We have previously observed that gastrin has a
cholecystokinin B (CCK-B) receptor-mediated
growth-promoting effect on the AR42J rat pancreatic acinar cell line
and that this effect is paralleled by induction of expression of the
early response gene c-fos. We
undertook these experiments to elucidate the mechanism for induction of
c-fos and the linkage of this action
to the trophic effects of gastrin. Gastrin (0.1-10 nM) dose
dependently induced luciferase activity in AR42J cells transfected with
a construct consisting of a luciferase reporter gene coupled to the
serum response element (SRE) of the
c-fos promoter. This effect was blocked by the specific CCK-B receptor antagonist D2 but not by the
specific CCK-A receptor antagonist L-364,718 or by pertussis toxin,
indicating that gastrin targets the SRE via specific CCK-B receptors
through a mechanism independent of
Gi. Inhibition of protein kinase C
(PKC) either by prolonged (24 h) exposure of the cells to the phorbol
ester 12-O-tetradecanoylphorbol
13-acetate (100 nM) or by incubation with the selective inhibitor
GF-109203X (3.5 µM) resulted in an 80% reduction in luciferase
activity. Similar results were observed in the presence of the specific extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor PD-98059 (50 µM). We measured ERK2 activity in AR42J cells via in-gel
kinase assays and observed that gastrin (1 pM-100 nM) induced ERK2
enzyme activity in a dose-dependent manner. Addition of GF-109203X and
PD-98059, either alone or in combination, produced, respectively, partial and total inhibition of gastrin-induced ERK2 activity. Gastrin
induction of ERK2 activity also resulted in a threefold increase in the
transcriptional activity of Elk-1, a factor known to bind to the
c-fos SRE and to be phosphorylated and
activated by ERK2. PD-98059 blocked the growth-promoting effect of
gastrin on the AR42J cells, demonstrating that this effect depends on activation of MEK. Our data lead us to conclude that the trophic actions of gastrin are mediated by ERK2-induced
c-fos gene expression via
PKC-dependent and -independent pathways.
cholecystokinin-B receptor; cellular proliferation; early response
genes; protein kinases; transcriptional regulation; c-fos; mitogen-activated protein
kinase; extracellular signal-regulated protein kinases
 |
INTRODUCTION |
ALTHOUGH CHARACTERIZED AS a stimulant of gastric acid
secretion (11), the peptide hormone gastrin also exerts
growth-promoting effects on normal and malignant gastrointestinal
tissues (16, 28, 29). Gastrin is an important growth factor for the
fetal pancreas (4, 34), and in the stomach it is a potent stimulant for
the growth of the gastric mucosa (33). In addition, recent reports have
indicated that gastrin induces the growth of colon carcinomas both in
vivo and in vitro, thus underscoring the importance of gastrin as a
growth factor for gastrointestinal neoplasms (23, 28).
The process of cellular proliferation is under the control of a complex
cascade of phosphorylation reactions that is triggered by the
interaction of growth factors with their specific cellular receptors
(14). One of the best-characterized pathways linked to the control of
cellular growth is known to involve the activation of the
serine-threonine protein kinase Raf through its interaction with the
small GTP binding protein Ras (14). Raf is responsible for the
phosphorylation and activation of a dual-specificity protein kinase
known as mitogen-activated protein kinase (MAPK) or extracellular signal-regulated protein kinase (ERK) kinase (MEK), which in turn phosphorylates both serine and tyrosine residues in a family of serine-threonine protein kinases known as MAPKs or ERKs (14). The ERKs
phosphorylate numerous cellular proteins, including transcription factors such as p62TCF or Elk-1,
that play an important role in the transcriptional regulation of the
promoter of the early response gene
c-fos through the serum response
element (SRE) (14, 18). Thus c-fos
appears to be an important central node for the propagation of
mitogenic signals from second messengers to the nucleus and for the
activation or repression of the next set of genes in the biological
programs initiated by the extracellular signals (14, 20).
