Prostaglandin E2 Protects Gastric Mucosal Cells from Apoptosis via EP2 and EP4 Receptor Activation
2003; Elsevier BV; Volume: 278; Issue: 15 Linguagem: Inglês
10.1074/jbc.m212097200
ISSN1083-351X
AutoresTatsuya Hoshino, Shinji Tsutsumi, Wataru Tomisato, Hyun-Jung Hwang, Tomofusa Tsuchiya, Tohru Mizushima,
Tópico(s)Genomics, phytochemicals, and oxidative stress
ResumoProstaglandin E2(PGE2) has a strong protective effect on the gastric mucosain 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. Prostaglandin E2(PGE2) has a strong protective effect on the gastric mucosain 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. prostaglandins 8-(4-chlorophenylthio)-cAMP protein kinase A phosphatidylinositol 3-kinase 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide non-steroidal anti-inflammatory drugs N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide aminomethylcoumarin 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid 1,4-piperazinediethanesulfonic acid Prostaglandins (PGs),1one of the major groups of chemical mediators in the mammalian body, are involved in numerous physiological reactions, such as inflammation and cellular differentiation (1Srinivasan B.D. Kulkarni P.S. Prog. Clin. Biol. Res. 1989; 312: 229-249PubMed Google Scholar). 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 (2Keogh J.P. Allen A. Garner A. Clin. Exp. Pharmacol. Physiol. 1997; 24: 844-849Crossref PubMed Scopus (27) Google Scholar, 3Kauffman Jr., G.L. Reeve Jr., J.J. Grossman M.I. Am. J. Physiol. 1980; 239: G44-G48PubMed Google Scholar), inhibition of gastric motility (4Suzuki K. Araki H. Mizoguchi H. Furukawa O. Takeuchi K. Digestion. 2001; 63: 92-101Crossref PubMed Scopus (49) Google Scholar), inhibition of acid secretion (5Yokotani K. Okuma Y. Osumi Y. Br. J. Pharmacol. 1996; 117: 653-656Crossref PubMed Scopus (42) Google Scholar), amelioration of mucosal blood flow (6Morris G.P. Fallone C.A. Pringle G.C. MacNaughton W.K. J. Clin. Gastroenterol. 1998; 27, Suppl.1: 53-63Crossref Scopus (16) Google Scholar), inhibition of free radical and enzyme release from neutrophils (7Gryglewski R.J. Szczeklik A. Wandzilak M. Biochem. Pharmacol. 1987; 6: 4209-4213Crossref Scopus (97) Google Scholar), and vascular, luminal, and/or extrinsic and intrinsic neural mechanisms (8Pihan G. Majzoubi D. Haudenschild C. Trier J.S. Szabo S. Gastroenterology. 1986; 91: 1415-1426Abstract Full Text PDF PubMed Scopus (0) Google Scholar). 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 (9Tomisato W. Tsutsumi S. Rokutan K. Tsuchiya T. Mizushima T. Am. J. Physiol. 2001; 281: G1092-G1100Crossref PubMed Google Scholar). For example, a stimulated rate of apoptosis of gastric mucosal cells was reported at the onset of gastric ulceration (10Konturek P.C. Brzozowski T. Konturek S.J. Pajdo R. Konturek J.E. Kwiecien S. Taut A. Hahn E.G. J. Physiol. Pharmacol. 1999; 50: 211-225PubMed Google Scholar). Apoptosis associated withHelicobacter pylori infection was suggested to be involved in the development of atrophic gastritis caused by H. pyloriinfection (11Moss S.F. Calam J. Agarwal B. Wang S. Holt P.R. Gut. 1996; 38: 498-501Crossref PubMed Scopus (385) Google Scholar). 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 (9Tomisato W. Tsutsumi S. Rokutan K. Tsuchiya T. Mizushima T. Am. J. Physiol. 