Title: Cellular Prostaglandin E2 Production by Membrane-bound Prostaglandin E Synthase-2 via Both Cyclooxygenases-1 and -2
Abstract: Current evidence suggests that two forms of prostaglandin (PG) E synthase (PGES), cytosolic PGES and membrane-bound PGES (mPGES) -1, preferentially lie downstream of cyclooxygenase (COX) -1 and -2, respectively, in the PGE2 biosynthetic pathway. In this study, we examined the expression and functional aspects of the third PGES enzyme, mPGES-2, in mammalian cells and tissues. mPGES-2 was synthesized as a Golgi membrane-associated protein, and spontaneous cleavage of the N-terminal hydrophobic domain led to the formation of a truncated mature protein that was distributed in the cytosol with a trend to be enriched in the perinuclear region. In several cell lines, mPGES-2 promoted PGE2 production via both COX-1 and COX-2 in the immediate and delayed responses with modest COX-2 preference. In contrast to the marked inducibility of mPGES-1, mPGES-2 was constitutively expressed in various cells and tissues and was not increased appreciably during tissue inflammation or damage. Interestingly, a considerable elevation of mPGES-2 expression was observed in human colorectal cancer. Collectively, mPGES-2 is a unique PGES that can be coupled with both COXs and may play a role in the production of the PGE2 involved in both tissue homeostasis and disease. Current evidence suggests that two forms of prostaglandin (PG) E synthase (PGES), cytosolic PGES and membrane-bound PGES (mPGES) -1, preferentially lie downstream of cyclooxygenase (COX) -1 and -2, respectively, in the PGE2 biosynthetic pathway. In this study, we examined the expression and functional aspects of the third PGES enzyme, mPGES-2, in mammalian cells and tissues. mPGES-2 was synthesized as a Golgi membrane-associated protein, and spontaneous cleavage of the N-terminal hydrophobic domain led to the formation of a truncated mature protein that was distributed in the cytosol with a trend to be enriched in the perinuclear region. In several cell lines, mPGES-2 promoted PGE2 production via both COX-1 and COX-2 in the immediate and delayed responses with modest COX-2 preference. In contrast to the marked inducibility of mPGES-1, mPGES-2 was constitutively expressed in various cells and tissues and was not increased appreciably during tissue inflammation or damage. Interestingly, a considerable elevation of mPGES-2 expression was observed in human colorectal cancer. Collectively, mPGES-2 is a unique PGES that can be coupled with both COXs and may play a role in the production of the PGE2 involved in both tissue homeostasis and disease. Biosynthesis of prostaglandin (PG) 1The abbreviations used are: PG, prostaglandin; AA, arachidonic acid; PGES, PGE synthase; mPGES, membrane-bound PGES; cPGES, cytosolic PGES; PLA2, phospholipase A2; COX, cyclooxygenase; IL-1β, interleukin-1β; TNFα, tumor necrosis factor α; IFN, interferon; RA, rheumatoid arthritis; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; ER, endoplasmic reticulum; FITC, fluorescein isothiocyanate; RT, reverse transcriptase; FL, full-length; HEK, human embryonic kidney. E2, which is produced by a variety of cells and tissues and exhibits diverse bioactivities, is mediated by three enzymatic reactions involving phospholipase A2 (PLA2), cyclooxygenase (COX), and PGE synthase (PGES). In this biosynthetic pathway, arachidonic acid (AA) released from membrane phospholipids by cytosolic or secretory PLA2s is converted to PGH2 by COX-1 or COX-2 and is then isomerized to PGE2 by terminal PGES enzymes. The constitutive COX-1 mainly promotes immediate PG production elicited by agonists promptly mobilizing intracellular Ca2+, a situation in which a burst release of AA occurs (1Murakami M. Nakatani Y. Tanioka T. Kudo I. Prostaglandins Other Lipid Mediat. 