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• W2167029659 abstract "Here we report the molecular identification of membrane-bound glutathione (GSH)-dependent prostaglandin (PG) E2 synthase (mPGES), a terminal enzyme of the cyclooxygenase (COX)-2-mediated PGE2 biosynthetic pathway. The activity of mPGES was increased markedly in macrophages and osteoblasts following proinflammatory stimuli. cDNA for mouse and rat mPGESs encoded functional proteins that showed high homology with the human ortholog (microsomal glutathioneS-transferase-like 1). mPGES expression was markedly induced by proinflammatory stimuli in various tissues and cells and was down-regulated by dexamethasone, accompanied by changes in COX-2 expression and delayed PGE2 generation. Arg110, a residue well conserved in the microsomal GSHS-transferase family, was essential for catalytic function. mPGES was functionally coupled with COX-2 in marked preference to COX-1, particularly when the supply of arachidonic acid was limited. Increased supply of arachidonic acid by explosive activation of cytosolic phospholipase A2 allowed mPGES to be coupled with COX-1. mPGES colocalized with both COX isozymes in the perinuclear envelope. Moreover, cells stably cotransfected with COX-2 and mPGES grew faster, were highly aggregated, and exhibited aberrant morphology. Thus, COX-2 and mPGES are essential components for delayed PGE2 biosynthesis, which may be linked to inflammation, fever, osteogenesis, and even cancer. Here we report the molecular identification of membrane-bound glutathione (GSH)-dependent prostaglandin (PG) E2 synthase (mPGES), a terminal enzyme of the cyclooxygenase (COX)-2-mediated PGE2 biosynthetic pathway. The activity of mPGES was increased markedly in macrophages and osteoblasts following proinflammatory stimuli. cDNA for mouse and rat mPGESs encoded functional proteins that showed high homology with the human ortholog (microsomal glutathioneS-transferase-like 1). mPGES expression was markedly induced by proinflammatory stimuli in various tissues and cells and was down-regulated by dexamethasone, accompanied by changes in COX-2 expression and delayed PGE2 generation. Arg110, a residue well conserved in the microsomal GSHS-transferase family, was essential for catalytic function. mPGES was functionally coupled with COX-2 in marked preference to COX-1, particularly when the supply of arachidonic acid was limited. Increased supply of arachidonic acid by explosive activation of cytosolic phospholipase A2 allowed mPGES to be coupled with COX-1. mPGES colocalized with both COX isozymes in the perinuclear envelope. Moreover, cells stably cotransfected with COX-2 and mPGES grew faster, were highly aggregated, and exhibited aberrant morphology. Thus, COX-2 and mPGES are essential components for delayed PGE2 biosynthesis, which may be linked to inflammation, fever, osteogenesis, and even cancer. prostaglandin prostaglandin E2 synthase membrane-bound prostaglandin E2 synthase cytosolic prostaglandin E2 synthase cyclooxygenase cytosolic phospholipase A2 thromboxane synthase hematopoietic prostaglandin D2 synthase leukotriene C4 synthase lipopolysaccharide glutathione glutathione S-transferase microsomal GST microsomal glutathioneS-transferase-like 1 membrane-associated proteins involved in eicosanoid and GSH metabolism arachidonic acid phosphate-buffered saline interleukin tumor necrosis factor fetal calf serum human embryonic kidney fluorescein isothiocyanate reverse transcriptase polymerase chain reaction rapid amplification of cDNA ends Tris-buffered saline plus Tween 20 The two kinetically distinct prostaglandin (PG)1 biosynthetic responses, the immediate and delayed phases, imply the recruitment of different sets of the biosynthetic enzymes whose expression and activation are tightly regulated by post-receptor transmembrane signaling. In