Prostaglandin E2 Protects Gastric Mucosal Cells from
Apoptosis via EP2 and EP4 Receptor
Activation*
Tatsuya
Hoshino
,
Shinji
Tsutsumi
,
Wataru
Tomisato
,
Hyun-Jung
Hwang
,
Tomofusa
Tsuchiya
, and
Tohru
Mizushima
§¶
From the
Faculty of Pharmaceutical Sciences, Okayama
University and § PRESTO, Japan Science and Technology
Corporation, Okayama 700-8530, Japan
Received for publication, November 27, 2002, and in revised form, January 27, 2003
 |
ABSTRACT |
Prostaglandin E2
(PGE2) has a strong protective effect on the gastric mucosa
in vivo; however, the molecular mechanism of a direct
cytoprotective effect of PGE2 on gastric mucosal cells has
yet to be elucidated. Although we reported previously that PGE2 inhibited gastric irritant-induced apoptotic DNA
fragmentation in primary cultures of guinea pig gastric mucosal cells,
we show here that PGE2 inhibits the
ethanol-dependent release of cytochrome c from
mitochondria. Of the four main subtypes of PGE2 receptors, we also demonstrated, using subtype-specific agonists, that
EP2 and EP4 receptors are involved in the
PGE2-mediated protection of gastric mucosal cells from
ethanol-induced apoptosis. Activation of EP2 and
EP4 receptors is coupled with an increase in cAMP, for
which a cAMP analogue was found here to inhibit the ethanol-induced apoptosis. The increase in cAMP is known to activate both protein kinase A (PKA) and phosphatidylinositol 3-kinase pathways. An inhibitor
of PKA but not of phosphatidylinositol 3-kinase blocked the
PGE2-mediated protection of cells from ethanol-induced
apoptosis, suggesting that a PKA pathway is mainly responsible for the
PGE2-mediated inhibition of apoptosis. Based on these
results, we considered that PGE2 inhibited gastric
irritant-induced apoptosis in gastric mucosal cells via induction of an
increase in cAMP and activation of PKA, and that this effect was
involved in the PGE2-mediated protection of the gastric
mucosa from gastric irritants in vivo.
 |
INTRODUCTION |
Prostaglandins (PGs),1
one of the major groups of chemical mediators in the mammalian body,
are involved in numerous physiological reactions, such as inflammation
and cellular differentiation (1). PGs, especially PGE2,
also have strong cytoprotective effects on the gastric mucosa as a
consequence of various indirect mechanisms that include increased
epithelial mucus production and bicarbonate secretion (2, 3),
inhibition of gastric motility (4), inhibition of acid secretion (5),
amelioration of mucosal blood flow (6), inhibition of free radical and
enzyme release from neutrophils (7), and vascular, luminal, and/or
extrinsic and intrinsic neural mechanisms (8). In contrast, it is still
unclear as to whether or not PGE2 directly protects gastric
mucosal cells from various gastric irritants.