2002; 68–69: 383-399Crossref PubMed Scopus (234) Google Scholar, 2Morita I. Prostaglandins Other Lipid Mediat. 2002; 68–69: 165-175Crossref PubMed Scopus (354) Google Scholar, 3Smith W.L. DeWitt D.L. Garavito R.M. Annu. Rev. Biochem. 2000; 69: 145-182Crossref PubMed Scopus (2521) Google Scholar, 4Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 5Smith W.L. Langenbach R. J. Clin. Invest. 2001; 107: 1491-1495Crossref PubMed Scopus (539) Google Scholar). The inducible COX-2 is essential for delayed PG generation induced by proinflammatory stimuli, during which AA is gradually supplied over long periods, and also promotes immediate PG production if it already exists in cells primed by particular stimuli (1Murakami M. Nakatani Y. Tanioka T. Kudo I. Prostaglandins Other Lipid Mediat. 2002; 68–69: 383-399Crossref PubMed Scopus (234) Google Scholar, 2Morita I. Prostaglandins Other Lipid Mediat. 2002; 68–69: 165-175Crossref PubMed Scopus (354) Google Scholar, 3Smith W.L. DeWitt D.L. Garavito R.M. Annu. Rev. Biochem. 2000; 69: 145-182Crossref PubMed Scopus (2521) Google Scholar, 4Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 5Smith W.L. Langenbach R. J. Clin. Invest. 2001; 107: 1491-1495Crossref PubMed Scopus (539) Google Scholar). 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In addition, selective coupling with various terminal PG synthases has also been shown to influence crucially the utilization of the two COX isoforms during the different phases of cell activation (14Murakami M. Naraba H. Tanioka T. Semmyo N. Nakatani Y. Kojima F. Ikeda T. Fueki M. Ueno A. Oh-Ishi S. Kudo I. J. Biol. Chem. 2000; 275: 32783-32792Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar, 15Tanioka T. Nakatani Y. Semmyo N. Murakami M. Kudo I. J. Biol. Chem. 2000; 275: 32775-32782Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar, 16Ueno N. Murakami M. Tanioka T. Fujimori K. Tanabe T. Urade Y. Kudo I. J. Biol. Chem. 2001; 276: 34918-34927Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). PGES enzymes, which lie downstream of COXs, occur in multiple forms in mammalian cells (1Murakami M. Nakatani Y. Tanioka T. Kudo I. Prostaglandins Other Lipid Mediat. 2002; 68–69: 383-399Crossref PubMed Scopus (234) Google Scholar). Among them, a perinuclear membrane-bound form of PGES belonging to the MAPEG (for membrane-associated proteins involved in eicosanoid and glutathione metabolism) family, which we herein call mPGES-1, has been well characterized by several recent studies (14Murakami M. Naraba H. Tanioka T. Semmyo N. Nakatani Y. Kojima F. Ikeda T. Fueki M. Ueno A. Oh-Ishi S. Kudo I. J. Biol. Chem. 2000; 275: 32783-32792Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar, 17Jakobsson P.J. Thoren S. Morgenstern R. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7220-7225Crossref PubMed Scopus (905) Google Scholar, 18Mancini J.A. Blood K. Guay J. Gordon R. Claveau D. Chan C.C. Riendeau D. J. Biol. Chem. 2001; 276: 4469-4475Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 19Stichtenoth D.O. Thoren S. Bian H. Peters-Golden M. Jakobsson P.J. Crofford L.J. J. Immunol. 2001; 167: 469-474Crossref PubMed Scopus (254) Google Scholar, 20Yamagata K. Matsumura K. Inoue W. Shiraki T. Suzuki K. Yasuda S. Sugiura H. Cao C. Watanabe Y. Kobayashi S. J. Neurosci. 2001; 21: 2669-2677Crossref PubMed Google Scholar, 21Filion F. Bouchard N. Goff A.K. Lussier J.G. Sirois J. J. Biol. Chem. 2001; 276: 34323-34330Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 22Kamei D. Murakami M. Nakatani Y. Ishikawa Y. Ishii T. Kudo I. J. Biol. Chem. 2003; 278: 19396-19405Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 23Uematsu S. Matsumoto M. Takeda K. Akira S. J. Immunol. 