and the PKA-signaling pathways,
which leads to cumulative phosphorylation and activation of CREB. We
propose that bFGF in combination with neurotransmitters/neuropeptides
coupling to the PKA-signaling pathway play an important role in the
control of CCK gene expression.
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INTRODUCTION
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Cholecystokinin (CCK) is an important neuroendocrine peptide
expressed in the endocrine I cells of the small intestine and in
central and peripheral neurons (for a review see Ref. 1). CCK is the
most abundant neuropeptides in the mammalian brain (2, 3), and in
humans particularly high levels are expressed in the neocortical
and hippocampal neurons, although significant quantities occur in
almost any region of the brain (for a review see Ref. 4). Whereas the
role of intestinal CCK in the release of pancreatic enzymes and
contraction of the gall bladder is well established, the role of
cerebral CCK is not entirely understood. Consistent with its widespread
expression, however, CCK has been proposed to regulate a variety of
central nervous system functions, including feeding behavior,
anxiety, analgesia, and memory functions. Despite the physiological
significance of CCK, the factors involved in the control of CCK
transcription are essentially unknown.
The proximal upstream regulatory sequences of the human CCK gene
exhibit at least three conserved and functional elements (5, 6).
A Sp1 site is located close to the putative TATA box, and further
upstream the promoter exhibits a combined cAMP response element
(CRE)/12-O-tetradecanoylphorbol-13-acetate response element
(TRE) and an E box (Fig. 1
). The CRE/TRE
motif exhibits a core sequence of 5'-CTGCGTCA-3', which is identical to
the TRE(-296) of the c-fos gene (7) and the CRE-2 element
of the proenkephalin gene (8, 9). CRE/TRE elements are recognized by
numerous transcription factors, including members of the CRE-binding
protein (CREB)/activating transcription factor (ATF) and the AP-1
family of transcription factors (for review see Refs. 10, 11). The
factors readily form heterodimers that posses distinct activating
potentials and are stimulated by different signaling pathways.
Therefore, the CRE/TRE element is likely to play a key role in the
control of promoter activity and consequently CCK gene expression.

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Figure 1. Structure of the Proximal Upstream Regulatory
Domain of the Human CCK Gene
The TATA box, the Sp1(-39), the CRE/TRE(-80), and the E box(-97)
elements are indicated.
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In this study we have examined the effects of basic fibroblast growth
factor (bFGF) and forskolin on CCK gene expression. bFGF has previously
been demonstrated to stimulate the proenkephalin gene promoter in
combination with cAMP (12), indicating that bFGF may play a role in the
control of neuropeptide synthesis. We show that bFGF and forskolin
stimulate the CCK gene promoter via phosphorylation of CREB bound to a
conserved CRE/TRE in the proximal promoter region. Activation proceeds
through the p38 mitogen-activated protein kinase (MAPK), the
extracellular signal-regulated kinase (ERK) MAPK, and the protein
kinase A (PKA)-signaling pathways, leading to a cumulative
phosphorylation and activation of CREB.
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RESULTS
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Activation of the CCK Gene Promoter by bFGF and Forskolin
To examine the effect of bFGF and forskolin on CCK gene promoter
activity, human SK-N-MC cells were transfected with the CCK gene
promoter constructs CCK-200, CCK-100, or CCK-67 and treated with 25
ng/ml bFGF, 10 µM forskolin, or both (Fig. 2A
). bFGF stimulated chloramphenicol
acetyl transferase (CAT) activity of the constructs CCK-200 and CCK-100
about 2-fold, whereas, forskolin increased CAT activity 5-fold. No
effect of bFGF or forskolin was observed on the CCK-67 construct. The
combined treatment with bFGF and forskolin was followed by a 12-fold
increase in CAT activity. The effect of bFGF was dose dependent, and
half-maximal stimulation was obtained at a concentration of 0.8
nM bFGF (Fig. 2B
). Since the response element appeared to
be located in the region between -100 and -67 bp, which contains the
previously identified CRE/TRE element (5, 6), this element was mutated
(CTGCGTCA
CTGCTGAA). Mutation reduced activation 75%,
indicating that the CRE/TRE element is the major element mediating the
effects of bFGF and forskolin.

