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Cholesteryl ester hydrolase activity is abolished in HSL macrophages but unchanged in macrophages lacking KIAA1363

2010; Elsevier BV; Volume: 51; Issue: 10 Linguagem: Inglês

10.1194/jlr.m004259

ISSN

1539-7262

Autores

Marlene Buchebner, Thomas Pfeifer, Nora Rathke, Prakash G. Chandak, Achim Lass, Renate Schreiber, Adelheid Kratzer, Robert Zimmermann, Wolfgang Sattler, Harald Koefeler, Eleonore Fröhlich, Gerhard M. Kostner, Ruth Birner‐Gruenberger, Kyle P. Chiang, Guenter Haemmerle, Rudolf Zechner, Sanja Levak‐Frank, Benjamin F. Cravatt, Dagmar Kratky,

Tópico(s)

Adipose Tissue and Metabolism

Resumo

Cholesteryl ester (CE) accumulation in macrophages represents a crucial event during foam cell formation, a hallmark of atherogenesis. Here we investigated the role of two previously described CE hydrolases, hormone-sensitive lipase (HSL) and KIAA1363, in macrophage CE hydrolysis. HSL and KIAA1363 exhibited marked differences in their abilities to hydrolyze CE, triacylglycerol (TG), diacylglycerol (DG), and 2-acetyl monoalkylglycerol ether (AcMAGE), a precursor for biosynthesis of platelet-activating factor (PAF). HSL efficiently cleaved all four substrates, whereas KIAA1363 hydrolyzed only AcMAGE. This contradicts previous studies suggesting that KIAA1363 is a neutral CE hydrolase. Macrophages of KIAA1363−/− and wild-type mice exhibited identical neutral CE hydrolase activity, which was almost abolished in tissues and macrophages of HSL−/− mice. Conversely, AcMAGE hydrolase activity was diminished in macrophages and some tissues of KIAA1363−/− but unchanged in HSL−/− mice. CE turnover was unaffected in macrophages lacking KIAA1363 and HSL, whereas cAMP-dependent cholesterol efflux was influenced by HSL but not by KIAA1363. Despite decreased CE hydrolase activities, HSL−/− macrophages exhibited CE accumulation similar to wild-type (WT) macrophages. We conclude that additional enzymes must exist that cooperate with HSL to regulate CE levels in macrophages. KIAA1363 affects AcMAGE hydrolase activity but is of minor importance as a direct CE hydrolase in macrophages. Cholesteryl ester (CE) accumulation in macrophages represents a crucial event during foam cell formation, a hallmark of atherogenesis. Here we investigated the role of two previously described CE hydrolases, hormone-sensitive lipase (HSL) and KIAA1363, in macrophage CE hydrolysis. HSL and KIAA1363 exhibited marked differences in their abilities to hydrolyze CE, triacylglycerol (TG), diacylglycerol (DG), and 2-acetyl monoalkylglycerol ether (AcMAGE), a precursor for biosynthesis of platelet-activating factor (PAF). HSL efficiently cleaved all four substrates, whereas KIAA1363 hydrolyzed only AcMAGE. This contradicts previous studies suggesting that KIAA1363 is a neutral CE hydrolase. Macrophages of KIAA1363−/− and wild-type mice exhibited identical neutral CE hydrolase activity, which was almost abolished in tissues and macrophages of HSL−/− mice. Conversely, AcMAGE hydrolase activity was diminished in macrophages and some tissues of KIAA1363−/− but unchanged in HSL−/− mice. CE turnover was unaffected in macrophages lacking KIAA1363 and HSL, whereas cAMP-dependent cholesterol efflux was influenced by HSL but not by KIAA1363. Despite decreased CE hydrolase activities, HSL−/− macrophages exhibited CE accumulation similar to wild-type (WT) macrophages. We conclude that additional enzymes must exist that cooperate with HSL to regulate CE levels