Recent reports have indicated that gastrin can induce the ERKs and that
this effect is paralleled by its ability to induce the early response
gene c-fos (26, 30, 31). However,
little is known about the signal-transduction pathways that target the ERKs and c-fos in response to gastrin
and whether this effect is responsible for stimulation of cellular
growth. Thus we undertook these studies to elucidate the mechanisms for
induction of c-fos and the linkage of
this action to the trophic effects of gastrin. Our data indicate that
gastrin, binding to specific cholecystokinin (CCK)-B receptors on the
AR42J cells, induces a cascade of phosphorylation reactions that target
and activate the ERKs via both protein kinase C (PKC)-dependent and
-independent pathways. The ERKs, in turn, appear to induce
c-fos gene expression through
phosphorylation and transcriptional activation of Elk-1. The net result
of this cascade of events is stimulation of cellular proliferation.
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MATERIAL AND METHODS |
Plasmids. 5×Gal-Luc (13) was a gift
from M. Karin (San Diego, CA); Gal4-ElkC (18) was a gift from R. Treisman (London, UK). SRE-Luc (35) was obtained from J. Pessin (Iowa
City, IA), and pCMV-
Gal was a gift from M. Uhler (Ann Arbor, MI).
Cell culture, transient transfection, and luciferase
assays. For our experiments, we used the rat exocrine
pancreatic cell line AR42J, which is known to express receptors for
both gastrin (G17) and glycine-extended progastrin-processing
intermediates (G-Gly) (27). The cells (obtained from American Type
Culture Collection, Rockville, MD) were grown at 37°C in 35-mm
dishes in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in 5%
CO2-95%
O2. Subconfluent AR42J cells were
transfected with 5 µg of the luciferase reporter plasmid and, where
indicated, with 0.5 µg of the expression vectors. Transfections were
carried out using lipofectamine (GIBCO-BRL, Grand Island, NY), as
previously described (31). The day after transfection, the media were
removed, and the cells were fed with serum-free media (DMEM) for 24 h,
then incubated for 5 h with or without human gastrin
heptadecapeptide (Bachem, Torrance, CA) (0.1-10 nM)
or 12-O-tetradecanoylphorbol
13-acetate (TPA, Sigma, St. Louis, MO; 100 nM). In some experiments,
bisindolylmaleimide I (GF-109203X, 3.5 µM; Calbiochem, La Jolla, CA),
PD-98059 (50 µM; a gift from Dr. Alan Saltiel, Park Davis
Pharmaceutical Research, Ann Arbor, MI), L-364,718 (10 nM; a gift from
Merck Sharp & Dohme, Rahway, NJ), or D2 (10 nM; a gift from Dr. Yoshi
Goto, Daiichi Pharmaceuticals, Tokyo, Japan) was added 30 min before
the addition of gastrin. In additional studies, the cells were also
preincubated for either 16 h in 200 ng/ml pertussis toxin (Sigma) or
for 24 h with TPA (100 nM) prior to the addition of gastrin.
Bisindolylmaleimide I, TPA, L-364,718, and PD-98059 were
dissolved in dimethyl sulfoxide (DMSO, Sigma). All other test
substances were dissolved in water. Control experiments were performed
by incubating the cells in either incubation buffer or vehicle without
the test substances. At the end of the incubation period, the cells
were washed and lysed, and luciferase assays were performed as
previously described (31). Luciferase activity was expressed as
relative light units and normalized for
-galactosidase
activity.
-Galactosidase activity was measured by the luminescent
light derived from 10 µl of each sample incubated in 100 µl of
Lumi-Gal 530 (Lumigen, Southfield, MI) and used to correct the
luciferase assay data for transfection efficiency.
Immunoprecipitations and in-gel ERK2
assay. Immunoprecipitations and in-gel ERK2 assays were
performed according to previously described techniques (25, 31).
