Departments of 1 Internal Medicine and 2 Pediatrics, University of Michigan Medical Center, Ann Arbor, Michigan 48109
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ABSTRACT |
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Gastrin (G17) has a CCKB receptor-mediated growth-promoting effect on the AR42J rat acinar cell line that is linked to induction of both mitogen-activated protein kinase (MAPK) and c-fos gene expression. We investigated the mechanisms that regulate the growth factor action of G17 on the rat pituitary adenoma cell line GH3. Both AR42J and GH3 cells displayed equal levels of CCKB receptor expression and similar binding kinetics of 125I-labeled G17. G17 stimulation of cell proliferation was identical in both cell lines. G17 stimulation of GH3 cell proliferation was completely blocked by the CCKB receptor antagonist D2 but not by the MEK inhibitor PD-98059 or the protein kinase C inhibitor GF-109203X, which completely inhibited G17 induction of AR42J cell proliferation. G17 induced a c-fos SRE-luciferase reporter gene plasmid more than fourfold in the AR42J cells, whereas it had no effect in the GH3 cells. In contrast to what we observed in the AR42J cells, G17 failed to stimulate MAPK activation and Shc tyrosyl phosphorylation and association with the adapter protein Grb2. Epidermal growth factor induced the MAPK pathway in the GH3 cells, demonstrating the integrity of this signaling system. G17 induced Ca2+ mobilization in both the GH3 and AR42J cells. The calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide inhibited AR42J cell proliferation by 20%, whereas it completely blocked G17 induction of GH3 cell growth. The Ca2+ ionophore ionomycin stimulated GH3 cell proliferation to a level similar to that observed in response to G17, but it had no effect on AR42J cell proliferation. Thus there are cell type specific differences in the requirement of the MAPK pathway for the growth factor action of G17. Whereas in the AR42J cells G17 stimulates cell growth through activation of MAPK and c-fos gene expression, in the GH3 cells, G17 fails to activate MAPK, and it induces cell proliferation through Ca2+-dependent signaling pathways. Furthermore, induction of Ca2+ mobilization in the AR42J cells appears not to be sufficient to sustain cell proliferation.
cellular proliferation; early response genes; protein kinases; transcriptional regulation; c-fos; mitogen-activated protein kinases; extracellular signal-regulated protein kinases
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INTRODUCTION |
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THE PEPTIDE HORMONE GASTRIN is a potent growth factor for both normal and malignant gastrointestinal tissues (23-26, 28, 29, 32, 35). Gastrin is known to interact with two separate cellular receptors known as CCKA and CCKB that are members of the seven-transmembrane receptor family (36).
One of the best-characterized pathways that is responsible for the propagation of mitogenic signals from the cell surface to the nucleus is that involving the activation of the extracellular signal-regulated protein kinases (ERKs) or mitogen-activated protein kinases (MAPKs). The ERKs are important elements in a signaling cascade that includes upstream protein kinases such as Raf and MAPK/ERK kinase (MEK) (5, 9, 19). Activation of the ERKs is known to target numerous cellular proteins, including downstream protein kinases, such as 90-kDa S6 kinase (RSK) (5, 9, 19) and transcription factors like Elk-1 and Sap-1a, which regulate the activity of the promoter of the early response gene c-fos (33). The protooncogene c-fos is expressed within minutes of cell activation (6, 16, 21, 33) and plays an important role in the process of transmission, amplification, and integration of the extracellular signals (6, 16, 21, 33).
The molecular mechanisms responsible for the growth-promoting effect of gastrin have been the focus of numerous investigations. Gastrin is known to stimulate inositol phospholipid turnover and protein kinase C (PKC) activation and to induce intracellular Ca2+ release (10, 11). Gastrin also induces tyrosine kinase activity, and it stimulates both the ERKs and the early response genes c-fos and c-jun (8, 17, 20, 24, 30-32). In a recent study, we have reported that gastrin stimulates the growth of the rat pancreatic adenocarcinoma cell line AR42J through induction of the ERKs and c-fos (32). In addition, gastrin has been found to activate the ERKs in both AGS gastric cancer cells and in rat fibroblasts stably transfected with the CCKB receptor (17, 24), suggesting that activation of the MAPK pathway represents an important mechanism for the transduction of mitogenic signals through the activation of gastrin/CCKB receptors.