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Figure 2. Activation of the CCK Gene Promoter by bFGF and
Forskolin
A, SK-N-MC cells were transfected with the CCK -200, -100, -67, or
-100 CRE/TRE constructs and incubated with 25 ng/ml bFGF, 10
µM forskolin, or both as indicated. After 6 h, CAT
and luciferase activity were measured. The results are presented as
fold activation over basal (unstimulated cells) and represents the
mean ± SEM of three experiments. B, Dose-response
curve of the additive bFGF activation. SK-N-MC cells were transfected
with the CCK-100 construct and treated with 10 µM
forskolin and increasing concentrations of bFGF as indicated. CAT and
luciferase activity were measured after 6 h. The results were
normalized as above and are mean ± SEM of three
experiments.
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CREB and ATF-1 Bind to the CRE/TRE in the CCK Gene Promoter
To identify the transcription factors involved in promoter
activation, we examined the binding of members of the CREB/ATF and AP-1
family of transcription factors to the CRE/TRE element by
electrophoretic mobility shift assay and supershift analysis.
Incubation of crude nuclear extracts from unstimulated SK-N-MC cells
with the CCK(-85)-(--66) oligonucleotide, comprising the CRE/TRE,
identified a single retarded DNA-protein complex (Fig. 3A
). The binding pattern remained
unchanged, when nuclear extracts from bFGF-stimulated,
forskolin-stimulated, or bFGF/forskolin stimulated-SK-N-MC cells were
used. The subsequent supershift analysis with antibodies recognizing
CREB, ATF-1, ATF-3, the Jun family (c-Jun, JunB, JunD), and the Fos
family (c-Fos, FosB, Fra-1, Fra-2) showed that only CREB and ATF-1 were
associated with the CRE/TRE in both unstimulated and stimulated SK-N-MC
cells (Fig. 3B
).

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Figure 3. CREB and ATF-1 Bind to the CRE/TRE Element and Are
Phosphorylated upon Stimulation with bFGF and Forskolin
A, SK-N-MC cells were treated with 25 ng/ml bFGF, 10 µM
forskolin, or both for 6 h, after which crude nuclear extracts
were prepared. [ -32P]ATP-labeled CCK(-85)-(-66)
probe was incubated without nuclear extract (lane 1), with nuclear
extract from unstimulated cells (lane 2), with nuclear extract from
bFGF-stimulated cells (lane 3), with nuclear extract from
forskolin-stimulated cells (lane 4), or with nuclear extract from bFGF-
and forskolin-stimulated cells (lane 5). B, Binding of CREB and ATF-1
to the CRE/TRE element. Crude nuclear extracts from unstimulated cells,
cells treated with 25 ng/ml bFGF, 10 µM forskolin, or
both, were incubated with [ -32P]ATP-labeled
CCK(-85)-(-66) probe and antibodies recognizing CREB (lane 2), ATF-1
(lane 3), ATF-3 (lane 4), the Jun family (c-Jun, JunB, JunD) (lane 5),
and the Fos family (c-Fos, FosB, Fra-1, Fra-2) (lane 6), respectively.
Supershifts are indicated with an asterisk. C, Increased
phosphorylation of CREB and ATF-1 upon stimulation by bFGF and
forskolin. SK-N-MC cells were unstimulated (lane 1), stimulated with
either 25 ng/ml bFGF (lane 2), 10 µM forskolin (lane 3),
or both (lane 4). Western blot analysis was performed on cell extracts
using either anti-phospho-CREB/ATF-1, anti-CREB, or anti-ATF-1
antibodies.
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Since the binding of CREB and ATF-1 remained unchanged, we examined
whether activation was associated with phosphorylation of CREB and
ATF-1 using an antibody specific for the serine-133-phosphorylated form
of CREB and the serine-63-phosphorylated form of ATF-1 (Fig. 3C
).
Addition of bFGF or forskolin increased phosphorylation of CREB and
ATF-1, and stimulation with both agents further increased in the level
of phosphorylation. The relative immunoreactivity of ATF-1 was
approximately 5-fold lower than that of CREB (data not shown). In
agreement with the supershift analysis, the Western analysis with
antibodies recognizing both phosphorylated and dephosphorylated forms
of CREB or ATF-1 showed that the total amount of CREB or ATF-1 was
unchanged upon stimulation (Fig. 3C
). The functional significance of
the serine-133 phosphorylation was further examined by coexpression of
GAL4-CREB fusion proteins and the GAL4-TATA-luciferase reporter plasmid
(Fig. 4
). The GAL4 reporter alone was
unaffected by stimulation but coexpression of GAL4-CREB resulted in a
4-fold stimulation by bFGF, a 10-fold stimulation by forskolin, and a
22-fold stimulation by bFGF and forskolin. In contrast, coexpression of
GAL4-CREB(Ala-133), in which serine-133 is mutated to alanine, was
insensitive to stimulation by either bFGF, forskolin, or both. We infer
that transcriptional activation by bFGF and forskolin involves
phosphorylation of CREB serine-133 and ATF-1 serine-63.