Subconfluent AR42J cells were cultured for 24 h in serum-free DMEM,
then incubated for 5 min with or without gastrin (1 pM-100 nM) or TPA
(100 nM). In some experiments, GF-109203X (3.5 µM),
bisindolylmaleimide V (3.5 µM), or PD-98059 (0.1 mM) were added 30 min before the addition of gastrin. Control experiments were performed
by incubating the cells in either incubation buffer or vehicle without
the test substances. The cells were lysed in 500 µl of lysis buffer
[10 mM KPO4 (pH 7.4), 1 mM
EDTA, 5 mM ethylene glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic
acid (EGTA), 10 mM MgCl2, 50 mM
-glycerophosphate, 1 mM sodium orthovanadate
(Na3VO4),
2 mM dithiothreitol, 40 µg/ml phenylmethylsulfonyl
fluoride, 10 nM okadaic acid, 0.8 µg/ml leupeptin, 10 mg/ml p-nitrophenylphosphate, and 10 µg/ml aprotinin], transferred into Microfuge tubes, and spun at
16,000 g for 20 min at 4°C. Protein concentrations were measured by the Bradford method (3). Equal
amounts of protein from each treatment group (1,000 µg) were
incubated with 10 µl of an anti-ERK2-specific antibody (Santa Cruz
Biotechnology, Santa Cruz, CA) and mixed on a rotating platform for 3 h
at 4°C. Control experiments were conducted by mixing aliquots of
the samples with identical volumes of nonimmune sera. Equal aliquots of
protein A-agarose (Santa Cruz Biotechnology) were then added to the
tubes, and the solutions were mixed for one additional hour. After
centrifugation, the pellets were washed four times with lysis buffer.
The samples were resuspended in 60 µl of electrophoresis buffer
[for 10 ml:1 ml glycerol, 0.5 ml 2-mercaptoethanol, 3 ml 10%
sodium dodecyl sulfate (SDS), 1.25 ml 1 M
tris(hydroxymethyl)aminomethane (Tris) buffer, 2 ml 0.1% bromophenol
blue, and 0.6 g urea], boiled for 5 min, and applied to a 10%
SDS-polyacrylamide gel containing 0.5 mg/ml myelin basic protein
(Sigma). After electrophoresis, the gel was washed with two changes of 20% 2-propanol in 50 mM Tris (pH 8.0) for 1 h and then
with two changes of 50 mM Tris (pH 8.0) containing 5 mM
2-mercaptoethanol for 1 h. The enzyme was denatured by
incubating the gel with two changes of 6 M guanidine hydrochloride for
1 h and then renaturated with five changes of 50 mM Tris (pH 8.0)
containing 0.04% Tween 40 and 5 mM 2-mercaptoethanol for 1 h. The
kinase reaction was performed in conditions inhibitory to cyclic
nucleotide-dependent protein kinase and
Ca2+-dependent protein kinases by
incubating the gel at 25°C for 1 h with 40 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (pH 8.0) containing 0.5 mM EGTA, 10 mM
MgCl2, 2 µM protein kinase inhibitor peptide, 40 µM ATP, and 2.5 µCi/ml of
[
-32P]ATP (6,000 Ci/mmol). After incubation, the gel was washed with a 5%
(wt/vol) trichloroacetic acid solution containing 1% (wt/vol) sodium pyrophosphate, dried, and subjected to autoradiography.
Proliferation studies. These studies
were conducted according to previously described techniques (27).
Briefly, the AR42J cells were grown in 35-mm dishes in DMEM
supplemented with 10% FBS in 5%
CO2 at 37°C. Subconfluent
AR42J cells were cultured for 24 h in serum-free DMEM containing 0.2 mM
unlabeled thymidine. After being washed with serum-free medium, the
cells were treated with gastrin (1 nM) or G-Gly (1 nM) for 18 h. In
some experiments, PD-98059 (50 µM) was added 30 min prior to the
addition of gastrin and G-Gly. Control experiments were performed by
incubating the cells in either incubation buffer or vehicle without the
test substances. DNA synthesis was estimated by measurement of
[3H]thymidine
incorporation into the trichloroacetic acid-precipitable material
according to the method of Seva et al. (27).
[3H]thymidine (0.1 µCi/ml, 10 Ci/mmol) was added during the last hour of the treatment
period. Cells were washed with serum-free media to remove
unincorporated
[3H]thymidine, and the
DNA was precipitated with 5% trichloroacetic acid at 4°C for 15 min. Precipitates were washed twice with 95% ethanol, dissolved in 1 ml of 0.1 N NaOH, and analyzed in a liquid scintillation counter
(Beckman Instruments, Palo Alto, CA).
Data analysis. Data are presented as
means ± SE, wherein n is equal to
the number of separate transfections performed with the AR42J cells.
Statistical analysis was performed using Student's t-test.