Gastropathy, such as gastric ulcer and gastritis, is caused by damage
to the gastric mucosa due to its exposure to various gastric irritants
such as ethanol and acids. It appears that these gastric irritants
damage the gastric mucosa by inducing not only necrosis but also
apoptosis in gastric mucosal cells (9). For example, a stimulated rate
of apoptosis of gastric mucosal cells was reported at the onset of
gastric ulceration (10). Apoptosis associated with
Helicobacter pylori infection was suggested to be involved
in the development of atrophic gastritis caused by H. pylori
infection (11). We recently reproduced such gastric irritant-induced
apoptosis in vitro by using primary cultures of guinea pig
gastric mucosal cells. Various gastric irritants (non-steroidal
anti-inflammatory drugs (NSAIDs), ethanol, hydrogen peroxide, and
hydrochloric acid) induced apoptotic DNA fragmentation, chromatin
condensation, and caspase activation (9, 12-14). We also found that
these gastric irritants induced apoptosis through a common pathway in
which mitochondrial dysfunction plays an important role (13). In order
to examine the direct cytoprotective effect of PGE2 on
gastric mucosal cells, we investigated previously the effect of
PGE2 on this gastric irritant-induced apoptosis in cultured guinea pig gastric mucosal cells, and we found that PGE2
inhibited the apoptosis caused by various gastric irritants (ethanol,
hydrogen peroxide, and hydrochloric acid) (15). The molecular mechanism governing this inhibitory effect of PGE2 on apoptosis has,
however, yet to be elucidated. For example, although PGE2
receptors have been pharmacologically subdivided into four main
subtypes (EP1, EP2, EP3, and
EP4) (16), the EP subtype involved in the
PGE2-mediated inhibition of apoptosis in gastric mucosal
cells has not been revealed. In this study, we examine the molecular
mechanism of this PGE2-mediated inhibition of
ethanol-induced apoptosis in gastric mucosal cells, and we suggest that
PGE2 inhibits gastric irritant-induced apoptosis through
EP2- and EP4-mediated increases in cAMP and
activation of protein kinase A (PKA).
 |
EXPERIMENTAL PROCEDURES |
Chemicals, Media, and Animals--
Fetal bovine serum and
trypsin were purchased from Invitrogen. RPMI 1640 was obtained from
Nissui Pharmaceutical Co. (Tokyo, Japan). Pronase E and type I
collagenase were purchased from Kaken Pharmaceutical Co. (Kyoto, Japan)
and Nitta Gelatin Co. (Osaka, Japan), respectively. pCPT-cAMP (a cAMP
analogue), wortmannin, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide
(H-89), PGE2, and
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were
from Sigma. Peptides for the assay of caspases were from Peptide
Institute, Inc. (Osaka, Japan). ONO-DI-004 (an EP1
agonist), ONO-AE1-259-01 (an EP2 agonist), ONO-NT-012 (an EP3 agonist), and ONO-AE1-329 (an EP4 agonist)
were gifts kindly provided by Ono Pharmaceutical Co., Ltd. (Osaka,
Japan). Anti-cytochrome c antibody was from Pharmingen.
Antibodies against actin, caspase-3, caspase-8, and caspase-9 were from
Santa Cruz Biotechnology (Santa Cruz, CA). Male guinea pigs (4 weeks of
age) were purchased from Shimizu Co., Ltd. (Kyoto, Japan). All
experiments and procedures described here were approved by the Animal
Care Committee of Okayama University.
Preparation and Culture of Gastric Mucosal Cells--
Gastric
mucosal cells were isolated from guinea pig fundic glands as described
previously (17). Isolated gastric mucosal cells (1 × 106 cells/dish) were cultured for 48 h in RPMI 1640 containing 0.3% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin in type I collagen-coated plastic culture plates
(Iwaki) under the conditions of 5% CO2, 95% air at
37 °C. After removing non-adherent cells by washing with RPMI 1640, cells that were attached to the plate at about 50% confluence were
used. Guinea pig gastric mucosal cell preparations cultured under these
conditions have been characterized previously (17, 18), with the
majority (about 90%) of cells being identified as pit cells.
Treatment of Cells with Ethanol and PGE2--
Cells
were preincubated with PGE2 for 2 h and were then
exposed to ethanol by replacement of the entire bathing medium with fresh medium containing ethanol and the same concentration of PGE2. For monitoring cell viability, cells were incubated
with MTT solution at a final concentration of 1 mg/ml for 2 h.
Isopropyl alcohol and hydrochloric acid were added to the cells at
final concentrations of 50% and 20 mM, respectively. The
absorbance of each sample at 570 nm was determined by a
spectrophotometer using a reference wavelength of 630 nm (19).