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Figure 4. CREB serine-133 Is Necessary for Activation by bFGF
and Forskolin
SK-N-MC cells were transfected with 5 x GAL4-TATA-luciferase
reporter plasmid and GAL4-CREB or GAL4-CREB(Ala-133) expression vectors
as indicated. Six hours before harvesting the cells were stimulated
with either 25 ng/ml bFGF, 10 µM forskolin, or both as
indicated. Luciferase activity was measured after 6 h. The results
are stated as fold activation over basal (GAL4-TATA-luciferase) and
represents the mean ± SEM of three experiments.
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CREB Activates the CCK Gene Promoter
To directly establish the functional significance of CREB and
ATF-1 in promoter activation, CCK-100 reporter plasmid, CREB, and ATF-1
were coexpressed in murine F9 cells, which in the undifferentiated
state have been shown to contain low levels of functional CREB and
therefore are suitable for CREB studies (13). Coexpression of CREB with
or without PKA was followed by a 4- and 10-fold increase in promoter
activity, respectively (Fig. 5A
). The significance of endogenous CREB,
moreover, was examined by expression of a dominant negative CREB
mutant, KCREB, in SK-N-MC cells. The mutant exhibits a point mutation
within the DNA-binding domain that leads to the formation of inactive
heterodimers with endogenous CREB and ATF-1 (14). As shown in Fig. 5C
, stimulation by bFGF and forskolin, as well as forskolin alone, was
inhibited with 6070% by the expression of KCREB. ATF-1 had no
significant effect on promoter activity alone or in combination with
PKA (Fig. 5A
) or on CREB/PKA activation (data not shown) in F9 cells.
In SK-N-MC cells, ATF-1 caused a dose-dependent repression of basal and
PKA-induced transcription (Fig. 5B
). The results indicate that CREB is
the major trans-activating factor involved in bFGF and
forskolin-stimulated CCK transcription.

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Figure 5. Stimulation of the CCK Gene Promoter by CREB
A, F9 cells were transfected with CCK-100 reporter plasmid and CREB or
ATF-1 expression vectors in the absence or presence of PKA expression
vector as indicated. After 48 h, the cells were harvested and
analyzed for CAT and luciferase activity. The results are stated as
fold activation over basal (CCK-100) and represents the mean ±
SEM of three experiments. B, SK-N-MC cells were transfected
with CCK-100 reporter plasmid and the indicated amount of ATF-1
expression vector in the absence or presence of PKA expression vector
as indicated. The results are stated as fold activation over basal
(CCK-100) and represent the mean ± SEM of two
experiments. C, Effect of KCREB on CCK promoter stimulation. SK-N-MC
cells were cotransfected with CCK-100 reporter plasmid and KCREB or
CREB expression vectors as indicated. Six hours before harvesting, the
cells were stimulated with 10 µM forskolin or 10
µM forskolin and 25 ng/ml bFGF, after which CAT and
luciferase activity were measured. The results represent the mean
± SEM of three experiments.
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bFGF Induction of the CCK Gene Promoter Requires the Ras-Signaling
Pathway
To depict the signaling pathways that mediates CCK gene promoter
activation, CCK-100 reporter plasmid and a dominant negative Ras
mutant, Ha-Ras (Asn-17), which competes with endogenous cellular
p21Ras for upstream activators (15, 16), were cotransfected
into SK-N-MC cells (Fig. 6A
).
Coexpression of Ha-Ras (Asn-17) inhibited bFGF stimulation 90%, and
the combined bFGF and forskolin stimulation by 40%, but had no effect
on forskolin stimulation, indicating that Ha-Ras (Asn-17) interferes
with bFGF signaling. Furthermore, we examined the effect of oncogenic
Ras on CCK gene promoter activity. Cotransfection of oncogenic Ras and
the catalytic subunit of PKA caused a synergistic activation of CCK
gene expression (Fig. 6B
), similar to the activation observed with bFGF
and forskolin. We infer that Ras is required for bFGF activation of the
CCK gene promoter.