Interestingly, in addition to this well-established growth-promoting effect on gastrointestinal tissues, gastrin has been reported to stimulate the proliferation of several cell lines derived from hematopoietic, pituitary, lung, and renal neoplasms as well (15, 19, 29, 37). In particular, gastrin is a potent growth factor for the rat pituitary adenoma cell line GH3 (37). Recent reports have demonstrated that the GH3 cells express CCKB receptors and that they are able to synthesize and secrete gastrin into the culture medium (37). Currently, little is known about the molecular mechanisms that regulate GH3 cell proliferation in response to gastrin. Furthermore, considerable cell type specific differences have been reported with regard to the ability of CCKB receptors to activate intracellular signal transduction pathways (4, 20, 18). For example, stimulation of CCKB receptors in the AR42J cells leads to inhibition of cAMP generation (4), whereas it increases cAMP levels in colon cancer cells (18). Thus we undertook these studies to dissect the signal transduction pathways activated by gastrin in the GH3 cells. In particular, we examined if gastrin would induce MAPK and c-fos gene expression and whether this effect would be involved in the growth-promoting effect of gastrin on these cells. Our data indicate that there are clear cell type specific differences in the requirement of the MAPK pathway for the growth factor action of gastrin. Although in the AR42J cells the trophic effect of gastrin is mediated by MAPK activation and c-fos gene expression, in the GH3 cells, gastrin fails to activate MAPK and induces cell proliferation through signaling pathways that are dependent on Ca2+ mobilization.
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MATERIALS AND METHODS |
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Plasmids.
SRE-Luc (38) 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 and the rat pituitary adenoma cell line
GH3, which are both known to
express receptors for gastrin (4, 37). The cells (obtained from
American Type Culture Collection, Rockville, MD) were grown at 37°C
in 35-mm dishes in DMEM supplemented with 10% fetal bovine serum (FBS)
in 5% CO2-95%
O2. Subconfluent cells were
transfected with 5 µg of the luciferase reporter plasmids and with
0.5 µg of the pCMV-Gal expression vector. Transfections were
carried out using Lipofectamine (GIBCO, Grand Island, NY) as previously
described (31, 32). 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 10 nM human gastrin heptadecapeptide
(Bachem, Torrance, CA) and 10 nM epidermal growth factor (EGF;
Becton-Dickinson, Bedford, MA). At the end of the incubation period,
the cells were washed and lysed, and luciferase assays were performed
as previously described (31, 32). Luciferase activity was expressed as
relative light units.
-Galactosidase activity was measured by the
luminescent light derived from 10 µl of each sample incubated in 100 µl Lumi-Gal 530 (Lumigen, Southfield, MI) and used to normalize the
luciferase assay data for transfection efficiency.
Northern blots. After different periods of incubation with either 10 nM gastrin or 10 nM EGF, the AR42J and the GH3 cells were lysed with TRIzol (GIBCO) according to the manufacturer's instructions. Northern blot hybridization assays were performed as previously described (31). Equal amounts of each RNA sample, with ethidium bromide (10 mg/ml) in a final volume of 20 µl, were electrophoresed on a 1.25% agarose gel containing formaldehyde, and the RNA was transferred from the gel to nitrocellulose filters. The ethidium bromide-stained ribosomal RNA bands in the gel were photographed before and after transfer to ensure that equivalent amounts of RNA were loaded onto each lane and that no residual RNA was left on the gel. The probes used for hybridization analysis were c-fos and glyceraldehyde-3-phosphate dehydrogenase cDNA, obtained from American Type Culture Collection. The cDNA were labeled with [32P]dCTP by the random priming procedure, and the nitrocellulose filters were hybridized to the 32P-labeled cDNA probes as previously described (31).
Immunoprecipitations and in-gel MAPK assay.
