Involvement of Prostaglandin E2 in Production of Amyloid-β Peptides Both in Vitro and in Vivo
2007; Elsevier BV; Volume: 282; Issue: 45 Linguagem: Inglês
10.1074/jbc.m703087200
ISSN1083-351X
AutoresTatsuya Hoshino, Tadashi Nakaya, Takashi Homan, Ken‐ichiro Tanaka, Yukihiko Sugimoto, Wataru Araki, Masami Narita, Shuh Narumiya, Toshiharu Suzuki, Tohru Mizushima,
Tópico(s)Alzheimer's disease research and treatments
ResumoAmyloid-β peptides (Aβ), generated by proteolysis of the β-amyloid precursor protein (APP) by β- and γ-secretases, play an important role in the pathogenesis of Alzheimer disease (AD). Inflammation is also believed to be integral to the pathogenesis of AD. Here we show that prostaglandin E2 (PGE2), a strong inducer of inflammation, stimulates the production of Aβ in cultured human embryonic kidney (HEK) 293 or human neuroblastoma (SH-SY5Y) cells, both of which express a mutant type of APP. We have demonstrated using subtype-specific agonists that, of the four main subtypes of PGE2 receptors (EP1–4), EP4 receptors alone or EP2 and EP4 receptors together are responsible for this PGE2-stimulated production of Aβ in HEK293 or SH-SY5Y cells, respectively. An EP4 receptor antagonist suppressed the PGE2-stimulated production of Aβ in HEK293 cells. This stimulation was accompanied by an increase in cellular cAMP levels, and an analogue of cAMP stimulated the production of Aβ, demonstrating that increases in the cellular level of cAMP are responsible for the PGE2-stimulated production of Aβ. Immunoblotting experiments and direct measurement of γ-secretase activity suggested that PGE2-stimulated production of Aβ is mediated by activation ofγ-secretase but not of β-secretase. Transgenic mice expressing the mutant type of APP showed lower levels of Aβ in the brain, when they were crossed with mice lacking either EP2 or EP4 receptors, suggesting that PGE2-mediated activation of EP2 and EP4 receptors is involved in the production of Aβ in vivo and in the pathogenesis of AD. Amyloid-β peptides (Aβ), generated by proteolysis of the β-amyloid precursor protein (APP) by β- and γ-secretases, play an important role in the pathogenesis of Alzheimer disease (AD). Inflammation is also believed to be integral to the pathogenesis of AD. Here we show that prostaglandin E2 (PGE2), a strong inducer of inflammation, stimulates the production of Aβ in cultured human embryonic kidney (HEK) 293 or human neuroblastoma (SH-SY5Y) cells, both of which express a mutant type of APP. We have demonstrated using subtype-specific agonists that, of the four main subtypes of PGE2 receptors (EP1–4), EP4 receptors alone or EP2 and EP4 receptors together are responsible for this PGE2-stimulated production of Aβ in HEK293 or SH-SY5Y cells, respectively. An EP4 receptor antagonist suppressed the PGE2-stimulated production of Aβ in HEK293 cells. This stimulation was accompanied by an increase in cellular cAMP levels, and an analogue of cAMP stimulated the production of Aβ, demonstrating that increases in the cellular level of cAMP are responsible for the PGE2-stimulated production of Aβ. Immunoblotting experiments and direct measurement of γ-secretase activity suggested that PGE2-stimulated production of Aβ is mediated by activation ofγ-secretase but not of β-secretase. Transgenic mice expressing the mutant type of APP showed lower levels of Aβ in the brain, when they were crossed with mice lacking either EP2 or EP4 receptors, suggesting that PGE2-mediated activation of EP2 and EP4 receptors is involved in the production of Aβ in vivo and in the pathogenesis of AD. Alzheimer disease (AD) 2The abbreviations used are: AD, Alzheimer disease; Aβ, amyloid-β peptides; APP, β-amyloid precursor