Title: Protection against Oxidative Stress-induced Hepatic Injury by Intracellular Type II Platelet-activating Factor Acetylhydrolase by Metabolism of Oxidized Phospholipids in Vivo
Abstract: Membrane phospholipids are susceptible to oxidation, which is involved in various pathological processes such as inflammation, atherogenesis, neurodegeneration, and aging. One enzyme that may help to remove oxidized phospholipids from cells is intracellular type II platelet-activating factor acetylhydrolase (PAF-AH (II)), which hydrolyzes oxidatively fragmented fatty acyl chains attached to phospholipids. Overexpression of PAF-AH (II) in cells or tissues was previously shown to suppress oxidative stress-induced cell death. In this study we investigated the functions of PAF-AH (II) by generating PAF-AH (II)-deficient (Pafah2-/-) mice. PAF-AH (II) was predominantly expressed in epithelial cells such as kidney proximal and distal tubules, intestinal column epithelium, and hepatocytes. Although PAF-AH activity was almost abolished in the liver and kidney of Pafah2-/- mice, Pafah2-/- mice developed normally and were phenotypically indistinguishable from wild-type mice. However, mouse embryonic fibroblasts derived from Pafah2-/- mice were more sensitive to tert-butylhydroperoxide treatment than those derived from wild-type mice. When carbon tetrachloride (CCl4) was injected into mice, Pafah2-/- mice showed a delay in hepatic injury recovery. Moreover, after CCl4 administration, liver levels of the esterified form of 8-iso-PGF2α, a known in vitro substrate of PAF-AH (II), were higher in Pafah2-/- mice than in wild-type mice. These results indicate that PAF-AH (II) is involved in the metabolism of esterified 8-isoprostaglandin F2α and protects tissue from oxidative stress-induced injury. Membrane phospholipids are susceptible to oxidation, which is involved in various pathological processes such as inflammation, atherogenesis, neurodegeneration, and aging. One enzyme that may help to remove oxidized phospholipids from cells is intracellular type II platelet-activating factor acetylhydrolase (PAF-AH (II)), which hydrolyzes oxidatively fragmented fatty acyl chains attached to phospholipids. Overexpression of PAF-AH (II) in cells or tissues was previously shown to suppress oxidative stress-induced cell death. In this study we investigated the functions of PAF-AH (II) by generating PAF-AH (II)-deficient (Pafah2-/-) mice. PAF-AH (II) was predominantly expressed in epithelial cells such as kidney proximal and distal tubules, intestinal column epithelium, and hepatocytes. Although PAF-AH activity was almost abolished in the liver and kidney of Pafah2-/- mice, Pafah2-/- mice developed normally and were phenotypically indistinguishable from wild-type mice. However, mouse embryonic fibroblasts derived from Pafah2-/- mice were more sensitive to tert-butylhydroperoxide treatment than those derived from wild-type mice. When carbon tetrachloride (CCl4) was injected into mice, Pafah2-/- mice showed a delay in hepatic injury recovery. Moreover, after CCl4 administration, liver levels of the esterified form of 8-iso-PGF2α, a known in vitro substrate of PAF-AH (II), were higher in Pafah2-/- mice than in wild-type mice. These results indicate that PAF-AH (II) is involved in the metabolism of esterified 8-isoprostaglandin F2α and protects tissue from oxidative stress-induced injury. Oxidative stress has been implicated in a number of human diseases including atherosclerosis, cancer, neurodegenerative disorders, and aging (1Halliwell B. Gutteridge J.M. Methods Enzymol. 1990; 186: 1-85Crossref PubMed Scopus (4466) Google Scholar, 2Southorn P.A. Powis G. Mayo Clin. Proc. 1988; 63: 390-408Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar, 3Ames B.N. Science. 