Assay for Caspase Activation--
The activities of caspase-3,
caspase-8, and caspase-9 were determined as described previously (20,
21). Briefly, cells were collected by centrifugation and suspended in
extraction buffer (50 mM PIPES (pH 7.0), 50 mM
KCl, 5 mM EGTA, 2 mM MgCl2, and 1 mM DTT). Suspensions were sonicated and centrifuged, after
which the supernatants were incubated with fluorogenic peptide
substrates (Ac-DEVD-MCA (caspase-3), Ac-IETD-MCA (caspase-8), and
Ac-LEHD-MCA (caspase-9)) in reaction buffer (100 mM
HEPES-KOH (pH 7.5), 10% sucrose, 0.1% CHAPS, and 1 mg/ml bovine serum
albumin) for 15 min at 37 °C. The release of aminomethylcoumarin
(AMC) was determined using a fluorescence spectrophotometer. One unit
of protease activity was defined as the amount of enzyme required for
releasing 1 pmol of AMC/min.
Pro-caspase-3, caspase-8, and caspase-9 cleavage was monitored by
immunoblotting with specific antibodies against these caspases. Cell
lysates (20 µg of protein) were applied to 12 (for caspase-3 and
actin detection) or 8% (for caspase-8 and caspase-9 detection) polyacrylamide gels containing SDS and subjected to electrophoresis, and then proteins were immunoblotted with the anti-caspase-3, caspase-8, caspase-9, or actin antibody.
Release of Cytochrome c from Mitochondria--
After ethanol
treatment, cells were washed in fractionation buffer (250 mM sucrose, 20 mM HEPES/KOH (pH 8.0), 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, and 1 mM EGTA) and resuspended in the
same buffer supplemented with protease inhibitors. After homogenization on ice using a tight fitting Dounce homogenizer, nuclei were spun down
at 3,000 rpm for 10 min. The supernatant was further spun at 14,000 rpm
for 25 min for separation into cytosolic and mitochondrial fractions.
The mitochondrial fraction was then resuspended in buffer D (50 mM Tris/HCl (pH 7.2), 150 mM NaCl, 1% Nonidet
P-40, 1% sodium deoxycholate, 0.05% SDS). Protein concentrations in these fractions were determined by the Bradford method. Samples (4 µg) were applied to 15% polyacrylamide gels containing SDS and
subjected to electrophoresis, and then proteins were immunoblotted with
the anti-cytochrome c or anti-actin antibody.
Statistical Analysis--
All values are expressed as the
means ± S.E. A Student's t test for unpaired results
was performed for the evaluation of differences between the groups.
Differences were considered to be significant for values of
p < 0.05.
 |
RESULTS |
Effect of PGE2 on Ethanol-induced Apoptosis--
We
reported previously (15) that PGE2 inhibited apoptosis
induced by the treatment of guinea pig gastric mucosal cells in primary
culture with 3% ethanol for 4 h. In this study, conditions were
changed slightly for inducing apoptosis in that gastric mucosal cells
were incubated with 4% ethanol for 6 h. Fig.
1 shows that PGE2 inhibited
ethanol-induced apoptosis under these conditions. In this way,
PGE2 (0.1-10 µM) attenuated an
ethanol-induced decrease in cell viability (Fig. 1). In this
experiment, cells were first incubated with PGE2
(preincubation step) and then with ethanol in the presence of the same
concentration of PGE2 (incubation step). We confirmed that
PGE2 must be present in both steps in order to inhibit
efficiently the ethanol-induced apoptosis (data not shown), as
described previously (15). This method of PGE2 treatment
was thus used in all subsequent experiments.

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Fig. 1.
Effect of PGE2 on ethanol-induced
apoptosis. Cultured gastric mucosal cells were preincubated with
the indicated concentrations of PGE2 for 2 h. Cells
were further incubated for 6 h with 4% ethanol (EtOH)
in the presence of the same concentration of PGE2 as in the
preincubation step. Cell viability was determined by the MTT assay.
