Molecular Identification of Cytosolic Prostaglandin E2 Synthase That Is Functionally Coupled with Cyclooxygenase-1 in Immediate Prostaglandin E2Biosynthesis
2000; Elsevier BV; Volume: 275; Issue: 42 Linguagem: Inglês
10.1074/jbc.m003504200
ISSN1083-351X
AutoresToshihiro Tanioka, Yoshihito Nakatani, Natsuki Semmyo, Makoto Murakami, Ichiro Kudo,
Tópico(s)NF-κB Signaling Pathways
ResumoHere we report the molecular identification of cytosolic glutathione (GSH)-dependent prostaglandin (PG) E2 synthase (cPGES), a terminal enzyme of the cyclooxygenase (COX)-1-mediated PGE2 biosynthetic pathway. GSH-dependent PGES activity in the cytosol of rat brains, but not of other tissues, increased 3-fold after lipopolysaccharide (LPS) challenge. Peptide microsequencing of purified enzyme revealed that it was identical to p23, which is reportedly the weakly bound component of the steroid hormone receptor/hsp90 complex. Recombinant p23 expressed in Escherichia coli and 293 cells exhibited all the features of PGES activity detected in rat brain cytosol. A tyrosine residue near the N terminus (Tyr9), which is known to be critical for the activity of cytosolic GSHS-transferases, was essential for PGES activity. The expression of cPGES/p23 was constitutive and was unaltered by proinflammatory stimuli in various cells and tissues, except that it was increased significantly in rat brain after LPS treatment. cPGES/p23 was functionally linked with COX-1 in marked preference to COX-2 to produce PGE2 from exogenous and endogenous arachidonic acid, the latter being supplied by cytosolic phospholipase A2 in the immediate response. Thus, functional coupling between COX-1 and cPGES/p23 may contribute to production of the PGE2 that plays a role in maintenance of tissue homeostasis. Here we report the molecular identification of cytosolic glutathione (GSH)-dependent prostaglandin (PG) E2 synthase (cPGES), a terminal enzyme of the cyclooxygenase (COX)-1-mediated PGE2 biosynthetic pathway. GSH-dependent PGES activity in the cytosol of rat brains, but not of other tissues, increased 3-fold after lipopolysaccharide (LPS) challenge. Peptide microsequencing of purified enzyme revealed that it was identical to p23, which is reportedly the weakly bound component of the steroid hormone receptor/hsp90 complex. Recombinant p23 expressed in Escherichia coli and 293 cells exhibited all the features of PGES activity detected in rat brain cytosol. A tyrosine residue near the N terminus (Tyr9), which is known to be critical for the activity of cytosolic GSHS-transferases, was essential for PGES activity. The expression of cPGES/p23 was constitutive and was unaltered by proinflammatory stimuli in various cells and tissues, except that it was increased significantly in rat brain after LPS treatment. cPGES/p23 was functionally linked with COX-1 in marked preference to COX-2 to produce PGE2 from exogenous and endogenous arachidonic acid, the latter being supplied by cytosolic phospholipase A2 in the immediate response. Thus, functional coupling between COX-1 and cPGES/p23 may contribute to production of the PGE2 that plays a role in maintenance of tissue homeostasis. prostaglandin cytosolic prostaglandin E2 synthase prostaglandin E2 synthase cyclooxygenase cytosolic phospholipase A2 lipopolysaccharide glutathione glutathioneS-transferase microsomal GST1-like 1 arachidonic acid 1-chloro-2,4-dinitrobenzene interleukin tumor necrosis factor fetal calf serum bovine serum albumin polyacrylamide gel electrophoresis Chinese hamster ovary Dulbecco's modified Eagle's medium phosphate-buffered saline phospholipase A2 Biosynthesis of prostaglandin (PG)1 E2, the most common prostanoid with potent bioactivities, is regulated by three sequential steps of the cyclooxygenase (COX) pathway. Phospholipase A2 (PLA2) initiates this pathway by releasing arachidonic acid (AA) from membrane glycerophospholipids. Of more than 10 members of the PLA2 family characterized to date, cytosolic PLA2 (cPLA2) and several secretory PLA2s are involved in supplying AA to either of the two COX isozymes, COX-1 and COX-2, depending upon the phases of cell activation (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. 