immediate PG biosynthesis, which occurs within several minutes after stimulation with agonists that increase cytoplasmic Ca2+levels, cytosolic phospholipase A2 (cPLA2) is a prerequisite for supplying arachidonic acid (AA) to the constitutive cyclooxygenase (COX) isozyme, COX-1 (1Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 2Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 3Murakami M. Matsumoto R. Austen K.F. Arm J.P. J. Biol. Chem. 1994; 269: 22269-22275Abstract Full Text PDF PubMed Google Scholar, 4Kuwata H. Nakatani Y. Murakami M. Kudo I. J. Biol. Chem. 1998; 273: 1733-1740Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 5Uozumi N. Kume K. Nagase T. Nakatani N. Ishii S. Tashiro F. Komagata Y. Maki K. Ikuta K. Ouchi Y. 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Lam B.K. Sapirstein A. Bonventre J.V. Austen K.F. Arm J.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4803-4808Crossref PubMed Scopus (168) Google Scholar, 7Naraba H. Murakami M. Matsumoto H. Shimbara S. Ueno A. Kudo I. Oh-ishi S. J. Immunol. 1998; 160: 2974-2982PubMed Google Scholar, 8Murakami M. Kuwata H. Amakasu Y. Shimbara S. Nakatani Y. Atsumi G. Kudo I. J. Biol. Chem. 1997; 272: 19891-19897Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 9Murakami M. Kambe T. Shimbara S. Higashino K. Hanasaki K. Arita H. Horiguchi M. Arita M. Arai H. Inoue K. Kudo I. J. Biol. Chem. 1999; 274: 31435-31444Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 10Smith W.L. Garavito R.M. DeWitt D.L. J. Biol. Chem. 1996; 271: 33157-33160Abstract Full Text Full Text PDF PubMed Scopus (1843) Google Scholar). cPLA2 and several inducible secretory phospholipase A2 isozymes cooperatively contribute to supplying AA to COX-2 (1Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 2Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 4Kuwata H. Nakatani Y. Murakami M. Kudo I. J. Biol. Chem. 1998; 273: 1733-1740Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 5Uozumi N. Kume K. Nagase T. Nakatani N. Ishii S. Tashiro F. Komagata Y. Maki K. Ikuta K. Ouchi Y. Miyazaki J. Shimizu T. Nature. 1997; 390: 618-621Crossref PubMed Scopus (640) Google Scholar, 6Fujishima H. Sanchez Mejia R.O. Bingham C.O. Lam B.K. Sapirstein A. Bonventre J.V. Austen K.F. Arm J.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4803-4808Crossref PubMed Scopus (168) Google Scholar, 7Naraba H. Murakami M. Matsumoto H. Shimbara S. Ueno A. Kudo I. Oh-ishi S. J. Immunol. 1998; 160: 2974-2982PubMed Google Scholar, 8Murakami M. Kuwata H. Amakasu Y. Shimbara S. Nakatani Y. Atsumi G. Kudo I. J. Biol. Chem. 1997; 272: 19891-19897Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 9Murakami M. Kambe T. Shimbara S. Higashino K. Hanasaki K. Arita H. Horiguchi M. Arita M. Arai H. Inoue K. Kudo I. J. Biol. Chem. 1999; 274: 31435-31444Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 11Roshak A. Sathe G. Marshall L.A. J. Biol. Chem. 1994; 269: 25999-26005Abstract Full Text PDF PubMed Google Scholar, 12Shinohara H. Balboa M.A. Johnson C.A. Balsinde J. Dennis E.A. J. Biol. Chem. 1999; 274: 12263-12268Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 13Nakatani Y. Tanioka Y. Sunaga S. Murakami M. Kudo I. J. Biol. Chem. 2000; 275: 1161-1168Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). The preference of COX-2 over COX-1 in the delayed response is explained, at least in part, by the ability of COX-2 to metabolize lower levels of AA to PGH2 than those required for COX-1-directed catalysis (2Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 14Kulmacz R.J. Wang L.-H. J. Biol. Chem. 1995; 270: 24019-24023Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 15Shitashige M. Morita I. Murota S. Biochim. Biophys. Acta. 1998; 1389: 57-66Crossref PubMed Scopus (70) Google Scholar). When cells are first treated with proinflammatory stimuli and subsequently exposed to Ca2+ mobilizers, the inducible COX-2 can also promote the immediate response (priming or induced immediate response) (7Naraba H. Murakami M. Matsumoto H. Shimbara S. Ueno A. Kudo I. Oh-ishi S. J. Immunol. 