2001; 281: G1092-G1100Crossref PubMed Google Scholar, 12Takano T. Tsutsumi S. Tomisato W. Hoshino T. Tsuchiya T. Mizushima T. Dig. Dis. Sci. 2002; 47: 1546-1553Crossref PubMed Scopus (30) Google Scholar, 13Tsutsumi S. Tomisato W. Takano T. Rokutan K. Tsuchiya T. Mizushima T. Biochim. Biophys. Acta. 2002; 1589: 168-180Crossref PubMed Scopus (51) Google Scholar, 14Mizushima T. Tsutsumi S. Rokutan K. Tsuchiya T. Dig. Dis. Sci. 1999; 44: 510-514Crossref PubMed Scopus (43) Google Scholar). We also found that these gastric irritants induced apoptosis through a common pathway in which mitochondrial dysfunction plays an important role (13Tsutsumi S. Tomisato W. Takano T. Rokutan K. Tsuchiya T. Mizushima T. Biochim. Biophys. Acta. 2002; 1589: 168-180Crossref PubMed Scopus (51) Google Scholar). 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 PGE2inhibited the apoptosis caused by various gastric irritants (ethanol, hydrogen peroxide, and hydrochloric acid) (15Hoshino T. Takano T. Tsutsumi S. Tomisato W. Tsuchiya T. Mizushima T. Dig. Dis. Sci. 2002; 47: 2370-2379Crossref PubMed Scopus (18) Google Scholar). The molecular mechanism governing this inhibitory effect of PGE2 on apoptosis has, however, yet to be elucidated. For example, although PGE2receptors have been pharmacologically subdivided into four main subtypes (EP1, EP2, EP3, and EP4) (16Coleman R.A. Smith W.L. Narumiya S. Pharmacol. Rev. 1994; 46: 205-229PubMed Google Scholar), 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). 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 EP1agonist), 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. Gastric mucosal cells were isolated from guinea pig fundic glands as described previously (17Rokutan K. Yamada M. Torigoe J. Saito T. Am. J. Physiol. 1998; 275: G526-G533PubMed Google Scholar). 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 (17Rokutan K. Yamada M. Torigoe J. Saito T. Am. J. Physiol. 1998; 275: G526-G533PubMed Google Scholar, 18Tomisato W. Hoshino T. Tsutsumi S. Tsuchiya T. Mizushima T. Dig. Dis. Sci. 2002; 47: 2125-2133Crossref PubMed Scopus (13) Google Scholar), with the majority (about 90%) of cells being identified as pit cells. 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 (19Wolf B.B. Green D.R. J. Biol. Chem. 1999; 274: 20049-20052Abstract Full Text Full Text PDF PubMed Scopus (860) Google Scholar). The activities of caspase-3, caspase-8, and caspase-9 were determined as described previously (20He J. Whitacre C.M. Xue L.Y. Berger N.A. Oleinick N.L. Cancer Res. 1998; 58: 940-946PubMed Google Scholar,21Thornberry N.A. Rano T.A. Peterson E.P. Rasper D.M. Timkey T. Garcia-Calvo M. Houtzager V.M. Nordstrom P.A. Roy S. Vaillancourt J.P. Chapman K.T. Nicholson D.W. J. Biol. Chem. 1997; 272: 17907-17911Abstract Full Text Full Text PDF PubMed Scopus (1834) Google Scholar). Briefly, cells were collected by centrifugation and suspended in extraction buffer (50 mm PIPES (pH 7.0), 50 mmKCl, 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 mmHEPES-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. 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. 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 ofp < 0.05. We reported previously (15Hoshino T. Takano T. Tsutsumi S. Tomisato W. Tsuchiya T. Mizushima T. Dig. Dis. Sci. 2002; 47: 2370-2379Crossref PubMed Scopus (18) Google Scholar) 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 (15Hoshino T. Takano T. Tsutsumi S. Tomisato W. Tsuchiya T. Mizushima T. Dig. Dis. Sci. 2002; 47: 2370-2379Crossref PubMed Scopus (18) Google Scholar). This method of PGE2 treatment was thus used in all subsequent experiments. We reported previously (13Tsutsumi S. Tomisato W. Takano T. Rokutan K. Tsuchiya T. Mizushima T. Biochim. Biophys. Acta. 2002; 1589: 168-180Crossref PubMed Scopus (51) Google Scholar) 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 (15Hoshino T. Takano T. Tsutsumi S. Tomisato W. Tsuchiya T. Mizushima T. Dig. Dis. Sci. 2002; 47: 2370-2379Crossref PubMed Scopus (18) Google Scholar). 