in macrophages. KIAA1363 affects AcMAGE hydrolase activity but is of minor importance as a direct CE hydrolase in macrophages. Mobilization of cholesterol and fatty acids from cholesteryl ester (CE) and triacylglycerol (TG) stores of all cells and tissues requires lipolytic enzymes. Dysfunctional cellular hydrolysis affects lipid homeostasis, causes lipid storage disorders, and may contribute to the pathogenesis of atherosclerosis and obesity. Human and murine macrophages express cell surface scavenger receptors that bind and subsequently internalize modified LDL. An excessive and uncontrolled uptake of cholesterol results in foam cell formation, which is a hallmark of atherosclerosis. Cholesterol is esterified by acyl-CoA:cholesterol acyltransferase (ACAT)1 and stored as CE in lipid droplets. Conversely, CE hydrolases are required for the hydrolysis of lipid droplet-associated CE (1Brown M.S. Ho Y.K. Goldstein J.L. The cholesteryl ester cycle in macrophage foam cells. Continual hydrolysis and re-esterification of cytoplasmic cholesteryl esters.J. Biol. Chem. 1980; 255: 9344-9352Abstract Full Text PDF PubMed Google Scholar, 2Ho Y.K. Brown M.S. Goldstein J.L. Hydrolysis and excretion of cytoplasmic cholesteryl esters by macrophages: stimulation by high density lipoprotein and other agents.J. Lipid Res. 1980; 21: 391-398Abstract Full Text PDF PubMed Google Scholar). CE hydrolysis in macrophages has been studied for more than 40 years (3Goodman D.S. Cholesterol ester metabolism.Physiol. Rev. 1965; 45: 747-839Crossref PubMed Scopus (209) Google Scholar). In 1989, it was first reported that the neutral CE hydrolase activity in the mouse macrophage cell line WEH1 is catalyzed by hormone-sensitive lipase (HSL) (4Small C.A. Goodacre J.A. Yeaman S.J. Hormone-sensitive lipase is responsible for the neutral cholesterol ester hydrolase activity in macrophages.FEBS Lett. 1989; 247: 205-208Crossref PubMed Scopus (73) Google Scholar). In RAW264.7 macrophages, neutral CE hydrolase activity was nearly completely blocked with an anti-HSL antibody, whereas overexpression of HSL increased CE hydrolysis in these cells (5Escary J.L. Choy H.A. Reue K. Schotz M.C. Hormone-sensitive lipase overexpression increases cholesteryl ester hydrolysis in macrophage foam cells.Arterioscler. Thromb. Vasc. Biol. 1998; 18: 991-998Crossref PubMed Scopus (55) Google Scholar). Adenoviral-mediated overexpression of HSL in human THP-1 macrophages resulted in complete elimination of CE stores (6Okazaki H. Osuga J. Tsukamoto K. Isoo N. Kitamine T. Tamura Y. Tomita S. Sekiya M. Yahagi N. Iizuka Y. et al.Elimination of cholesterol ester from macrophage foam cells by adenovirus-mediated gene transfer of hormone-sensitive lipase.J. Biol. 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Tsutsumi O. et al.Targeted disruption of hormone-sensitive lipase results in male sterility and adipocyte hypertrophy, but not in obesity.Proc. Natl. Acad. Sci. USA. 2000; 97: 787-792Crossref PubMed Scopus (504) Google Scholar, 9Contreras J.A. Hormone-sensitive lipase is not required for cholesteryl ester hydrolysis in macrophages.Biochem. Biophys. Res. Commun. 2002; 292: 900-903Crossref PubMed Scopus (31) Google Scholar), arguing against a dominant role of HSL as a CE hydrolase in macrophages. Very recently, however, these authors reported on reduced neutral CE hydrolase activity in HSL−/− MPM (10Sekiya M. Osuga J. Nagashima S. Ohshiro T. Igarashi M. Okazaki H. Takahashi M. Tazoe F. Wada T. Ohta K. et al.Ablation of neutral cholesterol ester hydrolase 1 accelerates atherosclerosis.Cell Metab. 