1998; 160: 2974-2982PubMed Google Scholar, 16Chen Q.-R. Miyaura C. Higashi S. Murakami M. Kudo I. Saito S. Hiraide T. Shibasaki Y. Suda T. J. Biol. Chem. 1997; 272: 5952-5968Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Understanding COX-2-dependent biological responses has received much attention in the past few years, because numerous pharmacological, biological and genetic studies have suggested that this inducible COX isozyme is involved in various human diseases, including inflammation and cancer (10Smith W.L. Garavito R.M. DeWitt D.L. J. Biol. Chem. 1996; 271: 33157-33160Abstract Full Text Full Text PDF PubMed Scopus (1843) Google Scholar, 17Harada Y. Hatanaka K. Kawamura M. Saito M. Ogino M. Majima M. Ohno T. Ogino K. Yamamoto K. Taketani Y. Yamamoto S. Katori M. Prostaglandins. 1996; 51: 19-33Crossref PubMed Scopus (97) Google Scholar, 18Langenbach R. Morham S.G. Tiano H.F. Loftin C.D. Ghanayem B.I. Chulada P.C. Mahler J.F. Lee C.A. Goulding E.H. Kluckman K.D. Ledford A. Lee C.A. Smithies O. 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Vane J.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7563-7568Crossref PubMed Scopus (1420) Google Scholar). In many cells, the main PG species produced during the delayed response is PGE2. Indeed, among the several PGs produced by macrophages, only the level of PGE2 was increased during the delayed response (7Naraba H. Murakami M. Matsumoto H. Shimbara S. Ueno A. Kudo I. Oh-ishi S. J. Immunol. 1998; 160: 2974-2982PubMed Google Scholar, 25Fournier T. Fadok V. Henson P.M. J. Biol. Chem. 1997; 272: 31065-31072Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar,26Matsumoto H. Naraba H. Murakami M. Kudo I. Yamaki K. Ueno A. Oh-ishi S. Biochem. Biophys. Res. Commun. 1997; 230: 110-114Crossref PubMed Scopus (144) Google Scholar). Moreover, in vivo studies have shown that COX-2 inhibitors reduce PGE2 more profoundly than other PGs (17Harada Y. Hatanaka K. Kawamura M. Saito M. Ogino M. Majima M. Ohno T. Ogino K. Yamamoto K. Taketani Y. Yamamoto S. Katori M. Prostaglandins. 1996; 51: 19-33Crossref PubMed Scopus (97) Google Scholar). Thus, the COX-2-dependent pathway may be more selectively linked to the terminal PGE2 synthase (PGES). More importantly, several recent studies have suggested that PGES activity is increased during the period when COX-2-dependent delayed PGE2 generation is ongoing (7Naraba H. Murakami M. Matsumoto H. Shimbara S. Ueno A. Kudo I. Oh-ishi S. J. Immunol. 1998; 160: 2974-2982PubMed Google Scholar, 26Matsumoto H. Naraba H. Murakami M. Kudo I. Yamaki K. Ueno A. Oh-ishi S. Biochem. Biophys. Res. Commun. 1997; 230: 110-114Crossref PubMed Scopus (144) Google Scholar). PGES activity has been detected in both cytosolic and membrane-associated fractions of various cells and tissues (27Watanabe K. Kurihara K. Hayaishi O. Biochem. Biophys. Res. Commun. 1997; 235: 148-152Crossref PubMed Scopus (71) Google Scholar, 28Ogorochi T. Ujihara M. Narumiya S. J. Neurochem. 1987; 48: 900-909Crossref PubMed Scopus (56) Google Scholar, 29Watanabe K. Kurihara K. Suzuki T. Biochim. Biophys. Acta. 1999; 1439: 406-414Crossref PubMed Scopus (96) Google Scholar, 30Jakobsson P.-J. Thoren S. Morgenstern R. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7220-7225Crossref PubMed Scopus (893) Google Scholar). In most cases the enzyme requires glutathione (GSH) for catalytic activity (27Watanabe K. Kurihara K. Hayaishi O. Biochem. Biophys. Res. Commun. 1997; 235: 148-152Crossref PubMed Scopus (71) Google Scholar, 28Ogorochi T. Ujihara M. Narumiya S. J. Neurochem. 1987; 48: 900-909Crossref PubMed Scopus (56) Google Scholar, 30Jakobsson P.