Abstract
23 min readWe examined the in vivo role of membrane-bound prostaglandin E synthase (mPGES)-1, a terminal enzyme in the PGE2-biosynthetic pathway, using mPGES-1 knockout (KO) mice. Comparison of PGES activity in the membrane fraction of tissues from mPGES-1 KO and wild-type (WT) mice indicated that mPGES-1 accounted for the majority of lipopolysaccharide (LPS)-inducible PGES in WT mice. LPS-stimulated production of PGE2, but not other PGs, was impaired markedly in mPGES-1-null macrophages, although a low level of cyclooxygenase-2-dependent PGE2 production still remained. Pain nociception, as assessed by the acetic acid writhing response, was reduced significantly in KO mice relative to WT mice. This phenotype was particularly evident when these mice were primed with LPS, where the stretching behavior and the peritoneal PGE2 level of KO mice were far less than those of WT mice. Formation of inflammatory granulation tissue and attendant angiogenesis in the dorsum induced by subcutaneous implantation of a cotton thread were reduced significantly in KO mice compared with WT mice. Moreover, collagen antibody-induced arthritis, a model for human rheumatoid arthritis, was milder in KO mice than in WT mice. Collectively, our present results provide unequivocal evidence that mPGES-1 contributes to the formation of PGE2 involved in pain hypersensitivity and inflammation. We examined the in vivo role of membrane-bound prostaglandin E synthase (mPGES)-1, a terminal enzyme in the PGE2-biosynthetic pathway, using mPGES-1 knockout (KO) mice. Comparison of PGES activity in the membrane fraction of tissues from mPGES-1 KO and wild-type (WT) mice indicated that mPGES-1 accounted for the majority of lipopolysaccharide (LPS)-inducible PGES in WT mice. LPS-stimulated production of PGE2, but not other PGs, was impaired markedly in mPGES-1-null macrophages, although a low level of cyclooxygenase-2-dependent PGE2 production still remained. Pain nociception, as assessed by the acetic acid writhing response, was reduced significantly in KO mice relative to WT mice. This phenotype was particularly evident when these mice were primed with LPS, where the stretching behavior and the peritoneal PGE2 level of KO mice were far less than those of WT mice. Formation of inflammatory granulation tissue and attendant angiogenesis in the dorsum induced by subcutaneous implantation of a cotton thread were reduced significantly in KO mice compared with WT mice. Moreover, collagen antibody-induced arthritis, a model for human rheumatoid arthritis, was milder in KO mice than in WT mice. Collectively, our present results provide unequivocal evidence that mPGES-1 contributes to the formation of PGE2 involved in pain hypersensitivity and inflammation. Prostaglandin (PG) 1The abbreviations used are: PG, prostaglandin; BMD, bone mineral density; CAIA, collagen antibody-induced arthritis; CIA, collagen-induced arthritis; COX, cyclooxygenase; cPGES, cytosolic PGES; cPLA2α, cytosolic phospholipase A2α; HDC, histidine decarboxylase; KO, knockout; LPS, lipopolysaccharide; mPGES, membrane-bound PGES; PGES, PGE synthase; siRNA, small interfering RNA; TBS, Tris-buffered saline; TRAP, tartrate-resistant acid phosphatase; VEGF, vascular endothelial cell growth factor; WT, wild-type. E2 is the most common prostanoid, being produced by a variety of cells and tissues, and has a broad range of biological activity. Recent advances in this research field have led to molecular identification and characterization of various enzymes involved in the biosynthesis of PGE2, including phospholipase A2 (PLA2), cyclooxygenase (COX) and terminal PGE synthase (PGES) (1Murakami M. Kudo I. Prog. Lipid Res. 2004; 43: 3-35Crossref PubMed Scopus (310) Google Scholar). Each of these three enzymatic steps can be rate limiting for PGE2 biosynthesis and involves multiple enzymes/isozymes that can act in different phases of cell activation. The PGE2 produced thus far is then released from the cells and acts on the four types of PGE receptor, EP1, EP2, EP3, and EP4, which are coupled with trimeric G protein signaling (2Sugimoto Y. Narumiya S. Ichikawa A. Prog. Lipid Res. 2000; 39: 289-314Crossref PubMed Scopus (168) Google Scholar). PGES, which catalyzes the conversion of PGH2 to PGE2, exists as membrane-associated and cytosolic enzymes. Two of them are membrane-bound enzymes and have been designated as mPGES-1 and mPGES-2 (3Murakami M. Nakatani Y. Tanioka T. Kudo I. Prostaglandins Other Lipid Mediat. 