1983; 221: 1256-1264Crossref PubMed Scopus (2746) Google Scholar, 4Harman D. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 7124-7128Crossref PubMed Scopus (1594) Google Scholar). Polyunsaturated fatty acid-containing lipids are recognized as targets of oxidative damage, readily undergoing peroxidation upon exposure to free radicals. Peroxidation of lipids can greatly alter the physicochemical properties of membrane lipid bilayers, resulting in severe cellular dysfunction. In addition, a variety of lipid byproducts evolve from lipid peroxidation, some of which can exert adverse biological effects (5Kinnunen P.K. Chem. Phys. Lipids. 1991; 57: 375-399Crossref PubMed Scopus (183) Google Scholar, 6Uchida K. Prog. Lipid Res. 2003; 42: 318-343Crossref PubMed Scopus (976) Google Scholar, 7Fruhwirth G.O. Loidl A. Hermetter A. Biochim. Biophys. Acta. 2007; 1772: 718-736Crossref PubMed Scopus (215) Google Scholar). Therefore, peroxidized and oxidized phospholipids have to be promptly removed in vivo. It is assumed that these chemically modified lipids are preferentially hydrolyzed by cellular phospholipase A2 that mainly hydrolyzes fatty acyl chains attached to the sn-2 position of phosphoglycerides, the position where polyunsaturated fatty acyl chains are generally attached. One of the candidate enzymes responsible for the release of oxidized fatty acyl chains from the sn-2 position of membrane phospholipids is thought to be platelet-activating factor acetylhydrolase (PAF-AH) 2The abbreviations used are: PAF-AHplatelet-activating factor (PAF)-acetylhydrolasePAF-AH (II)intracellular type II PAF-AHMEFmouse embryonic fibroblastst-BuOOHtert-butylhydroperoxide8-iso-PGF2α8-iso-prostaglandin F2αPCphosphatidylcholineES cellsembryonic stem cells. (8Nigam S. Schewe T. Biochim. Biophys. Acta. 2000; 1488: 167-181Crossref PubMed Scopus (176) Google Scholar). platelet-activating factor (PAF)-acetylhydrolase intracellular type II PAF-AH mouse embryonic fibroblasts tert-butylhydroperoxide 8-iso-prostaglandin F2α phosphatidylcholine embryonic stem cells. PAF-AHs were originally identified as enzymes that hydrolyze the acetyl group attached to the sn-2 position of PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), which is a potent signaling phospholipid involved in diverse physiological and pathological events such as inflammation, anaphylaxis, reproduction, and fetal development (9Prescott S.M. Zimmerman G.A. Stafforini D.M. McIntyre T.M. Annu. Rev. Biochem. 2000; 69: 419-445Crossref PubMed Scopus (589) Google Scholar). Three types of PAF-AH have been identified in mammals, namely the intracellular types I and II and a plasma type (10Stafforini D.M. Prescott S.M. Zimmerman G.A. McIntyre T.M. Biochim. Biophys. Acta. 1996; 1301: 161-173Crossref PubMed Scopus (101) Google Scholar, 11Stafforini D.M. McIntyre T.M. Zimmerman G.A. Prescott S.M. J. Biol. Chem. 1997; 272: 17895-17898Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). PAF-AHs comprise groups VII and VIII of the PLA2 superfamily of proteins (12Dennis E.A. J. Biol. Chem. 1994; 269: 13057-13060Abstract Full Text PDF PubMed Google Scholar, 13Six D.A. Dennis E.A. Biochim. Biophys. Acta. 2000; 1488: 1-19Crossref PubMed Scopus (1223) Google Scholar). The initial studies on PAF-AH were performed on plasma PAF-AH (10Stafforini D.M. Prescott S.M. Zimmerman G.A. McIntyre T.M. Biochim. Biophys. Acta. 1996; 1301: 161-173Crossref PubMed Scopus (101) Google Scholar, 14Tjoelker L.W. Stafforini D.M. Biochim. Biophys. Acta. 2000; 1488: 102-123Crossref PubMed Scopus (150) Google Scholar, 15Karabina S.A. Ninio E. Biochim. Biophys. Acta. 2006; 1761: 1351-1358Crossref PubMed Scopus (54) Google Scholar, 16Karasawa K. Biochim. Biophys. Acta. 2006; 1761: 1359-1372Crossref PubMed Scopus (93) Google Scholar). Plasma PAF-AH is a 45-kDa secretory enzyme (17Tjoelker L.W. Wilder C. Eberhardt C. Stafforini D.M. Dietsch G. Schimpf B. Hooper S. Trong H.L. Cousens L.S. Zimmerman G.A. Yamada Y. McIntyre T.M. Prescott S.M. Gray P.W. Nature. 