-J. Thoren S. Morgenstern R. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7220-7225Crossref PubMed Scopus (893) Google Scholar). In an effort to identify PGES isoforms, we have succeeded in identification of the GSH-dependent cytosolic PGES (cPGES/p23), as shown in an accompanying paper (31Tanioka T. Nakatani Y. Semmyo N. Murakami M. Kudo I. J. Biol. Chem. 2000; 275: 32775-32782Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar). However, this enzyme is constitutively expressed in a wide variety of cells and tissues and shows preferential functional coupling with COX-1. The linkage between the three constitutive enzymes of the biosynthetic cascade (i.e. cPLA2, COX-1, and cPGES/p23) implies that this pathway is crucial for the production of the PGE2 required for maintenance of tissue homeostasis. In view of the fact that PGE2 is often produced via the COX-2-dependent pathway (1Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 2Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 3Murakami M. Matsumoto R. Austen K.F. Arm J.P. J. Biol. Chem. 1994; 269: 22269-22275Abstract Full Text PDF PubMed Google Scholar, 7Naraba H. Murakami M. Matsumoto H. Shimbara S. Ueno A. Kudo I. Oh-ishi S. J. Immunol. 1998; 160: 2974-2982PubMed Google Scholar, 8Murakami M. Kuwata H. Amakasu Y. Shimbara S. Nakatani Y. Atsumi G. Kudo I. J. Biol. Chem. 1997; 272: 19891-19897Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 9Murakami M. Kambe T. Shimbara S. Higashino K. Hanasaki K. Arita H. Horiguchi M. Arita M. Arai H. Inoue K. Kudo I. J. Biol. Chem. 1999; 274: 31435-31444Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 11Roshak A. Sathe G. Marshall L.A. J. Biol. Chem. 1994; 269: 25999-26005Abstract Full Text PDF PubMed Google Scholar, 12Shinohara H. Balboa M.A. Johnson C.A. Balsinde J. Dennis E.A. J. Biol. Chem. 1999; 274: 12263-12268Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 16Chen Q.-R. Miyaura C. Higashi S. Murakami M. Kudo I. Saito S. Hiraide T. Shibasaki Y. Suda T. J. Biol. Chem. 1997; 272: 5952-5968Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 17Harada Y. Hatanaka K. Kawamura M. Saito M. Ogino M. Majima M. Ohno T. Ogino K. Yamamoto K. Taketani Y. Yamamoto S. Katori M. Prostaglandins. 1996; 51: 19-33Crossref PubMed Scopus (97) Google Scholar,26Matsumoto H. Naraba H. Murakami M. Kudo I. Yamaki K. Ueno A. Oh-ishi S. Biochem. Biophys. Res. Commun. 1997; 230: 110-114Crossref PubMed Scopus (144) Google Scholar), we looked for another PGES that is induced by proinflammatory stimuli and shows selective coupling with COX-2. While this study was under way, Jakobsson et al. (30Jakobsson P.-J. Thoren S. Morgenstern R. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7220-7225Crossref PubMed Scopus (893) Google Scholar) reported that human microsomal GST-like 1 (MGST-L1), a member of the MAPEG (membrane-associated proteins involved in eicosanoid and GSH metabolism) superfamily (32Jakobsson P.-J. Morgenstern R. Mancini J. Ford-Hunchinton A. Persson B. Protein Sci. 1999; 8: 689-692Crossref PubMed Scopus (298) Google Scholar), exhibits significant PGES activity. Moreover, the expression of this enzyme has been shown to increase after stimulation with interleukin (IL)-1 in A549 cells (30Jakobsson P.-J. Thoren S. Morgenstern R. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7220-7225Crossref PubMed Scopus (893) Google Scholar). In the present study, we show that MGST-L1 is identical to the membrane-associated PGES (mPGES), which we have originally detected in lipopolysaccharide (LPS)-stimulated macrophages (7Naraba H. Murakami M. Matsumoto H. Shimbara S. Ueno A. Kudo I. Oh-ishi S. J. Immunol. 