2002; 68-69: 383-399Crossref PubMed Scopus (234) Google Scholar, 4Murakami M. Naraba H. Tanioka T. Semmyo N. Nakatani Y. Kojima F. Ikeda T. Fueki M. Ueno A. Oh-Ishi S. Kudo I. J. Biol. 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Mancini J.A. J. Immunol. 2003; 70: 4738-4744Crossref Scopus (170) Google Scholar, 11Thoren S. Weinander R. Saha S. Jegerschold C. Pettersson P.L. Samuelsson B. Hebert H. Hamberg M. Morgenstern R. Jakobsson P.J. J. Biol. Chem. 2003; 278: 22199-22209Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 12Kamei 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, 13Han R. Tsui S. Smith T.J. J. Biol. Chem. 2002; 277: 16355-16364Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 14Naraba 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, 15Tanikawa 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, 16Murakami M. Nakashima K. Kamei D. Masuda S. Ishikawa Y. Ishii T. Ohmiya Y. Watanabe K. Kudo I. J. Biol. Chem. 2003; 278: 37937-37947Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). mPGES-1 is a glutathione (GSH)-requiring perinuclear protein belonging to the MAPEG (for membrane-associated proteins involved in eicosanoid and GSH metabolism) family (4Murakami 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, 5Jakobsson P.J. Thoren S. Morgenstern R. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7220-7225Crossref PubMed Scopus (905) Google Scholar, 6Mancini 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). This enzyme is induced markedly by proinflammatory stimuli, is down-regulated by antiinflammatory glucocorticoids, and is functionally coupled with COX-2 in marked preference to COX-1. Induction of mPGES-1 expression has also been observed in various systems in which COX-2-derived PGE2 has been implicated to play a critical role, such as inflammation, fever, pain, female reproduction, tissue repair, and cancer (4Murakami 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, 5Jakobsson P.J. Thoren S. Morgenstern R. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7220-7225Crossref PubMed Scopus (905) Google Scholar, 6Mancini 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, 7Stichtenoth 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, 8Yamagata 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, 9Filion 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, 10Claveau D. Sirinyan M. Guay J. Gordon R. Chan C.C. Bureau Y. Riendeau D. Mancini J.A. J. Immunol. 2003; 70: 4738-4744Crossref Scopus (170) Google Scholar, 11Thoren S. Weinander R. Saha S. Jegerschold C. Pettersson P.L. Samuelsson B. Hebert H. Hamberg M. Morgenstern R. Jakobsson P.J. J. Biol. Chem. 2003; 278: 22199-22209Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 12Kamei 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). Inducible expression of mPGES-1 is in part regulated by the mitogen-activated protein kinase pathways (13Han R. Tsui S. Smith T.J. J. Biol. Chem. 2002; 277: 16355-16364Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), where the kinases may switch on the inducible transcription factor Egr-1 that in turn binds to the proximal GC box in the mPGES-1 promoter, leading to mPGES-1 transcription (14Naraba 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-2, which has a catalytic glutaredoxin- or thioredoxin-like domain and is activated by various thiol reagents, is synthesized as a Golgi membrane-associated protein, and the proteolytic removal of the N-terminal hydrophobic domain leads to the formation of a mature cytosolic enzyme (15Tanikawa 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, 16Murakami M. Nakashima K. Kamei D. Masuda S. Ishikawa Y. Ishii T. Ohmiya Y. Watanabe K. Kudo I. J. Biol. Chem. 2003; 278: 37937-37947Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). This enzyme is rather constitutively expressed in various cells and tissues and is functionally coupled with both COX-1 and COX-2 (16Murakami M. Nakashima K. Kamei D. Masuda S. Ishikawa Y. Ishii T. Ohmiya Y. Watanabe K. Kudo I. J. Biol. Chem. 2003; 278: 37937-37947Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). Cytosolic PGES (cPGES), a GSH-requiring enzyme constitutively expressed in a wide variety of cells, is