P < 0.05 was considered significant.
 |
RESULTS |
We first investigated the effect of increasing concentrations of
gastrin on SRE-luciferase activity. For these experiments, we
cotransfected the AR42J cells with luciferase reporter plasmids containing the c-fos SRE upstream of
the thymidine kinase (TK) gene minimal promoter and the luciferase
reporter gene together with the pCMV-
Gal expression vector. As shown
in Fig. 1, gastrin dose
dependently stimulated SRE-luciferase activity (1.76 ± 0.08-fold induction over control, n = 3; 3.24 ± 0.4-fold induction over control,
n = 3; and 3.46 ± 0.87-fold induction over control, n = 3, in the presence of 0.1, 1, and 10 nM gastrin,
respectively). Gastrin interacts with at least two
different cellular receptor subtypes, designated CCK-A and CCK-B,
respectively (5, 15). Thus we investigated which receptor subtype
mediates the stimulatory action of gastrin on SRE-luciferase activity.
As depicted in Fig. 2, 10 nM gastrin
induced SRE transcriptional activity, and this effect was blocked by
the highly selective CCK-B receptor antagonist D2 (3.73 ± 0.75-fold
induction over control, n = 3, in the
presence of gastrin vs. 1.36 ± 0.14-fold induction over control,
n = 3, in the presence of gastrin in
combination with 10 nM D2). In contrast, no inhibition was observed in
the presence of the selective CCK-A receptor antagonist L-364,718 (4.03 ± 0.57-fold induction over control,
n = 3, in the presence of gastrin vs.
4.23 ± 0.49-fold induction over control,
n = 3, in the presence of gastrin in
combination with 10 nM L-364,718), suggesting that the CCK-B receptor
mediates gastrin induction of c-fos
gene transcription. Treatment of the cells with either the D2 compound
or L-364,718 alone did not have any significant effect on SRE
transcriptional activity (1.11 ± 0.13-fold induction over
control, n = 3, and 1.23 ± 0.18-fold induction over control, n = 3, in the presence of D2 and L-364,718, respectively). As previously
reported, cells transfected with a plasmid containing the TK gene
minimal promoter but devoid of the SRE exhibited no induction in
response to treatment with gastrin (31).

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Fig. 1.
Effect of human gastrin heptadecapeptide (G17) on serum response
element (SRE) transcriptional activity in AR42J cells. Cells were
cotransfected with plasmids SRE-Luc and pCMV- Gal and treated with
increasing concentrations of G17 (0.1-10 nM). Data are expressed
as multiple-fold induction over control and are means ± SE. Numbers
in parentheses are number of separate transfections performed with the
AR42J cells (Figs. 1-5 and 9).
* P < 0.05. RLU, relative
light units.
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Fig. 2.
Effect of D2 compound and of L-364,718 on G17-stimulated SRE
transcriptional activity in AR42J cells. Cells were cotransfected with
plasmids SRE-Luc and pCMV- Gal and treated with 10 nM G17 alone and
in combination with either 10 nM D2 or 10 nM L-364,718. Data are
expressed as multiple-fold induction over control and are means ± SE. * P < 0.05. NS, not
significant.
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To characterize the signaling pathways that target the
c-fos SRE in response to stimulation
with gastrin, we performed studies in which we preincubated the AR42J
cells with 200 ng/ml pertussis toxin, an agent known to ADP ribosylate
and thereby inactivate some GTP binding proteins such as
Gi. Pertussis toxin pretreatment failed to inhibit gastrin stimulation of SRE-luciferase activity (4 ± 0.6-fold induction over control,
n = 3, in the presence of gastrin vs. 4.23 ± 0.67-fold induction over control,
n = 3, in the presence of gastrin in
combination with pertussis toxin) (Fig. 3),
indicating that this effect of gastrin is mediated by a mechanism independent of Gi. Treatment of
the cells with pertussis toxin alone exhibited a 1.14 ± 0.15-fold
induction over control (n = 3).

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Fig. 3.
Effect of pertussis toxin (PT) on G17-stimulated SRE transcriptional
activity in AR42J cells. Cells were cotransfected with plasmids SRE-Luc
and pCMV- Gal and treated with 10 nM G17 alone and in combination
with 200 ng/ml PT. Data are expressed as multiple-fold induction over
control and are means ± SE.