Immunoprecipitations and in-gel MAPK assays were performed according to
previously described techniques (31, 32). Briefly, after 5 min of
incubation with either gastrin or EGF, the cells were lysed in 500 µl
of lysis buffer [10 mM KPO4
(pH 7.4), 1 mM EDTA, 5 mM 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. Equal amounts
of proteins from each treatment group (1,000 µg) were incubated with
an ERK2 specific antibody (Santa Cruz Biotechnology, Santa Cruz, CA)
and mixed on a rotating platform for 3 h at 4°C. Protein
concentrations were measured by the Bradford method (3). Control
experiments were conducted by mixing aliquots of the samples with
identical volumes of preimmune sera. Aliquots of protein A agarose
(Santa Cruz Biotechnology) were then added, 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 of 10% SDS, 1.25 ml of 1 M Tris buffer, 2 ml
of 0.1% bromphenol 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 (MBP; Sigma, St. Louis, MO). 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 HCl 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
HEPES (pH 8.0) containing 0.5 mM EGTA, 10 mM
MgCl2, 2 µM cAMP-dependent
protein kinase inhibitor peptide (Sigma), 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) TCA
solution containing 1% (wt/vol) sodium pyrophosphate, dried, and
subjected to autoradiography.
[3H]thymidine incorporation. These studies were conducted according to previously described techniques (25, 32). Briefly, the AR42J and the GH3 cells were grown in 35-mm dishes in DMEM supplemented with 10% FBS in 5% CO2 at 37°C. Subconfluent cells were cultured for 24 h in serum-free DMEM containing 0.2 mM unlabeled thymidine. After the cells were washed with serum-free medium, they were treated with either gastrin (0.1-10 nM) or with the Ca2+ ionophore ionomycin (0.1 µM) for 18 h. In some experiments, the MEK inhibitor PD-98059 (25 µM) (New England Biolabs, Beverly, MA) (1), the PKC inhibitor GF-109203X (1 µM) (Calbiochem, La Jolla, CA), or the calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7; 1 µM) (Calbiochem) was added 30 min before the addition of gastrin. Control experiments were performed by incubating the cells in either incubation buffer or vehicle (0.01% DMSO) without the test substances. DNA synthesis was estimated by measurement of [3H]thymidine incorporation into the TCA-precipitable material according to the method of Seva et al. (25). [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% TCA 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).
Cell growth. Cells were seeded onto six-well plates at a concentration of 10,000 cells/ml. After 24 h, the media were changed to serum-free media containing 1 nM gastrin. Media were changed every 24 h to new serum-free, peptide-containing media, and the cells were counted using a Coulter counter (Coulter Electronics, Hialeah, FL) after 4 days of stimulation.
Western blots. The CCKB receptor was immunoprecipitated from 1,000 µg of either GH3 or AR42J cell lysates with an antibody directed against the NH2 terminus of the receptor (antibody 9421, a gift from John Walsh, Los Angeles, CA). Grb2 was immunoprecipitated from each treatment group using a specific anti-Grb2 antibody (Santa Cruz Biotechnology). Immunoprecipitations and SDS-PAGE were carried out as previously described above. The gels were transferred on a Immobilon-P transfer membrane (Millipore, Bedford, MA) in 25 mM Tris, 150 mM glycine, and 20% methanol. After transfer, the membranes were blocked in 10 ml TBST (20 mM Tris, 0.15 M NaCl, and 0.3% Tween) and 5% dry milk for 2 h and then incubated for 1 h at 37°C in 10 ml TBST, 5% dry milk, containing antibody 9421 (1:10,000). At the end of the incubation period, the membranes were washed in TBST for 30 min at room temperature and then incubated for 1 h in TBST, 5% dry milk, containing protein A directly conjugated to horseradish peroxidase (HRP) (1:2,500) (Amersham Life Science, Arlington Heights, IL). For the anti-phosphotyrosine Western blots, the membranes were blocked in 10 ml TBST and 5% BSA for 2 h and incubated for 20 min at 37°C in TBST, 1% BSA, containing a specific anti-phosphotyrosine antibody directly conjugated to HRP (RC20, Transduction Laboratories, Lexington, KY) (1:2,500). At the end of the incubation period, the membranes were washed in TBST for 15-30 min at room temperature and then exposed to the Amersham enhanced chemiluminescence detection system (Amersham Life Science) according to the manufacturer's instructions.