protein; PS, presenilin; CHO, Chinese hamster ovary; COX, cyclooxygenase; CTF, C-terminal fragment; DAPT, N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester; EIA, enzyme immunoassay; Epac, exchange protein directly activated by cAMP; H-89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide; HEK, human embryonic kidney; NF-κB, nuclear factor-κB; NSAIDs, non-steroidal anti-inflammatory drugs; pCPT-cAMP, 8-(4-chlorophenylthio)-cAMP; pCPT-O-Me-cAMP, 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′-5′-cyclic monophosphate; PI3K, phosphatidylinositol 3-kinase; PGE2, prostaglandin E2; PGs, prostaglandins; Rock, Rho kinase; PKA, protein kinase A; RT, reverse transcriptase; sELISA, sandwich enzyme-linked immunosorbent; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid. 2The abbreviations used are: AD, Alzheimer disease; Aβ, amyloid-β peptides; APP, β-amyloid precursor protein; PS, presenilin; CHO, Chinese hamster ovary; COX, cyclooxygenase; CTF, C-terminal fragment; DAPT, N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester; EIA, enzyme immunoassay; Epac, exchange protein directly activated by cAMP; H-89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide; HEK, human embryonic kidney; NF-κB, nuclear factor-κB; NSAIDs, non-steroidal anti-inflammatory drugs; pCPT-cAMP, 8-(4-chlorophenylthio)-cAMP; pCPT-O-Me-cAMP, 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′-5′-cyclic monophosphate; PI3K, phosphatidylinositol 3-kinase; PGE2, prostaglandin E2; PGs, prostaglandins; Rock, Rho kinase; PKA, protein kinase A; RT, reverse transcriptase; sELISA, sandwich enzyme-linked immunosorbent; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid. is the leading cause of adult onset dementia, with a dramatic increase in the incidence of AD apparent in our rapidly aging society. AD is characterized pathologically by the accumulation of tangles and senile plaques. Senile plaques are composed of the amyloid-β peptides (Aβ) Aβ40 and Aβ42 (1Hardy J. Selkoe D.J. Science. 2002; 297: 353-356Crossref PubMed Scopus (10847) Google Scholar, 2Mattson M.P. Nature. 2004; 430: 631-639Crossref PubMed Scopus (2452) Google Scholar). Aβ is generated by secretase-dependent proteolysis of the β-amyloid precursor protein (APP). Prior to proteolysis, APP undergoes modifications, such as glycosylation and phosphorylation. To generate Aβ40 and Aβ42, APP is first cleaved by β-secretase and then by γ-secretase. For the cleavage of APP, β-secretase competes with α-secretase, which produces non-amyloidogenic peptides (3Sisodia S.S. St. George-Hyslop P.H. Nat. Rev. Neurosci. 2002; 3: 281-290Crossref PubMed Scopus (486) Google Scholar, 4Selkoe D.J. Nature. 1999; 399: A23-A31Crossref PubMed Scopus (1520) Google Scholar). The γ-secretase is an aspartyl protease complex composed of four core components, including presenilin (PS) 1 and PS2 (5Haass C. EMBO J. 2004; 23: 483-488Crossref PubMed Scopus (482) Google Scholar). The early onset familial AD is linked to three genes, APP, PS1, and PS2 (5Haass C. EMBO J. 2004; 23: 483-488Crossref PubMed Scopus (482) Google Scholar, 6Price D.L. Sisodia S.S. Borchelt D.R. Science. 1998; 282: 1079-1083Crossref PubMed Scopus (224) Google Scholar), strongly suggesting that the production of Aβ, which reflects the proteolysis of APP by secretases (particularly γ-secretase), is a key factor in the pathogenesis of AD. Therefore, cellular factors that stimulate the production of Aβ may be good drug targets for the prevention and treatment of AD. It has been repeatedly suggested that inflammation is important in the pathogenesis of AD. Chronic inflammation, which is indicated by accumulation of microglia around senile plaques and elevated levels of inflammatory cytokines, chemokines, proteases, and reactive oxygen species, has been observed in the brains of AD patients (7Townsend K.P. Pratico D. FASEB J. 2005; 19: 1592-1601Crossref PubMed Scopus (205) Google Scholar). Furthermore, trauma to the brain and ischemia, both of which can activate inflammation, are major risk factors for AD (8Ikonomovic M.D. Uryu K. Abrahamson E.E. Ciallella J.R. Trojanowski J.Q. Lee V.M. Clark R.S. Marion D.W. Wisniewski S.R. DeKosky S.T. Exp. Neurol. 