2002; 168: 5811-5816Crossref PubMed Scopus (276) Google Scholar, 24Han R. Tsui S. Smith T.J. J. Biol. Chem. 2002; 277: 16355-16364Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 25Naraba H. Yokoyama C. Tago N. Murakami M. Kudo I. Fueki M. Oh-Ishi S. Tanabe T. J. Biol. Chem. 2002; 277: 28601-28608Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). The expression of mPGES-1 is up-regulated by proinflammatory stimuli and down-regulated by anti-inflammatory glucocorticoids, often in accordance with that of COX-2 (14Murakami M. Naraba H. Tanioka T. Semmyo N. Nakatani Y. Kojima F. Ikeda T. Fueki M. Ueno A. Oh-Ishi S. Kudo I. J. Biol. Chem. 2000; 275: 32783-32792Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar, 17Jakobsson P.J. Thoren S. Morgenstern R. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7220-7225Crossref PubMed Scopus (905) Google Scholar, 18Mancini J.A. Blood K. Guay J. Gordon R. Claveau D. Chan C.C. Riendeau D. J. Biol. Chem. 2001; 276: 4469-4475Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 19Stichtenoth D.O. Thoren S. Bian H. Peters-Golden M. Jakobsson P.J. Crofford L.J. J. Immunol. 2001; 167: 469-474Crossref PubMed Scopus (254) Google Scholar, 20Yamagata K. Matsumura K. Inoue W. Shiraki T. Suzuki K. Yasuda S. Sugiura H. Cao C. Watanabe Y. Kobayashi S. J. Neurosci. 2001; 21: 2669-2677Crossref PubMed Google Scholar, 21Filion F. Bouchard N. Goff A.K. Lussier J.G. Sirois J. J. Biol. Chem. 2001; 276: 34323-34330Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 22Kamei D. Murakami M. Nakatani Y. Ishikawa Y. Ishii T. Kudo I. J. Biol. Chem. 2003; 278: 19396-19405Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 23Uematsu S. Matsumoto M. Takeda K. Akira S. J. Immunol. 2002; 168: 5811-5816Crossref PubMed Scopus (276) Google Scholar, 24Han R. Tsui S. Smith T.J. J. Biol. Chem. 2002; 277: 16355-16364Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 25Naraba H. Yokoyama C. Tago N. Murakami M. Kudo I. Fueki M. Oh-Ishi S. Tanabe T. J. Biol. Chem. 2002; 277: 28601-28608Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). mPGES-1 displays functional coupling with COX-2 in marked preference to COX-1, even though COX-1/mPGES-1 coupling can occur if a large amount of AA is supplied (14Murakami M. Naraba H. Tanioka T. Semmyo N. Nakatani Y. Kojima F. Ikeda T. Fueki M. Ueno A. Oh-Ishi S. Kudo I. J. Biol. Chem. 2000; 275: 32783-32792Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar). A recent mPGES-1 gene targeting study clearly demonstrated that COX-2-mediated PGE2 production by lipopolysaccharide-stimulated mouse macrophages depends largely on this enzyme (23Uematsu S. Matsumoto M. Takeda K. Akira S. J. Immunol. 2002; 168: 5811-5816Crossref PubMed Scopus (276) Google Scholar). The expression of mPGES-1 in vivo has been reported to be associated with various physiological and pathological events, such as inflammation, cancer, and reproduction (18Mancini J.A. Blood K. Guay J. Gordon R. Claveau D. Chan C.C. Riendeau D. J. Biol. Chem. 2001; 276: 4469-4475Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 19Stichtenoth D.O. Thoren S. Bian H. Peters-Golden M. Jakobsson P.J. Crofford L.J. J. Immunol. 2001; 167: 469-474Crossref PubMed Scopus (254) Google Scholar, 20Yamagata K. Matsumura K. Inoue W. Shiraki T. Suzuki K. Yasuda S. Sugiura H. Cao C. Watanabe Y. Kobayashi S. J. Neurosci. 2001; 21: 2669-2677Crossref PubMed Google Scholar, 21Filion F. Bouchard N. Goff A.K. Lussier J.G. Sirois J. J. Biol. Chem. 2001; 276: 34323-34330Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 22Kamei D. Murakami M. Nakatani Y. Ishikawa Y. Ishii T. Kudo I. J. Biol. Chem. 2003; 278: 19396-19405Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 23Uematsu S. Matsumoto M. Takeda K. Akira S. J. Immunol. 2002; 168: 5811-5816Crossref PubMed Scopus (276) Google Scholar). A cytosolic form of PGES (cPGES) is ubiquitously and constitutively expressed in cells and is an accessory protein for the molecular chaperone Hsp90 (15Tanioka T. Nakatani Y. Semmyo N. Murakami M. Kudo I. J. Biol. Chem. 2000; 275: 32775-32782Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar, 26Tanioka T. Nakatani Y. Kobayashi T. Tsujimoto M. Oh-ishi S. Murakami M. Kudo I. Biochem. Biophys. Res. Commun. 