1995; 374: 549-553Crossref PubMed Scopus (476) Google Scholar) that is associated mainly with low density lipoproteins and high density lipoproteins in plasma (18Stafforini D.M. McIntyre T.M. Carter M.E. Prescott S.M. J. Biol. Chem. 1987; 262: 4215-4222Abstract Full Text PDF PubMed Google Scholar, 19Tselepis A.D. Dentan C. Karabina S.A. Chapman M.J. Ninio E. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1764-1773Crossref PubMed Scopus (193) Google Scholar). Plasma PAF-AH has marked selectivity for phospholipids with short acyl chains at the sn-2 position; with chains longer than nine carbons there was essentially no measurable activity (20Stafforini D.M. Prescott S.M. McIntyre T.M. J. Biol. Chem. 1987; 262: 4223-4230Abstract Full Text PDF PubMed Google Scholar, 21Stremler K.E. Stafforini D.M. Prescott S.M. McIntyre T.M. J. Biol. Chem. 1991; 266: 11095-11103Abstract Full Text PDF PubMed Google Scholar, 22Min J.H. Jain M.K. Wilder C. Paul L. Apitz-Castro R. Aspleaf D.C. Gelb M.H. Biochemistry (Mosc). 1999; 38: 12935-12942Crossref Scopus (64) Google Scholar). Interestingly, the suitability of a phospholipid as a substrate for plasma PAF-AH increases if the sn-2 acyl group has an oxidized functionality, such as an aldehydic or carboxylic group (21Stremler K.E. Stafforini D.M. Prescott S.M. McIntyre T.M. J. Biol. Chem. 1991; 266: 11095-11103Abstract Full Text PDF PubMed Google Scholar, 23Stremler K.E. Stafforini D.M. Prescott S.M. Zimmerman G.A. McIntyre T.M. J. Biol. Chem. 1989; 264: 5331-5334Abstract Full Text PDF PubMed Google Scholar). These unusual sn-2 acyl groups are generated by oxidative cleavage of long-chain polyunsaturated fatty acyl groups. Recently it has been reported that oxidatively modified phospholipids such as esterified F2-isoprostanes (24Stafforini D.M. Sheller J.R. Blackwell T.S. Sapirstein A. Yull F.E. McIntyre T.M. Bonventre J.V. Prescott S.M. Roberts L.J. J. Biol. Chem. 2006; 281: 4616-4623Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar) and phospholipid hydroperoxides (25Kriska T. Marathe G.K. Schmidt J.C. McIntyre T.M. Girotti A.W. J. Biol. Chem. 2007; 282: 100-108Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) are good substrates for plasma PAF-AH, suggesting that residue length is not the only factor involved in substrate recognition. In animal models recombinant plasma PAF-AH was effective in treating acute pancreatitis (26Hofbauer B. Saluja A.K. Bhatia M. Frossard J.L. Lee H.S. Bhagat L. Steer M.L. Gastroenterology. 1998; 115: 1238-1247Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), asthma (27Henderson W.R. Lu J. Poole K.M. Dietsch G.N. Chi E.Y. J. Immunol. 2000; 164: 3360-3367Crossref PubMed Scopus (76) Google Scholar), and anaphylactic shock (28Fukuda Y. Kawashima H. Saito K. Inomata N. Matsui M. Nakanishi T. Eur. J. Pharmacol. 2000; 390: 203-207Crossref PubMed Scopus (35) Google Scholar). Adenovirus-mediated gene transfer of plasma PAF-AH into the liver in apolipoprotein E-deficient mice reduced the level of oxidized low density lipoproteins in the blood (29Theilmeier G. De Geest B. Van Veldhoven P.P. Stengel D. Michiels C. Lox M. Landeloos M. Chapman M.J. Ninio E. Collen D. Himpens B. Holvoet P. FASEB J. 2000; 14: 2032-2039Crossref PubMed Scopus (136) Google Scholar, 30Quarck R. De Geest B. Stengel D. Mertens A. Lox M. Theilmeier G. Michiels C. Raes M. Bult H. Collen D. Van Veldhoven P. Ninio E. Holvoet P. Circulation. 