2004; 190: 192-203Crossref PubMed Scopus (340) Google Scholar). Prostaglandins (PGs), one of the major groups of chemical mediators in the mammalian body, are potent inducers of inflammation (9Srinivasan B.D. Kulkarni P.S. Prog. Clin. Biol. Res. 1989; 312: 229-249PubMed Google Scholar). Cyclooxygenase (COX) is essential for the synthesis of PGs and has two subtypes, COX-1 and COX-2. COX-1 is expressed constitutively in most cell types, whereas expression of COX-2 is induced by various factors including inflammatory cytokines and is responsible for the progression of inflammation (10Vane J. Nature. 1994; 367: 215-216Crossref PubMed Scopus (696) Google Scholar, 11Smith C.J. Zhang Y. Koboldt C.M. Muhammad J. Zweifel B.S. Shaffer A. Talley J.J. Masferrer J.L. Seibert K. Isakson P.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13313-13318Crossref PubMed Scopus (725) Google Scholar). Elevated levels of PGE2, a major proinflammatory product of COX, and overexpression of COX-2 have been observed in the brains of AD patients (12Kitamura Y. Shimohama S. Koike H. Kakimura J. Matsuoka Y. Nomura Y. Gebicke-Haerter P.J. Taniguchi T. Biochem. Biophys. Res. Commun. 1999; 254: 582-586Crossref PubMed Scopus (210) Google Scholar, 13Yasojima K. Schwab C. McGeer E.G. McGeer P.L. Brain Res. 1999; 830: 226-236Crossref PubMed Scopus (236) Google Scholar, 14Montine T.J. Sidell K.R. Crews B.C. Markesbery W.R. Marnett L.J. Roberts L.J. 2nd Morrow Neurology. 1999; 53: 1495-1498Crossref PubMed Google Scholar). It has also been reported that the extent of COX-2 expression correlates with the amount of Aβ and the degree of progression of AD pathogenesis (15Ho L. Purohit D. Haroutunian V. Luterman J.D. Willis F. Naslund J. Buxbaum J.D. Mohs R.C. Aisen P.S. Pasinetti G.M. Arch. Neurol. 2001; 58: 487-492Crossref PubMed Scopus (173) Google Scholar). Furthermore, transgenic mice that constitutively overexpress COX-2 have been reported to show stimulation of aging-dependent neural apoptosis and memory dysfunction (16Andreasson K.I. Savonenko A. Vidensky S. Goellner J.J. Zhang Y. Shaffer A. Kaufmann W.E. Worley P.F. Isakson P. Markowska A.L. J. Neurosci. 2001; 21: 8198-8209Crossref PubMed Google Scholar). These previous studies suggest that COX-2 and PGE2 are important in the pathogenesis of AD and are therefore good targets for potential AD drugs. Supporting this notion, epidemiological studies have revealed that prolonged use of non-steroidal anti-inflammatory drugs (NSAIDs), inhibitors of COX, delays the onset and reduces the risk of AD (17in t'Veld, B. A., Ruitenberg, A., Hofman, A., Launer, L. J., van Duijn, C. M., Stijnen, T., Breteler, M. M., and Stricker, B. H. (2001) N. Engl. J. Med. 345, 1515–1521Google Scholar). In an animal model of AD, administration of some NSAIDs decreased the amount of Aβ and senile plaques and suppressed microglial activation (18Lim G.P. Yang F. Chu T. Chen P. Beech W. Teter B. Tran T. Ubeda O. Ashe K.H. Frautschy S.A. Cole G.M. J. Neurosci. 2000; 20: 5709-5714Crossref PubMed Google Scholar, 19Eriksen J.L. Sagi S.A. Smith T.E. Weggen S. Das P. McLendon D.C. Ozols V.V. Jessing K.W. Zavitz K.H. Koo E.H. Golde T.E. J. Clin. Investig. 