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Figure 6. bFGF Stimulates the CCK Gene Promoter via a
Ras-Dependent Signaling Pathway
A, SK-N-MC cells were cotransfected with CCK-100 reporter plasmid and
the dominant negative Ras(Asn-17) expression vector as indicated. Six
hours before harvesting, the cells were stimulated with 25 ng/ml bFGF,
10 µM forskolin, or both and subsequently analyzed for
CAT and luciferase activity. The results are stated as fold activation
over basal (CCK-100) and represent the mean ± SEM of
three experiments. B, Synergistic activation of the CCK gene promoter
by oncogenic Ras and PKA. SK-N-MC cells were cotransfected with CCK-100
reporter plasmid, oncogenic Ras(Val-12), and PKA expression vectors as
indicated. CAT and luciferase activity were measured after 6 h.
The results are stated as fold activation over basal (CCK-100) and
represent the mean ± SEM of three experiments.
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bFGF Stimulates CCK Transcription via the p38- and the ERK-MAPK
Pathways
To identify putative downstream targets of bFGF-Ras signaling,
SK-N-MC cells transfected with CCK-100 reporter construct were
stimulated with bFGF in the presence of kinase inhibitors. Neither
inhibitors of protein kinase C (chelerythrine chloride), protein kinase
A (H-89), phosphoinositide 3-kinase (wortmannin), p70 S6 kinase
(rapamycin), or calmodulin kinase (KN-62) inhibited stimulation (data
not shown). Therefore, the role of the MAPK pathways, including the p38
and the ERK MAPKs, was examined (Fig. 7A
). The p38 MAPK inhibitor SB203580 (17, 18) and the MAP/ERK kinase 1 (MEK1) inhibitor PD098059 (19) selectively
prevented the cumulative effect of bFGF. Since these results indicated
that bFGF would activate both the p38 MAPK- and the ERK MAPK-signaling
pathways, the effect of bFGF on ERK and p38 phosphorylation was
examined. Western blot analysis using antibodies recognizing p38
phosphorylated on threonine-180/tyrosine-182, and ERK1/2 phosphorylated
on threonine-202/tyrosine-204 (Fig. 7B
), showed that both p38 and
ERK1/2 phosphorylation was increased after stimulation with bFGF,
whereas the total amount of p38 or ERK1/2 was unchanged. bFGF-induced
CREB phosphorylation was also examined (Fig. 7D
). Pretreatment of the
cells with either SB203580 or PD098059 reduced phosphorylation, whereas
combined treatment with SB203580 and PD098059 completely blocked
phosphorylation. Finally, the bFGF-stimulated GAL4-CREB transcription
was also inhibited by both SB203580 and PD098059 (Fig. 7E
). Whereas
separate addition of SB203580 and PD098059 caused a partial inhibition,
both inhibitors almost completely blocked bFGF-induced CREB
activity.

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Figure 7. Activation of the CCK Gene Promoter by bFGF and
Forskolin Involves the p38 and the MEK/ERK MAPK Signaling Pathways
A, SK-N-MC cells were transfected with CCK-100 reporter plasmid and
6 h before harvesting, the cells were stimulated with 25 ng/ml
bFGF or 10 µM forskolin or 10 µM forskolin
and 25 ng/ml bFGF in the presence of 10 µM SB203580, 20
µM PD098059, or both as indicated. The cells were
analyzed for CAT and luciferase activity, and results are stated as
fold activation over basal and represent the mean ±
SEM of three experiments. B, bFGF increase phosphorylation
of p38 and ERK1/2. SK-N-MC cells were incubated in culture medium (lane
1) or stimulated with 25 ng/ml bFGF (lane 2) or incubated with either
10 µM SB203580 (p38) or 20 µM PD098059
(ERK1/2) before addition of 25 ng/ml bFGF (lane 3). Cell extracts were
examined by Western analysis with anti-phospho-p38, anti-p38,
anti-phospho-ERK1/2, or anti-ERK1/2 antibodies as indicated. C,
Forskolin increases phosphorylation of p38 and ERK1/2. SK-N-MC cells
were incubated in culture medium (lane 1) or 10 µM
forskolin (lane 2) before cell extracts were examined by Western
analysis with anti-phospho-p38, anti-p38, anti-phospho-ERK1/2, or
anti-ERK1/2 antibodies as indicated. D, SB203580 and PD098059 inhibit
bFGF-induced CREB phosphorylation. Western blot analysis with
anti-phospho-CREB, anti-CREB, or anti-ATF-1 antibodies was performed on
unstimulated SK-N-MC cells (lane 1), cells stimulated with 25 ng/ml
bFGF (lane 2), and cells pretreated with 10 µM of
SB203580 (lane 3), 20 µM of PD098059 (lane 4), or both
(lane 5) before stimulation with 25 ng/ml bFGF. E, GAL4-CREB-dependent
transcription is inhibited by SB203580 and PD098059. SK-N-MC cells were
cotransfected with 5 x GAL4-TATA-luciferase reporter plasmid and
GAL4-CREB expression vector. Seven hours before harvesting the cells
were preincubated with 25 µM SB203580 and/or 50
µM PD098059 as indicated for 60 min. The cells were then
stimulated with 25 ng/ml bFGF and subsequently analyzed for luciferase
activity. The results are stated as fold activation over basal
(CCK-100) and represent the mean ± SEM of three
experiments.