1998; 160: 2974-2982PubMed Google Scholar). mPGES/MGST-L1 expression is strongly induced in several cells and tissues related to the inflammatory response in vitro and in vivo. Coexpression experiments clearly demonstrate that mPGES/MGST-L1 is preferentially linked with COX-2, promoting delayed and induced immediate PGE2 biosynthesis. Furthermore, sustained expression of both COX-2 and mPGES/MGST-L1 leads to aberrant cell growth. Our results indicate the presence of two segregated PGE2-biosynthetic routes, the cPLA2-COX-1-cPGES/p23 and cPLA2-COX-2-mPGES/MGST-L1 pathways, in the same cell. Harlan Sprague-Dawley rats and C57BL/6 mice were purchased from Japan SLC (Hamamatsu, Japan). Rabbits (New Zealand White, 1-kg body weight, female) were from Saitama Experimental Animal Supply (Saitama, Japan). The goat anti-human COX-2 and rabbit anti-human cPLA2 antibodies were purchased from Santa Cruz. The rabbit anti-rat hematopoietic PGD2 synthase (hPGDS) antibody was a generous gift from Dr. Y. Urade (Osaka Bioscience Institute, Osaka, Japan). cDNA probes for human COX-1, human COX-2, and mouse COX-2 were described previously (2Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 3Murakami M. Matsumoto R. Austen K.F. Arm J.P. J. Biol. Chem. 1994; 269: 22269-22275Abstract Full Text PDF PubMed Google Scholar). Human cPGES/p23 cDNA was described in the accompanying paper (31Tanioka T. Nakatani Y. Semmyo N. Murakami M. Kudo I. J. Biol. Chem. 2000; 275: 32775-32782Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar). Rat thromboxane synthase (TXS) cDNA was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) using rat platelet mRNA as a template using 5′- and 3′-primers corresponding the N- and C-terminal 23-base pair nucleotide sequences. The touchdown PCR condition was 94 °C for 30 s and then 30 cycles of 94 °C for 5 s and 68 °C for 4 min using Advantage cDNA polymerase mix (CLONTECH). Superscript II RNase H-reverse transcriptase, RNase H, Taq DNA polymerase, dNTP mixture, LipofectAMINE Plus reagent, Opti-MEM, RPMI 1640 medium, and TRIzol reagent were obtained from Life Technologies, Inc.. Bacterial LPS (E. coli O111:B4), dexamethasone, fetal calf serum (FCS), GSH, and mouse anti-FLAG epitope monoclonal antibody were purchased from Sigma. Freund's complete and incomplete adjuvants, thioglycollate, and bactopeptone were from Difco Laboratories. PGH2, rabbit anti-human COX-1, and anti-mouse COX-2 polyclonal antibodies, and the enzyme immunoassay kits for PGE2 and TXB2 were from Cayman Chemical. AA was purchased from NuChek Prep. Oligonucleotide primers were from Amersham Pharmacia Biotech. The plasmid pGEM-T easy was purchased from Promega. Geneticin, hygromycin, zeocin, and the mammalian expression vectors pCR3.1, pCDNA3.1/hyg(+), and pCDNA3.1/zeo(+) were from Invitrogen. A23187 was purchased from Calbiochem. Human and mouse interleukin (IL)-1βs and mouse tumor nectoris factor (TNF) α were from Genzyme. Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG, FITC-rabbit anti-goat IgG, and FITC-goat anti-rabbit IgG antibodies, and horseradish peroxidase-conjugated anti-rabbit and mouse IgGs were purchased from Zymed Laboratories Inc.Cy3-conjugated donkey anti-rabbit IgG antibody was from Chemicon. Other reagents were obtained from Wako Pure Chemical Industries. Computational analysis on the isolated cDNAs and related sequences were performed using the GENETYX program (Software Development). Culture of human embryonic kidney (HEK) 293 cells (1Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 2Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar), mouse osteoblastic MC3T3-E1 cells (8Murakami M. Kuwata H. Amakasu Y. Shimbara S. Nakatani Y. Atsumi G. Kudo I. J. Biol. Chem. 1997; 272: 19891-19897Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) and rat calvaria osteoblasts (33Higashi S. Ohishi H. Kudo I. Inflammation Res. 2000; 49: 102-111Crossref PubMed Scopus (21) Google Scholar) was described previously. To prepare macrophages, 5% (w/v) Bactopeptone in saline (5 ml/100 g of body weight) was injected