2003; 303: 1018-1023Crossref PubMed Scopus (63) Google Scholar). This enzyme, in cooperation with Hsp90, converts COX-1-derived PGH2 to PGE2 in a situation where high concentrations of AA are promptly supplied by strong activation of cytosolic PLA2. Recently, a third PGES, which shows broad specificity of thiol requirement, was purified from the microsomal fraction of bovine heart (27Watanabe K. Kurihara K. Suzuki T. Biochim. Biophys. Acta. 1999; 1439: 406-414Crossref PubMed Scopus (97) Google Scholar), and the whole sequences of the human and monkey enzymes were determined (28Tanikawa N. Ohmiya Y. Ohkubo H. Hashimoto K. Kangawa K. Kojima M. Ito S. Watanabe K. Biochem. Biophys. Res. Commun. 2002; 291: 884-889Crossref PubMed Scopus (276) Google Scholar). This enzyme, designated mPGES-2, has an N-terminal hydrophobic domain followed by a glutaredoxin or thioredoxin homology region. The N-terminal 22 amino acid sequence of the purified bovine enzyme was identical to residues 88–109 of the full-length mPGES-2 protein predicted from its cDNA, suggesting the occurrence of proteolytic cleavage at this position. The full-length and N-terminally truncated mPGES-2 proteins expressed by a bacterial expression system displayed similar PGES activity in vitro (28Tanikawa N. Ohmiya Y. Ohkubo H. Hashimoto K. Kangawa K. Kojima M. Ito S. Watanabe K. Biochem. Biophys. Res. Commun. 2002; 291: 884-889Crossref PubMed Scopus (276) Google Scholar), indicating that the N-terminal hydrophobic region is dispensable for its enzymatic function. However, it has remained obscure whether this protein indeed acts as a functional PGES in mammalian cells and, if so, how the expression of this protein is regulated. In this paper, we report the cellular PGE2 biosynthetic function and tissue expression of mPGES-2 in comparison with those of mPGES-1. We provide evidence that mPGES-2 undergoes N-terminal cleavage during protein maturation in the Golgi membrane, is coupled with both COX-1 and COX-2 leading to PGE2 production, and is expressed relatively constitutively, rather than inducibly, in various cells and tissues. Moreover, immunohistochemical analyses of mPGES-1 and mPGES-2 in human tissues with various diseases allowed us to speculate on their general roles in human pathophysiology. Materials—Culture of human embryonic kidney (HEK) 293 cells, human colon adenocarcinoma HCA-7 cells, human lung epithelial BEAS-2B cells, and rat fibroblastic 3Y1 cells in RPMI1640 medium (Nissui Pharmaceutical Co.) supplemented with 10% fetal calf serum was described previously (14Murakami M. Naraba H. Tanioka T. Semmyo N. Nakatani Y. Kojima F. Ikeda T. Fueki M. Ueno A. Oh-Ishi S. Kudo I. J. Biol. Chem. 2000; 275: 32783-32792Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar, 22Kamei D. Murakami M. Nakatani Y. Ishikawa Y. Ishii T. Kudo I. J. Biol. Chem. 2003; 278: 19396-19405Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 29Murakami M. Masuda S. Shimbara S. Bezzine S. Lazdunski M. Lambeau G. Gelb M.H. Matsukura S. Kokubu F. Adachi M. Kudo I. J. Biol. Chem. 2003; 278: 10657-10667Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 30Kuwata H. Nakatani Y. Murakami M. Kudo I. J. Biol. Chem. 1998; 273: 1733-1740Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). The cDNAs for human mPGES-1 (14Murakami M. Naraba H. Tanioka T. Semmyo N. Nakatani Y. Kojima F. Ikeda T. Fueki M. Ueno A. Oh-Ishi S. Kudo I. J. Biol. Chem. 2000; 275: 32783-32792Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar), human COX-1 and COX-2 (4Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar), and monkey mPGES-2 (28Tanikawa N. Ohmiya Y. Ohkubo H. Hashimoto K. Kangawa K. Kojima M. Ito S. Watanabe K. Biochem. Biophys. Res. Commun. 