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Since we observed a minor (
20%) decrease in forskolin stimulation
after incubation with SB203580 and PD098059 (Fig. 7A
), we examined
whether forskolin leads to activation of the p38 and ERK. Figure 7C
shows the Western analysis of p38 and ERK in SK-N-MC cells
stimulated with forskolin. Whereas the total level of the proteins
remained unchanged, an increase in the phosphorylation of both p38 and
ERK was observed.
We infer that bFGF stimulates CCK gene expression via the p38- and the
ERK MAPK-signaling pathways, whereas, forskolin activation involves PKA
and p38 and ERK MAPK.
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DISCUSSION
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Synergy between growth factors and neurotransmitters in control of
neuropeptide expression may be important for both differentiation and
survival of endocrine cells and neurons and their neurophysiological
actions. Here we report that bFGF and forskolin activate the CCK gene
promoter via phosphorylation of CREB bound to a conserved CRE/TRE in
the proximal promoter region. Activation proceeds through the p38
MAPK-, the Ras/ERK MAPK-, and the PKA-signaling pathways, which
converge and phosphorylate CREB on serine-133 (Fig. 8
).

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Figure 8. Model of the Signaling Pathways Leading to
Cumulative Activation of the CCK Gene Promoter by bFGF and Forskolin
Receptor binding of bFGF is followed by stimulation of Ras that
activates the MEK/ERK pathway and the p38 MAPK pathway. The downstream
activators of ERK and p38 MAPK are likely to be the family of pp90
ribosomal S6 kinases (RSK) and the MAPK-activated protein (MAPKAP)
kinase 2, respectively, which have been demonstrated to phosphorylate
CREB at serine-133 (30 33 36 39 ). Forskolin or putative
neurotransmitter substances or neuropeptides stimulate adenylate
cyclase activity and cAMP production, after which PKA is activated and
phosphorylates CREB at serine-133. Moreover, the PKA pathway cross-talk
with the p38 and ERK MAPK. Ultimately, trans-activation
is accomplished by association of CREB with CBP and activation of
transcription.
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The CCK CRE/TRE motif consists of a core consensus sequence of
5'-CTGCGTCA-3', which, like the CRE-2 element of the proenkephalin
gene, may bind CREB/ATF and factors belonging to the c-Jun and c-Fos
family of transcription factors (6). bFGF and cAMP have previously been
demonstrated to activate the proenkephalin promoter via binding of
ATF-3/c-Jun heterodimers (12). The mechanism of CCK gene promoter
activation is different, since stimulation is mainly mediated by CREB.
Promoter activation correlated to CREB serine-133 phosphorylation (13),
which is essential for the association of the CREB-binding protein
(CBP) required for trans-activation (20, 21, 22). CREB and the
catalytic subunit of PKA, moreover, were shown to directly activate CCK
gene expression in F9 cells, and coexpression of a dominant negative
CREB mutant, which forms inactive heterodimers with wild-type CREB and
ATF-1 (14), inhibited activation by bFGF and forskolin. ATF-1 also
bound to the CCK CRE/TRE and, like CREB, it was phosphorylated upon
stimulation with bFGF and forskolin. We did not, however, observe any
promoter activation by ATF-1 even after coexpression with PKA. The
reason for the distinct behavior of CREB and ATF-1 in this system is
unclear but it has recently been suggested that ATF-1 can reduce
promoter activation by heterodimerizing with CREB-1 (23). Factors
belonging to the Jun or Fos family of transcription factors were not
detected in the band-shift analysis, supporting the conception that CCK
CRE/TRE and the identical proenkephalin CRE-2 are likely to have a
preference for CREB, because they exhibit a C residue
(CTGCGTCA) in the core of the element (9, 24). It should be
noted, however, that CREB has been recently demonstrated to regulate
Fos transcription (25), and some of the dominant negative effect of
KCREB may be related to Fos. Finally, CCK promoter activation by
forskolin/cAMP may involve the direct recruitment of CBP (26). This may
explain why bFGF, which fails to induce cAMP, is a weaker activator of
CREB- dependent transcription than forskolin, even though both factors
promote phosphorylation of CREB on serine 133.