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) (21Thornberry N.A. Rano T.A. Peterson E.P. Rasper D.M. Timkey T. Garcia-Calvo M. Houtzager V.M. Nordstrom P.A. Roy S. Vaillancourt J.P. Chapman K.T. Nicholson D.W. J. Biol. Chem. 1997; 272: 17907-17911Abstract Full Text Full Text PDF PubMed Scopus (1834) Google Scholar, 22Nicholson D.W. Ali A. Thornberry N.A. Vaillancourt J.P. Ding C.K. Gallant M. Gareau Y. Griffin P.R. Labelle M. Lazebnik Y.A. Munday N.A. Raju S.M. Smulson M.E. Yamin T. Yu V.L. Miller D.K. Nature. 1995; 376: 37-43Crossref PubMed Scopus (3768) Google Scholar), 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 (13Tsutsumi S. Tomisato W. Takano T. Rokutan K. Tsuchiya T. Mizushima T. Biochim. Biophys. Acta. 2002; 1589: 168-180Crossref PubMed Scopus (51) Google Scholar). 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 (13Tsutsumi S. Tomisato W. Takano T. Rokutan K. Tsuchiya T. Mizushima T. Biochim. Biophys. Acta. 2002; 1589: 168-180Crossref PubMed Scopus (51) Google Scholar). PGE2 suppressed the cleavage of each of all these pro-caspases (Fig. 2B). The release of cytochrome c from mitochondria activates caspase-9 in collaboration with Apaf-1 (23Cain K. Bratton S.B. Langlais C. Walker G. Brown D.G. Sun X.M. Cohen G.M. J. Biol. Chem. 2000; 275: 6067-6070Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). We reported previously (13Tsutsumi S. Tomisato W. Takano T. Rokutan K. Tsuchiya T. Mizushima T. Biochim. Biophys. Acta. 2002; 1589: 168-180Crossref PubMed Scopus (51) Google Scholar) 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. 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 cytochromec 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 cytochromec-independent activation of caspase-9, which was reported recently (24Morishima N. Nakanishi K. Takenouchi H. Shibata T. Yasuhiko Y. J. Biol. Chem. 2002; 277: 34287-34294Abstract Full Text Full Text PDF PubMed Scopus (788) Google Scholar, 25Iordanov M.S. Ryabinina O.P. Wong J. Dinh T.H. Newton D.L. Rybak S.M. Magun B.E. Cancer Res. 2000; 60: 1983-1994PubMed Google Scholar) in other cell types. PGE2 receptors have been pharmacologically subdivided into four main subtypes, EP1, EP2, EP3, and EP4 (16Coleman R.A. Smith W.L. Narumiya S. Pharmacol. Rev. 1994; 46: 205-229PubMed Google Scholar). 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 EP2agonist) 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 (26Suzawa T. Miyaura C. Inada M. Maruyama T. Sugimoto Y. Ushikubi F. Ichikawa A. Narumiya S. Suda T. Endocrinology. 2000; 141: 1554-1559Crossref PubMed Scopus (318) Google Scholar) 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 (4Suzuki K. Araki H. Mizoguchi H. Furukawa O. Takeuchi K. Digestion. 2001; 63: 92-101Crossref PubMed Scopus (49) Google Scholar, 26Suzawa T. Miyaura C. Inada M. Maruyama T. Sugimoto Y. Ushikubi F. Ichikawa A. Narumiya S. Suda T. Endocrinology. 2000; 141: 1554-1559Crossref PubMed Scopus (318) Google Scholar, 27Ikari A. Sakai H. Tanaka A. Ikeda A. Inoue K. Takeguchi N. Jpn. J. Physiol. 