Departments of 1 Internal Medicine and 2 Pediatrics, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0682
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ABSTRACT |
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Gastrin (G17) has a CCK-B receptor-mediated growth-promoting effect on the AR42J rat acinar cell line. We examined whether G17 inhibits apoptosis induced by serum withdrawal of AR42J cells and CHO-K1 cells stably expressing CCK-B receptors (CHO-K1/CCK-B cells). Cellular apoptosis was measured by flow cytometry and the terminal deoxynucleotidyltransferase-mediated dUTP-FITC nick end-labeling method. Serum withdrawal induced AR42J and CHO-K1/CCK-B cell apoptosis. Addition of 10 nM G17 reversed these effects. We examined the action of G17 (10 nM) on phosphorylation and activation of protein kinase B/Akt, a kinase known to promote cell survival. Akt phosphorylation and activation were measured by kinase assays and Western blots with an anti-phospho-Akt antibody. G17 stimulated Akt phosphorylation and activation. G17 induction of Akt phosphorylation was inhibited by the phosphoinositide 3-kinase (PI 3-kinase) inhibitors LY-294002 (10 µM) and wortmannin (200 nM) but not by the mitogen-activated protein kinase kinase 1 inhibitor PD-98059 (50 µM). To study the role of p38 kinase in G17 signaling to Akt, we examined the effect of G17 on p38 kinase activation and phosphorylation using kinase assays and Western blots with an anti-phospho-p38 kinase antibody. G17 induced p38 kinase activity at doses and with kinetics similar to those observed for Akt induction. The p38 kinase inhibitor SB-203580 inhibited G17 induction of Akt phosphorylation and activation at a concentration (10 µM) 10-fold higher than necessary to block p38 kinase (1 µM), suggesting the possible involvement of kinase activities other than p38 kinase. Transduction of AR42J cells with the adenoviral vector Adeno-dn Akt, which overexpresses an inhibitor of Akt, reversed the antiapoptotic action of G17. In conclusion, G17 promotes AR42J cell survival through the induction of Akt via PI 3-kinase and SB-203580-sensitive kinase activities.
protein kinases; phosphoinositide 3-kinase; Akt kinase; p38 kinase
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INTRODUCTION |
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ALTHOUGH CHARACTERIZED AS a stimulant of gastric acid secretion (10), the peptide hormone gastrin also exerts growth-promoting effects on normal and malignant gastrointestinal tissues (8, 10, 15, 18, 26, 30, 31, 33-36). Gastrin is an important growth factor for the fetal pancreas (5, 37), and in the stomach it is a potent stimulant for the growth of the gastric mucosa (10, 38). Studies (38) conducted in transgenic mice overexpressing amidated gastrin have indicated that gastrin leads to thickening of the fundic mucosa and to multifocal hyperplasia with an increased bromodeoxyuridine proliferation index. Together, these observations confirm the notion that gastrin plays an important role in the regulation of gastrointestinal mucosal growth. Reports (18, 26, 30, 38) have indicated that gastrin induces the growth of colonic and gastric carcinomas both in vivo and in vitro, underscoring the importance of gastrin as a growth factor for gastrointestinal neoplasms.
The intracellular signal transduction pathways activated by gastrin to induce cellular proliferation have been the focus of numerous investigations. Gastrin induces protein tyrosine kinase activity, stimulates phosphoinositide 3-kinase (PI 3-kinase), and activates the extracellular signal-regulated protein kinases (ERKs) or mitogen-activated protein kinases (MAPKs) (8, 10, 16, 40). Indeed, work from our (33-35) laboratory has demonstrated that gastrin stimulates the growth of rat pancreatic adenocarcinoma cells (AR42J) through induction of the ERKs and of the early response gene c-fos through PKC-dependent and -independent mechanisms.
Because growth factors are known to induce both cellular growth and survival (12), we have undertaken studies to examine the role of gastrin in the regulation of cellular apoptosis. We hypothesize that gastrin might be an important physiological inhibitor of apoptosis and that this effect might contribute to its ability to stimulate cellular growth and proliferation.