Although the PKA-signaling pathway, leading to phosphorylation of
CREB, is well established (for a review see Ref. 27), the events after
growth factor stimulation are less well characterized. Receptor binding
of bFGF is followed by receptor dimerization, autophosphorylation, and
activation of phospholipase C (28), as well as the MAPK-signaling
pathways in a Ras-dependent manner (29, 30, 31). Since coexpression
of Ha-Ras (Asn-17) inhibited activation by bFGF and oncogenic Ras
caused a synergistic activation of the promoter together with the
catalytic subunit of PKA, we inferred that CCK gene promoter activation
was associated with activation of the MAPK pathways. Mammalian cells
contain at least three well characterized MAPK cascades, which regulate
the activity of the ERK MAPKs, the Ras/MEK/ERK pathway, the
stress-activated protein kinase/c-Jun amino-terminal kinase (SAPK/JNK)
MAPKs, and the p38 MAPKs (for a review see Ref. 32). While the ERK MAPK
cascade has been implicated in the activation by various growth
factors, the SAPK/JNK and the p38 MAPK pathways were originally shown
to mediate stress responses. However, recent studies have shown that
the p38 MAPK pathway can also be activated by growth factors (30, 33).
Stimulation of CCK transcription and CREB phosphorylation by bFGF was
inhibited by the p38 MAPK inhibitor SB203580 and the MEK1 inhibitor
PD098059, indicating that both the p38 MAPK pathway and the Ras/MEK/ERK
pathway are involved in promoter activation. Likewise, we could
demonstrate that bFGF stimulated phosphorylation of both ERK and p38,
which are associated with activation of the kinases (34, 35).
Inhibition of GAL4-CREB transcription and CREB phosphorylation,
moreover, was additive, indicating that the p38 MAPK pathway and the
Ras/MEK/ERK pathway proceed in parallel. This is in agreement with
recent data showing that nerve growth factor-mediated phosphorylation
of CREB also proceeds via the p38 MAPK pathway and the Ras/MEK/ERK
pathways (36). The signaling pathways that activate p38 are not
completely understood, but since signaling can be inhibited by a
dominant Ras mutant, it is possible that Ras controls p38 via
activation of Rho (37, 38). The downstream activators of ERK and p38
MAPK are likely to be the family of pp90 ribosomal S6 kinases (RSK) and
the MAPK-activated protein kinase 2 (MAPKAP kinase-2), respectively,
which have all been demonstrated to phosphorylate CREB at serine-133
(30, 33, 36, 39).
Forskolin stimulation involved not only the classical PKA pathway since
about 20% of the stimulation can be attributed to activation of p38
and ERK. Although only a minor stimulation proceeded via these
pathways, they may be physiologically relevant under certain
conditions. In fact, cAMP and PKA have recently been demonstrated to
stimulate differentiation of PC12 cells via activation of ERK (40). We
find that forskolin phosphorylates p38 more potently than ERK, and this
pathway is therefore likely to mediate additional effects of cAMP and
PKA. Moreover, the results predict that additive effects of growth
factors and cAMP may occur at the level of p38 and ERK.
The CCK gene promoter CRE/TRE is conserved from shark and bullfrog to
man and is likely to play a central role in the control of CCK gene
expression. Our data indicate that it serves to integrate signals from
a variety of extracellular factors, including growth and neurotrophic
factors as well as transmitters, and generate a finely tuned output of
CCK RNA. Signaling from the MAPK- and PKA-signaling pathways are
integrated by CREB, localized in situ on the CCK CRE/TRE.
CREB has been reported to play a key role in both neurotrophin
responses and memory functions (41, 42, 43, 44) and, based on a comparison with
the physiological roles of CCK, it is also likely that CREB could be
involved in regulation of feeding behavior and anxiety control.