functionally linked to COX-1, not COX-2, to promote immediate PGE2 production (17Tanioka 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). This enzyme is regulated by formation of a complex with Hsp90, a molecular chaperone (18Tanioka 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). In addition, two cytosolic GSH-S-transferases (μ2 and μ3) have the ability to catalyze the isomerization of PGH2 to PGE2, at least in vitro (19Beuckmann C.T. Fujimori K. Urade Y. Hayaishi O. Neurochem. Res. 2000; 25: 733-738Crossref PubMed Scopus (81) Google Scholar). The importance of PGE2 in various pathophysiological events dictates the necessity to understand the role of each PGES enzyme in vivo. In fact, biochemical and cell biological analyses have led to the proposal that among the PGES enzymes identified so far, mPGES-1 may be most critically responsible for the production of the PGE2 implicated in various pathophysiological events. An initial study with mPGES-1 knock-out (KO) mice has reported the essential role of mPGES-1 in lipopolysaccharide (LPS)-stimulated delayed PGE2 production by macrophages, although these mice are fertile, develop normally after birth, and retain LPS-stimulated production of various cytokines (20Uematsu S. Matsumoto M. Takeda K. Akira S. J. Immunol. 2002; 168: 5811-5816Crossref PubMed Scopus (276) Google Scholar). In this study, we used mPGES-1 KO mice to analyze the role of mPGES-1 in inflammation-associated pain hypersensitivity, tissue granulation accompanying angiogenesis, and arthritis induced by collagen antibody. Animals—Male C57BL/6 mice were obtained from Saitama Animal Center. The mPGES-1 KO mice and littermate wild-type (WT) mice (C57BL/6 × 129/SvJ background) were described previously (20Uematsu S. Matsumoto M. Takeda K. Akira S. J. Immunol. 2002; 168: 5811-5816Crossref PubMed Scopus (276) Google Scholar). Male mice (7 weeks old) were used in each experiment. Mice were housed in microisolator cages in a pathogen-free barrier facility, and all experiments were performed under approved institutional guidance. Agents—LPS (Escherichia coli 0111:B4), goat anti-mouse vascular endothelial cell growth factor (VEGF), and indomethacin were purchased from Sigma. Mouse anti-human cPLA2α monoclonal antibody and goat anti-human COX-1 and COX-2 polyclonal antibodies were purchased from Santa Cruz Biotechnology. Rabbit antibodies against human mPGES-1 (12Kamei 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), mPGES-2 (16Murakami M. Nakashima K. Kamei D. Masuda S. Ishikawa Y. Ishii T. Ohmiya Y. Watanabe K. Kudo I. J. Biol. Chem. 2003; 278: 37937-37947Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar), and cPGES (17Tanioka 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) were prepared as described previously. Rabbit anti-mouse histidine decarboxylase (HDC) antibody was donated by Dr. S. Tanaka (Kyoto University) (21Tanaka S. Nemoto K. Yamamura E. Ichikawa A. J. Biol. Chem. 1998; 273: 8177-8182Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Enzyme immunoassay kits for PGE2, 6-keto-PGF1α (a stable end product of PGI2), PGF2α, and thromboxane B2 (a stable end product of thromboxane A2) and the COX-2 inhibitor NS-398 were purchased from Cayman Chemicals. The COX-1 inhibitor valeryl salicylate was a generous gift from Dr. W. Smith (University of Michigan). Oligonucleotides were purchased from Bex. Measurement of PGES Activity—PGES activity was measured by assessment of conversion of PGH2 to PGE2 as reported previously (4Murakami 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, cell or tissue homogenates were centrifuged at 100,000 × g for 1hat4 °C, and the membrane fractions were used as an enzyme source. An aliquot (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 glutathione and 5 μg of indomethacin. After stopping the reaction by the addition of 100 mm FeCl2, the PGE2 content of the reaction mixture was quantified by use of the enzyme immunoassay kit. Preparation and Activation of Peritoneal Macrophages—Peritoneal cells were recovered from mice that had received thioglycollate medium (Difco) (1 ml/20 g of body weight) 4 days before (22Naraba H. Murakami M. Matsumoto H. Shimbara S. Ueno A. Kudo I. Oh-ishi S. J. Immunol. 