A signal transduction pathway involving PI 3-kinase and protein kinase B (PKB)/Akt has recently been shown (6, 7) to play an important role in the inhibition of cellular apoptosis. Akt is homologous to the PKA and PKC families of protein kinases (6, 7). In vivo, the activity of Akt is regulated by growth factors and serum through the induction of PI 3-kinase (6, 7). Phosphorylation of Akt appears to be critical for Akt activation (6, 7). The major phosphorylation sites required for activation of Akt have been identified as threonine 308 and serine 473, which are the targets of the phosphoinositide-dependent kinases I (PDK1) and II (PDK2), respectively (6, 7). Recent investigations (6, 7) have suggested that growth factors induce the activation of PI 3-kinase, which in turn leads to the production of phosphatidylinositol-3,4-bisphosphate at the cell membrane. Akt binds to this lipid, dimerizes, and is stabilized in a partially active state (6, 7). The location at the membrane and the dimerization appear to enhance the ability of Akt to be phosphorylated and fully activated (6, 7).
Akt appears to send numerous survival signals in response to growth factors such as epidermal growth factor (EGF), nerve growth factor, platelet-derived growth factor, insulin growth factor-1, insulin, and interleukin-3 (6, 7). A recent study (22) has shown that ligands for seven transmembrane G protein-coupled receptors, such as carbamylcholine, can also activate Akt in both PC-12 and COS-7 cells, stably expressing either the M1 or M2 muscarinic receptor. The mechanisms that regulate the activation of Akt in response to growth factor stimulation have only been partially characterized. In addition, the intracellular signal transduction pathways that mediate the antiapoptotic actions of gastrin are unknown. Accordingly, we undertook these studies to investigate the action of gastrin on cellular apoptosis induced by serum withdrawal and to dissect the signal transduction pathways responsible for this effect.
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MATERIAL AND METHODS |
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Plasmids and adenoviral vectors.
Bacteria transformed with the expression plasmid for
glutathione-S-transferase-activated transcription
factor-2-(1-109)
[GST-ATF2-(1-109)] were a gift of J. Han (Scripps
Research Institute, La Jolla, CA). The replication-defective adenoviral
vector expressing dominant-negative, hemoagglutinin (HA)-tagged Akt
(with serine 473 and threonine 308 mutated to alanine) under the
control of the cytomegalovirus (CMV) promoter (Adeno-dnAkt) was a gift
of K. Walsh (Tufts University, Boston, MA) (11). The
adenoviral vector expressing the -galactosidase enzyme under the
control of the CMV promoter (AD.CMV-
-gal) was previously described
(3).
Cell culture and infection.
For our experiments, we used both the rat exocrine pancreatic cell line
AR42J (obtained from American Type Culture Collection, Rockville, MD),
which is known to express receptors for both gastrin (G17) and EGF
(8), and CHO-K1 cells stably expressing the CCK-B receptor
(CHO-K1/CCK-B cells) (a gift of I. Song, University of Michigan). The
AR42J cells were grown at 37°C in DMEM supplemented with 10% fetal
bovine serum (FBS) and 1% sodium pyruvate. The CHO-K1/CCK-B cells were
grown at 37°C in DMEM supplemented with 10% FBS and 0.05% Geneticin
(GIBCO BRL, Grand Island, NY). Both cell types were maintained in 5%
CO2-95%O2. For the apoptosis studies, the
cells were cultured in serum-free medium (either DMEM or Ham's F-12
nutrient mixture; GIBCO BRL) for 72 h. In the experiments
performed with CHO-K1/CCK-B cells, gastrin (G17) (10 nM) (Bachem,
Torrance, CA) was added at the time of serum starvation. In the AR42J
cell studies, the cells were serum starved, transduced when indicated
with 600 multiplicities of infection (MOI) of either the adenoviral
vector expressing dominant-negative Akt or that expressing
-galactosidase for 16 h, and then treated with or without
gastrin for an additional 56 h. Control experiments were performed
in nontransduced AR42J cells according to an identical protocol. For
all other studies, the cells were starved for 24 h in serum-free
medium and then treated for different time periods with either gastrin
(1-100 nM) or EGF (10 nM). In some experiments, PD-98059 (50 µM;
New England Biolabs, Beverly, MA) (2), SB-203580 (0.1-10 µM;
Calbiochem, La Jolla, CA) (17, 39), LY-294002 (10 µM;
Calbiochem), and wortmannin (200 nM; Calbiochem) were added 30 min
before the addition of gastrin. PD-98059, SB-203580, LY-294002, and
wortmannin were dissolved in DMSO (Sigma Chemical, St. Louis, MO). 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.