Values are means ± S.D.; n = 3. **,
p < 0.01.
|
|
We reported previously (13) that various gastric irritants activated
caspase-3, caspase-8, and caspase-9 in gastric mucosal cells. We also
reported that PGE2 suppressed the ethanol-mediated activation of caspase-3 (15). We therefore examined here the effect of
PGE2 on the activation of various caspases, including caspase-3, where caspase activities were examined by the use of fluorogenic peptide substrates (Ac-DEVD-MCA (for caspase-3),
Ac-IETD-MCA (for caspase-8), and Ac-LEHD-MCA (for caspase-9)). Since
these peptides can be cleaved by other caspases (for example, caspase-7 can recognize and cleave Ac-DEVD-MCA) (21, 22), it is more appropriate
to describe our findings in terms of caspase-3-, caspase-8-, or
caspase-9-like activity, instead of caspase-3, caspase-8, or caspase-9
activity, respectively. Ethanol treatment clearly activated all of the
caspases tested (Fig. 2A), as
has been found previously (13). PGE2 suppressed the
activation of not only caspase-3-like activity but also that of
caspase-9- and caspase-8-like activities (Fig. 2A). By
employing immunoblotting experiments, using specific antibodies against
these caspases, we observed pro-caspase-3, pro-caspase-8, and
pro-caspase-9 cleavage by ethanol treatment (Fig. 2B), as
has been found previously (13). PGE2 suppressed the
cleavage of each of all these pro-caspases (Fig. 2B).

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Fig. 2.
Effect of PGE2 on
caspase-activation by ethanol. Cultured gastric mucosal cells were
preincubated with the indicated concentrations of PGE2 for
2 h. Cells were further incubated for 6 h with 4% ethanol
(EtOH) in the presence of the same concentration of
PGE2 as in the preincubation step. Cell lysates were
prepared, and caspase-3-, -8-, or -9-like activities were measured by a
fluorometric assay using Ac-DEVD-MCA, Ac-IETD-MCA, and Ac-LEHD-MCA for
each of the caspases, respectively. One unit of protease activity was
defined as the amount of enzyme required for releasing 1 pmol of
AMC/min. Values are means ± S.D.; n = 3. *,
p < 0.05 (A). Pro-caspase-3, caspase-8, and
caspase-9 cleavage was monitored by immunoblotting with specific
antibodies against these caspases. The relative intensity of each band
to control was shown. For control experiment, the amount of actin in
each sample was monitored (B).
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The release of cytochrome c from mitochondria activates
caspase-9 in collaboration with Apaf-1 (23). We reported previously (13) that various gastric irritants, including ethanol, stimulated the
release of cytochrome c from mitochondria. Therefore,
PGE2-mediated suppression of ethanol-induced activation of
caspase-9-like activity (Fig. 3) suggests
that PGE2 inhibits the ethanol-stimulated release of
cytochrome c from mitochondria.

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Fig. 3.
Effect of PGE2 on ethanol-induced
release of cytochrome c from mitochondria.
Cultured gastric mucosal cells were preincubated with PGE2
(1 µM) for 2 h. Cells were further incubated for
6 h with 4% ethanol (EtOH) in the presence of the same
concentration of PGE2 as in the preincubation step. After
subcellular fractionation, cytosolic and mitochondrial fractions were
analyzed by immunoblotting with an antibody against cytochrome
c. As control, the amount of actin in cytosolic fraction was
monitored by immunoblotting with an antibody against actin
(A). The relative amounts of cytochrome c to
actin in cytosolic fractions were determined and expressed
(B).
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We therefore examined the effect of PGE2 on the
ethanol-dependent release of cytochrome c from
the mitochondria. As shown in Fig. 3, the amount of cytochrome
c in the cytosolic or mitochondrial fraction was increased
or decreased, respectively, by the ethanol treatment, suggesting that
ethanol stimulated the release of cytochrome c from the
mitochondria. In the presence of PGE2, however, the ethanol-induced release of cytochrome c from the
mitochondria was inhibited (Fig. 3). Therefore, it would appear that
the target of PGE2 for inhibiting apoptosis is located
upstream of mitochondrial dysfunction in the ethanol-induced apoptosis
pathway. Although the inhibition of ethanol-induced caspase-9
activation by PGE2 was partial (Fig. 2), that of release of
cytochrome c was almost complete (Fig. 3), suggesting that
the ethanol-induced apoptosis partially involves cytochrome
c-independent activation of caspase-9, which was reported
recently (24, 25) in other cell types.