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). The constitutive COX-1 is mainly utilized in immediate PGE2 biosynthesis, which occurs within several minutes after stimulation with Ca2+ mobilizers, whereas the inducible COX-2 mediates the delayed PGE2 biosynthesis, which lasts for several hours following proinflammatory stimuli. Although COX-1 and COX-2 have been reported to exhibit subtle differences in AA requirements in that COX-2 is favored over COX-1 at low AA concentrations (3Murakami 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, 4Kulmacz R.J. Wang L.-H. J. Biol. Chem. 1995; 270: 24019-24023Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 5Smith W.L. Garavito R.M. DeWitt D.L. J. Biol. Chem. 1996; 271: 33157-33160Abstract Full Text Full Text PDF PubMed Scopus (1851) Google Scholar) and subcellular localizations (6Morita I. Schindler M. Regier M.K. Otto J.C. Hori T. DeWitt D.L. Smith W.L. J. Biol. Chem. 1995; 270: 10902-10908Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar), their functional segregation in the PGE2 biosynthetic response cannot be fully explained only by these aspects. The activity of PGES, which catalyzes conversion of COX-derived PGH2 to PGE2, has been detected in both cytosolic and microsomal fractions of various cells, and in most, if not all, cases it requires glutathione (GSH) for optimal activity (7Tanaka Y. Ward S. Smith W.L. J. Biol. Chem. 1987; 262: 1374-1381Abstract Full Text PDF PubMed Google Scholar, 8Watanabe K. Kurihara K. Hayaishi O. Biochem. Biophys. Res. Commun. 1997; 235: 148-152Crossref PubMed Scopus (72) Google Scholar, 9Ogorochi T. Ujihara M. Narumiya S. J. Neurochem. 1987; 48: 900-909Crossref PubMed Scopus (56) Google Scholar). Although several groups have attempted to purify this critical enzyme to near homogeneity for the last 20 years (7Tanaka Y. Ward S. Smith W.L. J. Biol. Chem. 1987; 262: 1374-1381Abstract Full Text PDF PubMed Google Scholar, 8Watanabe K. Kurihara K. Hayaishi O. Biochem. Biophys. Res. Commun. 1997; 235: 148-152Crossref PubMed Scopus (72) Google Scholar, 9Ogorochi T. Ujihara M. Narumiya S. J. Neurochem. 1987; 48: 900-909Crossref PubMed Scopus (56) Google Scholar), such trials have been unsuccessful. The PGES enzyme purified from human brain cytosol was identified as a GSH S-transferase (GST), which converts PGH2 to PGE2, PGD2, and PGF2α nonspecifically (9Ogorochi T. Ujihara M. Narumiya S. J. Neurochem. 1987; 48: 900-909Crossref PubMed Scopus (56) Google Scholar). GSH-independent PGES with a molecular mass of 31 kDa was recently purified from bovine heart (10Watanabe K. Kurihara K. Suzuki T. Biochim. Biophys. Acta. 1999; 1439: 406-414Crossref PubMed Scopus (97) Google Scholar). Interestingly, PGES activity has been shown to be strongly induced by proinflammatory stimuli in macrophages (11Naraba H. Murakami M. Matsumoto H. Shimbara S. Ueno A. Kudo I. Oh-ishi S. J. Immunol. 1998; 160: 2974-2982PubMed Google Scholar, 12Fournier T. Fadok V. Henson P.M. J. Biol. Chem. 1997; 272: 31065-31072Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). More recently, microsomal GST1-like 1 (MGST1-L1), a member of the MAPEG (membrane-associated proteins involved in eicosanoid and glutathione metabolism) superfamily, has been shown to exhibit significant PGES activity (13Jakobsson P.-J. Thoren S. Morgenstern R. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7220-7225Crossref PubMed Scopus (895) Google Scholar, 14Jakobsson P.