1998; 160: 2974-2982PubMed Google Scholar). The peritoneal cells were seeded into 6- or 12-well plates (Iwaki Glass) at a cell density of 106 cells/ml in 2 ml (for 6-well plates) or 1 ml (for 12-well plates) of RPMI medium (Nissui) supplemented with 10% (v/v) fetal calf serum. After incubation for 2 h in a CO2 incubator, the supernatants and nonadherent cells were removed. More than 90% of adherent cells were macrophages. Then the cells were incubated with or without 10 μg/ml LPS in medium containing 2% serum for appropriate periods. The supernatants were taken for enzyme immunoassay for prostanoids, and the cells were subjected to Western blotting (see below). Experiments with mPGES-1 Small Interfering RNA (siRNA)—Two synthetic hairpin-forming oligonucleotides directed at mPGES-1, 5′-GATCCCGGCCTTTGCCAACCCCGAGTTCAAGAGACTCGGGGTTGGCAAAGGCCTTTTTTGGAAA-3′ (sense) and 3′-AGCTTTTCCAAAAAAGGCCTTTGCCAACCCCGAGTCTCTTGAACTCGGGGTTGGCAAAGGCCGG-5′ (antisense), both of which harbored BamHI and HindIII sites at their 5′- and 3′-ends, respectively, were annealed, cut with BamHI and XbaI, and then ligated into the BamHI/XbaI-digested pRNA-U6.1/Hygro vector (GenScript) using T4 ligase (Takara Biomedicals). After transformation into DH5α-competent cells (TOYOBO), the plasmid was extracted and purified using the Endofree Plasmid Maxi Kit (Qiagen). Transfection of the plasmid into peritoneal macrophages was performed by lipofection, as described previously (4Murakami 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, 5 μg of the plasmid was mixed with 10 μl of LipofectAMINE 2000 (Invitrogen) in 100 μl of Opti-MEM (Invitrogen) for 30 min and then added to macrophages in 0.5 ml of Opti-MEM in 12-well plates. After incubation for 24 h, the medium was replaced with 1 ml of fresh culture medium and then incubated with or without 10 μg/ml LPS in medium containing 2% fetal calf serum for 16 h. Acetic Acid Writhing Reaction—The writhing reaction was induced in mice by intraperitoneal injection of 0.9% (v/v) acetic acid solution at a dose of 5 ml/kg, as described previously (23Matsumoto H. Naraba H. Ueno A. Fujiyoshi T. Murakami M. Kudo I. Oh-ishi S. Eur. J. Pharmacol. 1998; 352: 47-52Crossref PubMed Scopus (44) Google Scholar, 24Ueno A. Matsumoto H. Naraba H. Ikeda Y. Ushikubi F. Matsuoka T. Narumiya S. Sugimoto Y. Ichikawa A. Oh-ishi S. Biochem. Pharmacol. 2001; 62: 157-160Crossref PubMed Scopus (58) Google Scholar). In one group of animals, LPS (10 μg/0.1 ml of saline/mouse) was given intraperitoneally 18 h before the injection of acetic acid solution. A suspension of 1 mg/ml indomethacin in 1% (w/v) sodium carboxymethylcellulose solution was injected subcutaneouslys into mice (at final dose of 10 mg/kg indomethacin/mouse) 30 min before the injection of acetic acid solution. The number of writhing responses was counted every 5 min. For measurement of prostanoids, mice were sacrificed 15 min after acetic acid injection, and their peritoneal cavities were washed twice with Hanks' balanced salt solution (Nissui) containing 10 μm indomethacin. The pooled peritoneal fluids were adjusted to pH 3.0 with 1 n HCl and passed through Sep-Pak C18 cartridges (Waters), and the retained PGs were eluted from the cartridges with 8 ml of methanol, as described previously (23Matsumoto H. Naraba H. Ueno A. Fujiyoshi T. Murakami M. Kudo I. Oh-ishi S. Eur. J. Pharmacol. 1998; 352: 47-52Crossref PubMed Scopus (44) Google Scholar, 24Ueno A. Matsumoto H. Naraba H. Ikeda Y. Ushikubi F. Matsuoka T. Narumiya S. Sugimoto Y. Ichikawa A. Oh-ishi S. Biochem. Pharmacol. 2001; 62: 157-160Crossref PubMed Scopus (58) Google Scholar). A trace amount of [3H]PGE2 (Cayman Chemicals) was added to the samples before passage through the cartridges to calibrate the recovery of PGs. The solvent of the samples was evaporated, and then PGs were dissolved in an aliquot of buffer and assayed with commercial enzyme immunoassay kits for each PG. Cotton Thread-induced Granulation Tissue Formation—Cotton threads (Araiwa Co.) were washed overnight with ethyl acetate and dried at room temperature before being cut into 1-cm lengths (3 mg weight), and sterilized by dry heat at 180 °C for 2 h. The cotton threads were implanted subcutaneously into the dorsum of anesthetized mice by using a 13-gauge implant needle (Natume), as described previously (25Ghosh A.K. Hirasawa N. Ohtsu H. Watanabe T. Ohuchi K. J. Exp. Med. 2002; 195: 973-982Crossref PubMed Scopus (99) Google Scholar). After appropriate