Identification of EP Receptors Involved in the
PGE2-mediated Protection of Cells from Ethanol-induced
Apoptosis--
PGE2 receptors have been pharmacologically
subdivided into four main subtypes, EP1, EP2,
EP3, and EP4 (16). We used agonists specific
for each EP receptor in order to identify EP receptors involved in the
PGE2-mediated protection of gastric mucosal cells from
ethanol-induced apoptosis. Both ONO-AE1-259-01 (an EP2
agonist) and ONO-AE1-329 (an EP4 agonist) suppressed the
ethanol-induced decrease in cell viability (Fig.
4, B and D). The
extent of the suppression was similar to that achieved by
PGE2 (Fig. 1). We also found that each of these agonists
suppressed the ethanol-mediated activation of caspase-3-, caspase-8-,
and caspase-9-like activities to much the same extent as did
PGE2 (Fig. 5A).
Furthermore, each of these agonists suppressed the cleavage of
pro-caspase-3, pro-caspase-8, and pro-caspase-9 (Fig. 5B).
Based on findings reported previously (26) using these agonists
(ONO-AE1-259-01 and ONO-AE1-329), it is reasonable to postulate that,
for the concentrations of each of them used in the experiments
described in Figs. 4 and 5, they act as specific agonists for the
EP2 or EP4 receptor. On the other hand, neither
ONO-DI-004 (an EP1 agonist) nor ONO-NT-012 (an
EP3 agonist) suppressed the ethanol-induced decrease in
cell viability (Fig. 4, A and C). We also found
that neither of these agonists suppressed the ethanol-mediated
activation of caspase-3-, caspase-8-, and caspase-9-like activities
(Fig. 5A) and ethanol-mediated cleavage of pro-caspase-3,
pro-caspase-8, and pro-caspase-9 (Fig. 5B). On the evidence
presented in previous papers (4, 26, 27) where these agonists
(ONO-DI-004 and ONO-NT-012) were used, the concentrations employed in
the experiments whose results are described in Figs. 4 and 5 should
have been enough to activate their respective EP receptor.
Consequently, the results provided in Figs. 4 and 5 suggest that
EP2 and EP4 but not EP1 and
EP3 receptors are involved in the PGE2-mediated
protection of gastric mucosal cells from ethanol-induced apoptosis.

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Fig. 4.
Effects of EP receptor agonists on
ethanol-induced apoptosis. Cultured gastric mucosal cells were
preincubated with indicated the concentrations of ONO-DI-004
(EP1 agonist) (A), ONO-AE1-259-01
(EP2 agonist) (B), ONO-NT-012 (EP3
agonist) (C), ONO-AE1-329 (EP4 agonist)
(D), or PGE2 (1 µM) for 2 h.
Cells were further incubated for 6 h with 4% ethanol
(EtOH) in the presence of the same concentration of each
agonist or PGE2 as in the preincubation step. Cell
viability was determined by the MTT assay. Values are means ± S.D.; n = 3. *, p < 0.05; **,
p < 0.01; ***, p < 0.001.
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Fig. 5.
Effects of EP receptor agonists on
ethanol-induced caspase activation. Cultured gastric mucosal cells
were preincubated with ONO-DI-004 (EP1 agonist; 10 µM), ONO-AE1-259-01 (EP2 agonist; 1 µM), ONO-NT-012 (EP3 agonist; 1 µM), ONO-AE1-329 (EP4 agonist; 1 µM), or PGE2 (1 µM) for 2 h and then further incubated for 6 h with 4% ethanol
(EtOH) in the presence of the same concentration of each
agonist or PGE2 as that used in the preincubation step.