Although this study has focused on the effects of bFGF, the mechanism
may be extended to nerve growth factor and brain-derived neurotrophic
factor and other growth factors in the gastrointestinal channel, which
have been demonstrated to activate the MAPK pathways in a similar way
as bFGF (33, 41). Stimulation of CCK gene expression by bFGF and
neurotransmitters, coupling to the PKA-signaling pathway, may be of
importance in several situations. bFGF is produced in vast areas of the
central nervous system (45) and could play a role in CCK production in
late development. In the rat, adult bFGF levels and patterns of
distribution are reached at postnatal day 28 (46), and this coincides
with the production of transmitter-active CCK peptides (47). Moreover,
unlike most neurotransmitters, bFGF and other growth factors are not
released from stored granules in short bursts, but are secreted in a
constitutive manner over longer time periods. During adult life it may
be envisioned that the function of bFGF is mainly to up-regulate the
response of the CCK gene promoter to neuropeptides and smaller
neurotransmitters that couple to the PKA-signaling pathway. Finally, it
is possible that cumulative actions may be relevant during regeneration
in which bFGF and CCK have been demonstrated to be expressed at high
levels (48, 49).
In conclusion, we show that bFGF and forskolin stimulate CCK
transcription via the p38 MAPK-, ERK MAPK-, and PKA-signaling pathways
and enhanced phosphorylation and activation of CREB. We propose that
bFGF or other growth and neurotrophic factors, in combination with
neurotransmitter/neuropeptide coupling to the PKA signal pathway, plays
an important role in the control of endocrine and neuronal CCK gene
expression.
 |
MATERIALS AND METHODS
|
---|
Plasmid Constructions
The human CCK gene promoter constructs, CCK-200, CCK-100,
CCK-67, and 100
CRE/TRE, consist of 5'-promoter fragments inserted
into the pCAT vector (Promega, Madison, WI) as recently described (6).
Thymidine kinase (TK)-luciferase was constructed by inserting a
HindIII-BglII fragment of pRL-TK (Promega)
containing the TK promoter into pGL3 basic (Promega). The mammalian
expression vectors encoding CREB and KCREB (14), Ha-Ras (Asn-17) (15),
oncogenic Ras (Val-12) (50), and the catalytic subunit of PKA (51) were
kind gifts from Richard H. Goodman, Larry A. Feigh, Robert A. Weinberg,
and G. Stanley McKnight, respectively. Full-length ATF-1 cDNA was
amplified from SK-N-MC cell RNA using the oligonucleotide primers
5'-GTCGTAGCGGCCGCTTATGGAAGATTCCCACAAGAGTACCACG-3' and
5'-CAGCTGAAGCTTAATCAAACACTTTTATTGGAATAAAGATC-3'
and subcloned into pcDNA3.1+ (Invitrogen, Carlsbad, CA). The sequence
was verified by sequencing. The 5 x GAL4-TATA-luciferase reporter
plasmid, the GAL4-CREB4283, and the
GAL4-CREB4283(Ala-133) expression vectors (52, 53) were
kind gifts from Richard A. Maurer.
Cell Culture and Transient DNA Transfections
Human SK-N-MC neuroblastoma cells were maintained as described
(6). One day before transfection 2.5 x 106 SK-N-MC
cells were seeded in 100-mm culture dishes. Five micrograms of CCK
reporter plasmid, 2 µg TK-luciferase, 10 µg CREB, 10 µg KCREB, or
1 µg PKA expression vector when indicated, and pBluescript
(Stratagene, La Jolla, CA) to a total of 20 µg were cotransfected
using the calcium phosphate coprecipitation method (54). For the GAL4
assay, 5 µg 5 x GAL4-TATA-luciferase reporter plasmid, 2 µg
GAL4-CREB or GAL4-CREB(Ala-133) expression vector, 0.5 µg pRL-TK
(Promega), and pBluescript (Stratagene) to a total of 20 µg were
cotransfected. Six hours before harvesting, the cells were stimulated
with either 25 ng/ml bFGF (Amersham, Arlington Heights, IL), 10
µM forskolin (Sigma Chemical Co., St. Louis, MO), or
both. SB203580 (1025 µM) (Calbiochem, San Diego, CA),
2050 µM PD098059 (Calbiochem), or dimethylsulfoxide
were added together with the stimulant when indicated. Murine F9 cells
were cultured in DMEM (Life Technologies, Gaithersburg, MD) containing
15% FBS and 1.0 mM sodium pyruvate at 10% CO2
and 37 C. Cells were seeded at 1 x 106/100-mm culture
dish coated with 0.1% gelatin 1 day before transfection. Each plate
was transfected with 30 µg of DNA, including 5 µg CCK-100 reporter
plasmid, 2 µg TK-luciferase, 10 µg CREB or ATF-1 expression vector,
5 µg PKA expression vector, and pBluescript (Stratagene) as described
above. At the end of incubation the cells were harvested and analyzed
for CAT and luciferase activity. All values were normalized to
luciferase activity.