intraperitoneally into Harlan Sprague-Dawley rats and 4% thioglycollate (w/v) solution was injected intraperitoneally into C57BL/6 mice (1 ml/20 g body weight). The peritoneal exudate cells of rats and mice were collected on day 4 by washing the cavity with ice-cold Ca2+/Mg2+-free Hanks' balanced salt solution. The cells were washed twice and plated onto six-well plastic plates (Corning) at a density of 4.5 × 106 cells/well in 2 ml of RPMI 1640 medium containing 10% (v/v) FCS. After 2 h of incubation at 37 ?C in a humidified atmosphere of 5% CO2 and 95% air, non-adherent cells were removed by rinsing. Then RPMI 1640 medium containing 10% FCS was added to the adherent cells and used as macrophages. The cells were incubated in the medium with or without 10 μg/ml LPS for up to 24 h. After incubation, PGE2 and TXB2 accumulated in the supernatants were measured by the enzyme immunoassay kits, and PGD2 was quantified by high performance liquid chromatography, as described previously (26Matsumoto H. Naraba H. Murakami M. Kudo I. Yamaki K. Ueno A. Oh-ishi S. Biochem. Biophys. Res. Commun. 1997; 230: 110-114Crossref PubMed Scopus (144) Google Scholar). In some experiments, the cells were incubated with LPS in the presence of 10 μmdexamethasone. Total RNA was extracted from mouse and rat peritoneal macrophages incubated with LPS for 12 h by using TRIzol reagent. The RT reaction was carried out by using the Superscript preamplification system (Life Technologies) according to the manufacturer's instructions. RNA (1 μg) was mixed with 1 μl of 50 μg/ml random hexamer oligonucleotides and 200 units of reverse transcriptase in a total volume of 20 μl, and incubated for 50 min at 42 °C. The PCR primers 5′-ATC AAG ATG TAC GTG GTG GC-3′ (sense) and 5′-GAG CTG GGC CAG GGT GTA GG-3′ (antisense), designed on the basis of the reported cDNA sequence of human mPGES (MGST-L1) (30Jakobsson P.-J. Thoren S. Morgenstern R. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7220-7225Crossref PubMed Scopus (893) Google Scholar), were used for PCR amplification. PCR was performed by adding both primers (0.2 μm for each) and an appropriate amount of template DNA to 25 μl of PCR buffer (20 mmTris-HCl, pH 8.4, 50 mm KCl, 1.5 mmMgCl2, and 0.05% W-1) containing 0.5 units ofTaq DNA polymerase and 0.2 mm dNTP. The reaction was carried out with 36 cycles of 30 s of denaturation at 94 °C, 30 s of annealing at 58 °C, and 45 s of extension at 72 °C using a DNA thermal cycler (PerkinElmer Life Sciences). The amplified DNA fragments were directly subcloned into the TA cloning vector pGEM and sequenced by an autofluorometric DNA sequencer DSQ-1000L (Shimadzu) using the Thermo Sequenase cycle sequencing kit (Amersham Pharmacia Biotech). The cDNA fragments flanking the 3′-end regions of mouse and rat PGES cDNAs were obtained by the 3′-RACE method. The first strand cDNA from the total RNA isolated from LPS-treated macrophages was synthesized by the RT reaction as follows. After denaturation at 70 °C for 10 min, 1.2 μg of RNA was mixed with 500 nm(dT)17-adaptor primer (5′-GGC CAC GCG TCG ACT AGT AC(dT)17-3′) and 200 units of reverse transcriptase (Life Technologies, Inc.) and incubated for 50 min at 42 °C in 20 μl of reaction mixture (20 mm Tris, pH 8.4, 50 mmKCl, 2.5 mm MgCl2, 0.5 mm dNTP, and 10 mm dithiothreitol). After heating at 70 °C for 15 min, the reaction mixture was further incubated with 2 units of RNase H at 37 °C for 20 min. The materials obtained were used for nested PCR amplification with the adapter primer (5′-GGC CAC GCG TCG ACT AGT AC-3′) and the gene-specific primers for mouse (5′-TGT CAT CAC AGG CCA GAT-3′) or rat (5′-TGT CAT CAC AGG CCA AGT-3′) mPGES. These gene-specific primers were designed on the basis of the nucleotide sequence data of the partial cDNA fragments obtained above. 