Cell lysates were prepared, and caspase-3-, -8-, or -9-like activities
were measured by a fluorometric assay using Ac-DEVD-MCA, Ac-IETD-MCA,
and Ac-LEHD-MCA, respectively. One unit of protease activity was the
amount of enzyme required for releasing 1 pmol of AMC/min. Values are
means ± S.D.; n = 3. *, p < 0.05; **, p < 0.01 (A). Pro-caspase-3,
caspase-8, and caspase-9 cleavage was monitored by immunoblotting with
specific antibodies against these caspases. The relative intensity of
each band to control was shown. For control experiment, the amount of
actin in each sample was monitored (B).
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Signal Transduction Pathway for PGE2-mediated
Protection of Cells from Ethanol-induced Apoptosis--
The
intracellular signaling pathways differ among the various EP receptor
subtypes; EP1 is coupled to Ca2+ mobilization,
whereas EP3 receptor activation inhibits adenylate cyclase
activity, in contrast to EP2 and EP4 receptor
activation in which adenylate cyclase activity is stimulated (16).
Therefore, the results described above suggest that stimulation of
adenylate cyclase activity (increase in cAMP) is involved in the
PGE2-mediated protection of gastric mucosal cells from
ethanol-induced apoptosis. To test this hypothesis, a cAMP analogue,
pCPT-cAMP, was used. As shown in Fig. 6,
pCPT-cAMP attenuated the ethanol-induced apoptosis in a
dose-dependent manner. The concentration of pCPT-cAMP
required for expressing this activity was much the same as that for
other cAMP-dependent phenomena (28-30). Therefore, it
seems that activation of adenylate cyclase coupled with activation of
EP2 and EP4 receptors plays an important role
in the signal transduction mechanism associated with the
PGE2-mediated protection of gastric mucosal cells from ethanol-induced apoptosis.

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Fig. 6.
Effect of cAMP analogue on ethanol-induced
apoptosis. Cultured gastric mucosal cells were preincubated with
the indicated concentrations of pCPT-cAMP or PGE2 (1 µM) for 2 h and then further incubated for 6 h
with 4% ethanol (EtOH) in the presence of the same
concentration of pCPT-cAMP or PGE2 as that used in the
preincubation step. Cell viability was determined by the MTT assay.
Values are means ± S.D.; n = 3. **,
p < 0.01.
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The increase in cAMP then activates two types of kinase, PKA and
phosphatidylinositol 3-kinase (PI3K), that are important for various
signal transduction mechanisms related to apoptosis (31). On this
basis, we next examined the involvement of these kinases in the
PGE2-mediated protection of gastric mucosal cells from
ethanol-induced apoptosis, using an inhibitor for each kinase. H-89 (an
inhibitor of PKA) blocked the PGE2-mediated protection of
cells from ethanol-induced apoptosis in a dose-dependent
manner (Fig. 7A). It is
reasonable to consider that H-89 used in the experimental results
described in Fig. 7A acts as a specific inhibitor for PKA
based on the results of previous papers where similar concentrations of
this inhibitor were used (32). H-89, at the concentrations used in the
experimental results shown in Fig. 7A, did not affect cell
viability in the absence of ethanol and PGE2 or in the
presence of ethanol only (data not shown). On the other hand,
wortmannin (an inhibitor of PI3K) did not affect the PGE2-mediated protection of gastric mucosal cells from
ethanol-induced apoptosis (Fig. 7B). Higher concentrations
of wortmannin (more than 20 nM) showed toxicity in the
absence of ethanol (data not shown). Based on previous papers (33)
where wortmannin was used, the concentrations of this drug used in the
experiments described here should have been enough to inhibit PI3K.
Therefore, it seems that PKA rather than PI3K is involved in the
PGE2-mediated protection of cells from ethanol-induced
apoptosis in gastric mucosal cells.

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Fig. 7.