2001; 103: 2495-2500Crossref PubMed Scopus (201) Google Scholar). These suppressive effects of plasma PAF-AH are thought to be due to its ability to hydrolyze PAF and PAF-like oxidized phospholipids. Four percent of the Japanese population lack plasma PAF-AH, and such a deficiency or decrease in plasma PAF-AH activity may be associated with severe asthma (31Stafforini D.M. Numao T. Tsodikov A. Vaitkus D. Fukuda T. Watanabe N. Fueki N. McIntyre T.M. Zimmerman G.A. Makino S. Prescott S.M. J. Clin. Investig. 1999; 103: 989-997Crossref PubMed Scopus (112) Google Scholar, 32Kruse S. Mao X.Q. Heinzmann A. Blattmann S. Roberts M.H. Braun S. Gao P.S. Forster J. Kuehr J. Hopkin J.M. Shirakawa T. Deichmann K.A. Am. J. Hum. Genet. 2000; 66: 1522-1530Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 33Miwa M. Miyake T. Yamanaka T. Sugatani J. Suzuki Y. Sakata S. Araki Y. Matsumoto M. J. Clin. Investig. 1988; 82: 1983-1991Crossref PubMed Scopus (277) Google Scholar), atopy (32Kruse S. Mao X.Q. Heinzmann A. Blattmann S. Roberts M.H. Braun S. Gao P.S. Forster J. Kuehr J. Hopkin J.M. Shirakawa T. Deichmann K.A. Am. J. Hum. Genet. 2000; 66: 1522-1530Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), stroke, and various cardiovascular diseases (34Hiramoto M. Yoshida H. Imaizumi T. Yoshimizu N. Satoh K. Stroke. 1997; 28: 2417-2420Crossref PubMed Scopus (128) Google Scholar, 35Yamada Y. Yokota M. Biochem. Biophys. Res. Commun. 1997; 236: 772-775Crossref PubMed Scopus (45) Google Scholar, 36Yamada Y. Ichihara S. Fujimura T. Yokota M. Metabolism. 1998; 47: 177-181Abstract Full Text PDF PubMed Scopus (148) Google Scholar, 37Satoh N. Asano K. Naoki K. Fukunaga K. Iwata M. Kanazawa M. Yamaguchi K. Am. J. Respir. Crit. Care Med. 1999; 159: 974-979Crossref PubMed Scopus (36) Google Scholar). Intracellular type II PAF-AH (PAF-AH (II)) is a monomeric 40-kDa enzyme. The amino acid sequence of PAF-AH (II) does not show any similarity to any subunit of PAF-AH (I) but significant identity with plasma PAF-AH. PAF-AH (II) is N-myristoylated at the N terminus and is distributed in both the cytosol and membranes like other N-myristoylated proteins (38Matsuzawa A. Hattori K. Aoki J. Arai H. Inoue K. J. Biol. Chem. 1997; 272: 32315-32320Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). PAF-AH (II) exhibits a substrate specificity that is very similar to plasma PAF-AH. PAF-AH (II) can hydrolyze phospholipids with short to medium length sn-2 acyl chains including truncated chains derived from oxidative cleavage of long-chain polyunsaturated fatty acyl groups. However, the activity of this enzyme toward phospholipids with two long (14-18 carbons) fatty acyl chains is negligible (39Min J.H. Wilder C. Aoki J. Arai H. Inoue K. Paul L. Gelb M.H. Biochemistry (Mosc). 2001; 40: 4539-4549Crossref Scopus (64) Google Scholar, 40Inoue T. Sugimoto A. Suzuki Y. Yamamoto M. Tsujimoto M. Inoue K. Aoki J. Arai H. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13233-13238Crossref PubMed Scopus (16) Google Scholar). We and another group have demonstrated that overexpression of PAF-AH (II) suppresses oxidative stress-induced cell death. Moreover, PAF-AH (II) translocates from the cytosol to the membrane during oxidative stress (38Matsuzawa A. Hattori K. Aoki J. Arai H. Inoue K. J. Biol. Chem. 1997; 272: 32315-32320Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 41Marques M. Pei Y. Southall M.D. Johnston J.M. Arai H. Aoki J. Inoue T. Seltmann H. Zouboulis C.C. Travers J.B. J. Investig. Dermatol. 