1999; 49: 365-372Crossref PubMed Scopus (24) Google Scholar) 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.Figure 5Effects 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).View Large Image Figure ViewerDownload (PPT) 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 (16Coleman R.A. Smith W.L. Narumiya S. Pharmacol. Rev. 1994; 46: 205-229PubMed Google Scholar). 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 (28Webster C.R. Anwer M.S. Hepatology. 1998; 27: 1324-1331Crossref PubMed Scopus (106) Google Scholar, 29Webster C.R. Usechak P. Anwer M.S. Am. J. Physiol. 2002; 283: G727-G738Crossref PubMed Scopus (65) Google Scholar, 30Rideout H.J. Larsen K.E. Sulzer D. Stefanis L. J. Neurochem. 2001; 78: 899-908Crossref PubMed Scopus (251) Google Scholar). 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. 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 (31Li J. Yang S. Billiar T.R. J. Biol. Chem. 2000; 275: 13026-13034Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). 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 (32Chijiwa T. Mishima A. Hagiwara M. Sano M. Hayashi K. Inoue T. Naito K. Toshioka T. Hidaka H. J. Biol. Chem. 1990; 265: 5267-5272Abstract Full Text PDF PubMed Google Scholar). 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 (33Powis G. Bonjouklian R. Berggren M.M. Gallegos A. Abraham R. Ashendel C. Zalkow L. Matter W.F. Dodge J. Grindey G. Vlahos C.J. Cancer Res. 1994; 54: 2419-2423PubMed Google Scholar) 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. 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 (34Takeuchi K. Yagi K. Kato S. Ukawa H. Gastroenterology. 1997; 113: 1553-1559Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 35Watabe A. Sugimoto Y. Honda A. Irie A. Namba T. Negishi M. Ito S. Narumiya S. Ichikawa A. J. Biol. Chem. 1993; 268: 20175-20178Abstract Full Text PDF PubMed Google Scholar); the stimulation of mucin production by PGE2 is mediated by EP4 receptor (36Hassan S. Kinoshita Y. Min D. Nakata H. Kishi K. Matsushima Y. Asahara M. Wang H.Y. Okada A. Maekawa T. Matsui H. Chiba T. Digestion. 1996; 57: 196-200Crossref PubMed Scopus (67) Google Scholar); the increase in gastric mucosal blood flow by PGE2 is mediated by EP3receptor (37Araki H. Ukawa H. Sugawa Y. Yagi K. Suzuki K. Takeuchi K. Aliment. Pharmacol. Ther. 2000; 14 Suppl. 1: 116-124Crossref PubMed Scopus (89) Google Scholar); and inhibition of acid secretion by PGE2 is mediated by EP2/EP3 receptors (5Yokotani K. Okuma Y. Osumi Y. Br. J. Pharmacol. 1996; 117: 653-656Crossref PubMed Scopus (42) Google Scholar, 38Naribayashi-Inomoto Y. Ding M. Nakata H. Narumiya S. Sugimoto Y. Honda A. Ichikawa A. Chiba T. Kinoshita Y. Gastroenterology. 1995; 109: 341-347Abstract Full Text PDF PubMed Scopus (28) Google Scholar). 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 (15Hoshino T. Takano T. Tsutsumi S. Tomisato W. Tsuchiya T. Mizushima T. Dig. Dis. Sci. 2002; 47: 2370-2379Crossref PubMed Scopus (18) Google Scholar) 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 (39Houchen C.W. Sturmoski M.A. Anant S. Breyer R.M. Stenson W.F. Am. J. Physiol. 2003; 284: G490-G498Crossref PubMed Scopus (61) Google Scholar) 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 (9Tomisato W. Tsutsumi S. Rokutan K. Tsuchiya T. Mizushima T. Am. J. Physiol. 