2003; 112: 440-449Crossref PubMed Scopus (0) Google Scholar, 20Yan Q. Zhang J. Liu H. Babu-Khan S. Vassar R. Biere A.L. Citron M. Landreth G. J. Neurosci. 2003; 23: 7504-7509Crossref PubMed Google Scholar). Furthermore, in cultured cells, treatment with NSAIDs decreased the amount of Aβ (21Weggen S. Eriksen J.L. Das P. Sagi S.A. Wang R. Pietrzik C.U. Findlay K.A. Smith T.E. Murphy M.P. Bulter T. Kang D.E. Marquez-Sterling N. Golde T.E. Koo E.H. Nature. 2001; 414: 212-216Crossref PubMed Scopus (1322) Google Scholar, 22Weggen S. Eriksen J.L. Sagi S.A. Pietrzik C.U. Ozols V. Fauq A. Golde T.E. Koo E.H. J. Biol. Chem. 2003; 278: 31831-31837Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). Therefore, NSAIDs have attracted much attention as a new class of drugs for the treatment of AD. However, clinical use of NSAIDs is associated with various side effects, such as gastrointestinal complications (23Hawkey C.J. Gastroenterology. 2000; 119: 521-535Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar) and cardiovascular thrombotic events (24Singh D. Br. Med. J. 2004; 329: 816Crossref PubMed Scopus (62) Google Scholar, 25Ray W.A. Griffin M.R. Stein C.M. N. Engl. J. Med. 2004; 351: 2767Crossref PubMed Scopus (50) Google Scholar). These side effects are mainly due to an NSAID-induced nonspecific decrease in the levels of various types of prostanoids and eicosanoids and the inhibition of signal transduction mediated by their receptors. Identification of specific prostanoids and eicosanoids, or of their receptors that are involved in the anti-AD activity of NSAIDs, is therefore important for the development of new types of drugs for AD with a reduced risk of side effects. Based on the studies described above, it is reasonable to hypothesize that PGE2 increases the amount of Aβ. In fact, it was recently reported that PGE2 stimulates the production of Aβ in Chinese hamster ovary (CHO) cells (26Qin W. Ho L. Pompl P.N. Peng Y. Zhao Z. Xiang Z. Robakis N.K. Shioi J. Suh J. Pasinetti G.M. J. Biol. Chem. 2003; 278: 50970-50977Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). However, the molecular mechanism governing this stimulation has remained unclear. For example, whereas PGE2 receptors have been pharmacologically subdivided into four main subtypes (EP1, EP2, EP3, and EP4) (27Coleman R.A. Smith W.L. Narumiya S. Pharmacol. Rev. 1994; 46: 205-229PubMed Google Scholar), the EP subtype involved in this PGE2-stimulated production of Aβ has not been identified. In the present study, we have confirmed that PGE2 stimulates the production of Aβ in human embryonic kidney (HEK) 293 and human neuroblastoma (SH-SY5Y) cells. Experiments with EP agonists and antagonists have revealed that, depending on cell type, the EP4 receptor alone or the EP2 and EP4 receptors together are involved in the PGE2-stimulated production of Aβ. Furthermore, experiments with transgenic mice suggest that EP2 and EP4 receptors are also involved in the production of Aβ in vivo. Based on the results of the current study, we propose that antagonists for both EP2 and EP4 receptors may be therapeutically beneficial for the treatment of AD. Materials—Compounds used in this study are listed in Table 1. Dulbecco's modified Eagle's medium and Ham's F-12 medium were obtained from Nissui Pharmaceutical Co. The EIA (enzyme immunoassay) kit for cAMP measurement and the first-strand cDNA synthesis kit were from GE Healthcare. Lipofectamine (TM2000) and the pcDNA3.1 plasmid were purchased from Invitrogen. HilyMax was from Dojindo Laboratories. The plasmids pcDNA3.1/APPsw, pcDNA3/APP695 and pcDNA3/APP695 T668A were from our laboratory stocks (28Ando K. Iijima K.I. Elliott J.I. Kirino Y. Suzuki T. J. Biol. Chem. 2001; 276: 