-J. Morgenstern R. Mancini J. Ford-Hunchinton A. Persson B. Protein Sci. 1999; 8: 689-692Crossref PubMed Scopus (302) Google Scholar). In this study, we report the molecular identification of cytosolic PGES (cPGES), a GSH-requiring enzyme that is expressed ubiquitously in a wide variety of cells and tissues. Importantly, this enzyme is capable of converting COX-1-, but not COX-2-, derived PGH2 to PGE2 efficiently. Our present results, together with identification of the inducible membrane-associated PGES that is preferentially coupled with COX-2 as described in the accompanying paper (15Murakami 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 (856) Google Scholar), revealed that segregated utilization of the biosynthetic enzymes in different phases of PG production also occurs at the step of terminal synthases. Wistar rats (7 weeks old, male) were purchased from Japan Bio-Supply center (Tokyo, Japan). Rabbits (New Zealand White, 1-kg body weight, female) were from Saitama Experimental Animal Supply (Saitama, Japan). Human embryonic kidney 293 cells were obtained from Japanese Cancer Resources Bank. Rat fibroblastic 3Y1 cells were donated by Dr. Y. Uehara (National Institute of Infectious Disease, Tokyo, Japan). Human cervix epithelial HeLa cells, human stomach MKN45 cells, human glial U251 cells, human fibroblastic WI38 cells, human neuroblastoma GOTO cells, Chinese hamster ovary (CHO) cells, mouse osteoblastic MC3T3-E1 cells, and mouse fibroblastic L929 cells were obtained from the RIKEN Cell Bank. GOTO, HEK293, CHO, MKN45 and L929 were cultured in RPMI 1640 medium (Nissui Pharmaceutical) containing 10% fetal calf serum (FCS; Bioserum), WI38. U251 and 3Y1 in DMEM (Nissui Pharmaceutical) containing 10% FCS, and MC3T3-E1 in α-minimal essential medium (Dainippon Pharmaceutical) containing 10% FCS. The goat anti-human COX-2 and rabbit anti-human cPLA2 antibodies were purchased from Santa Cruz. The rabbit anti-mouse COX-1 antibody was provided by Dr. W. L. Smith (Michigan State University, East Lansing, MI). 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, 16Murakami M. Matsumoto R. Austen K.F. Arm J.P. J. Biol. Chem. 1994; 269: 22269-22275Abstract Full Text PDF PubMed Google Scholar). LipofectAMINE Plus, LipofectAMINE 2000, Opti-MEM, and TRIzol reagent were obtained from Life Technologies. Bacterial LPS (Salmonella minnesota Re 595), 1-chloro-2,4-dinitrobenzene (CDNB), indomethacin, and GSH were purchased from Sigma. Ethacrynic acid, 1,2-dichloro-4-nitrobenzene, andp-nitrophenethyl bromide were from Wako. Freund's complete and incomplete adjuvants were from Difco Laboratories. AA, PGH2, rabbit anti-human COX-1 antibody, and the enzyme immunoassay kits for PGE2 were from Cayman Chemical. Oligonucleotide primers were from Amersham Pharmacia Biotech. Geneticin, hygromycin, and the mammalian expression vectors pCR3.1 and pCDNA3.1/hyg(+) were purchased from Invitrogen. A23187 was purchased from Calbiochem. Human and mouse interleukin (IL)-1β and tumor necrosis factor (TNF) α were from Genzyme. Fluorescein isothiocyanate-conjugated goat anti-rabbit IgG and horseradish peroxidase-conjugated anti-rabbit and mouse IgGs were purchased fromZymed Laboratories Inc. Other reagents were obtained from Wako Pure Chemical Industries. Computational analysis on the protein and cDNA sequences were performed using the GENETYX program (Software Development). PGES activities in cell lysates were measured by assessment of conversion of PGH2to PGE2 as previously reported (11Naraba H. Murakami M. Matsumoto H. Shimbara S. Ueno A. Kudo I. Oh-ishi S. J. Immunol. 1998; 160: 2974-2982PubMed Google Scholar). The cells were scraped off from the dishes and disrupted by sonication using Branson Sonifier (10 s, three times, 1-min interval) in 400 μl of 10 mmTris-HCl (pH 8.0). After centrifugation of the sonicates at 1,700 × g for 10 min at 4 °C, the supernatants were used as the enzyme source. An aliquot of each lysate (10 μg of protein equivalents) was incubated with 0.5 μg of PGH2 for 30 s at 24 °C in 0.1 ml of 0.1 m Tris-HCl (pH 8.0), containing 1 mm GSH and 5 μg of indomethacin. After terminating the reaction by the addition of 100 mmFeCl2, PGE2 contents in the supernatants were quantified by use of the enzyme immunoassay kit. Protein concentrations were determined by the protein assay kit (Pierce) using bovine serum albumin (BSA) as a standard. Brains obtained from 10 Wistar rats 48 h after intravenous injection of 150 μg/kg LPS