periods, the mice were anesthetized and killed, and the granulation tissues were dissected together with the cotton threads and weighed. As required for the experiments, indomethacin (5 mg/kg) or vehicle was injected intraperitoneally every day. The isolated granulation tissues were washed, cut into small pieces with scissors, and homogenized with a Polytron homogenizer in a homogenizing buffer comprising 20 mm Tris-HCl (pH 7.4), 250 mm sucrose, 0.5 mm EDTA, 10 μm indomethacin, 1 μm phenylmethylsulfonyl fluoride, and 0.5% (v/v) Triton X-100. The obtained tissue homogenates were centrifuged at 3,000 rpm for 5 min, and 200-μl aliquots of the supernatants were centrifuged again at 14,000 × g for 30 min at 4 °C. Then, the hemoglobin concentrations in the supernatants were determined spec-trophotometrically by measuring the absorbance at 540 nm with a hemoglobin assay kit (Wako). PGs were extracted from the homogenates with Sep-Pak C18 cartridges and quantified by enzyme immunoassay, as described above. Western Blotting—Aliquots of samples (20-μg protein equivalents) were subjected to SDS-PAGE using 7.5% (for cPLA2α and COXs) or 12.5% (for PGESs) gels under reducing conditions. The separated proteins were electroblotted onto nitrocellulose membranes (Schleicher & Schuell) with a semidry blotter (MilliBlot-SDE system; Millipore). After blocking with 3% (w/v) skim milk in Tris-buffered saline (TBS (pH 7.4)) containing 0.05% Tween 20 (TBS-Tween), the membranes were probed with the respective antibodies (1:5,000 dilution for cPLA2α, COX-2, and PGESs; 1:1,000 dilution for VEGF; 1:2,500 dilution for HDC, and 1:20,000 dilution for COX-1 in TBS-Tween) for 2 h, followed by incubation with horseradish peroxidase-conjugated anti-mouse (for cPLA2α), anti-rabbit (for PGESs and HDC), or anti-goat (for COXs and VEGF) IgG antibody (1:5,000 dilution in TBS-Tween) for 2 h, and were visualized with the ECL Western blot system (PerkinElmer Life Sciences), as described previously (4Murakami 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). Immunohistochemistry—Formalin-fixed, paraffin-embedded sections of the granulation tissue sections were incubated with Target Retrieval Solution (DAKO) as required, incubated for 10 min with 3% (v/v) H2O2, washed three times with TBS for 5 min each, incubated for 30 min with 5% (v/v) skim milk, washed three times with TBS-Tween for 5 min each, and incubated for 2 h with anti-mPGES-1 antibody in TBS (1:100 dilution). After five washes, the sections were treated with the CSA system staining kit (DAKO) followed by counterstaining with hematoxylin and eosin, as described previously (12Kamei 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). Arthritis Model—Arthritis was induced in mPGES-1 KO and WT mice by the modified method of Terato et al. (26Terato K. Hasty K.A. Reife R.A. Cremer M.A. Kang A.H. Stuart J.M. J. Immunol. 1992; 148: 2103-2108PubMed Google Scholar, 27Terato K. Harper D.S. Griffiths M.M. Hasty D.L. Ye X.J. Cremer M.A. Seyer J.M. Autoimmunity. 1995; 22: 137-147Crossref PubMed Scopus (156) Google Scholar). Briefly, mice were injected intraperitoneally with 10 mg of anti-type II collagen monoclonal antibodies (Immuno-Biological Laboratory) on day 0. On days 2 and 7, 50 μg of LPS (100 μl of 500 μg/ml solution in saline) was injected intraperitoneally followed by an intermittent LPS injection every 3 days to the end of the experiments. As a control, 2.5 or 0.1 ml of saline was injected in place of the antibodies or LPS, respectively. The clinical severity of arthritis was graded on a 0-3 scale as follows: 0, normal; 1, swelling of ankle or wrist, or limited to digits; 2, swelling of the entire paw; 3, maximal swelling. Each limb was graded by a single blinded observer, allowing a maximum arthritis score of 12 for each animal. On day 28, the mice were anesthetized with ketamine/xylazine solution and a radiograph was taken with a soft x-ray apparatus (CMB-2; SOFTEX). After perfusion of mice with 4% (w/v) buffered paraformaldehyde, fore and right hind limbs were removed, decalcified in 10% (w/v) EDTA, and embedded in paraffin, and 5-μm sections were stained with hematoxylin and eosin or toluidine blue. Bone resorption was evaluated by the tartrate-resistant acid phosphatase (TRAP) staining of the carpometacarpal joints. TRAP-positive cells were stained at pH 5.0 in