40353-40361Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 29Tomita S. Kirino Y. Suzuki T. J. Biol. Chem. 1998; 273: 19304-19310Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The plasmid pEGFP-N1 was obtained from Clontech. Antibodies against actin or Thr-668 phosphorylated APP were obtained from Santa Cruz or Cell Signaling, respectively. An antibody against EP2 receptor was from Cayman Chemical. Fetal bovine serum, PGE2, LY294002, pCPT-cAMP, pCPT-O-Me-cAMP, G418, 3-isobutyl-1-methylxanthine, H-89 and an antibody against the C-terminal fragment of APP were from Sigma. DI-004, AE1-259, AE-248, AE1-329, 8713, AE3-240, and AE3-208 were from our laboratory stocks. The RNeasy kit was from Qiagen. The APP-derived fluorescent substrate of γ-secretase (Nma-Gly-Gly-Val-Val-Ile-Ala-Thr-Val-Lys(Dnp)-d-Arg-d-Arg-d-Arg-NH2) and DAPT were from the Peptide Institute Inc. Taq DNA Polymerase was from TAKARA.TABLE 1Compounds used in this studyCompoundPrimary actionDI-004EP1 agonistAE1-259EP2 agonistAE-248EP3 agonistAE1-329EP4 agonist8713EP1 antagonistAE3-240EP3 antagonistAE3-208EP4 antagonistpCPT-cAMPcAMP analogueLY294002PI3K inhibitorH-89PKA inhibitorpCPT-O-Me-cAMPEpac activatorDAPTγ-Secretase inhibitor Open table in a new tab Animals—APP23 transgenic mice were a gift from Dr. M. Staufenbiel; these mice were generated as previously described (30Sturchler-Pierrat C. Abramowski D. Duke M. Wiederhold K.H. Mistl C. Rothacher S. Ledermann B. Burki K. Frey P. Paganetti P.A. Waridel C. Calhoun M.E. Jucker M. Probst A. Staufenbiel M. Sommer B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13287-13292Crossref PubMed Scopus (1243) Google Scholar). APP23 mice were crossed with EP2–/– mice (31Hizaki H. Segi E. Sugimoto Y. Hirose M. Saji T. Ushikubi F. Matsuoka T. Noda Y. Tanaka T. Yoshida N. Narumiya S. Ichikawa A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10501-10506Crossref PubMed Scopus (403) Google Scholar) to generate APPsw/EP2+/– mice and these mice were again crossed to EP2–/– mice to generate APPsw/EP2–/– mice. Parallel crosses were made between APPsw mice and C57BL/6 mice (wild type mice for EP2–/– mice) to generate APPsw/EP2+/+ control mice. Most EP4–/– mice die in the C57BL/6 background. Therefore, survivors of the F2 progenies of EP4–/– mice in the mixed genetic background of 129/Ola and C57BL/6 were intercrossed and the resulting female survivors were used as described (32Kabashima K. Saji T. Murata T. Nagamachi M. Matsuoka T. Segi E. Tsuboi K. Sugimoto Y. Kobayashi T. Miyachi Y. Ichikawa A. Narumiya S. J. Clin. Investig. 2002; 109: 883-893Crossref PubMed Scopus (407) Google Scholar, 33Segi E. Sugimoto Y. Yamasaki A. Aze Y. Oida H. Nishimura T. Murata T. Matsuoka T. Ushikubi F. Hirose M. Tanaka T. Yoshida N. Narumiya S. Ichikawa A. Biochem. Biophys. Res. Commun. 1998; 246: 7-12Crossref PubMed Scopus (271) Google Scholar). APP23 mice were crossed to these EP4–/– mice to generate APPsw/EP4+/– mice and these were crossed to EP4–/– mice to generate APPsw/EP4–/– mice. Parallel crosses were made between APPsw mice and mice in the mixed genetic background of 129/Ola and C57BL/6 to generate APPsw/EP4+/+ control mice. The experiments and procedures described here were carried out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institute of Health, and were approved by the Animal Care Committee of Kumamoto University. Cell Culture—HEK293 or SH-SY5Y cells were cultured in Dulbecco's modified Eagle's medium or Dulbecco's modified Eagle's medium/Ham's F-12 medium, respectively, supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin in a humidified atmosphere of 95% air, 5% CO2 at 37 °C. HEK293 and SH-SY5Y cells expressing APPsw were from our laboratory stocks (34Hoshino T. Nakaya T. Araki W. Suzuki K. Suzuki T. Mizushima T. Biochem. J. 2007; 402: 581-589Crossref PubMed Scopus (107) Google Scholar). For transient expression of each gene, cells were seeded 24 h before the transfection in 24-well plates at a density of 1.5 × 105 cells/well. The transfection was carried out using Lipofectamine (TM2000) or HilyMax according to the manufacturer's instructions. Cells were used for experiments after a 24-h recovery period. Transfection efficiency was determined in parallel plates by transfection of cells with pEGFP-N1 control vector. Transfection efficiencies were greater than 90% in all experiments. The stable transfectants expressing each gene were selected by immunoblotting or real-time RT-PCR analyses. Positive clones were maintained in the presence of 200 μg/ml G418. Immunoblotting Analysis—Whole cell extracts were prepared as described previously (35Hoshino T. Tsutsumi S. Tomisato W. Hwang H.J. Tsuchiya T. Mizushima T. J. Biol. Chem. 2003; 278: 12752-12758Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). For detection of the C-terminal fragment (CTF) α and CTFβ, membrane fractions were prepared as described previously (36Gu Y. Misonou H. Sato T. Dohmae N. Takio K. Ihara Y. J. Biol. Chem. 2001; 276: 35235-35238Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). For detection of CTFγ, the membrane fractions were incubated for 2 h at 37°C. The protein concentration of each sample was determined by the Bradford method (37Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214321) Google Scholar). Samples were applied to polyacrylamide SDS gels (Tris-Tricine gel for the detection of APP and Tris glycine gel for other proteins), and subjected to electrophoresis, after which proteins were immunoblotted with their respective antibodies. Sandwich Enzyme-linked Immunosorbent Assay (sELISA) for Aβ and EIA for cAMP—Cells were cultured for 24 h and the conditioned medium was subjected to sELISA using three types of specific monoclonal antibodies, as described previously (29Tomita S. Kirino Y. Suzuki T. J. Biol. Chem. 1998; 273: 19304-19310Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 34Hoshino T. Nakaya T. Araki W. Suzuki K. Suzuki T. Mizushima T. Biochem. J. 2007; 402: 581-589Crossref PubMed Scopus (107) Google Scholar). The amount of Aβ in mouse brain was determined as described previously (38Iwata N. Mizukami H. Shirotani K. Takaki Y. Muramatsu S. Lu B. Gerard N.P. Gerard C. Ozawa K. Saido T.C. J. Neurosci. 2004; 24: 991-998Crossref PubMed Scopus (213) Google Scholar). Briefly, the brain hemispheres were homogenized in 50 mm Tris-HCl buffer, pH 7.6, containing 150 mm NaCl with a homogenizer (Polytron) and centrifuged at 200,000 × g for 20 min at 4 °C. The supernatant, defined as the soluble fraction, was taken and guanidine-HCl added to give a final concentration of 0.5 m before sELISA. The pellet was solubilized by sonication in 6 m guanidine-HCl buffer. The solubilized pellet was centrifuged at 200,000 × g for 20 min at 4 °C, and the resulting supernatant was diluted and termed the insoluble fraction. The amounts of Aβ40 and Aβ42 in each fraction were determined by sELISA. Cells were pre-treated for 30 min with 0.5 m 3-isobutyl-1-methylxanthine (an inhibitor of phosphodiesterase) and further cultured for 10 min with or without PGE2, EP agonists, or EP antagonist. Cells were lysed with ice-cold 100% ethanol and centrifuged. The supernatants were dried, re-suspended in the assay buffer, and applied to the EIA kit for measurement of cAMP, according to the manufacturer's instructions. RT-PCR Analysis—Total RNA was extracted from cells using an RNeasy kit according to the manufacturer's protocols. Samples (10 μg of RNA) were reverse transcribed using a first-strand cDNA synthesis kit according to the manufacturer's instructions. Synthesized cDNA was amplified by PCR (TAKARA PCR Thermal Cycler) using TAKARA Taq DNA polymerase, and reaction products were analyzed by agarose gel electrophoresis. PCR cycle conditions were 2 min at 50 °C, followed by 10 min at 95 °C and finally 35 cycles at each of 95 °C for 20 s, 60 °C for 60 s, and 72 °C for 60 s. Primers were designed using the Primer3 Web site (www.frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). The sequences of the primers (written as target cDNA: forward primer and reverse primer) were: EP1, 5′-accttctttggcggctct-3′ and 5′-gcacgacaccaccatgatac-3′; EP2, 5′-ccacctcattctcctggcta-3′ and 5′-cgacaacagaggactgaacg-3′; EP3, 5′-agcttatggggatcatgtgc-3′ and 5′-tctgcttctccgtgtgtgtc-3′; EP4, 5′-tgcgagtattcgtcaaccag-3′ and 5′-ggtctaggatggggttcaca-3′; and actin, 5′-ggacttcgagcaagagatgg-3′ and 5′-agcactgtgttggcgtacag-3′. γ-Secretase-mediated Peptide Cleavage Assay—We performed the assay as previously reported (39Farmery M.R. Tjernberg L.O. Pursglove S.E. Bergman A. Winblad B. Naslund J. J. Biol. Chem. 2003; 278: 24277-24284Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 40Ni Y. Zhao X. Bao G. Zou L. Teng L. Wang Z. Song M. Xiong J. Bai Y. Pei G. Nat. Med. 2007; 12: 1390-1396Crossref Scopus (185) Google Scholar). Solubilized membranes were re-suspended and incubated overnight at 37 °C in 200 μl of assay buffer containing 50 mm Tris-HCl, pH 6.8, 2 mm EDTA, 0.25% CHAPSO (w/v), and 10 μm APP-derived fluorescent substrate of γ-secretase. We measured fluorescence using a plate reader (Fluorstar Galaxy) with an excitation wavelength of 355 nm and an emission wavelength of 440 nm. Statistical Analysis—All values are expressed as the mean ± S.E. One-way analysis of variance followed by Tukey multiple comparison test or Student's t test for unpaired results was used for evaluation of differences among more than three groups or for the evaluation of differences between two groups, respectively. Differences were considered to be significant for values of p < 0.05. Stimulation of Aβ Production by PGE2—We used HEK293 cells that stably express a form of APP with double mutations (K651N/M652L), known as the "Swedish" mutations (APPsw) (29Tomita S. Kirino Y. Suzuki T. J. Biol. Chem. 1998; 273: 19304-19310Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). These mutations elevate cellular and secreted levels of Aβ (29Tomita S. Kirino Y. Suzuki T. J. Biol. Chem. 1998; 273: 19304-19310Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The amount of these peptides in conditioned medium was determined using a sELISA. Treatment of cells with PGE2 increased the levels of Aβ (Aβ40 and Aβ42) in the conditioned medium (Fig. 1, A and B), a similar result to that observed for CHO cells (26Qin W. Ho L. Pompl P.N. Peng Y. Zhao Z. Xiang Z. Robakis N.K. Shioi J. Suh J. Pasinetti G.M. J. Biol. Chem. 2003; 278: 50970-50977Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). We concluded that this increase is due to stimulation of production of Aβ because, after treatment of the cells with PGE2, not only did the amount of secreted Aβ increase but the amount of Aβ in the cells also increased and because pulse label experiments with [35S]methionine showed that treatment of cells with PGE2 increased generation of CTFs of APP that are co-generated by γ-secretase (CTFγ) with Aβ (data not shown). Although it is not clear whether comparison of concentrations of PGE2 between in vivo and in vitro is reasonable or not, the concentrations of PGE2 required for stimulation of Aβ production (1–10 nm) are within the same range of PGE2 concentrations that are observed in the human brain (41Iwamoto N. Kobayashi K. Kosaka K. J. Neurol. 