Binding studies. Binding studies with iodinated human [Leu15]gastrin (sp act ~1.5 µCi/pmol) were done as previously described (24) on isolated cells that were detached in PBS containing 0.02% EDTA. Cells (2 × 106) were incubated with 60 pM 125I-labeled [Leu15]gastrin in a total volume of 1 ml of Krebs-HEPES buffer supplemented with 0.5% BSA, 0.03% soybean trypsin inhibitor, and 0.05% bacitracin, at 37°C until equilibrium. Specific binding was calculated as the difference between the total amount of label bound and the amount of label remaining bound in the presence of 1 µM gastrin. Receptor numbers were calculated as previously described (25) using Scatchard analysis according to the Ligand program of Munson and Rodbard (22).
Data analysis. Data are presented as means ± SE, and n is the number of separate transfections performed with the AR42J cells. Statistical analysis was performed using Student's t-test. P values < 0.05 were considered to be significant.
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RESULTS |
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Both the AR42J and the GH3 cells
are known to express specific binding sites for gastrin (4, 37). To
compare the level of CCKB receptor
expression between the AR42J and the
GH3 cells, we performed Western
blots using a specific CCKB
receptor antibody. As shown in Fig. 1, both
the AR42J and the GH3 cells
exhibited identical levels of CCKB
receptor expression. In addition, displacement of
125I-[Leu15]gastrin
binding by unlabeled gastrin was identical in both cell lines (Fig.
2), demonstrating that the AR42J and the
GH3 cells express equal levels of
CCKB receptors with similar
affinities. Scatchard analysis confirmed that both cell lines expressed
equal levels of functional CCKB
receptors [39,569 ± 4,571 and 44,575 ± 5,572 (SE)
CCKB receptors
(n = 3) in the
GH3 and the AR42J cells, respectively].
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We then examined the effect of gastrin on
GH3 cell proliferation. Gastrin (1 nM) stimulated GH3 cell
proliferation, assessed as increase in total cell number, and this
effect was similar to that observed in the AR42J cells [1.48 ± 0.063-fold induction over control (SE,
n = 5) and 1.52 ± 0.058-fold
induction over control (SE, n = 5) in
the GH3 and the AR42J cells,
respectively] (Fig.
3A). In
agreement with previously reported experiments conducted in the AR42J
cells (25), gastrin induced
[3H]thymidine
incorporation into the GH3 cells
with a maximal effect detected at doses ranging between 0.1 and 1 nM
[1.59 ± 0.08-, 1.54 ± 0.09-, and 1.37 ± 0.08-fold
induction over control (SE, n = 3) in
the presence of 0.1, 1, and 10 nM gastrin, respectively] (Fig.
3B).
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We then confirmed that gastrin induction of
GH3 cell proliferation was
mediated by specific CCKB
receptors. As depicted in Fig. 4, 1 nM
gastrin induced
[3H]thymidine
incorporation into the GH3 cells,
and this effect was blocked by the highly selective
CCKB receptor antagonist D2 [1.55 ± 0.07-fold induction over control (SE,
n = 3) in the presence of 1 nM gastrin
vs. 1.09 ± 0.08-fold induction over control (SE, n = 3) in the presence of gastrin in
combination with 10 nM D2]. Treatment of the cells with the D2
compound alone did not have any significant effect on
GH3 cell proliferation [1.01 ± 0.03-fold induction over control (SE,
n = 3)].
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Gastrin is a potent inducer of both
c-fos gene expression and MAPK
activation in the AR42J cells (8, 31, 32). Thus we undertook studies to
examine if gastrin would have similar actions in the
GH3 cells. As shown in Fig.