were homogenized in 100 ml of SET buffer comprising 20 mm Tris-HCl (pH 7.4), 250 mm sucrose, 5 mm EDTA, and 1 mm phenylmethylsulfonyl fluoride by using a Potter homogenizer. After centrifugation at 100,000 ×g, the supernatant was subjected to 60–80% ammonium sulfate precipitation. The precipitate obtained by centrifugation for 30 min at 10,000 × g at 4 °C was dissolved in SET buffer, dialyzed against 20 mm Tris-HCl (pH 7.4) containing 150 mm NaCl, 1 mm EDTA, 0.1 mmphenylmethylsulfonyl fluoride, 1 μm leupeptin, and 1 μm antipain, and then applied to a DEAE-Sephacel ion-exchange column (1 × 7 cm) (Amersham Pharmacia Biotech) at a flow rate of 30 ml/h. The bound proteins were eluted with 25 mm Tris-HCl (pH 7.4) containing 1 mm EDTA and a linear gradient of 0.15–1 m NaCl. Fractions containing PGES activity were concentrated to 1 ml using Centriprep 10 (Amicon) and applied to a Superdex 200 gel filtration column (Amersham Pharmacia Biotech) equilibrated with 20 mm Tris-HCl (pH 7.4) containing 150 mm NaCl, 1 mm EDTA, and 0.1 mm phenylmethylsulfonyl fluoride at a flow rate of 0.5 ml/min. A 10-μl aliquot of each fraction was taken for PGES enzyme assay. The protein band on the first sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was visualized by Coomassie Brilliant Blue, cut out from the gel, and then digested in the second gel with 10 μg of V8 protease (Sigma). The resultant peptides were electrotransferred to a polyvinylidene difluoride membrane (Millipore), and the three major peptide fragments obtained were subjected to amino acid sequencing using an Applied Biosystems 473A protein sequencer, as described previously (17Nakatani 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). Total RNA from HeLa cells was subjected to a reverse transcription reaction using RNA PCR kit (avian myeloblastosis virus) (Takara Biomedicals). cDNA thus obtained was subjected to PCR using the human p23 (18Johnson J.L. Beito T.G. Krco C.J. Toft D.O. Mol. Cell. Biol. 1994; 14: 1956-1963Crossref PubMed Scopus (181) Google Scholar) primers 5′-ATGCAGCCTGCTTCTGCA-3′ and 5′-TTACTCCAGATCTGGCAT-3′ (94 °C for 30 s, 50 °C for 30 s, and 72 °C for 30 s, for 25 cycles). An amplified product of the expected size was subcloned into pCR3.1 (Invitrogen) and transfected into E. coliTop10F′ (Invitrogen). The plasmid was isolated and sequenced using a thermo sequenase fluorescent-labelled primer cycle sequencing kit with 7-deaza-dGTP (Amersham Pharmacia Biotech) and an autofluorometric DNA sequencer DSQ-1000L (Shimadzu). Human p23 cDNA (18Johnson J.L. Beito T.G. Krco C.J. Toft D.O. Mol. Cell. Biol. 1994; 14: 1956-1963Crossref PubMed Scopus (181) Google Scholar) was subcloned into pET21c (Novagen) and transformed into E. coli BL21 (DE3) (Stratagene). The cells were cultured until they reached the late lag phase, and 0.3 mmisopropyl-1-thio-β-d-galactopyranoside was added to induce (His)6-tagged protein. Bacterial cell pellets were lysed in 20 mm Tris-HCl (pH 8) containing 0.5 mNaCl, 10% glycerol, and 6 m guanidine HCl by stirring for 30 min at room temperature. After centrifugation at 15,000 ×g for 30 min at 4 °C, the resulting supernatants were applied to a nickel-nitrilotriacetic acid-agarose column (Qiagen) preequilibrated with 100 mm NiSO4 at a flow rate of 10 ml/h. After washing, the bound protein was eluted with the same buffer containing 20–60 mm imidazole in a stepwise manner. To obtain cPGES/p23 Y9N mutant, mismatched primer PCR was carried out using ex Taqpolymerase with cPGES/p23 cDNA as a template and the primers 5′-ATG CAG CCT GCT TCT GCA AAG TGG AAC G-3′ (the mutated base is underlined) and 5′-TTA CTC CAG ATC TGG CAT-3′. PCR conditions were 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 30 s, for 25 cycles. The fragment obtained was subcloned into pCR3.1 and sequenced. The cDNA flanking the entire open reading frame of human cPGES/p23 was subcloned into the mammalian expression vector pCDNA3.1/hyg(+) and transfected into 293 cells stably expressing human COX-1 or COX-2, which we had previously