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One of the best-characterized signal-transduction pathways activated by
gastrin on interaction with the CCK-B receptor involves PKC stimulation (5). Accordingly, we performed studies to
examine whether PKC mediated gastrin induction of SRE-luciferase
activity. As depicted in Fig.
4A, the
selective PKC inhibitor GF-109203X (3.5 µM) significantly inhibited,
although not completely, gastrin-stimulated SRE luciferase activity
(2.78 ± 0.19-fold induction over control, n = 3, in the presence of gastrin vs.
1.37 ± 0.21-fold induction over control,
n = 3, in the presence of gastrin in
combination with GF-109203X). Identical results were obtained when PKC
was inhibited by prolonged (24 h) incubation of the cells with 100 nM
TPA (data not shown). TPA (100 nM) stimulated SRE transcriptional activity, and this effect was blocked by 3.5 µM GF109203X (2.78 ± 0.07-fold induction over control, n = 4, in the presence of TPA vs. 0.79 ± 0.045-fold induction over
control, n = 4, in the presence of TPA
in combination with GF-109203X) (Fig.
4B), demonstrating that GF-109203X
is able to inhibit PKC completely in AR42J cells. Identical results
were obtained when the cells were preincubated for 24 h with 100 nM TPA
(data not shown). No induction of SRE transcriptional activity was
detected in the presence of either GF-109203X alone (0.97 ± 0.1-fold induction over control, n = 3) (Fig. 4A) or vehicle (0.1% DMSO)
(data not shown). Thus gastrin specifically targets the
c-fos SRE via both PKC-dependent and -independent pathways.

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Fig. 4.
Effect of protein kinase C (PKC) inhibitor GF-109203X (GF) on G17- and
12-O-tetradecanoylphorbol 13-acetate
(TPA)-stimulated SRE transcriptional activity in AR42J cells.
A: cells were cotransfected with
plasmids SRE-Luc and pCMV- Gal and treated with 10 nM G17 alone or in
combination with specific PKC inhibitor GF-109203X (3.5 µM).
B: effect of GF-109203X (3.5 µM) on
TPA (100 nM)-stimulated SRE transcriptional activity. Data are
expressed as multiple-fold induction over control; means ± SE.
* P < 0.01.
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Because activation of the c-fos SRE is
known to require induction of the ERKs and of their upstream activator,
MEK, we conducted experiments to examine the effect of the highly
specific MEK inhibitor PD-98059, known to induce complete ERK
inhibition at doses ranging between 50 and 100 µM (1). We observed,
as shown in Fig. 5, that PD-98059 (50 µM)
potently inhibited SRE-luciferase activity stimulated by gastrin (3.17 ± 0.64-fold induction over control, n = 4, in the presence of gastrin vs.
1.28 ± 0.26-fold induction over control, n = 4, in the presence of
gastrin in combination with PD-98059), confirming the involvement of
MEK in gastrin induction of the c-fos
SRE. Treatment of the cells with PD-98059 alone had no effect on
SRE luciferase activity (1.08 ± 0.22-fold induction over control,
n = 4).

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Fig. 5.
Effect of the extracellular signal regulated protein kinase kinase
(MEK) inhibitor PD-98059 (PD) on G17-stimulated SRE transcriptional
activity in AR42J cells. Cells were cotransfected with plasmids SRE-Luc
and pCMV- Gal and treated with 10 nM G17 alone or in combination with
50 µM PD-98059. Data are expressed as multiple-fold induction over
control and are means ± SE.
* P < 0.05.
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Because ERK2 activation is required for induction of the SRE in
response to growth factor stimulation (18), we tested the effect of
gastrin on ERK2 activity using in-gel kinase assays. Gastrin induced
ERK2 in a dose-dependent fashion with a maximal effect detected between
10
8 and
10
7 M (Fig.
6). We then examined whether inhibition of
PKC resulted in inhibition of gastrin-stimulated ERK2 activity. As
shown in Fig. 7, addition of 3.5 µM
GF-109203X produced partial inhibition of gastrin-stimulated ERK2
activity, whereas no effect was observed in the presence of the
inactive PKC inhibitor bisindolylmaleimide V (3.5 µM). In contrast,
GF-109203X completely inhibited the induction observed in the presence
of 0.1 µM TPA, confirming the notion that gastrin targets ERK2
through pathways that depend only partially on activation of PKC.