Effects of an inhibitor of PKA or PI3K on
PGE2-mediated protection of cells from ethanol-induced
apoptosis. Cultured gastric mucosal cells were preincubated with 1 µM PGE2 in the presence of indicated
concentrations of H-89 (PKA inhibitor) (A) or wortmannin
(PI3K inhibitor) (B) for 2 h. Cells were further
incubated for 6 h with 4% ethanol (EtOH) in the
presence of the same concentration of H-89 (A) or wortmannin
(B) and PGE2 as in the preincubation step. Cell
viability was determined by the MTT assay. Values are means ± S.D.; n = 3. *, p < 0.05; **,
p < 0.01; ***, p < 0.001 (versus ethanol- (only) treated group). #, p < 0.05; ##, p < 0.01 (versus ethanol and
PGE2-treated group).
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 |
DISCUSSION |
A number of clinically used anti-ulcer drugs are related in their
action to achieving increased intracellular levels of PGE2. Furthermore, NSAIDs, one of the major causes of gastric ulcers, are
thought to damage the gastric mucosa by inhibiting cyclooxygenase and
decreasing the levels of circulating PGE2 at the gastric
mucosa. Therefore, it is clear that PGE2 is one of the most
important protective factors for the gastric mucosa in vivo.
PGE2 protects the gastric mucosa both directly and
indirectly. Compared with indirect PGE2-protective
mechanisms, the mechanism of the direct protection is relatively
unclear. For example, EP receptors involved in the indirect protection
of the gastric mucosa by PGE2 have been revealed as
follows: inhibition of gastric motility and stimulation of bicarbonate
secretion by PGE2 are mediated by EP1 receptor (34, 35); the stimulation of mucin production by PGE2 is
mediated by EP4 receptor (36); the increase in gastric
mucosal blood flow by PGE2 is mediated by EP3
receptor (37); and inhibition of acid secretion by PGE2 is
mediated by EP2/EP3 receptors (5, 38). However,
at the present time we have had no concrete evidence as to the role of
EP receptors in the direct protection of the gastric mucosa by
PGE2.
In a continuation of our previous work (15) that showed that
PGE2 inhibited gastric irritant-induced apoptosis in
primary cultures of gastric mucosal cells, we examined this phenomenon here in order to understand the underlying molecular mechanism of the
direct protection of the gastric mucosa by PGE2. By using agonists specific for each receptor, we have identified that both EP2 and EP4 receptors are involved in the
PGE2-mediated protection of gastric mucosal cells from
ethanol-induced apoptosis. Relating this to our conclusions, it was
recently reported (39) that inhibition of irradiation-induced apoptosis
by PGE2 in intestinal epithelium of the jejunum was
mediated by the EP2 receptor, using a knockout mouse of
this receptor. Since we reported previously that all gastric irritants
tested (NSAIDs, ethanol, hydrogen peroxide, and hydrochloric acid)
induced apoptosis through a common pathway in which mitochondrial
dysfunction plays an important role (9, 13), we assumed that
PGE2 inhibited apoptosis by these gastric irritants other
than ethanol through EP2 and EP4 receptors.
Therefore, it appears that PGE2 protects the gastric mucosa
by various mechanisms (both direct and indirect) via different EP receptors.
The conclusion arrived at above is apparently not consistent with some
previous reports. The expression of EP receptors in the
gastrointestinal tract of mouse has been examined by in situ hybridization studies where it was shown that the expression of EP4 mRNA could be detected in gastric mucosal cells;
however, expression of EP2 mRNA was not detected in any
types of cells from the stomach by this method (40). We consider
that this experiment cannot exclude the possibility that
EP2 is weakly expressed in gastric mucosal cells, because
it was recently reported (39) that EP2 is expressed in the
stomach of the mouse, using RNase protection assay. Furthermore, it was
recently found that the gene encoding EP2 is inducible in
macrophages; EP2 mRNA was detected only after a change
of medium or following the addition of LPS to the medium (41).