2009; 10: 219-228Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Evidence was provided for neutral CE hydrolase activity by monocyte/macrophage serine esterase 1 (CES1) in human THP-1 monocytes and macrophages (11Ghosh S. Cholesteryl ester hydrolase in human monocyte/macrophage: cloning, sequencing, and expression of full-length cDNA.Physiol. Genomics. 2000; 2: 1-8Crossref PubMed Scopus (96) Google Scholar). This enzyme is related to the neutral CE hydrolase expressed in liver, and its overexpression causes cytoplasmic CE mobilization from ACAT1 stably expressing CHO cells (12Ghosh S. St. Clair R.W. Rudel L.L. Mobilization of cytoplasmic CE droplets by overexpression of human macrophage cholesteryl ester hydrolase.J. Lipid Res. 2003; 44: 1833-1840Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Its mouse ortholog, named carboxylesterase 3 (TG hydrolase, TGH), is expressed in macrophages at a low level, primarily in white adipose tissue, where it showed little TG and only detectable CE hydrolase activity (13Soni K.G. Lehner R. Metalnikov P. O'Donnell P. Semache M. Gao W. Ashman K. Pshezhetsky A.V. Mitchell G.A. Carboxylesterase 3 (EC 3.1.1.1) is a major adipocyte lipase.J. Biol. Chem. 2004; 279: 40683-40689Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Finally, a pancreatic CE hydrolase, [cholesterol esterase, bile salt-dependent lipase (CEL)] was reported to be also expressed in macrophages in aorta (14Shamir R. Johnson W.J. Morlock-Fitzpatrick K. Zolfaghari R. Li L. Mas E. Lombardo D. Morel D.W. Fisher E.A. Pancreatic carboxyl ester lipase: a circulating enzyme that modifies normal and oxidized lipoproteins in vitro.J. Clin. Invest. 1996; 97: 1696-1704Crossref PubMed Scopus (72) Google Scholar, 15Li F. Hui D.Y. Modified low density lipoprotein enhances the secretion of bile salt-stimulated cholesterol esterase by human monocyte-macrophages. species-specific difference in macrophage cholesteryl ester hydrolase.J. Biol. Chem. 1997; 272: 28666-28671Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Since CEL is expressed in human but not in murine macrophages, it was proposed that CEL plays a similar role in human macrophages as does HSL in mouse macrophages (16Hui D.Y. Howles P.N. Carboxyl ester lipase: structure-function relationship and physiological role in lipoprotein metabolism and atherosclerosis.J. Lipid Res. 2002; 43: 2017-2030Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Recently, the serine hydrolase KIAA1363 was identified as a new CE hydrolase by in silico searches of databases for proteins showing homology to known lipases (17Okazaki H. Igarashi M. Nishi M. Sekiya M. Tajima M. Takase S. Takanashi M. Ohta K. Tamura Y. Okazaki S. et al.Identification of neutral cholesterol ester hydrolase, a key enzyme removing cholesterol from macrophages.J. Biol. Chem. 2008; 283: 33357-33364Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Okazaki et al. (17Okazaki H. Igarashi M. Nishi M. Sekiya M. Tajima M. Takase S. Takanashi M. Ohta K. Tamura Y. Okazaki S. et al.Identification of neutral cholesterol ester hydrolase, a key enzyme removing cholesterol from macrophages.J. Biol. Chem. 2008; 283: 33357-33364Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) renamed KIAA1363 as neutral cholesterol ester hydrolase (NCEH) and suggested NCEH to be the key enzyme responsible for neutral CE hydrolase activity in murine macrophages (10Sekiya M. Osuga J. Nagashima S. Ohshiro T. Igarashi M. Okazaki H. Takahashi M. Tazoe F. Wada T. Ohta K. et al.Ablation of neutral cholesterol ester hydrolase 1 accelerates atherosclerosis.Cell Metab. 