Identical results were obtained when the cells were preincubated for 24 h with 0.1 µM TPA prior to the addition of either gastrin or TPA
(data not shown). To study the relative contribution of PKC and MEK to
the stimulatory effect of gastrin on ERK2 activation, we performed
studies in which we compared the inhibitory effects of GF-109203X and
PD-98059 alone with those of both agents combined. Addition of
GF-109203X (3.5 µM) and of a supramaximal dose of PD-98059 (100 µM), either alone or in combination, produced, respectively, partial
and total inhibition of gastrin-induced ERK2 activity (Fig.
8), suggesting that both MEK and PKC are
activated in response to gastrin stimulation.

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Fig. 6.
Effect of G17 on ERK2 activity in AR42J cells.
A: ERK2 in lysates from AR42J cells
stimulated with increasing concentrations of G17 was
immunoprecipitated, and its activity was measured by in-gel kinase
assays. B: linear transformation of
the densitometric analysis of the autoradiograms. Data are
representative of a single experiment.
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Fig. 7.
Effect of the PKC inhibitor GF-109203X on G17- and TPA-stimulated
extracellular signal regulated kinase-2 (ERK2) activity in AR42J cells.
A: ERK2 in lysates from AR42J cells
stimulated with 10 nM G17 and 0.1 µM TPA, alone or in combination
with either GF-109203X (3.5 µM) or bisindolylmaleimide V (3.5 µM),
was immunoprecipitated, and its activity was measured by in-gel kinase
assays. B: linear transformation of
the densitometric analysis of the autoradiograms. Data are
representative of a single experiment.
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Fig. 8.
Effect of the MEK inhibitor PD-98059 and of the PKC inhibitor
GF-109203X on G17-stimulated ERK2 activity in AR42J cells.
A: ERK2 in lysates from AR42J cells
stimulated with 10 nM G17, alone or in combination with either 100 µM
PD-98059 or 3.5 µM GF-109203X and in the presence of both 100 µM
PD-98059 and 3.5 µM GF109203X combined, was immunoprecipitated, and
its activity was measured by in-gel kinase assays.
B: linear transformation of the
densitometric analysis of the autoradiograms. Identical results were
obtained in one other separate experiment.
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ERK2 activation induces c-fos gene
expression by phosphorylation of the transcription factor Elk-1, which
binds to the c-fos SRE (18). To
examine whether this pathway mediated the effects of gastrin, we used a
yeast hybrid system involving cotransfection of the cells with a
chimeric Gal4-ElkC expression vector and the 5×Gal-luciferase
reporter plasmid. In this system, Gal4-ElkC transactivates and
stimulates luciferase activity only if the carboxy terminus of Elk-1 is
phosphorylated by ERK2. As depicted in Fig.
9, 10 nM gastrin induced a 3.22 ± 0.5-fold increase (n = 3) in 5×Gal-luciferase activity, demonstrating that gastrin
induction of the c-fos SRE depends, at
least in part, on ERK2 activation and Elk-1 phosphorylation.

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Fig. 9.
Effect of G17 on Elk-1 transcriptional activity. AR42J cells were
cotransfected with the Gal4-ElkC and the pCMV- Gal expression vectors
and the 5×Gal-luciferase reporter plasmids and treated with 10 nM
G17. Cotransfection of the cells with the expression vector lacking
Gal4-ElkC coding sequences and the 5×Gal-luciferase reporter plasmid
gave only background luciferase activity (data not shown). Data are
expressed as multiple-fold induction over control; means ± S.E.
* P < 0.05.
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We finally examined whether inhibition of MEK resulted in inhibition of
cellular proliferation. For these experiments, the AR42J cells were
incubated in the presence of gastrin, alone or in combination with
PD-98059. Gastrin (1 nM) induced
[3H]thymidine
incorporation into AR42J cells by 1.64 ± 0.2-fold (n = 6) over unstimulated control
cells, and this effect was inhibited in the presence of PD-98059 (1.11 ± 0.08-fold over control, n = 6),
demonstrating that activation of MEK and ERK is required for the
growth-promoting effect of gastrin (Fig.