5, gastrin failed to induce
c-fos gene expression in the
GH3 cells after 30 min of incubation, whereas it potently stimulated the expression of this gene
in the AR42J cells. Similarly, as shown in Fig.
6, gastrin failed to induce
c-fos gene expression in the
GH3 cells after both 5 and 60 min
of incubation, whereas 10 nM EGF potently induced c-fos in these cells. In addition, in
the GH3 cells, gastrin failed to
stimulate the transcriptional activity of the
c-fos SRE, a regulatory element known
to be the target of growth factor-activated signaling pathways (33)
[1.12 ± 0.06-fold induction over control (SE,
n = 6) in the presence of 10 nM
gastrin and 5.08 ± 0.71-fold induction over control (SE,
n = 6) in the presence of 10 nM
EGF] (Fig.
7B). In
contrast, SRE-luciferase activity was induced by both gastrin and EGF
in the AR42J cells [4.13 ± 0.89-fold induction over control
(SE, n = 7) in the presence of 10 nM
gastrin and 3.63 ± 1.04-fold induction over control (SE,
n = 7) in the presence of 10 nM
EGF] (Fig. 7A). Gastrin also
failed to induce the SRE-luciferase construct in the human embryonic
kidney cell line HEK 293, which is known to express
CCKB receptors (29) (data not
shown), demonstrating that lack of induction of the
c-fos SRE in response to gastrin can
occur in multiple cell types.
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Because MAPK activation is required for induction of the SRE in
response to gastrin stimulation in the AR42J cells (32), we tested the
effect of gastrin on MAPK activity in both the AR42J and the
GH3 cells using in-gel kinase
assays. In agreement with our previous observations (31, 32) and the
luciferase assay data, gastrin induced MAPK in the AR42J cells but
failed to induce this kinase in the
GH3 cells after 5 min of
incubation. In contrast, 10 nM EGF potently induced MAPK in the
GH3 cells, demonstrating the
integrity of this signaling pathway (Fig.
8, A and
B). Identical results were obtained
when we tested the effect of gastrin (10 nM) after 5, 30, and 60 min of
incubation and measured the activity of immunoprecipitated MAPK in an
in vitro kinase assay using MBP as substrate (data not shown).
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Because gastrin induction of the MAPK pathway in the AR42J cells
requires the phosphorylation of the adapter protein Shc and its
association with the adapter protein Grb2 (8), we tested the effect of
gastrin and EGF on Shc phosphorylation and association with Grb2 in the
GH3 cells. First, we confirmed
that gastrin induced phosphorylation of Shc in the AR42J cells. In
agreement with previously published observations (8), gastrin induced
the phosphorylation of the 46- and 52-kDa Shc isoforms (data not
shown). Immunoprecipitation of GH3
cells lysates with an anti-Grb2 antibody followed by Western blots with
an anti-phosphotyrosine antibody revealed the presence of three bands
of ~46, 52, and 66 kDa and of a larger band of 170 kDa in response to
stimulation with EGF (10 nM) but not with gastrin (10 nM) (Fig.
9A).
Identical results were observed when Shc immunoprecipitates were
blotted with an anti-phosphotyrosine antibody (data not shown).
Furthermore, blotting of Shc immunoprecipitates with anti-Shc anti-body
revealed the presence of three bands of ~46, 52, and 66 kDa in all
treatment groups (Fig. 9B). Thus
gastrin, in the GH3 cells, is
unable to induce Shc phosphorylation and association with Grb2, whereas
EGF can induce the tyrosyl phosphorylation of three major Shc isoforms
and of a larger 170-kDa protein that is likely to represent the EGF
receptor. These data demonstrate that gastrin is unable to activate the
MAPK pathway at multiple sites in the
GH3 cells.
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We then confirmed that gastrin induction of
GH3 cell proliferation is
independent of either PKC or MAPK stimulation. For these studies, we
examined the effect of specific inhibitors of either MAPK or PKC on
GH3 cell proliferation, assessed
as [3H]thymidine
incorporation into the cells. As shown in Fig.