2009; 10: 219-228Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 17Okazaki H. Igarashi M. Nishi M. Sekiya M. Tajima M. Takase S. Takanashi M. Ohta K. Tamura Y. Okazaki S. et al.Identification of neutral cholesterol ester hydrolase, a key enzyme removing cholesterol from macrophages.J. Biol. Chem. 2008; 283: 33357-33364Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Originally, Jessani et al. had characterized KIAA1363 as a serine hydrolase highly elevated in aggressive human cancer cells (18Jessani N. Liu Y. Humphrey M. Cravatt B.F. Enzyme activity profiles of the secreted and membrane proteome that depict cancer cell invasiveness.Proc. Natl. Acad. Sci. USA. 2002; 99: 10335-10340Crossref PubMed Scopus (289) Google Scholar) and showed that this enzyme was also enriched in primary human breast tumors (19Jessani N. Niessen S. Wei B.Q. Nicolau M. Humphrey M. Ji Y. Han W. Noh D.Y. Yates 3rd, J.R. Jeffrey S.S. et al.A streamlined platform for high-content functional proteomics of primary human specimens.Nat. Methods. 2005; 2: 691-697Crossref PubMed Scopus (205) Google Scholar). Nomura et al. (20Nomura D.K. Leung D. Chiang K.P. Quistad G.B. Cravatt B.F. Casida J.E. A brain detoxifying enzyme for organophosphorus nerve poisons.Proc. Natl. Acad. Sci. USA. 2005; 102: 6195-6200Crossref PubMed Scopus (47) Google Scholar) described KIAA1363 as a significant detoxifying enzyme for organophosphorus nerve poisons in brain. Most recently, KIAA1363 has been shown to regulate an ether lipid metabolic network in cancer cells, where the enzyme serves as a principal 2-acetyl monoalkylglycerol ether (AcMAGE) hydrolase (20Nomura D.K. Leung D. Chiang K.P. Quistad G.B. Cravatt B.F. Casida J.E. A brain detoxifying enzyme for organophosphorus nerve poisons.Proc. Natl. Acad. Sci. USA. 2005; 102: 6195-6200Crossref PubMed Scopus (47) Google Scholar, 21Chiang K.P. Niessen S. Saghatelian A. Cravatt B.F. An enzyme that regulates ether lipid signaling pathways in cancer annotated by multidimensional profiling.Chem. Biol. 2006; 13: 1041-1050Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). AcMAGE is the penultimate precursor in the de novo biosynthesis of platelet activating factor (PAF) (22Nomura D.K. Fujioka K. Issa R.S. Ward A.M. Cravatt B.F. Casida J.E. Dual roles of brain serine hydrolase KIAA1363 in ether lipid metabolism and organophosphate detoxification.Toxicol. Appl. Pharmacol. 2008; 228: 42-48Crossref PubMed Scopus (23) Google Scholar), a potent lipid mediator playing inflammatory and physiological roles in a variety of cells. Inhibition of KIAA1363 disrupted this metabolic pathway in tumor cells and impaired their invasion and growth in vivo (21Chiang K.P. Niessen S. Saghatelian A. Cravatt B.F. An enzyme that regulates ether lipid signaling pathways in cancer annotated by multidimensional profiling.Chem. Biol. 2006; 13: 1041-1050Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). The analysis of KIAA1363−/− mice confirmed that this enzyme is the primary AcMAGE hydrolase in brain, lung, heart, and kidney and was highly sensitive to inactivation by chlorpyrifos oxon, the bioactivated metabolite of the major insecticide chlorpyrifos (22Nomura D.K. Fujioka K. Issa R.S. Ward A.M. Cravatt B.F. Casida J.E. Dual roles of brain serine hydrolase KIAA1363 in ether lipid metabolism and organophosphate detoxification.Toxicol. Appl. Pharmacol. 