2002; 119: 913-919Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). These results strongly suggest that PAF-AH (II) functions as an antioxidant phospholipase. Unlike intracellular type I PAF-AH, PAF-AH (II) is conserved in many organisms, including mammals, frog, fishes, nematodes, and even in yeast. We have shown that the Caenorhabditis elegans orthologue of mammalian PAF-AH (II) is expressed in epithelial cells of C. elegans and plays an important role in epithelial morphogenesis (40Inoue T. Sugimoto A. Suzuki Y. Yamamoto M. Tsujimoto M. Inoue K. Aoki J. Arai H. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13233-13238Crossref PubMed Scopus (16) Google Scholar). Because PAF is not present in C. elegans (42Satouchi K. Hirano K. Sakaguchi M. Takehara H. Matsuura F. Lipids. 1993; 28: 837-840Crossref PubMed Scopus (69) Google Scholar), it is not likely that PAF-AH (II) functions as a PAF-degrading enzyme. In this study we generated PAF-AH (II)-deficient (Pafah2-/-) mice by targeted disruption to elucidate the physiological and pathological functions of intracellular PAF-AH (II) in mammals. Generation of Pafah2-/- Mice—Pafah2 genomic clones were isolated from a mouse 129/SvJ genomic library in the Lambda FIXII vector (Stratagene, La Jolla, CA). A replacement-type targeting vector was constructed; the short arm containing a 1.0-kilobase fragment in intron 7 and the long arm containing a 9.2-kilobase SpeI fragment spanning exons 10-11 were inserted into the XhoI and NotI sites, respectively, of the vector pPolIIshort-neobpA-HSVTK. Exons 8-9, which include catalytic motif (GXSXV), were replaced by a neomycin-resistant cassette. The targeting vectors were linearized and electroporated into E14Tg2a embryonic stem cells (ES cells) (43Hooper M. Hardy K. Handyside A. Hunter S. Monk M. Nature. 1987; 326: 292-295Crossref PubMed Scopus (925) Google Scholar). G418-resistant colonies were screened for homologous recombinants by PCR. Candidates of homologous recombinants were verified by Southern blot analysis using fragments at the 5′-end of the genes, external to the targeting vectors as probes. Chimeric mice were generated by injection of the ES cells into C57BL/6 blastocysts followed by transfer to foster mothers and back-crossed to C57BL/6 mice. Genotypes were determined by PCR and/or Southern blot analysis of the tail DNA samples. All experiments reported here were performed with mice derived from six to eight generations of backcross-breeding to C57BL/6 mice. Western Blot Analysis—Murine tissues were homogenized in quadruple volumes (w/v) of SET buffer (10 mm Tris-HCl, pH 7.4, 1 mm EDTA, 250 mm sucrose) with protease inhibitors (0.5 mm phenylmethylsulfonyl fluoride, 2 μg/ml pepstatin, 2 μg/ml leupeptin, 2 μg/ml aprotinin). After centrifugation at 1000 × g at 4 °C, the supernatants were used as the total protein extracts. The protein concentrations of samples were determined by the BCA assay (Pierce). Each total protein extract (20 μg/lane) was separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with 5% (w/v) skim milk (Wako Pure Chemical Industries, Osaka, Japan) in TTBS buffer (10 mm Tris-HCl, pH 7.4, 150 mm NaCl, 0.05% (w/v) Tween 20) and incubated with the specific monoclonal mouse anti-PAF-AH (II) antibody TI10 (44Bae K. Longobardi L. Karasawa K. Malone B. Inoue T. Aoki J. Arai H. Inoue K. Lee T.H. J. Biol. Chem. 2000; 275: 26704-26709Abstract Full Text Full Text PDF PubMed Google Scholar). After incubation with horseradish peroxidase-conjugated anti-mouse IgG antibody (GE Healthcare), PAF-AH (II) was detected by enhanced chemiluminescence (ECL Western blotting detection system, GE Healthcare). Histological and Immunohistochemical Analyses—Mice under anesthesia were perfused with phosphate-buffered saline. Tissues were dissected and fixed overnight in 4% paraformaldehyde/phosphate-buffered saline at 4 °C. Paraffin sections (5 μm) were