Immunoprecipitations and kinase assays.
Immunoprecipitations and kinase assays were performed according to
previously described techniques (25, 28, 35) with minor
modifications. The AR42J cells were lysed in 500 µl of lysis buffer
[50 mM HEPES, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EDTA,
1.5 mM MgCl2, 1 mM Na3VO4, 10 mM
NaF, 10 mM
Na4P2O7 · 10H2O,
1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (ICN
Biomedicals, Aurora, OH), 1 µg/ml leupeptin, and 1 µg/ml aprotinin]. The lysates were transferred into Microfuge tubes and spun
at 16,000 g for 20 min at 4°C. Equal amounts of protein from each treatment group (300 µg) were incubated with either an
anti-p38 kinase antibody (sc-535, Santa Cruz Biotechnology, Santa Cruz,
CA) or with a monoclonal anti-Akt kinase antibody (New England
Biolabs). Protein concentrations were measured by the Bradford method
(4). Aliquots of protein A-Sepharose (50 µl) (Pharmacia
Biotech, Piscataway, NJ) were then added, and the solutions were mixed
for an additional hour. After centrifugation, the pellets were washed
once with lysis buffer and twice with kinase buffer. Immunoprecipitated
p38 kinase was used to phosphorylate 2 µg of
GST-ATF2-(1-109). Kinase reactions were carried out
in 20 µl of kinase buffer (18 mM HEPES, pH 7.4, 10 mM magnesium
acetate, 50 µM ATP, and 2.5 µCi/sample [-32P]ATP)
at 30°C for 30 min. Reactions were terminated by addition of 20 µl
of 5× electrophoresis buffer (for 5 ml: 2.5 ml glycerol, 1.25 ml
2-mercaptoethanol, 0.5 g SDS, 1.043 ml 1.5 M Tris, pH 6.8, and
1.25 mg bromophenol blue). The samples were then boiled for 5 min and
applied to a 10% SDS-polyacrylamide gel, followed by staining with
Coomassie blue and destaining (25) to ensure that
identical amounts of proteins were loaded on the gel. Labeled phosphoproteins were visualized by autoradiography and quantitated by
scanning densitometry. GST-ATF2-(1-109) was expressed
and purified from Escherichia coli as previously described
(25). Immunoprecipitated Akt kinase was used to
phosphorylate glycogen synthase kinase-3
(GSK-3
). GSK-3
phosphorylation was measured by Western blot analysis with a specific
anti-phospho-GSK-3
/
antibody. Reactions were carried out
according to the instructions of the Akt kinase assay kit from New
England Biolabs.
Western blots.
AR42J cell lysates (80 µg) were loaded on 10% SDS-polyacrylamide
minigels and run at 20 A for 8 h. The gels were transferred on an
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 of TBST (20 mM Tris, 0.15M NaCl, and 0.3% Tween) and
5% dry milk for 1 h. The membranes were then incubated for
16-18 h at 4°C in 10 ml of TBST and 5% dry milk, containing
either a specific anti-phospho-Akt antibody that recognizes
phopshorylated serine 473 of Akt kinase (1:1,000) or a specific
anti-phospho-p38 antibody that recognizes phosphorylated threonine 180 and phosphorylated tyrosine 182 of p38 kinase (1:1,000) (New England
Biolabs). Control blots were performed using antibodies recognizing
either Akt kinase or p38 kinase independent of their phosphorylation
states (1:1,000) (New England Biolabs). For the GSK-3 phosphoblots,
the membranes were incubated for 16-18 h at 4°C in 10 ml TBST
and 5% BSA (Sigma Chemical) containing an anti-phospho-GSK-3
/
antibody (1:1,000) that recognizes phosphorylated serine 21 of GSK-3
and phosphorylated serine 9 of GSK-3
(New England Biolabs). 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 and 5%
dry milk, containing either protein A, directly conjugated to
horseradish peroxidase (HRP) (Amersham Life Science, Arlington Heights,
IL) (1:2,500), for the Akt blots or an HRP-conjugated anti-rabbit
secondary antibody (1:2,000) for the GSK-3
blots. The membranes were
washed in TBST for 30 min at room temperature and then exposed to the
Amersham enhanced chemiluminescence detection system according to the
manufacturer's instructions.