established (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), using LipofectAMINE Plus according to the manufacturer's instruction. Briefly, 1 μg of each plasmid was mixed with 4 μl of LipofectAMINE and 6 μl of Plus reagent in 200 μl of Opti-MEM, left for 15 min, and then added to cells that had attained 70% confluence in six-well plates (Iwaki Glass) in 1 ml of Opti-MEM. After incubation for 4 h, 2 ml of fresh culture medium was added. After 18 h, the medium was replaced with 2 ml of fresh medium, and the culture was continued for 3 days. In order to establish stable transfectants, cells transfected with each cDNA were cloned by limiting dilution in 96-well plates (Iwaki Glass) in culture medium supplemented with 50 μg/ml hygromycin. After 3–4 weeks of culture, single colonies were picked up and expanded. Expression of cPGES/p23 and each COX was assessed by RNA blotting and immunoblotting, as described below. All procedures were described in our previous reports (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. 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). Briefly, 293 cells (5 × 104/ml) were seeded into each well of 24- or 48-well plates in 1 and 0.5 ml of culture medium, respectively. After culture for 4 days, the cells were washed once with culture medium and then incubated with 250 μl (24-well plate) or 100 μl (48-well plate) of various concentrations of AA or 10 μmA23187 in medium containing 1% FCS for 30 min or 1 ng/ml IL-1β in medium containing 10% FCS for 4 h. The supernatants were subjected to the PGE2enzyme immunoassay. Activation of other cell lines was carried out in a similar way. The (His)6-tagged cPGES/p23 (500 μg) in 500 μl of phosphate-buffered saline was mixed with an equal volume of Freund's complete adjuvant and injected into rabbits. Immunization was repeated every 3 weeks with the same amounts of the antigen mixed with an equal volume of Freund's incomplete adjuvant. Serum titers were checked by the enzyme-linked immunosorbent assay (see below), followed by Western blotting (see below) using the purified recombinant (His)6-tagged cPGES/p23 and the lysate of HeLa cells. In the enzyme-linked immunosorbent assay, 1 μg/ml recombinant (His)6-tagged cPGES/p23 was coated on Immulon 2 plates (Dynatech Laboratories) (50 μl/well) overnight at 4 °C. Subsequent procedures were performed at room temperature. After washing with 10 mm Tris-HCl (pH 7.4) containing 0.05% Tween 20 and 150 mm NaCl (TBS-T), the plates were incubated for 1 h with 5% skim milk in PBS. After six washes with TBS-T, serial dilutions of rabbit antisera were added to the plates (50 μl/well) and incubated for 1 h. After 6 washes with TBS-T, the plates were incubated with horseradish peroxidase-conjugated anti-rabbit IgG (50 μl/well) at a 1:1,000 dilution for 1 h. After 6 washes, the plates were incubated with o-phenylenediamine. After terminating the reaction by adding 4 nH2SO4, absorbance at 490 nm was measured. Approximately equal amounts (∼10 μg) of the total RNAs obtained from the transfected 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 Pharmacia Biotech) by random priming (Takara Biomedicals). All hybridizations were carried out as described previously (19Murakami M. Nakatani Y. Kudo I. J. Biol. Chem. 1996; 271: 30041-30051Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Cell lysates (105 cell equivalents) or culture supernatants were subjected to SDS-PAGE using 15% (w/v) gels for cPGES/p23 and 10% gels for COXs under reducing conditions. The separated proteins were electroblotted onto nitrocellulose membranes (Schleicher & Schuell) using a semidry blotter (MilliBlot-SDE system; Millipore). The membranes were probed with the respective antibodies and visualized using the ECL Western blot system (PerkinElmer Life Sciences), as described previously (19Murakami M. Nakatani Y. Kudo I. J. Biol. Chem. 1996; 271: 30041-30051Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Cells were seeded onto cover glasses (Matsunami Glass) at 1 × 105 cells/ml and cultured for 1 