prepared and stained with hematoxylin and eosin. For immunohistochemistry, paraffin sections (5 μm) were boiled in a microwave oven in 10 mm sodium citrate buffer (pH 6.0) for antigen retrieval. Subsequent immunodetection was performed using the monoclonal antibody TI10 and Vector M.O.M. peroxidase immunodetection kit (Vector Laboratories, Burlingame, CA). The sections were also counterstained with hematoxylin. Enzyme Assays—Mouse tissue samples were homogenized in SET buffer followed by centrifugation at 100,000 × g for 1 h to obtain the soluble fraction. Mouse blood samples were obtained by retro-orbital bleeding. Plasma was prepared by centrifugation of the blood at 1800 × g for 10 min. Peritoneal exudate macrophages were suspended in SET buffer and disrupted by sonication for 3 periods of 4 s at 10-s intervals using a Branson Sonifer. The soluble fraction was prepared by centrifugation at 1000 × g for 20 min. PAF-AH activity of the tissue soluble fraction and the plasma were measured as previously described (45Hattori M. Arai H. Inoue K. J. Biol. Chem. 1993; 268: 18748-18753Abstract Full Text PDF PubMed Google Scholar). Oxidized phospholipid-hydrolyzing activity was measured as follows. The standard incubation system for assay comprised 50 mm Tris-HCl (pH 7.4), 5 mm EDTA, 10 nmol of 1-palmitoyl, 2-azeraoyl phosphatidylcholine (PC), and the sample in a total volume of 0.125 ml. After incubation for 15 min at 37 °C, the reaction was stopped by adding 1.25 ml of chloroform/methanol (4:1, v/v) followed by adding 0.125 ml of water and 6 nmol of 1,2-dimyristoyl PC as internal standard. The lipids in the organic phase were extracted and analyzed by a Quattro Micro tandem quadrupole mass spectrometer (Micromass, Manchester, UK) equipped with an electrospray ion source, as described previously (46Tanaka M. Kishi Y. Takanezawa Y. Kakehi Y. Aoki J. Arai H. FEBS Lett. 2004; 571: 197-204Crossref PubMed Scopus (98) Google Scholar). Lipid extracts were reconstituted in 2:1 chloroform/methanol, and the samples were introduced by means of a flow injector into the electrospray ion source chamber at a flow rate of 4 μl/min in a solvent system of acetonitrile/methanol/water (2:3:1; v/v) containing 0.1% (v/v) ammonium formate (pH 6.4). The mass spectrometer was operated in the positive and negative scan modes. The flow rate of the nitrogen drying gas was 12 liters/min at 80 °C. The capillary and cone voltages were set at 3.7 kV and 30 V, respectively. PAF Degradation Assay—PAF degradation by cultured peritoneal macrophages was measured as described (47Ohshima N. Ishii S. Izumi T. Shimizu T. J. Biol. Chem. 2002; 277: 9722-9727Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Peritoneal exudate macrophages were obtained by washing the peritoneal cavity with 5 ml of ice-cold phosphate-buffered saline 3 days after intraperitoneal injection of 2 ml of sterile 4% thioglycollate. After centrifugation at 250 × g, the cells were suspended in RPMI 1640 (Sigma) supplemented with 2 mm glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin (Invitrogen), and 10% heat-inactivated fetal bovine serum (Biowest, Nuaille, France). They were cultured in 24-well plates (0.5 × 106 cells/well) in 5% CO2 at 37 °C. After overnight incubation, non-adherent cells were removed by washing three times with phosphate-buffered saline. Cells were incubated for 2 h at 37 °C with Tyrode's buffer (140 mm NaCl, 2.7 mm KCl, 12 mm NaHCO3, 5.6 mm d-glucose, 0.49 mm MgCl2, and 0.37 mm NaH2PO4) containing 10 mm Hepes-NaOH (pH 7.4) and 0.1% of bovine serum albumin (BSA) (Hepes/Tyrode's/BSA) and then incubated with 2 nm [acetyl-3H]PAF (795.5 Gbq/mmol, PerkinElmer Life Sciences) in Hepes/Tyrode's/BSA for 30-120 min at 37 °C. The