2002; 291: 884-889Crossref PubMed Scopus (276) Google Scholar) were described previously. HEK293 cells stably expressing COX-1 or COX-2 were described previously (4Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 14Murakami M. Naraba H. Tanioka T. Semmyo N. Nakatani Y. Kojima F. Ikeda T. Fueki M. Ueno A. Oh-Ishi S. Kudo I. J. Biol. Chem. 2000; 275: 32783-32792Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar). AA and the enzyme immunoassay kit for PGE2 were purchased from Cayman Chemicals. The rabbit anti-human cPLA2α, goat anti-human COX-1, and goat antihuman COX-2 antibodies were purchased from Santa Cruz Biotechnology. The rabbit antibody for human mPGES-1 was prepared as described previously (22Kamei D. Murakami M. Nakatani Y. Ishikawa Y. Ishii T. Kudo I. J. Biol. Chem. 2003; 278: 19396-19405Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). The rabbit antibody for human group IIA PLA2 was provided by Dr. M. Gelb (University of Washington). Fluorescein isothiocyanate (FITC)-, Cy3-, and horseradish peroxidase-conjugated antibodies were purchased from Zymed Laboratories Inc.. Human interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) were purchased from Genzyme. A23187 was obtained from Calbiochem. LipofectAMINE 2000, Opti-MEM medium, TRIzol reagent, geneticin, hygromycin, blastcidin, the pcDNA3.1 series of mammalian expression vectors, the bacterial expression vector pTrc-HisA, and the ViraPower lentiviral expression system were obtained from Invitrogen. All primers for PCR were obtained from Greiner Japan. Preparation of Antibody against mPGES-2—cDNA for N-terminally truncated mPGES-2 (mPGES-2-del-N, which lacked the first 87 amino acids) subcloned into pTrc-His A (28Tanikawa N. Ohmiya Y. Ohkubo H. Hashimoto K. Kangawa K. Kojima M. Ito S. Watanabe K. Biochem. Biophys. Res. Commun. 2002; 291: 884-889Crossref PubMed Scopus (276) Google Scholar) was transformed into the competent cell BL21-D3 (Stratagene). After culture for an appropriate period with 0.5 mm isopropyl-β-d-(-)-thiogalactopyranoside, cells were spun down, freeze-thawed, and suspended in phosphate-buffered saline (PBS) containing 3 μg/ml leupeptin, 3 μg/ml antipain, 1 mm phenylmethylsulfonyl fluoride, and 1 mm dithiothreitol. After sonication and centrifugation for 10 min at 10,000 × g, the resulting supernatant was dialyzed against 20 mm Tris-HCl (pH 7.4) containing 150 mm NaCl (TBS) and 1 mm EDTA overnight. Then the dialyzed sample was applied to a nickel-nitrilotriacetic acid-agarose column (Novagen), and the bound proteins were eluted with 40–80 mm imidazole at a flow rate of 10 ml/h. Fractions containing pure His6-tagged mPGES-2 protein were collected and dialyzed against PBS. The purified protein gave a single band with a predicted size on SDS-PAGE followed by staining with Coomassie Brilliant Blue. New Zealand White rabbits (male, 1 kg; Saitama Animal Center) were immunized subcutaneously with the purified mPGES-2-del-N protein (0.3 mg each) mixed with Freund's complete adjuvant (Difco). After several booster immunizations with Freund's incomplete adjuvant (Difco) at 2-week intervals, blood was collected, and the serum titer was assayed by enzyme-linked immunosorbent assay and Western blotting with recombinant mPGES-2-del-N protein as purified above. The specific binding of the antibody to mPGES-2 was verified by immunoblotting using mPGES-2-transfected mammalian cell lines (see below) in comparison with parental cells. Transfection Studies—Transfection of cDNAs into HEK293 cells was performed by lipofection as described previously (4Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 14Murakami M. Naraba H. Tanioka T. Semmyo N. Nakatani Y. Kojima F. Ikeda T. Fueki M. Ueno A. Oh-Ishi S. Kudo I. J. Biol. Chem. 2000; 275: 