10A).
In contrast, as shown in Fig. 10B,
PD-98059 failed to inhibit AR42J cell proliferation stimulated by 1 nM
G-Gly (1.33 ± 0.03-fold induction over control, n = 5, in the presence of G-Gly vs.
1.34 ± 0.03-fold induction over control,
n = 5, in the presence of G-Gly in
combination with PD-98059), which, as previously reported, is known to
activate different signal-transduction pathways (31). Treatment of the cells with PD-98059 alone exhibited no significant effect on AR42J cell
proliferation.

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Fig. 10.
Effect of the MEK inhibitor PD-98059 (PD) on G17- and G17-Gly-
stimulated AR42J cellular proliferation.
[3H]thymidine incorporation into
AR42J cells was measured after treatment of the cells with 10 nM G17
(A) and 10 nM G17-Gly
(B), alone or in combination with 50 µM PD-98059. Data are expressed as multiple-fold induction over
control; means ± SE. * P < 0.05.
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 |
DISCUSSION |
Seven transmembrane receptors interact with heterotrimeric GTP binding
proteins to transduce extracellular signals to downstream effector
molecules (10, 21). A number of studies have recently indicated that
these receptors can signal through pathways that were initially
described in response to activation of receptor tyrosine kinases by
growth factors (2, 7, 12, 17, 22, 24, 25). One of the best
characterized of these pathways involves the activation of Ras, Raf,
and of the dual-specificity protein kinase MEK, which in turn
phosphorylates both serine and tyrosine residues in a family of
serine-threonine protein kinases known as MAPKs or ERKs (14). The ERKs
are proline-directed protein kinases that phosphorylate numerous
cellular proteins, including downstream protein kinases such as 90-kDa
S6 kinase (6, 9) and transcription factors like Elk-1 that regulate the
activity of the promoter of the early response gene
c-fos through the SRE (14, 18, 32).
Thus the ERKs, via activation of early gene function and of downstream
protein kinases, play a crucial role in the process of amplification,
integration, and transmission of the extracellular signals from the
cell surface to the nucleus.
The early response gene c-fos is a
member of a family of genes that are turned on within minutes of
cellular activation by growth factors, hormones, and neurotransmitters,
and it appears to be essential for the activation or repression of the
next set of genes in the biological programs initiated by the
extracellular signals (14, 20). The promoter of
c-fos has been the focus of extensive
investigations, and numerous DNA regulatory elements have been mapped
and characterized. One of these elements, the SRE, has been found to
receive inputs from growth factor-activated signal-transduction
pathways. The SRE is in fact regulated by numerous transcription
factors that are constitutively bound to the DNA and are rapidly
phosphorylated and activated in response to growth factor stimulation
(14, 32). Elk-1 in particular is a member of a well-characterized
family of transcription factors, which is known to interact with the
serum response factor to form a ternary complex that
plays an important role in the activation of
c-fos transcription (14, 32). In
response to growth factors, Elk-1 is rapidly phosphorylated and
transcriptionally activated by protein kinases such as ERK2, resulting
in stimulation of c-fos gene
expression (32). In some systems, the activation of this pathway
leads to induction of cellular growth and proliferation (19).
The gastrointestinal hormone gastrin interacts with two separate
cellular receptors known as CCK-A and CCK-B, respectively, which are
members of the seven transmembrane receptor family (5, 15). This
hormone has been found to be a potent growth factor for both normal and
neoplastic gastrointestinal tissues (16, 23, 28, 29, 33). In our study,
we have analyzed some of the signal-transduction pathways that are
activated by gastrin via interaction with CCK-B receptors and that
appear to be linked to stimulation of cellular proliferation. Gastrin
is known to induce mobilization of intracellular
Ca2+ and to activate PKC (5). In
addition, we and others have reported that gastrin can induce the ERKs
and stimulate c-fos gene expression via induction of SRE transcriptional activity (31). In the present study, we have confirmed, using highly specific CCK-B and CCK-A receptor antagonists, that the stimulatory effect of gastrin on c-fos gene transcription regulated by
the SRE is mediated by the CCK-B receptor subtype. In addition, we have
demonstrated that this effect of gastrin is independent of activation
of the GTP binding protein Gi,
since incubation of the cells with pertussis toxin failed to affect
gastrin induction of SRE transcriptional activity. Activation of the
ERKs and c-fos by seven transmembrane, G protein-coupled receptors has been reported to be both pertussis toxin sensitive and insensitive. Although the adrenergic
2A and the muscarinic M2
receptors, both known to couple to
Gi, are sensitive to treatment
with pertussis toxin (8, 12), the
Gq-coupled adrenergic
1B and the muscarinic M1
receptors are not (12), demonstrating that both
Gi-sensitive and -insensitive
pathways can target the ERKs.