Therefore, it is also possible that an unknown stimulus under our
culture conditions induces EP2 mRNA expression. In such
a case, the anti-apoptotic activity of PGE2 in normal gastric mucosa may be mainly mediated by EP4, whereas
EP2 could be involved in such activity only in the presence
of pathological conditions such as gastritis. Unfortunately, genes
encoding the EP receptors in guinea pig have not been cloned, meaning
that these possibilities cannot be further examined at present.
It has been reported that the EP1 receptor is mainly
responsible for the gastro-protective effect of PGE2
in vivo through the inhibition of gastric motility (37, 42).
In these reports, the authors examined an acute phase of gastric
lesions (1 h after administration of high doses of gastric irritants),
suggesting that they damaged gastric mucosal cells mainly through
necrosis (not apoptosis). We reported previously (15) that gastric
irritant-induced apoptosis, but not necrosis, was inhibited by
PGE2 in cultured gastric mucosal cells. Therefore, we
consider that PGE2 protects the gastric mucosa from
necrosis via an EP1-mediated inhibition of gastric motility
and from apoptosis via an EP2/EP4-mediated direct inhibition of apoptosis.
We have also revealed here a part of the intracellular signal
transduction pathway for the PGE2-mediated protection of
cells from ethanol-induced apoptosis in gastric mucosal cells. By using a cAMP analogue and inhibitors for PKA and PI3K, we propose that PGE2-activated EP2/EP4 receptors
stimulate adenylate cyclase activity, thus increasing cAMP, which then
activates PKA and brings about an inhibition of apoptosis. Inhibition
of apoptosis by cAMP has been reported for various cell types (43-46).
cAMP apparently inhibits apoptosis through activation of PKA- (28, 31)
or PI3K-mediated (28, 47) processes depending on the difference in cell
types. PKA can phosphorylate BAD (48, 49). Phosphorylated BAD is inert
for inhibiting Bcl-2, and Bcl-2 can suppress release of cytochrome
c from mitochondria (50). Therefore, activation of PKA by
PGE2 in our system may phosphorylate BAD, which could then explain the PGE2-mediated suppression of ethanol-induced
release of cytochrome c from mitochondria (Fig. 3). However,
since we could not detect the band of phosphorylated BAD in guinea pig gastric mucosal cells using antibodies directed against phosphorylated human BAD (data not shown), we are unable to be certain of this point
at this time. Alternatively, since PKA was reported to induce Bcl-2
expression via activation of cAMP-responsive element-binding protein
(51), it is also possible that the PGE2-mediated protection of cells from ethanol-induced apoptosis is explained by this mechanism.
 |
ACKNOWLEDGEMENTS |
We thank the Ono Pharmaceutical Co., Ltd.,
for providing us with EP receptor agonists. We also thank to Dr.
Ichikawa (Kyoto University) and Dr. Rokutan (University of Tokushima)
for helpful suggestions.
 |
FOOTNOTES |
*
This work was supported by grants-in-aid for scientific
research from the Ministry of Education, Culture, Sports, Science and
Technology, Japan.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
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¶
To whom correspondence should be addressed: Faculty of
Pharmaceutical Sciences, Okayama University, 1-1-1, Tsushima-naka, Okayama 700-8530, Japan. Tel./Fax: 81-86-251-7958;
E-mail: mizushima@pharm.okayama-u.ac.jp.
Published, JBC Papers in Press, January 29, 2003, DOI 10.1074/jbc.M212097200
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ABBREVIATIONS |
The abbreviations used are:
PGs, prostaglandins;
pCPT-cAMP, 8-(4-chlorophenylthio)-cAMP;
PKA, protein kinase A;
PI3K, phosphatidylinositol 3-kinase;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;
NSAIDs, non-steroidal anti-inflammatory drugs;
H-89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide;
AMC, aminomethylcoumarin;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
PIPES, 1,4-piperazinediethanesulfonic acid.
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