Amplification and purification of adenoviral vectors.
Briefly, recombinant adenoviruses expressing dominant negative Akt and
-galactosidase were amplified as described previously using 293 cells (3). The viruses were subsequently concentrated and
purified on a cesium chloride gradient (3). The
concentration of the recombinant adenoviruses was assessed on the basis
of the absorbance at 260 nm and on a limiting dilution plaque assay
(3).
Immunohistochemistry. The AR42J cells were transduced with the adenoviral vector expressing HA-tagged dominant- negative Akt and cultured on slides for 24 h. At the end of the incubation period the cells were fixed in 4% formalin-PBS. The slides were blocked for 30 min with 20% donkey serum and incubated for 2 h with a mouse monoclonal anti-HA antibody (1:500) (Babco). The cells were rinsed with PBS, and a 1:150 dilution of a FITC-conjugated donkey anti-mouse IgG secondary antibody (Jackson Immunoresearch Laboratories, West Grove, PA) was added for 1 h. After being washed with PBS, the cells were mounted in Vectashield (Vector Laboratories, Burlingame, CA) and visualized by fluorescence microscopy. In control experiments the cells were incubated with the FITC-conjugated secondary antibody without the primary anti-HA antibody.
Detection of adenoviral-delivered -galactosidase.
For identification of AR42J cells transduced with the adenoviral vector
expressing
-galactosidase, the cells were cultured on slides for
24 h and stained with 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside (X-gal) after 24 h of
infection. The cells were washed with PBS and then fixed in 0.5%
glutaraldehyde at room temperature for 10 min. After two washes with 1 mM MgCl2 in PBS, the cells were incubated overnight at
37°C in a solution consisting of 5 mM
K3Fe(CN)6, 5 mM
K4Fe(CN)6, and 2 mM MgCl2 in PBS
with 0.1% X-gal. At the end of the incubation, the cells were rinsed
with PBS and observed with a light transmission microscope.
Terminal deoxynucleotidyltransferase-mediated dUTP-FITC nick
end-labeling method.
The AR42J cells were cultured on coverslips and fixed in 4%
neutral-buffered formalin for 15 min followed by methanol for 10 min.
The cells were stored at 20°C for 24 h. Apoptosis was detected
using the terminal deoxynucleotidyltransferase-mediated dUTP-FITC nick
end-labeling (TUNEL) method as previously described (32).
The coverslips were washed with cold PBS and incubated with
biotin-conjugated dUTP and terminal transferase enzyme for 1 h at
37°C. After washing, the cells were incubated with FITC-avidin for
1 h at room temperature (light protected). The specimens were mounted in SlowFade containing 5 µg/ml propidium iodide and 0.05 mg/ml DNase-free RNase. The FITC-labeled DNA fragments in the apoptotic
cells were visualized using a fluorescence microscope. Two hundred
cells were counted blindly, and the positive cells were expressed as a
percentage of the total cells counted. This included adherent early
necrotic cells that stained with propidium iodide but not with FITC.
The negative control received only the label solution without terminal transferase.
Flow cytometric assays. DNA fragmentation was determined by DNA staining of isolated nuclei by flow cytometry (21). The cells were harvested by gentle scraping, washed, and lysed in a buffer containing of 0.1% sodium citrate, 0.01% Triton X-100, and 0.1 mg/ml propidium iodide. After incubation at 4°C, the nuclei were analyzed for DNA content by flow cytometry. Nonapoptotic nuclei were distinguished from apoptotic nuclei on the basis of their tight scatter profile. Flow cytometry was performed by the Biomedical Flow Cytometry Core Facility of the Cancer Center of the University of Michigan.
Cell viability assay. After 72 of culture in either 10% FBS or in serum-free medium, AR42J and CHO-B cell viability was measured by exposing the cells to 0.2% trypan blue (Sigma Chemical) for 10 min and counting the number of blue-positive cells per 200 cells in a blinded manner (32).