2008; 228: 42-48Crossref PubMed Scopus (23) Google Scholar). The aim of the present study was to compare CE hydrolase activities of HSL and KIAA1363 to reveal the importance of these enzymes as neutral CE hydrolases in murine macrophages and tissues. Our results demonstrate the functional presence of HSL as a neutral CE hydrolase in murine macrophages. In the absence of HSL in macrophages, a so far unknown mechanism compensates for the loss of HSL, resulting in unchanged cellular CE concentrations. In our hands, KIAA1363 significantly contributes to hydrolysis of AcMAGE but not to CE hydrolysis in macrophages, which contradicts data published recently (10Sekiya M. Osuga J. Nagashima S. Ohshiro T. Igarashi M. Okazaki H. Takahashi M. Tazoe F. Wada T. Ohta K. et al.Ablation of neutral cholesterol ester hydrolase 1 accelerates atherosclerosis.Cell Metab. 2009; 10: 219-228Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 17Okazaki H. Igarashi M. Nishi M. Sekiya M. Tajima M. Takase S. Takanashi M. Ohta K. Tamura Y. Okazaki S. et al.Identification of neutral cholesterol ester hydrolase, a key enzyme removing cholesterol from macrophages.J. Biol. Chem. 2008; 283: 33357-33364Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Animal experiments were performed in accordance with the standards established by the Austrian Federal Ministry of Education, Science and Culture, Division of Genetic Engineering and Animal Experiments (Vienna, Austria). HSL−/− (23Haemmerle G. Zimmermann R. Hayn M. Theussl C. Waeg G. Wagner E. Sattler W. Magin T.M. Wagner E.F. Zechner R. Hormone-sensitive lipase deficiency in mice causes diglyceride accumulation in adipose tissue, muscle, and testis.J. Biol. Chem. 2002; 277: 4806-4815Abstract Full Text Full Text PDF PubMed Scopus (488) Google Scholar, 24Haemmerle G. Zimmermann R. Strauss J.G. Kratky D. Riederer M. Knipping G. Zechner R. Hormone-sensitive lipase deficiency in mice changes the plasma lipid profile by affecting the tissue-specific expression pattern of lipoprotein lipase in adipose tissue and muscle.J. Biol. Chem. 2002; 277: 12946-12952Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar) and KIAA1363−/− mice (22Nomura D.K. Fujioka K. Issa R.S. Ward A.M. Cravatt B.F. Casida J.E. Dual roles of brain serine hydrolase KIAA1363 in ether lipid metabolism and organophosphate detoxification.Toxicol. Appl. Pharmacol. 2008; 228: 42-48Crossref PubMed Scopus (23) Google Scholar, 25Nomura D.K. Durkin K.A. Chiang K.P. Quistad G.B. Cravatt B.F. Casida J.E. Serine hydrolase KIAA1363: toxicological and structural features with emphasis on organophosphate interactions.Chem. Res. Toxicol. 2006; 19: 1142-1150Crossref PubMed Scopus (31) Google Scholar) (supplementary Fig. I) as well as wild-type (WT) littermates, all on a C57Bl/6 background, were obtained from inhouse breeding and maintained in a clean environment on a regular light-dark cycle (14 h light, 10 h dark). They received a standard mouse chow diet (Ssniff R/M H; ssniff, Soest, Germany) or Western-type diet (WTD; TD88137 mod, containing 21% fat and 0.2% cholesterol; ssniff, Soest, Germany). All mice have been backcrossed onto a C57Bl/6 background. Blood was taken from fed and fasted age-matched mice by retro-orbital bleeding and EDTA plasma was prepared within 20 min. Plasma concentrations of TG (DiaSys, Holzheim, Germany), total cholesterol (TC) (Greiner Diagnostics, Langenthal, Switzerland), and FC (DiaSys, Holzheim, Germany) were