day. After removing the supernatants, the cells were fixed with 10% (v/v) formalin in PBS for 30 min at 4 °C. The cells were then treated 0.2% (v/v) Triton X-100 for 2 min, washed six times, and incubated for 1 h with 3% (w/v) BSA in PBS (PBS-BSA). After three washes, the cells were incubated with rabbit anti-cPGES/p23 antibody (1:200 dilution) in PBS-BSA for 2 h, washed three times, and then incubated with fluorescein isothiocyanate-conjugated anti-rabbit IgG (1:100 dilution) in PBS-BSA for 1 h. After six washes, the coverslips were mounted on glass slides using Perma Fluor (Japan Tanner) and examined using a Fluoview laser fluorescence microscope (Olympus). Approximately 4 μg of cPGES/p23 cDNA subcloned into pCR3.1 in an inverse direction were incubated with 5 μl of LipofectAMINE 2000 reagent in 200 μl of Opti-MEM for 15 min at room temperature and then added to cells that had attained 60–80% confluence in six-well plates and been supplemented with 800 μl of Opti-MEM. After incubation for 6 h at 37 °C, 1 ml of DMEM supplemented with 2% FCS was added, and the culture was continued for another day. Then the cells were trypsinized, seeded into 24-well plates, and cultured for 2 days. After washing once with DMEM, the cells were stimulated for 30 min with 10 μmA23187 in DMEM or for 12 h with 1 ng/ml mouse IL-1β and mouse TNFα in DMEM containing 2% FCS. The supernatants were taken for PGE2 enzyme immunoassay, and the cells were subjected to PGES enzyme assay and immunoblotting. In an effort to identify PGES isoforms, we measured PGES activity, which converts PGH2 to PGE2, in the cytosol of various rat tissues before and 48 h after injection of LPS. All tissues examined contained significant PGES activity that was not affected by LPS, except that the activity in brain increased up to 3-fold 48 h after LPS challenge (Fig.1 A). This activity was stimulated markedly by GSH and was inhibited by CDNB, a substrate for several GST enzymes (20Salinas A.E. Wong M.G. Curr. Med. Chem. 1999; 6: 279-309PubMed Google Scholar) (Fig. 1 B). LPS-sensitive PGES activity in rat brain cytosol fraction was recovered in the 60–80% ammonium sulfate precipitated fraction (Fig.2 A). When this fraction was dialyzed and then applied to DEAE-Sephacel ion-exchange column chromatography, a single major peak of PGES activity was eluted with 0.5 m NaCl (Fig. 2 B). The activity obtained from LPS-treated rat brains was significantly higher than that obtained from untreated animals. When the fractions containing PGES activity were then applied to Superdex 200 gel filtration, there were three major peaks that exhibited significant PGES activity, among which only the activity eluted in fractions corresponding to a molecular mass of ∼50 kDa (around fraction 78) showed severalfold higher activity than that in replicate fractions purified from rats not treated with LPS (Fig.2 C). On SDS-PAGE, this activity comigrated with a major protein with an apparent molecular mass of 26 kDa, which was detected more faintly in the untreated group. The specific activity of the peak fraction purified from LPS-treated rat brains after gel filtration was estimated to be approximately 5 μmol/min/mg of protein. This activity showed dependence on GSH and was inhibited by CDNB (data not shown). There was no detectable GST activity toward several cytosolic GST substrates, such as CDNB, 1,2-dichloro-4-nitrobenzene,p-nitrophenethyl bromide, and ethacrynic acid (data not shown). On the other hand, the other two higher molecular weight PGES peaks, which were eluted in fractions 53 and 69 in both LPS-treated and -untreated groups (Fig. 2 C), were fairly insensitive to CDNB (data not shown). These results suggest that there are several proteins that exhibit PGES activity with different enzymatic properties in the cytosol. Peptide mapping of the 26-kDa protein revealed that the partial amino acid sequences (MDPASAKWYDRRDYVFIEFC, KSKLCFSCLG, and IDLFHCIDPN) were identical to those of the corresponding portions (1–20, 33–42, and 53–62, respectively) of human