medium was recovered, and the lipids were extracted by the Bligh and Dyer method (48Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (43132) Google Scholar). The radioactivity of liberated [3H]acetate in the aqueous phase was counted with a liquid scintillation counter (Tri-Carb 3100TR, PerkinElmer, Waltham, MA). Preparation of Mouse Embryonic Fibroblasts and Cell Viability Assay—Mouse embryonic fibroblasts (MEFs) were obtained from E13.5 embryos and cultured in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% heat-inactivated fetal bovine serum (Biowest, Nuaille, France), 2 mm glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen), and maintained in 5% CO2. Cells were plated at 2×104/well in 96-well plates and then exposed to varying concentrations of tert-butylhydroperoxide (t-BuOOH, Sigma) for 6 h. Eighteen hours after treatment, cell viability was measured using 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-8), which is converted to water-soluble formazan by mitochondrial dehydrogenase (Cell Counting Kit-8, Dojindo, Kumamoto, Japan). The relative number of surviving cells was determined in quadruplicate by estimating the value of untreated cells as 100%. To determine the distribution of PAF-AH (II) between the cytosol and membranes in MEFs, MEFs were harvested after the t-BuOOH treatment (100 μm) and disrupted by sonication. Cell lysates were centrifuged at 100,000 × g for 1 h at 4 °C to separate the cytosol (supernatant) and membrane (pellet) fractions. Both the cytosol and membrane fractions (7 μg protein) were analyzed by immunoblotting with the monoclonal antibody TI10 as described above. Measurement of 8-Isoprostaglandin F2α—8-Isoprostaglandin F2α (8-iso-PGF2α) was measured as previously described (49Yoshida Y. Itoh N. Hayakawa M. Piga R. Cynshi O. Jishage K. Niki E. Toxicol. Appl. Pharmacol. 2005; 208: 87-97Crossref PubMed Scopus (57) Google Scholar). Liver was homogenized (Polytron homogenizer, PT-3100) with saline (liver/saline = 1:3, w/w), and the aliquot (300 μl) was diluted with saline (1700 μl). Internal standard 8-iso-PGF2α-d4 (100 ng) and 1 ml of methanol were added to this solution followed by the reduction of hydroperoxides with excessive amount of sodium borohydride at room temperature for 5 min under nitrogen. Then the reduced sample was mixed with 1 m KOH in methanol (1 ml) under nitrogen and incubated for 30 min in the dark at 40 °C in a shaker. The sample was centrifuged (3000 × g, for 10 min, at 4 °C), and the supernatant was diluted with 4-fold volume of water (pH 3) and acidified (pH 3) using 2 n HCl. The acidified sample was centrifuged (3000 × g, for 10 min, at 4 °C), and the supernatant was subjected to the following solid phase extraction. C18 cartridge was preconditioned with 2 ml of methanol and 2 ml of water (pH 3). The sample was loaded to the cartridge at a flow rate of 1 ml/min, and the cartridge was washed with 10 ml of water (pH 3) and 10 ml of acetonitrile/water (15:85, by volume). An elution was performed with 4 ml of hexane/ethyl acetate/isopropyl alcohol (30:65:5, by volume) at a flow rate of 1 ml/min. The elute was then applied at a flow rate of 1 ml/min to NH2 cartridge preconditioned with hexane (5 ml). The cartridge was washed sequentially with 5 ml of hexane/ethyl acetate (30:70, by volume) and 5 ml of acetonitrile, and an elution was performed with 5 ml of ethyl acetate/methanol/acetic acid (10:85:5, by volume) at a flow rate of 1 ml/min. The solution was evaporated under nitrogen, and 30 μl of the silylating agent, N,O-bis(trimethylsilyl)trifluoroacetamide, was added to the dried residue. The solution was vigorously mixed by a vortex mixer for 1 min and incubated for 60 min at 60 °C to obtain the