determined enzymatically according to the manufacturer's protocols. Plasma samples (200 µl) of fasted mice were pooled and lipoproteins were isolated by fast protein liquid chromatography (FPLC) on a Pharmacia FPLC system equipped with a Superose 6 column (Amersham Biosciences, Piscataway, NJ) as described previously (26Haemmerle G. Lass A. Zimmermann R. Gorkiewicz G. Meyer C. Rozman J. Heldmaier G. Maier R. Theussl C. Eder S. et al.Defective lipolysis and altered energy metabolism in mice lacking adipose triglyceride lipase.Science. 2006; 312: 734-737Crossref PubMed Scopus (1021) Google Scholar). COS-7 (ATCC CRL-1651) cells were cultivated in a humidified incubator under standard conditions (5% CO2 at 37°C) in DMEM (Gibco, Invitrogen, Lofer, Austria) containing 10% FCS (Sigma-Aldrich Chemie, Vienna, Austria), 1% L-glutamine, and 1% streptomycin/penicillin. Thioglycolate-elicited MPM were isolated with 10 ml PBS three days after peritoneal injection of 3 ml 3% thioglycolate medium. MPM were centrifuged, washed with PBS, and cultured in 6-well plates in serum-free DMEM for 2 h. Thereafter, nonadherent cells were removed. Either MPM were used immediately for determination of lipid parameters or hydrolase activities, or they were cultured overnight in DMEM/10% LPDS for efflux experiments. Human LDL was isolated by density gradient ultracentrifugation (density 1.019–1.063 g/ml) in a near-vertical rotor. LDL was acetylated (acLDL) as described (27Basu S.K. Goldstein J.L. Anderson G.W. Brown M.S. Degradation of cationized low density lipoprotein and regulation of cholesterol metabolism in homozygous familial hypercholesterolemia fibroblasts.Proc. Natl. Acad. Sci. USA. 1976; 73: 3178-3182Crossref PubMed Scopus (823) Google Scholar). β-VLDL was isolated from WTD-fed apolipoprotein (apo)E−/− mice as described (28Van Eck M. Herijgers N. Yates J. Pearce N.J. Hoogerbrugge P.M. Groot P.H. Van Berkel T.J. Bone marrow transplantation in apolipoprotein E-deficient mice. Effect of ApoE gene dosage on serum lipid concentrations, (beta)VLDL catabolism, and atherosclerosis.Arterioscler. Thromb. Vasc. Biol. 1997; 17: 3117-3126Crossref PubMed Scopus (84) Google Scholar). LPDS was prepared from newborn bovine serum by ultracentrifugation. To achieve foam cell formation, MPM were incubated in the absence or presence of 100 µg acLDL/ml for 48 h and 72 h. Coding sequences of murine HSL and KIAA1363 were amplified by PCR from MPM cDNA using Advantage® cDNA Polymerase Mix (BD Biosciences Clontech, Palo Alto, CA). Primers were designed to create endonuclease cleavage sites (underlined): HSL forward: 5′- t ggtacc tatggatttacgcacgatgacaca-3′, HSL reverse 5′-c ctcgag cgttcagtggt gcagcaggcg-3′. KIAA1363 forward: 5′-cgggatccaggtcgtcatgcgtcctact-3′, KIAA1363 reverse: 5′-ccctcgagtcacaggttttgatccagcc-3′. PCR products, containing the complete open reading frames, were ligated into the eukaryotic expression vector pcDNA4/HisMax (Invitrogen Corp., Carlsbad, CA). A control pcDNA4/HisMax vector expressing β−galactosidase (LacZ) was obtained from the manufacturer (Invitrogen Corp., Carlsbad, CA). Total RNA from macrophages was isolated using RNeasy Mini Kit (Qiagen, Hilden, Germany). ABCA1 and ABCG1 mRNA levels were determined on a LightCycler 480 (Roche Diagnostics, Mannheim, Germany) using the QuantifastTM SYBR®GREEN PCR Kit (Qiagen) as described (29Kratzer A. Buchebner M. Pfeifer T. Becker T.M. Uray G. Miyazaki M. Miyazaki-Anzai S. Ebner B. Chandak P.G. Kadam R.S. et al.Synthetic LXR