2001; 281: G1092-G1100Crossref PubMed Google Scholar, 13Tsutsumi S. Tomisato W. Takano T. Rokutan K. Tsuchiya T. Mizushima T. Biochim. Biophys. Acta. 2002; 1589: 168-180Crossref PubMed Scopus (51) Google Scholar), 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 situhybridization 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 (40Morimoto K. Sugimoto Y. Katsuyama M. Oida H. Tsuboi K. Kishi K. Kinoshita Y. Negishi M. Chiba T. Narumiya S. Ichikawa A. Am. J. Physiol. 1997; 272: G681-G687PubMed Google Scholar). We consider that this experiment cannot exclude the possibility that EP2 is weakly expressed in gastric mucosal cells, because it was recently reported (39Houchen C.W. Sturmoski M.A. Anant S. Breyer R.M. Stenson W.F. Am. J. Physiol. 2003; 284: G490-G498Crossref PubMed Scopus (61) Google Scholar) 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 (41Ikegami R. Sugimoto Y. Segi E. Katsuyama M. Karahashi H. Amano F. Maruyama T. Yamane H. Tsuchiya S. Ichikawa A. J. Immunol. 2001; 166: 4689-4696Crossref PubMed Scopus (105) Google Scholar). 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 PGE2in vivo through the inhibition of gastric motility (37Araki H. Ukawa H. Sugawa Y. Yagi K. Suzuki K. Takeuchi K. Aliment. Pharmacol. Ther. 2000; 14 Suppl. 1: 116-124Crossref PubMed Scopus (89) Google Scholar, 42Takeuchi K. Araki H. Umeda M. Komoike Y. Suzuki K. J. Pharmacol. Exp. Ther. 2001; 297: 1160-1165PubMed Google Scholar). 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 (15Hoshino T. Takano T. Tsutsumi S. Tomisato W. Tsuchiya T. Mizushima T. Dig. Dis. Sci. 2002; 47: 2370-2379Crossref PubMed Scopus (18) Google Scholar) 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 (43Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1331Crossref PubMed Scopus (5019) Google Scholar, 44Nobes C.D. Tolkovsky A.M. Eur. J. Neurosci. 1995; 7: 344-350Crossref PubMed Scopus (71) Google Scholar, 45Messmer U.K. Lapetina E.G. Brune B. Mol. Pharmacol. 1995; 47: 757-765PubMed Google Scholar, 46Geier A. Weiss C. Beery R. Haimsohn M. Hemi R. Malik Z. Karasik A. J. Cell. Physiol. 1995; 163: 570-576Crossref PubMed Scopus (37) Google Scholar). cAMP apparently inhibits apoptosis through activation of PKA- (28Webster C.R. Anwer M.S. Hepatology. 1998; 27: 1324-1331Crossref PubMed Scopus (106) Google Scholar, 31Li J. Yang S. Billiar T.R. J. Biol. Chem. 2000; 275: 13026-13034Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) or PI3K-mediated (28Webster C.R. Anwer M.S. Hepatology. 1998; 27: 1324-1331Crossref PubMed Scopus (106) Google Scholar, 47Webster C.R. Anwer M.S. Am. J. Physiol. 1999; 277: G1165-G1172PubMed Google Scholar) processes depending on the difference in cell types. PKA can phosphorylate BAD (48Yusta B. Estall J. Drucker D.J. J. Biol. Chem. 2002; 277: 24896-24906Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 49Lizcano J.M. Morrice N. Cohen P. Biochem. J. 2000; 349: 547-557Crossref PubMed Scopus (256) Google Scholar). Phosphorylated BAD is inert for inhibiting Bcl-2, and Bcl-2 can suppress release of cytochromec from mitochondria (50Zha J. Harada H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2233) Google Scholar). 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 (51Chen W. Yu Y.L. Lee S.F. Chiang Y.J. Chao J.R. Huang J.H. Chiong J.H. Huang C.J. Lai M.Z. Yang-Yen H.F. Yen J.J. Mol. Cell. Biol. 2001; 21: 4636-4646Crossref PubMed Scopus (35) Google Scholar), it is also possible that the PGE2-mediated protection of cells from ethanol-induced apoptosis is explained by this mechanism. 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.
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