1989; 236: 80-84Crossref PubMed Scopus (72) Google Scholar). However, the fact that production of Aβ is stimulated by the concentration of PGE2, which is equivalent to that found physiologically is against the idea that PGE2-stimulated production of Aβ seen in vitro is involved in inflammation-stimulated development of AD. We also performed similar experiments in SH-SY5Y cells that stably express APPsw. As shown in Fig. 1, C and D, treatment with PGE2 also increased the level of Aβ in SH-SY5Y cells; however, the extent of stimulation of Aβ production in SH-SY5Y cells was not as dramatic as that seen in HEK293 cells (Fig. 1, A and B). We confirmed that PGE2 did not affect the cell growth and intracellular lactate dehydrogenase activity at concentrations used in Fig. 1 in both HEK293 and SH-SY5Y cells (supplemental Fig. S1). Identification of EP Receptors Involved in PGE2-stimulated Production of Aβ—We used agonists specific for each EP receptor (Table 1) to identify EP receptors involved in the PGE2-stimulated production of Aβ. Initially, we used HEK293 cells and examined the mRNA expression of each EP receptor by RT-PCR. As shown in Fig. 2A, mRNA for each of the EP receptors was detected, although the level of expression varied between them. We also confirmed the expression of EP2 receptor by immunoblotting experiments (Fig. 5A). Then, we examined the effect of agonists specific for each EP receptor on the production of Aβ in HEK293 cells. Treatment of cells with AE1-329 (an EP4 agonist) increased the level of Aβ in conditioned medium (Fig. 2, H and I). The amplitude of this increase was similar to that achieved with PGE2 (Fig. 1, A and B). Based on previously reported findings using AE1-329, it is reasonable to postulate that, for the concentrations used in the experiment described in Fig. 2, H and I, it acts as a specific agonist for the EP4 receptor (42Suzawa 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). In contrast, none of the other specific agonists, including DI-004 (an EP1 agonist), AE1-259 (an EP2 agonist), and AE-248 (an EP3 agonist), significantly affected the level of Aβ (Fig. 2, B–G). Based on previous reports, the concentrations of EP agonists employed in the experiments described in Fig. 2 should have been sufficient to activate their respective EP receptor (42Suzawa 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). We confirmed that each agonist did not affect the cell growth at these concentrations used in Fig. 2 (data not shown). Consequently, the results in Fig. 2 suggest that EP4 is responsible for the PGE2-stimulated production of Aβ in HEK293 cells. As described below, we suggested that EP2 receptor is not functional in HEK293 cells (Fig. 5A). Furthermore, we found that none of PGE2, DI-004, and AE-248 increased the intracellular Ca2+ levels (supplemental Fig. S2), suggesting that neither EP1 nor EP3 receptor is functional in HEK293 cells (both EP1 and EP3 receptors are coupled to Ca2+ mobilization (27Coleman R.A. Smith W.L. Narumiya S. Pharmacol. Rev. 1994; 46: 205-229PubMed Google Scholar, 43Ma H. Hara A. Xiao C.Y. Okada Y. Takahata O. Nakaya K. Sugimoto Y. Ichikawa A. Narumiya S. Ushikubi F. Circulation. 2001; 104: 1176-1180Crossref PubMed Scopus (95) Google Scholar)). Thus, we could not conclude that activation of these receptors (EP1, EP2, and EP3) does not affect the level of Aβ in general, based on the inability of DI-004, AE1-259, and AE-248 to affect the level of Aβ in HEK293 cells.FIGURE 5Involvement of the cellular level of cAMP in PGE2-stimulated produc
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