10A, gastrin stimulated AR42J cell proliferation, and this effect was inhibited by pretreatment of the cells with the specific MEK inhibitor PD-98059 [1.43 ± 0.054-fold induction over control (SE,
n = 5) in the presence of 1 nM gastrin
vs. 1.07 ± 0.045-fold induction over control (SE,
n = 5) in the presence of gastrin in
combination with 25 µM PD-98059]. In contrast, gastrin
stimulation of GH3 cell
proliferation was unaffected by PD-98059 [1.45 ± 0.04-fold induction over control (SE, n = 5) in
the presence of 1 nM gastrin vs. 1.36 ± 0.031-fold induction over
control (SE, n = 5) in the presence of
gastrin in combination with 25 µM PD-98059] (Fig. 10B). Treatment of either the AR42J
cells or the GH3 cells with PD-98059 alone did not have any significant effect on cell
proliferation [1.04 ± 0.09-fold induction over control (SE,
n = 5) and 1.02 ± 0.027-fold
induction over control (SE, n = 5),
respectively] (Fig. 10, A and
B). Similarly, gastrin-stimulated
AR42J cell proliferation was potently inhibited by pretreatment of the
cells with the specific PKC inhibitor GF-109203X (1 µM) [1.36 ± 0.017-fold induction over control (SE,
n = 5) in the presence of 1 nM gastrin
vs. 0.96 ± 0.052-fold induction over control (SE,
n = 5) in the presence of gastrin in
combination with 1 µM GF-109203X] (Fig.
11A),
whereas this compound failed to affect gastrin induction of
GH3 cell proliferation [1.37 ± 0.04-fold induction over control (SE,
n = 5) in the presence of 1 nM gastrin
vs. 1.28 ± 0.015-fold induction over control (SE, n = 5) in the presence of gastrin in
combination with 1 µM GF-109203X] (Fig.
11B). Treatment of either the AR42J
cells or the GH3 cells with
GF-109203X alone did not have any significant effect on cell proliferation [0.96 ± 0.029-fold induction over control (SE,
n = 5) and 1.01 ± 0.01-fold
induction over control (SE, n = 5)
respectively] (Fig. 11, A and
B).
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Ca2+ is an important and
well-established mediator of gastrin actions in the AR42J cells (4, 8).
In addition, in agreement with previously published studies (27),
gastrin was able to mobilize both intra- and extracellular
Ca2+ in our
GH3 cells (data not shown). Thus
we investigated the role of Ca2+
signaling in gastrin induction of
GH3 cell proliferation. First, we
tested the effect of the Ca2+
ionophore ionomycin (0.1 µM) and observed that this agent stimulated GH3 cell proliferation
[1.430.11-fold induction over control (SE, n = 3)] (Fig.
12B),
whereas it did not have any effect on AR42J cell proliferation
[1.06 ± 0.04-fold induction over control (SE, n = 8)] (Fig.
12A). Although ionomycin (0.1 µM)
was unable to stimulate AR42J cell proliferation, it induced
Ca2+ mobilization in the AR42J
cells (data not shown). We then examined the role of the calmodulin
inhibitor W-7 on gastrin induction of both
GH3 and AR42J cell proliferation.
W-7 is known to inhibit several
Ca2+-activated protein kinases
such as Ca2+/calmodulin kinase II
(39) that are involved in the regulation of cellular proliferation (2,
14). Although W-7 (1 µM) completely inhibited
GH3 cell proliferation [1.37 ± 0.04-fold induction over control (SE,
n = 4) in the presence of 1 nM gastrin
vs. 1.02 ± 0.025-fold induction over control (SE,
n = 4) in the presence of gastrin in
combination with W-7] (Fig.
12B), addition of this compound led
to a partial and modest inhibitory effect on AR42J cell proliferation
[1.72 ± 0.14-fold induction over control (SE, n = 8) in the presence of 1 nM gastrin
vs. 1.42 ± 0.12-fold induction over control (SE,
n = 8) in the presence of gastrin in
combination with W-7] (Fig.