[3H]Thymidine incorporation. These studies were conducted according to previously described techniques (29). Briefly, the AR42J 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 washing with serum-free medium, the cells were treated with 1 nM gastrin for an additional 24 h. Control experiments were performed by incubating the cells in incubation buffer. DNA synthesis was estimated by measurement of [3H]thymidine incorporation into the TCA precipitable material according to the method of Seva et al. (29). [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 medium to remove unincorporated [3H]thymidine, and 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).
Data analysis. Data are presented as means ± SE. Statistical analysis was performed using Student's t-test. P < 0.05 was considered significant.
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RESULTS |
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We investigated the effect of gastrin on apoptosis induced by
serum withdrawal. For these studies, AR42J cell apoptosis was measured
by quantitating DNA fragmentation with the TUNEL method. As shown in
Fig. 1, culture of the AR42J cells in
serum-free medium for 72 h increased AR42J cell apoptosis by
fourfold. Incubation of the cells in serum-free medium
containing 10 nM gastrin significantly inhibited this effect.
Measurement of AR42J cell apoptosis by flow cytometry yielded similar
results (data not shown). To confirm the validity of these observations
and to demonstrate the specific involvement of CCK-B receptors in the
antiapoptotic action of gastrin, we performed experiments using CHO-K1
cells stably expressing the CCK-B receptor (CHO-K1/CCK-B). CHO-K1/CCK-B
cell apoptosis was measured using flow cytometry. Serum withdrawal for
72 h induced CHO-K1/CCK-B cell apoptosis, whereas addition of 10 nM gastrin potently reversed this effect (Fig.
2). The specificity of this finding was
confirmed by the observation that gastrin had no effect on the
survival of wild-type CHO-K1 cells, which do not express CCK-B
receptors (data not shown). As indicated by the trypan blue exclusion
assays, serum starvation for 72 h did not significantly affect
both AR42J and CHO-K1/CCK-B cell viability. In particular, AR42J cell
viability was 94% in the presence of 10% FBS and 93% in serum-free
conditions. Similarly, CHO-K1/CCK-B cell viability was 97% and 96% in
the presence and absence of 10% FBS, respectively. Thus gastrin
inhibits serum withdrawal-induced apoptosis of both AR42J and
CHO-K1/CCK-B cells.
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To examine the signal transduction pathways activated by gastrin to
suppress AR42J cell apoptosis, we investigated the effect of gastrin on
Akt kinase, a signaling molecule known to play an important role in the
inhibition of cellular apoptosis (6, 7). First, we studied
the effect of gastrin on Akt phosphorylation using Western blots with a
specific anti-phospho-Akt antibody directed against phosphoserine 473 of Akt. Gastrin (10 nM) stimulated Akt phosphorylation, with a maximal
effect detected between 5 and 30 min of incubation (Fig.
3). Similar results were observed in the
presence of 100 nM gastrin, indicating that maximal phosphorylation of
Akt by gastrin is achieved at doses ranging between 10 and 100 nM (data
not shown). Total Akt levels were monitored by Western blot analysis
with an antibody recognizing Akt independent of its phosphorylation
state (Fig. 3).
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To confirm that phosphorylation of Akt correlates with Akt activation,
we conducted studies in which we examined the effect of gastrin on Akt
activity. For these experiments, Akt was immunoprecipitated with a
specific anti-Akt antibody and its activity was measured in an in vitro
kinase assay using GSK-3 as substrate. GSK-3
phosphorylation was
measured by Western blot analysis with a specific anti-phospho-GSK-3
/
antibody. As shown in Fig.
4, 10 nM gastrin induced Akt activation,
confirming the notion that phosphorylation of Akt on serine 473 correlates with Akt activation. Levels of immunoprecipitated Akt were
monitored by Western blot analysis with an antibody recognizing Akt
independent of its phosphorylation state (Fig. 4).
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Gastrin is known to induce PI 3-kinase in AR42J cells
(16). Because activation of PI 3-kinase is an important
step in the signaling pathway that leads to Akt activation in response
to growth factor stimulation (6, 7), we undertook studies
to examine whether gastrin induction of Akt phosphorylation requires activation of PI 3-kinase. As shown in Fig.
5, gastrin stimulated Akt
phosphorylation, and this effect was inhibited by both 10 µM
LY-294002 and 200 nM wortmannin, two specific and well-characterized inhibitors of PI 3-kinase, indicating that gastrin targets Akt through
PI 3-kinase-dependent signaling pathways.