In our study, we reported that inhibition of either PKC or MEK with the
use of highly selective and specific inhibitors led to only partial
inhibition of ERK2 activation. In the presence of both agents combined,
however, we observed that this inhibition was complete, suggesting that
both PKC-dependent and -independent and MEK-dependent pathways appear
to mediate gastrin action. Although PKC has been shown to induce c-Raf
directly and to increase Ras activation (6, 9), it is clear that
gastrin signals to ERK2 and c-fos
through pathways that are only partially dependent on activation of
PKC. Similar results were reported in the case of the
thyrotropin-releasing hormone (22) and the angiotensin II receptors
(24), which are known to target the ERKs through pathways that are, at
least in part, pertussis toxin insensitive and PKC independent. These
receptors can induce the activation of protein tyrosine kinases, which,
in turn, phosphorylate the adapter molecule Shc and allow the formation
of a complex with the adapter protein Grb2 and the mammalian guanine
nucleotide exchange factor mSos that leads to the activation of Ras
(22, 24). Furthermore, the observation that addition of supramaximal
doses of the MEK inhibitor PD-98059 leads to incomplete inhibition of
gastrin-stimulated ERK2 induction indicates that PKC does not totally
lie upstream of MEK but that it can target ERK2 via novel, yet
unidentified, signaling pathways.
In contrast to these findings, other investigators have examined the
trophic effects of gastrin in a fibroblast cell line stably transfected
with the human gastrin/CCK-B receptor and found that, in these cells,
gastrin stimulates both MAPK and Raf-1 kinase via PKC-independent
pathways (26), underscoring the complexity of the cellular activation
events that are initiated by the interaction of gastrin with its
specific cellular receptors.
We demonstrated that gastrin stimulation of the
c-fos SRE is mediated by activation of
ERK2 and induction of Elk-1 transcriptional activity. Thus gastrin can
activate a cascade of intracellular phosphorylation reactions that
involves PKC-dependent and -independent pathways that, in turn, induce
c-fos through Elk-1 transcriptional activation. The physiological relevance of this chain of events was
underscored by the finding that inhibition of the ERKs led to
inhibition of cellular proliferation, thus demonstrating that this
CCK-B receptor-linked signal-transduction pathway is essential for the
transmission of mitogenic signals. The specificity of this effect was
further confirmed by the observation that PD-98059 failed to inhibit
AR42J cell proliferation stimulated by G-Gly. This processing
intermediate of gastrin has been shown to induce AR42J cell
proliferation via signal-transduction pathways that do not involve
activation of the ERKs and induction of
c-fos (31).
Our data suggest a working model for the growth-promoting effect of
gastrin. Gastrin binding to specific CCK-B receptors on the AR42J cells
induces a complex cascade of reactions that targets and activates ERK2
via both PKC-dependent and -independent pathways. ERK2, in turn,
induces c-fos gene expression through
phosphorylation and transcriptional activation of Elk-1. The net result
of this cascade of events appears to be stimulation of cellular
proliferation. These data underscore the complexity of cellular
activation events initiated by ligand-receptor interaction on the AR42J
cell membrane.
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant RO1-DK-34306 and by funds from the
University of Michigan Gastrointestinal Peptide Research Center
(National Institute of Diabetes and Digestive and Kidney Diseases Grant
P30-DK-34933). A. Todisco is a recipient of an American
Gastroenterological Association Industry Research Scholar Award and a
Clinical Investigator Award from the National Institutes of Health
(National Institute of Diabetes and Digestive and Kidney Diseases Grant
K08-DK-02336).
Address for reprint requests: A. Todisco, 6520 MSRB I, Ann Arbor, MI
48109-0682.