12A). Treatment of either the AR42J
cells or the GH3 cells with W-7
alone did not have any significant effect on cell proliferation
[0.88 ± 0.07-fold induction over control (SE, n = 8) and 1.01 ± 0.015-fold
induction over control (SE, n = 4), respectively] (Fig. 12, A and
B). Thus these data indicate that gastrin stimulates GH3 cell
proliferation through pathways that are at least in part dependent on
Ca2+ signaling and that induction
of Ca2+ mobilization in the AR42J
cells might not be sufficient to sustain cell proliferation.
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DISCUSSION |
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The peptide hormone gastrin regulates numerous, complex cellular functions such as growth, proliferation, and secretion (10, 11, 13, 23-26, 28, 29, 32, 35). The intracellular mechanisms responsible for these effects have been the focus of extensive investigations.
Gastrin is known to activate multiple signal transduction pathways. In fact, this hormone stimulates the hydrolysis of phosphatidylinositol 4,5-bisphosphate by protein lipase C, leading to the generation of diacylglycerol and inositol 1,4,5-trisphosphate. These compounds, in turn, are responsible for the activation of PKC and for the mobilization of intracellular Ca2+ leading, in the AR42J cells, to the phosphorylation of Shc and to the activation of the MAPK pathway (4, 8, 10, 11). Gastrin also induces tyrosine kinase activity, and it stimulates the early response genes c-fos and c-jun (8, 17, 20, 24, 30-32). In a recent study, we have reported that gastrin stimulates AR42J cell growth through induction of the ERK and c-fos (32). Thus activation of the MAPK pathway represents an important mechanism for the transduction of mitogenic signals through the activation of gastrin/CCKB receptors.
A recent study indicated that in the stomach of the rodent Mastomys natalensis gastrin stimulates MAPK and Ras activation only in the ECL cells in which it induces cellular growth, whereas it fails to activate this signaling pathway in the parietal cells where gastrin is unable to stimulate cellular proliferation. In contrast to these findings, the ability of gastrin to induce inositol phospholipid turnover was found to be identical in both cell types (20). Thus, in the stomach of M. natalensis, cell type specific differences appear to regulate the ability of gastrin to induce the MAPK pathway, which, in turn, seems to be specifically linked to the growth-promoting effect of this hormone on the gastric ECL cells.
The complexity of these observations is further underscored by a recent report that investigated the ability of CCKB receptors transfected in Chinese hamster ovary cells (CHO) and Swiss 3T3 fibroblasts to induce cellular proliferation. According to this study, activation of CCKB receptors in CHO cells leads to inhibition of cellular proliferation, whereas it stimulates cellular growth in Swiss 3T3 fibroblasts. Although in the study induction of the MAPK pathway was not examined, activation of CCKB receptors in both cell lines led to identical levels of intracellular Ca2+ mobilization and polyphosphoinositide hydrolysis (12). Taken together, these data suggest that cellular context plays a crucial role in determining the type of biological response that follows activation of the CCKB receptor and that opposing biological responses could occur independently of significant differences in induction of signal transduction pathways.
In our study, we examined the signaling mechanisms that mediate the mitogenic effect of gastrin on the rat pituitary adenoma cell line GH3. Interestingly, we observed that gastrin stimulated the growth of these cells through MAPK- and c-fos-independent pathways. Thus, in contrast to other reports, gastrin was able to induce identical biological responses in both the AR42J and the GH3 cells through the activation of different signaling pathways.
Because gastrin has been shown to interact with receptors other then the CCKB receptor, we explored the possibility that gastrin might stimulate GH3 cell proliferation through interaction with either CCKA or non-A- non-B CCK receptors, which could be linked to different signaling systems (4, 25, 31). However, when we tested the effect of the highly specific CCKB receptor antagonist D2, we observed that this compound completely blocked gastrin induction of GH3 cell proliferation, thus strongly supporting the involvement of CCKB receptors in the growth-promoting effect of gastrin in the GH3 cells.