Preparation of Nuclear Extract and Electrophoretic Mobility Shift
Assays
Nuclear extracts from SK-N-MC cells were prepared as described
previously (55). The protein concentration was determined using the
Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA).
[
-32P]ATP labeled double-stranded oligonucleotides
(4 x 104 cpm), corresponding to the CCK gene promoter
5'-flanking sequence -85 to -66 relative to the transcription start
site (5'-CCAGTCTGCGTCAGCGTTGG-3'), were incubated with 5 µg of
nuclear extract in 10 mM HEPES (pH 7.9), 100 mM
KCl, 0.05 mM EDTA, 1 mM dithiothreitol,
2.5 mM MgCl2, 6% glycerol, 2 µg of (dI-dC),
in a total volume of 20 µl for 30 min at room temperature. For
supershift assays, 5 µl of anti-CREB (sc-271), 1 µl of anti-ATF-1
(sc-243x), 5 µl of anti-ATF-3 (sc-188), 1 µl of anti-Jun (sc-44x),
or 1 µl of anti-Fos (sc-253x) antibody (Santa Cruz Biotechnology,
Santa Cruz, CA) were added to the binding reaction and incubated for an
additional 60 min at 4 C before the loading of the gel. DNA-protein
complexes were analyzed on a 4% nondenaturing polyacrylamide gel in
0.5 x TBE buffer (1 x TBE is 130 mM Tris, 89
mM boric acid, and 2 mM EDTA) and exposed to
PhosphoImager (Fuji, Tokyo, Japan) for quantitative analysis.
Western Blot Analysis
For detection of proteins, approximately 2.5 x
106 SK-N-MC cells were grown in media containing 0.5%
serum for 2 days before stimulating with 25 ng/ml bFGF (Amersham), 10
µM forskolin (Sigma), or both for 15 min. SB203580 (10
µM) (Calbiochem), 20 µM PD098059
(Calbiochem), or dimethylsulfoxide were added 1 h before
stimulation when indicated. The cells were washed with cold PBS and
lysed by addition of 500 µl SDS loading buffer [100 mM
Tris-HCl (pH 6.8), 4% SDS, 0.2 M dithiothreitol, 0.2%
bromophenol blue, and 20% glycerol] followed by boiling for 5
min. Proteins were separated on 10% SDS polyacrylamide gels and
transferred to polyvinyl difluoride Immobilon-P membranes (Milipore,
Bedford, MA). After blocking with 5% nonfat dry milk in 10
mM Tris (pH 7.5), 100 mM NaCl, and 0.1% Tween
20 for 1 h at room temperature, filters were incubated with
anti-CREB (sc-271) (1:500 dilution) (Santa Cruz Biotechnology),
anti-ATF-1 (sc-243) (1:1000) (Santa Cruz Biotechnology), anti-p38
(1:1000 dilution) (New England Biolabs, Beverly, MA), anti-ERK1/2
(1:1000 dilution) (New England Biolabs), anti-phospho-CREB/ATF-1
(1:1000 dilution) (New England Biolabs), anti-phospho-p38 (1:1000
dilution) (New England Biolabs), or anti-phospho-ERK1/2 antibody
(1:1000 dilution) (New England Biolabs) in blocking solution overnight
at 4 C. After three washes in wash buffer [10 mM Tris (pH
7.5), 100 mM NaCl, and 0.1% Tween 20], the membranes were
incubated with conjugated antimouse or antirabbit IgG-horseradish
peroxidase (1:2000 dilution) (New England Biolabs) in blocking solution
for 1 h at room temperature. The membranes were then washed three
times with wash buffer, and detection of immunoreactive proteins was
performed with lumiGLO chemiluminescent reagent according to the
manufacturers instruction (New England Biolabs).
 |
ACKNOWLEDGMENTS
|
---|
Richard H. Goodman, Larry A. Feigh, Robert A. Weinberg, G.
Stanley McKnight, and Richard A. Maurer are gratefully acknowledged for
the gift of plasmids.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Finn Cilius Nielsen, Department of Clinical Biochemistry, Rigshospitalet, DK-2100 Copenhagen Ø. E-mail:
cilius{at}centrum.dk
This study was supported by grants from the Danish Medical Research
Council, the Danish Biotechnology Program for Signal Peptide Research,
the John and Birthe Meyer Foundation, and the NOVO Nordisk
Foundation.
Received for publication September 10, 1998.
Revision received November 13, 1998.
Accepted for publication December 8, 1998.
 |
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