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We further dissected the signal transduction pathways that regulate Akt
activation in response to gastrin stimulation. In particular, we
investigated the role of p38 kinase, a signaling molecule involved in
the regulation of cellular growth and survival (39), in
gastrin induction of Akt phosphorylation. First, we examined the effect
of gastrin on p38 kinase activity. p38 was immunoprecipitated from
AR42J cell lysates with a specific anti-p38 kinase antibody, and its
activity was measured in an vitro kinase assay using transcription
factor ATF2 as substrate. As shown in Fig.
6, gastrin potently induced p38 kinase
activity in a time- and dose-dependent fashion (Fig. 6) with a maximal
stimulatory effect detected between 10 and 100 nM after 5 min of
incubation. EGF (10 nM) failed to induce p38 kinase activity in AR42J
cells (data not shown).
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To define the role of p38 kinase in gastrin signaling to Akt, we
examined the effect of the p38 kinase inhibitor SB-203580 on gastrin
induction of Akt phosphorylation. Although SB-203580 is considered to
be a specific inhibitor of p38 kinase, a recent study (19)
has indicated that this agent can also inhibit the activity of PDK1.
Thus we conducted a detailed dose-response analysis of the effects of
SB-203580 (0.1-10 µM) on the phosphorylation of both Akt and p38
kinase. As shown in Fig. 7, 1 µM
SB-203580 completely inhibited p38 kinase phosphorylation. In contrast, a 10-fold higher concentration of SB-203580 (10 µM) was required to
achieve complete inhibition of Akt phosphorylation. Identical results
were observed when the effects of similar doses of SB-203580 were
tested on the kinase activities of both Akt and p38 kinase (data not
shown). These data indicate that SB-203580-sensitive kinase activities
other than p38 kinase might be involved in gastrin induction of Akt
phosphorylation.
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To further establish the specificity of the effect of SB-203580 on Akt
phosphorylation, we investigated whether this agent would also inhibit
the stimulatory action of EGF (10 nM). As indicated in Fig.
8, EGF induction of Akt phosphorylation
was unaffected by SB-203580 (10 µM). This suggests that gastrin, but
not EGF, activates SB-203580-sensitive kinase activities in the AR42J
cells.
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Gastrin induces MAPK in the AR42J cells (35, 40). Because
MAPK is known to be involved in the regulation of apoptosis, we
investigated the role of this kinase in gastrin induction of Akt
phosphorylation. As shown in Fig. 9,
gastrin (10 nM) induction of Akt phosphorylation was completely
inhibited by SB-203580 (10 µM) but not by the highly specific MAPK
kinase (MEK) inhibitor PD-98059 (50 µM), suggesting that gastrin
targets Akt via MEK-independent mechanisms.
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To confirm the role of Akt in the antiapoptotic action of gastrin, we
conducted studies in which we transduced the AR42J cells with the
AD.CMV--gal or Adeno-dnAkt adenoviral vector. First, we performed
experiments to demonstrate that AR-2J cells can be successfully
transduced with these adenoviral vectors. Figure 10B shows histochemical
staining of the AR42J cells for
-galactosidase after infection of
the cells with 600 MOI of the adenoviral vector AD.CMV-
-gal for
16 h. Examination of 100 cells in three separate slides revealed
that that 67 ± 0.88% (n = 3) of the cells were successfully infected with AD.CMV-
-gal. No staining was detected in
control, noninfected AR42J cells that were subjected to the same
staining protocol, as shown in Fig. 10A. Immunocytochemical staining of Adeno-dnAkt-transduced AR42J cells with an anti-HA mouse
monoclonal antibody and a donkey anti-mouse FITC-conjugated secondary
antibody indicated that ~70% of the AR42J cells were transduced by
this adenoviral vector (Fig. 10C). Control experiments conducted with AR42J cells transduced with the adenoviral vector AD.CMV-
-gal and incubated with both the primary and the secondary antibodies demonstrated only faint background fluorescence (Fig. 10D). Furthermore, infection with AD.CMV-
-gal did not
alter the function of the AR42J cells, because both infected and
noninfected cells showed identical growth responses to gastrin
stimulation. In particular, gastrin induced [3H]thymidine
incorporation into noninfected AR42J cells by 1.52 ± 0.1-fold
(n = 3) over unstimulated control cells and by
1.65 ± 0.07-fold (n = 3) into AR42J cells that
were infected with 600 MOI of the AD.CMV-
-gal adenoviral vector.