An important parameter that can influence the coupling of cellular
receptors with their specific signal transduction pathways is the level
of receptor expression (12). In our study, we showed, using Western
blots, binding assays, and Scatchard analysis, that both the
GH3 and the AR42J cells express
identical levels of functional CCKB receptors. These data suggest
that mechanisms other then receptor density must be responsible for the
lack of MAPK induction observed in the
GH3 cells. Another explanation for
our observation might involve the coupling of the
GH3-CCKB
receptor to families of G proteins such as
G12 or
G
13 which in some cell types
are unable to induce MAPK (34). Alternatively, because
seven-transmembrane receptors appear to require internalization for
efficient activation of the MAPK pathway (7), it would be intriguing to
speculate that the lack of MAPK induction might reflect abnormal
mechanisms of CCKB receptor
internalization in the GH3 cells.
Additional studies will be needed to further examine these interesting possibilities.
Changes in intracellular Ca2+ are known to affect numerous cellular functions such as growth and secretion (2, 10, 11, 14). One of the best-characterized intracellular targets for Ca2+ is calmodulin, a protein known to bind Ca2+ and to regulate the function of a large family of protein kinases known as Ca2+/calmodulin-dependent protein kinases (2, 14). These enzymes are widely distributed in eukaryotic tissues, and they are able to regulate the function of genes that are involved in the control of cellular growth (2, 14).
In our study, we observed that gastrin induced
Ca2+ mobilization in the
GH3 cells and that treatment of
these cells with a Ca2+ ionophore
induced cellular proliferation. Furthermore, we showed that the
calmodulin inhibitor W-7 abrogated the stimulatory effect of gastrin on
GH3 cell growth. Thus, in the
GH3 cells, mobilization of
Ca2+ in response to gastrin
stimulation appears to be an important mediator for the integration and
transmission of mitogenic signals. In the present study, we did not
examine the precise nuclear targets of these
Ca2+-activated signaling pathways,
although we speculate that it might involve the transcriptional
activation of nuclear proteins such as CREB, ATF-1, and C/EBP (14).
Definition of the particular Ca2+/calmodulin-dependent protein
kinases involved in this process and elucidation of their specific
nuclear targets will require further investigations.
We also observed that, in the AR42J cells, ionomycin induced Ca2+ mobilization, whereas it had no effect on stimulation of cell proliferation. Furthermore, we noted that gastrin induction of AR42J cell proliferation was only partially inhibited by addition of the calmodulin inhibitor W-7, which was able to block completely the stimulatory effect of gastrin on GH3 cell growth. Taken together, these data suggest that although Ca2+ mobilization is an important element in gastrin signaling, in the AR42J cells it appears to be unable to sustain, by itself, induction of cell proliferation. It could be possible that in the AR42J cells, which exhibit intact PKC and MAPK signal transduction pathways, Ca2+ mobilization is one of the many events that are activated by gastrin. Induction of cell proliferation may therefore require the coordinate interaction of multiple signaling pathways. Additional studies will be needed to examine these interesting possibilities in more detail.
In conclusion, our study indicates that there are cell type specific differences in the requirement of both the MAPK pathway and of Ca2+ mobilization for the growth factor action of gastrin. Although in the AR42J cells gastrin stimulates cell growth through activation of MAPK and c-fos gene expression, in the GH3 cells, gastrin fails to activate MAPK, and it induces cell proliferation through signaling pathways that are dependent on Ca2+ mobilization. Furthermore, induction of Ca2+ mobilization in the AR42J cells appears not to be sufficient to sustain cell proliferation.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grants RO1-DK-33500 and RO1-DK-34306 and funds from the University of Michigan Gastrointestinal Peptide Research Center (NIDDK Grant P30-DK-34933). A. Todisco is a recipient of an American Gastroenterological Association Industry Research Scholar Award, a Clinical Investigator Award from the National Institutes of Health (NIDDK Grant K08-DK-02336), and a grant from the Charles E. Culpeper Foundation Health Program.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. Todisco, 6520 MSRB I, Ann Arbor, MI 48109-0682 (E-mail: atodisco{at}umich.edu).
Received 30 July 1998; accepted in final form 17 February 1999.
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