trimethylsilyl esters and ethers. This solution was diluted with 70 μl of acetone, and then an aliquot of this sample was injected into the gas chromatograph (GC 6890 N, Agilent Technologies Co. Ltd.) equipped with a quadrupole mass spectrometer (5973 Network, Agilent Technologies Co. Ltd.). A fused-silica capillary column (HP-5MS, 5% phenyl methyl siloxane, 30 m × 0.25 mm, Agilent Technologies Co. Ltd.) was used. Helium was used as the carrier gas at a flow rate of 1.2 ml/min. Temperature programming was performed from 60 to 280 °C at 10 °C/min. The injector temperature was set at 250 °C, and temperatures of the transfer line to the mass detector and ion source were 250 and 230 °C, respectively. Electron energy was set at 70 eV. The identification of 8-iso-PGF2α was conducted by their retention times and mass patterns (m/z = 571, 481), and ions at 481 were selected for quantification 8-iso-PGF2α using the internal standard 8-iso-PGF2α-d4 (m/z = 485). Carbon Tetrachloride (CCl4)-induced Acute Hepatic Injury—Mice were injected intraperitoneally with 0.3 or 1 ml/kg body weight of CCl4 dissolved in olive oil (total volume 10 ml/kg body weight). At 0, 12, 24, 36, 48, and 72 h after injection, blood was collected by retro-orbital bleeding under light ether anesthesia with heparinized capillary tubes. Plasma was obtained by centrifugation at 1800 × g for 10 min. Plasma alanine transferase activity was evaluated using a Transaminase CII Testwako kit (Wako Pure Chemical Industries, Osaka, Japan). At each time point livers were excised and subjected to histological examination. Statistics—Statistical analyses were performed with Student's t test setting the significance at p < 0.05. PAF-AH (II) Is Expressed in Epithelial Tissues—Western blot analysis showed that PAF-AH (II) was expressed essentially in all the tissues, most abundantly in the liver, kidney, intestine, and testis (Fig. 1A). Immunohistochemical studies revealed that PAF-AH (II) was predominantly expressed in epithelial cells such as hepatocytes, kidney tubules, Bowman's capsule epithelium, and intestinal column epithelium (Fig. 1B). In contrast, endothelial and interstitial cells showed low expression of PAF-AH (II). No staining of PAF-AH (II) was detected in the tissues of null mutant mice (Fig. 1B). Immunofluorescence double staining of kidney sections with antibodies to PAF-AH (II) and nephron segment markers (aquaporin-1 and aquaporin-2) revealed that PAF-AH (II) was expressed in proximal tubules, distal tubules, and thick ascending limbs of Henle but not in the collecting ducts or thin loops of Henle (supplemental figure). Generation of Pafah2-/- Mice—To investigate the role of PAF-AH (II) in vivo, we generated PAF-AH (II) mutant mice by targeted disruption. Mice lacking the PAF-AH (II) gene (Pafah2) were produced by homologous recombination in embryonic stem cells using the strategy outlined in Fig. 2. The targeting vector was constructed to replace exons 8-9 of the Pafah2 gene with a neomycin-resistance gene. Targeted embryonic stem cell clones and subsequent germ line transmissions were detected by PCR and Southern blot analysis (Fig. 2B). Western blot analysis of kidney homogenates revealed that PAF-AH (II) protein was absent in homozygous mutants, and heterozygous mutants expressed about half the amount of PAF-AH (II) protein detected in wild-type mice (Fig. 2C), suggesting that the expression of PAF-AH (II) from the normal allele is not altered due to decreased amounts of total PAF-AH (II) protein. Quantitative real-time PCR analysis of kidney mRNA revealed that Pafah2 mRNA was absent in homozygous mutants (data not shown). Together, these results indicate that the introduced mutation eliminated expression of PA