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Using the TUNEL method, we observed that transduction of the AR42J
cells with the adenoviral vector expressing dominant-negative Akt, but
not with that expressing -galactosidase, reversed the antiapoptotic
action of gastrin, underscoring the functional importance of Akt in
gastrin-mediated inhibition of AR42J cell apoptosis (Fig.
11). Serum starvation-induced apoptosis
of AD.CMV-
-gal-transduced AR42J cells was identical to that of
nontransduced cells, indicating that AD.CMV-
-gal. does not affect
AR42J cell apoptosis (Figs. 1 and 11). Similarly, transduction of the
AR42J cells with AD.CMV-
-gal failed to affect AR42J cell viability
(data not shown).
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DISCUSSION |
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The peptide hormone gastrin regulates numerous complex cellular functions such as growth, proliferation, and secretion (8, 10, 15, 18, 26, 30, 31, 33-36). In this study, we have demonstrated that gastrin is also a potent inhibitor of cellular apoptosis.
Apoptotic cell death plays an important role in the process of regulated growth and development of multicellular organisms (12, 13). Cells are targeted by both death-inducing and survival-promoting agents. The ultimate fate of a cell is therefore determined by the integration of these complex and opposing signals. Cells undergoing apoptosis exhibit specific morphological changes such as DNA fragmentation, chromatin condensation, membrane blebbing, cell shrinkage, and disassembly (12, 13).
The molecular mechanisms that regulate cellular apoptosis have been the focus of numerous investigations. In particular, phosphorylation and dephosphorylation of cellular proteins appear to be important mechanisms for the transmission and integration of both death-inducing and survival-promoting signals. Numerous protein kinases and phosphatases have been shown to initiate complex programs of cellular activation that lead to either the induction or inhibition of cellular apoptosis (12, 14, 20, 23, 27, 39, 41).
PKB/Akt, in particular, is known to induce cell survival (6, 7, 9). Akt phosphorylates several cellular proteins that are known to play an important role in the induction of apoptosis. Akt phosphorylates the proapoptotic proteins Bad (6, 7, 9) and caspase 9 in vivo (6, 7). Akt also phosphorylates and inactivates nuclear proteins such as those belonging to the family of forkhead/winged-helix transcription factors that appear to be important for the transcription and expression of proapoptotic molecules such as Fas ligand (7). Accordingly, the Akt signaling pathway appears to function as a crucial element for the transmission of survival signals in multiple cell types.
In addition to Akt, some of the best characterized signal transduction
pathways known to modulate cellular apoptosis are the MAPK, c-Jun
NH2-terminal kinase, and p38 kinase signaling cascades (39). The specific intracellular targets of these kinases
have been only partially characterized. The role of p38 kinase in the regulation of cellular apoptosis appears to be complex. Although in
some systems activation of p38 kinase is linked to induction of
apoptosis (39), in others, such as NIH/3T3 cells
(27), Jurkat cells (23), and cardiac myocytes
(41), induction of p38 kinase appears to have a protective
effect. Furthermore, the kinetics of p38 kinase activation appear to
play an important role in the regulation of cell survival. A recent
report (27) has suggested that in some systems tumor
necrosis factor- (TNF-
) induces p38 kinase activity with biphasic
kinetics. Although the first phase of activation appears to be
transient and to protect cells from TNF-
-induced apoptosis, the
second phase has no effect on cell survival.