3′-RACE-PCR was performed by 36 cycles of 30 s of denaturation at 94 °C, 30 s of annealing at 54 °C, and 45 s of extension at 72 °C. The nested PCR products were subcloned into the pGEM vector and sequenced. 5′-RACE was conducted using the 5′-RACE system version 2.0 (Life Technologies, Inc) according to the manufacturer's instructions. The 5′-gene-specific primers used for this RACE were based on the nucleotide sequence data obtained above (5′-TCG ATT AAG GCG TGG GCT-3′ for mouse and 5′-GGA GCG AAT GCG GGG-3′ for rat). Total RNAs (0.5 μg) from LPS-treated mouse and rat macrophages were reverse-transcribed using 5′-gene-specific primers as described above for the 3′-RACE. The first strand products were isolated using a GlassMax DNA isolation spin cartridge (Life Technologies, Inc.). A 10-μl portion of cDNA was heated at 94 °C for 2 min and incubated with 0.4 units of terminal deoxynucleotidyltransferase at 37 °C for 10 min in 25 μl of reaction mixture (10 mm Tris, pH 8.4, 25 mmKCl, 1 mm MgCl2, and 0.2 mm dCTP). The first PCR was carried out using the 5′-gene-specific primers for mouse (5′-TTG TCT CCA TGT CGT TGC-3′) or rat (5′-TCG TCT CCA TGT CGT TGC-3′) mPGES and the anchor primer (5′-CUA CUA CUA CUA GGC CAC GCG TCG ACT AGT ACG GGI IGG GII GGG IIG-3′) under the same amplification condition as for the 3′-RACE. Subsequently, a 1-μl aliquot of the first PCR product was subjected to the second PCR amplification using the upstream 5′-gene-specific primers (5′-ATC TGG CCT GTG ATG ACA-3′ for mouse and 5′-ACT TGG CCT GTG ATG ACA-3′ for rat) and the universal amplification primer (5′-CUA CUA CUA CUA GGC CAC GCG TCG ACT AGT AC-3′). The PCR products were subcloned into the pGEM vector and sequenced. The cDNA encoding the open reading frame of human PGES was amplified by RT-PCR as described above using total RNA obtained from human umbilical vein endothelial cells as a template and the following oligonucleotide primers designed from the human mPGES (MGST-L1) cDNA sequence (30Jakobsson P.-J. Thoren S. Morgenstern R. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7220-7225Crossref PubMed Scopus (893) Google Scholar): sense, 5′-ATG CCT GCC CAC AGC CTG-3′; and antisense, 5′-TCA CAG GTG GCG GGC CGC-3′. To obtain the C-terminally FLAG-epitope-tagged human mPGES cDNA, PCR was conducted using the sense primer (see above) and the antisense primer 5′-TCACTTGTCATCGTCGTCCTTGTAGTC CAG GTG GCG GGC CGC TTC-3′ (the underlined sequence corresponds to the FLAG epitope). The amplified product was subcloned into pCR3.1 and sequenced. Site-specific mutations were introduced by mismatched primer PCR reactions with Advantage cDNA polymerase mix using human mPGES cDNA as a template, as described previously (34Murakami M. Nakatani Y. Kudo I. J. Biol. Chem. 1996; 271: 30041-30051Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). In order to obtain the R110S mutant, a product obtained from the PCR using the mutated sense primer 5′-CTC GTG GGCAGT GTG GCA CAC-3′, in which Arg110 was replaced by Ser at the underlined site, and the C terminus antisense primer that was tagged with the FLAG epitope (underlined) 5′-TCACTTGTCATCGTCGTCCTTGTAGTC CAG GTG GCG GGC CGC T-3′ (C-FLAG primer) was mixed with another product obtained from the PCR using the N terminus sense primer 5′-ATG CCT GCC CAC AGC CTG-3′ (N-primer) and the mutated antisense primer 5′-GTG TGC CAC ACT GCC CAC GAG-3′. After annealing, the second PCR was carried out using the N-primer and C-FLAG primer. The mutants R70S (Arg70 replaced by Ser) and Y117F (Tyr117replaced by Phe) were prepared by using the same strategy. The sequences of the m" @default.
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• W2167029659 title "Regulation of Prostaglandin E2 Biosynthesis by Inducible Membrane-associated Prostaglandin E2 Synthase That Acts in Concert with Cyclooxygenase-2" @default.
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