32783-32792Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar). Briefly, 1 μg of plasmid (the N-terminally FLAG-tagged full-length mPGES-2 (mPGES-2-FL) in pCMV-Tag2 (Stratagene), FLAG-tagged, N-terminally truncated mPGES-2 (mPGES-2-del-N) in pCDNA3.1/hyg (+), and COX-1 or -2 in pCDNA3.1/neo (+)) was mixed with 2 μl of LipofectAMINE 2000 in 100 μl of Opti-MEM medium for 30 min and then added to cells that had attained 40–60% confluence in 12-well plates (Iwaki Glass) containing 0.5 ml of Opti-MEM. After incubation for 6 h, the medium was replaced with 1 ml of fresh culture medium. After overnight culture, the medium was replaced with 1 ml of fresh medium, and culture was continued at 37 °C in an incubator flushed with 5% CO2 in humidified air. To obtain stable transfectants, the cells were cloned by limiting dilution in 96-well plates in culture medium containing appropriate antibiotics (5 μg/ml hygromycin or 1 mg/ml geneticin). After culture for 3–4 weeks, wells containing a single colony were chosen, and the expression of each protein was monitored by RNA blotting and/or Western blotting. The established clones were expanded and used for the experiments described below. To establish BEAS-2B cells stably expressing COX-1 or COX-2, their cDNAs in pCDNA3.1/neo (+) were transfected with LipofectAMINE 2000, and the geneticin-resistant clones were selected in a similar way. C-terminally FLAG-tagged mPGES-1 and N-terminally FLAG-tagged mPGES-2-del-N cDNAs were transfected into BEAS-2B and 3Y1 cells with the ViraPower lentiviral expression system according to the manufacturer's instructions. Briefly, the FLAG-tagged mPGES-1 or mPGES-2-del-N cDNA insert was amplified by PCR with the Advantage cDNA polymerase mix (Clontech) and was subcloned into the pLenti6/V4-D-TOPO vector (Invitrogen). The resulting plasmid was transfected into 293FT cells (Invitrogen) with LipofectAMINE 2000, and an aliquot of the supernatant harvested 3 days after transfection was then added to BEAS-2B and 3Y1 cells. The cells were cultured in the presence of 30 μg/ml blastcidin, and the surviving cells that expressed appropriate levels of mPGES-1 or mPGES-2-del-N proteins were used in subsequent studies. Construction of mPGES-2 Mutant—To construct the catalytically inactive mutant mPGES-2-CS, in which cysteine residues in the thioredoxin/glutaredoxin motif have been replaced by serine residues (31Watanabe K. Ohkubo H. Niwa H. Tanikawa N. Koda N. Ito S. Ohmiya Y. Biochem. Biophys. Res. Commun. 2003; 306: 577-581Crossref PubMed Scopus (32) Google Scholar), mismatched PCR was conducted with the Advantage cDNA polymerase mix, as reported previously (14Murakami M. Naraba H. Tanioka T. Semmyo N. Nakatani Y. Kojima F. Ikeda T. Fueki M. Ueno A. Oh-Ishi S. Kudo I. J. Biol. Chem. 2000; 275: 32783-32792Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar). The primers used for construction of the mutant were as follows. FLAG-del-N primer, 5′-CACCATGGACTACAAGGACGACGATGACAAGGAGCGCTCAGCAGTGCAGCTC-3′ (FLAG sequence underlined), m2-3′ primer, 5′-TCAGTGCGCTGGGGAGGCCTCG-3′; CS-S primer, 5′-TACAAGACGAGTCCCTTCAGCAGCAAGG-3′; and CS-AS primer, 5′-CCTTGCTGCTGAAGGGACTCGTCTTGTA-3′. The first PCR was performed with a set of FLAG-del-N and CS-AS primers and a set of CS-S and m2-3′ primers with mPGES-2 cDNA as a template. The resulting fragments were mixed, annealed, and then subjected to a second PCR with a set of FLAG-del-N and m2-3′ primers. The PCR conditions were 25 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s. The resulting fragment was purified by the Wizard SV gel and PCR clean-up system (Promega), subcloned into the pLenti6/V4-D-TOPO vector, and sequenced with an autofluorometric DNA sequencer 310 Genetic Analyzer (Applied Biosystems) to confirm the mutation. The plasmid was then transfected into COX-2-expressing HEK293 cells with the