We have demonstrated that gastrin stimulates both the activity and phosphorylation of p38 kinase with kinetics similar to those observed in systems in which activation of p38 kinase is linked to induction of cell survival (27). Furthermore, we have reported that gastrin induction of Akt phosphorylation could be effectively and specifically blocked by 10 µM SB-203580, a well-characterized inhibitor of p38 kinase. Indeed, it has been suggested that the p38 kinase signal transduction pathway can phosphorylate Akt in vitro (1) and that SB-203580 can inhibit, although weakly, the activation of Akt stimulated by the expression of a constitutively active form of the small GTP-binding protein Rac (24). However, in our study, we have also observed that the concentration of SB-203580 necessary to affect gastrin stimulation of Akt phosphorylation is 10-fold higher than that required to block the stimulatory action of gastrin on p38 kinase activity. Accordingly, these findings raise the possibility that SB-203580-sensitive kinases other than p38 kinase could be activated by gastrin to induce Akt in AR42J cells. Interestingly, similar results were observed in a recent study (19) conducted in a murine T cell line in which SB-203580 was found to block interleukin-2-stimulated Akt activation through inhibition of PDK1, independently of p38 kinase. Thus SB-203580 might inhibit the stimulatory effect of gastrin on Akt through its ability to block PDK1 activation.
Alternatively, the difference in the concentrations of SB-203580
required to inhibit Akt and p38 kinase might reflect the presence of
several isoforms of p38 kinase in the AR42J cells. Mammalian cells are
known to express multiple isoforms of p38 kinase (p38 ,
,
2,
, and
) (17, 39). Whereas some of these isoforms are highly sensitive to SB-203580 (p38
and
2), others are either partially (p38
) or completely
resistant to inhibition by SB-203580 (p38
and
)
(17). The antibodies used in our studies predominantly
recognize p38 kinase-
, commonly referred to as p38 kinase. In
addition, although p38 kinase-
and -
appear to share numerous
similarities, one study (23) has indicated that p38
kinase-
might play a greater role in the inhibition of cellular
apoptosis than the other isoforms of p38 kinase. Accordingly, it is
possible that in our system gastrin might activate p38 kinase-
,
which is only partially inhibited by SB-203580, to stimulate Akt and
inhibit AR42J cell apoptosis. This possibility would explain the fact
that a higher concentration of SB-203580 was required to inhibit the
stimulatory effect of gastrin on Akt activation. It is clear that
additional studies are necessary to characterize in more detail the
specific isoforms of p38 kinase that are activated by gastrin in the
AR42J cells and to gain more insight into the role of PDK1 in the
antiapoptotic action of gastrin.
We have also demonstrated that MEK1 does not play any role in either the phosphorylation or activation of Akt. This observation is in agreement with studies (9) demonstrating that the MAPK pathway is not involved in the activation of Akt in other cellular systems.
Ligands for seven transmembrane G protein-coupled receptors, such as
carbamylcholine, can activate Akt in PC-12 and COS-7 cells stably
expressing either the M1 or M2 muscarinic
receptor through signaling pathways that involve PI 3-kinase and
-subunits of GTP-binding proteins (22). In our
study, we have demonstrated that gastrin inhibits cellular apoptosis
induced by serum withdrawal through the activation of specific
gastrin/CCK-B receptors. We have also dissected the signal transduction
pathways responsible for this effect using AR42J cells, which express
endogenous gastrin/CCK-B receptors, as our model. In this system we
have demonstrated that gastrin induces Akt phosphorylation through the
activation of PI 3-kinase and SB-203580-sensitive kinase activities
that need to be further characterized. In addition, we have
demonstrated that gastrin inhibits AR42J cell apoptosis through a
signal transduction pathway that involves the activation of Akt,
because the antiapoptotic action of gastrin was inhibited by an
adenoviral vector expressing a dominant-negative Akt gene.
In conclusion, gastrin is a potent inhibitor of cellular apoptosis through its ability to induce PKB/Akt. This novel action of gastrin is likely to represent an important mechanism responsible for the growth-promoting effect of the hormone on both normal and neoplastic gastrointestinal tissues.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. John Wiley for helpful advice and Bill Malone and Daniel Miller for technical assistance.
![]() |
FOOTNOTES |
---|
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants RO1-DK-34306 and RO1-DK-47398 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 the recipient of an American Gastroenterological Association Industry Research Scholar Award, a Clinical Investigator Award from the National Institutes of Health (National Institute of Diabetes and Digestive and Kidney Diseases Grant K08-DK-02336), and a grant from the Charles E. Culpeper Foundation Health Program.
Address for reprint requests and other correspondence: A. Todisco, 6520 MSRB I, Ann Arbor, MI 48109-0682 (E-mail: atodisco{at}umich.edu).
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. Section 1734 solely to indicate this fact.
Received 22 February 2000; accepted in final form 29 August 2000.
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