the presence of l(+)-tartaric acid using naphthol AS-MX phosphate (Sigma) in N,N-dimethylformamide as the substrate. The specimens were subjected to histomorphometric analyses using a semiautomated system (Osteoplan II; Carl Zeiss), and measurements were made at a magnification of ×400. Osteoclast number and eroded surface were measured at the carpometacarpal joints in the metacarpal bones. After perfusion fixation, left femora and tibiae were excised and bone mineral density (BMD) was measured by dual energy x-ray absorptiometry using a bone mineral analyzer (DCS-600; Aloka Co.). To evaluate bone destruction of the knee joint area, BMDs of four equal longitudinal divisions in femur and tibia were measured, and the BMD around the knee joint was expressed by the sum of those in the distal one-fourth of femur and in the proximal one-fourth of tibia (28Azuma Y. Oue Y. Kanatani H. Ohta T. Kiyoki M. Komoriya K. J. Pharmacol. Exp. Ther. 1998; 286: 128-135PubMed Google Scholar). On day 6, all four paws including wrist or ankle joints were collected and homogenized in a homogenizing buffer. Then, aliquots were taken for PGE2 enzyme immunoassay and Western blotting, as described above. Other Methods—Protein concentrations were determined by a bicinchoninic acid protein assay kit (Pierce) with bovine serum albumin (Sigma) as a standard. Data were analyzed by Student's t test. Results are expressed as the mean ± S.E., with p = 0.05 as the limit of significance. mPGES-1 Is a Major Inducible PGES in Vivo—Fig. 1A illustrates PGES enzymatic activities in the membrane fraction of various tissues from mPGES-1 KO and WT mice with or without 24-h treatment with LPS. Basal PGES activities (i.e. without LPS administration) in the brain, heart, intestine, and ear were similar between WT and KO mice, whereas the activity in the kidney of WT mice was about 4-fold higher than that of KO mice (Fig. 1A). Treatment of WT mice with LPS resulted in 3-8-fold increases in PGES activity over the basal levels of the individual tissues. In tissues of mPGES-1 KO mice, LPS-stimulated increases in PGES activity over the basal levels were only modest, although <2-fold increases were consistently observed in all tissues examined (Fig. 1A). Immunoblotting of the membrane fractions of individual tissues revealed that the expression of mPGES-1 was markedly induced in tissues of WT mice, whereas no mPGES-1 protein was detected in tissues of KO mice (Fig. 1B). Constitutive expression of mPGES-1 was seen only in the kidney of WT mice (Fig. 1B), in agreement with a recent immunohistochemical study demonstrating the expression of mPGES-1 in the epithelia of distal tubules and medullary collecting ducts in this organ (29Guan Y. Zhang Y. Schneider A. Riendeau D. Mancini J.A. Davis L. Komhoff M. Breyer R.M. Breyer M.D. Am. J. Physiol. 2001; 281: F1173-F1177Crossref PubMed Scopus (52) Google Scholar). In the brain (Fig. 1B), heart, and ear (data not shown), LPS-induced COX-2 expression in KO mice was similar to that in WT mice. In the intestine and kidney, COX-2 expression in LPS-treated KO mice was significantly lower than that in replicate WT mice (Fig. 1B), suggesting that mPGES-1-derived PGE2 amplifies COX-2 induction in these tissues. Thus, mPGES-1 represents a major inducible membrane-associated PGES in various tissues of LPS-treated mice. However, the fact that substantial levels of PGES activity still exist in tissues of KO mice implies the presence of another membrane-bound PGES that is expressed rather constitutively. Evaluation of the Role of mPGES-1 in PGE2Production by LPS-stimulated Macrophages—Accumulating evidence suggests that delayed PGE2 production by LPS-stimulated macrophages depends on inducible COX-2 and mPGES-1 (4Murakami 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, 20Uematsu S. Matsumoto M. Takeda K. Akira S. J. Immunol. 2002; 168: 5811-5816Crossref PubMed Scopus (276) Google Scholar, 22Naraba H. Murakami M. Matsumoto H. Shimbara S. Ueno A. Kudo I. Oh-ishi S. J. Immunol. 1998; 160: 2974-2982PubMed Google Scholar). To reevaluate the contribution of mPGES-1 to this event, we first took advantage of siRNA technology. As shown in Fig. 2A, ex vivo stimulation of C57BL/6 mouse-derived thioglycollate-induced peritoneal macrophages with LPS led to a marked increase
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