p23, a cytosolic protein that is the weakly bound component of the steroid hormone receptor/hsp90 complex with a putative chaperone function (18Johnson J.L. Beito T.G. Krco C.J. Toft D.O. Mol. Cell. Biol. 1994; 14: 1956-1963Crossref PubMed Scopus (181) Google Scholar, 21Hutchinson K.A. Stancato L.F. Qwens-Grillo J.K. Johnson J.L. Krishna P. Toft D.O. Pratt W.B. J. Biol. Chem. 1995; 270: 18841-18847Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). We therefore isolated the full-length human p23 cDNA from HeLa cells and expressed it inE. coli as a C-terminally (His)6-tagged protein. The recombinant protein purified by nickel-chelating column had significant PGES activity in the presence of GSH and was inhibited by CDNB (Fig. 3 A), whereas formation of other PGs was negligible (data not shown). TheK m and V max values of the recombinant protein for PGH2 were estimated to be 14 μm and 190 nmol/min/mg of protein, respectively, in ourin vitro assay system. GST activity was undetected when CDNB, 1,2-dichloro-4-nitrobenzene, p-nitrophenethyl bromide, and ethacrynic acid were used as substrates (data not shown). Furthermore, PGES activity in lysate of p23-transfected HEK293 cells was stimulated markedly by GSH as compared with mock-transfected cells, and was inhibited by CDNB (Fig. 3 B). Thus, we conclude that p23 indeed possesses PGES activity, and therefore designate it cPGES/p23 (c stands for cytosolic) hereafter. Although the homology between cPGES/p23 and other known cytosolic GSTs (including hematopoietic PGD2 synthase (Ref. 22Kanaoka Y. Ago H. Inagaki E. Nanayama T. Miyano M. Kikuno R. Fujii Y. Eguchi N. Toh H. Urade Y. Hayaishi O. Cell. 1997; 90: 1085-1095Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar)) is low (∼20%), near the N terminus cPGES/p23 has a tyrosine residue (Tyr9) that is conserved in several other cytosolic GSTs as well as hematopoietic PGD2 synthase (Fig.4 A). The tyrosine residue in this position serves as a GSH acceptor, thereby being essential for catalytic activity (20Salinas A.E. Wong M.G. Curr. Med. Chem. 1999; 6: 279-309PubMed Google Scholar, 22Kanaoka Y. Ago H. Inagaki E. Nanayama T. Miyano M. Kikuno R. Fujii Y. Eguchi N. Toh H. Urade Y. Hayaishi O. Cell. 1997; 90: 1085-1095Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). As shown in Fig. 4 B, the cPGES/p23 mutant in which Tyr9 was replaced by Asn exhibited virtually no PGES activity when transfected into HEK293 cells. RNA blot analysis showed that cPGES/p23 was most abundantly expressed in the testis, and was also expressed in various tissues of the rat (Fig.5 A). In most tissues, expression was unchanged following LPS treatment. Exceptionally, cPGES/p23 mRNA expression in brain was increased approximately 3-fold after treatment with LPS (Fig. 5 A), a result consistent with increased PGES activity in brain cytosol fraction (Fig.1 A). Immunoblotting using anti-cPGES/p23 antibody confirmed the increased expression of cPGES/p23 protein in LPS-treated rat brain cytosol fraction (Fig. 5 B). cPGES/p23 mRNA was detected in the kidney only faintly (Fig. 5 A), whereas PGES activity in the kidney cytosol was higher than that in other tissues (Fig.1 A), suggesting that there are other types of PGES in this tissue. cPGES/p23 was expressed constitutively and was not altered significantly by stimulation with cytokines (TNFα and IL-1β) in all cell lines examined (Fig. 5 C). Confocal microscopic analysis using an anti-cPGES antibody revealed that cPGES is located in the cytosol of these cells (Fig. 6).Figure 6Subcellular distribution of cPGES/p23.Cells were fixed with folmalin, permeabilized, and then incubated sequentially with rabbit anti-cPGES/p23 antibody and fluorescein isothiocyanate-conjugated anti-rabbit IgG. The cells were mounted, and their fluorescence was visualized using a laser scanning confocal microscope.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To assess whether cPGES/p23 plays a role in PGE2 production by live cells, human
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