ViraPower lentiviral expression system, and virus-infected cells were selected by culturing with blastcidin, as noted above. RNA Blotting—Approximately equal amounts (5–10 μg as required for experiments) of total RNA obtained from the cells were applied to separate lanes of 1.2% (w/v) formaldehyde-agarose gels, electrophoresed, and transferred to Immobilon-N membranes (Millipore). The resulting blots were then probed with the respective cDNA probes that had been labeled with [32P]dCTP (Amersham Biosciences) by random priming (Takara Biomedicals). All hybridizations were carried out as described previously (4Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 14Murakami M. Naraba H. Tanioka T. Semmyo N. Nakatani Y. Kojima F. Ikeda T. Fueki M. Ueno A. Oh-Ishi S. Kudo I. J. Biol. Chem. 2000; 275: 32783-32792Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar). Western Blotting—Cell lysates were subjected to SDS-PAGE using 7.5 (for COXs) or 12.5% (for mPGESs) gels under reducing conditions. The separated proteins were electroblotted onto nitrocellulose membranes (Schleicher & Schuell) with a semi-dry blotter (MilliBlot-SDE system; Millipore). After blocking with 3% (w/v) skim milk in TBS containing 0.05% Tween 20 (TBS/Tween), the membranes were probed with the respective antibodies (1:5,000 dilution in TBS/Tween for COX-2, mPGES-1, and mPGES-2; 1:20,000 dilution for COX-1 and FLAG epitope) for 2 h, followed by incubation with horseradish peroxidase-conjugated anti-rabbit (for mPGES-1 and mPGES-2), anti-goat (for COXs), or anti-mouse (for FLAG) antibody (1:5,000 dilution in TBS/Tween) for 2 h, and were visualized with the ECL Western blot system (PerkinElmer Life Sciences) (4Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 14Murakami M. Naraba H. Tanioka T. Semmyo N. Nakatani Y. Kojima F. Ikeda T. Fueki M. Ueno A. Oh-Ishi S. Kudo I. J. Biol. Chem. 2000; 275: 32783-32792Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar). Cell Activation—Cells (2–5 × 104 cells/ml) were seeded into 48-well plates and cultured for 3–4 days to confluence. To assess immediate PGE2 production, cells were stimulated for 30 min with 10 μm A23187 in serum-free medium. To assess delayed PGE2 production, HEK293 cells were incubated for 4 h with 1 ng/ml human IL-1β (4Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 14Murakami M. Naraba H. Tanioka T. Semmyo N. Nakatani Y. Kojima F. Ikeda T. Fueki M. Ueno A. Oh-Ishi S. Kudo I. J. Biol. Chem. 2000; 275: 32783-32792Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar), 3Y1 cells for 24 h with mouse IL-1β (30Kuwata H. Nakatani Y. Murakami M. Kudo I. J. Biol. Chem. 1998; 273: 1733-1740Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar), and BEAS-2B cells for 4 h with 103 units/ml TNFα in culture medium. Aliquots of the supernatants were taken for the PGE2 enzyme immunoassay. Confocal Laser Microscopy—Cells grown in subconfluency on glass bottom dishes (Matsunami) pre-coated with 5 μg/ml fibronectin (Sigma) were fixed with 3% paraformaldehyde for 30 min in PBS. After three washes with PBS, the fixed cells were sequentially treated with 1% (w/v) bovine serum albumin and 1% (w/v) saponin in PBS (blocking solution) for 1 h, with first antibodies (1:500 dilution for FLAG, 1:200 dilution for COX-1 and mPGES-2, and 1:100 dilution for COX-2) for 2 h in blocking solution, and then with species-matched FITC-conjugated second antibodies (1:200 dilution) for 2 h in blocking solution. For double immunostaining with organelle markers of the endoplasmic reticulum (ER) and the Golgi apparatus, anti-GRP78 and anti-GM130 antibodies, respectively, from the Organelle Sampler Kit (Transduction Laboratories) were used as first antibodies (1:250 dilution for both), followed by incubation with Cy3-conjugated second antibody (1:100 dilution). After six washes with PBS, specific immunofluores