agonist attenuates plaque formation in apoE−/− mice without inducing liver steatosis and hypertriglyceridemia.J. Lipid Res. 2009; 50: 312-326Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Data are displayed as expression ratios normalized to cyclophilin A as reference gene. Primer sequences are available upon request. Before transfection, COS-7 cells were collected in the logarithmic growth phase. Cells (150,000 cells/well) were seeded in 6-well plates and cultured overnight. Transient transfection of COS-7 cells with pcDNA4/HisMax encoding His-tagged KIAA1363, HSL, and LacZ was performed using Metafectene™ (Biontex, Munich, Germany). For that purpose, 2.5 µg of purified DNA (Qiagen HiSpeed™, Qiagen, Valencia, CA) per well were incubated with 10 μl Metafectene™ for 20 min at room temperature in serum-free DMEM in a total volume of 100 μl. Subsequently, cells were incubated with the DNA/metafectene complex for 4 h in serum-free medium. Afterwards, the medium was replaced by DMEM containing 10% FCS. Then 48 h after transfection, cells were washed twice with PBS, scraped in lysis buffer (100 mM potassium phosphate, 250 mM sucrose, 1 mM EDTA, 1 mM dithiothreitol, 20 µg/ml leupeptin, 2 µg/ml antipain, 1 µg/ml pepstatin, pH 7.0), and used for lysate preparations. A total of 40 µg protein from various cellular fractions were separated on a 10% SDS-PAGE and transferred to a nitrocellulose membrane. For detection of HSL protein, anti-HSL polyclonal antibody (Cell Signaling Technology, Danvers, MA) was used at a dilution of 1:800. For detection of KIAA1363 protein, blots were incubated with an anti-KIAA1363 polyclonal antibody (21Chiang K.P. Niessen S. Saghatelian A. Cravatt B.F. An enzyme that regulates ether lipid signaling pathways in cancer annotated by multidimensional profiling.Chem. Biol. 2006; 13: 1041-1050Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) at a dilution of 1:1000. Anti-ABCA1 (Abcam, Cambridge, UK) and anti-ABCG1 (Epitomics, Burlingam, CA) antibodies were diluted 1:1000 and 1:2000, respectively. Specifically bound immunoglobulin was detected in a second reaction with a horseradish peroxidase-labeled IgG conjugate and visualized by enhanced chemoluminescence detection (ECL plus, Amersham Bioscience, Piscataway, NJ) on an AGFA Curix Ultra X Ray film (Siemens, Graz, Austria). p-Nitrophenolvalerate (PNPV) esterase activity (30Kienesberger P.C. Lass A. Preiss-Landl K. Wolinski H. Kohlwein S.D. Zimmermann R. Zechner R. Identification of an insulin-regulated lysophospholipase with homology to neuropathy target esterase.J. Biol. Chem. 2008; 283: 5908-5917Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) and AcMAGE hydrolase activity (22Nomura D.K. Fujioka K. Issa R.S. Ward A.M. Cravatt B.F. Casida J.E. Dual roles of brain serine hydrolase KIAA1363 in ether lipid metabolism and organophosphate detoxification.Toxicol. Appl. Pharmacol. 2008; 228: 42-48Crossref PubMed Scopus (23) Google Scholar) were assayed as described. For determination of CE, TG, and diacylglycerol (DG) hydrolase activities, freshly prepared substrates were used as follows. CE substrate contained 20 nmol cholesteryl oleate/assay, cholesteryl [1-14C]oleate (50,000 cpm/nmol) (Amersham Biosciences, Piscataway, NJ), 35.5 µg mixed micelles of phosphatidylcholine (PC) and phosphatidylinositol (PI) (3:1, w:w), and 4 µM sodium taurocholate. Alternatively, CE hydrolase activities in macrophages were assayed using 35.5 µg mixed micelles of PC and PI (3:1, w:w) or 35.5 µg PC and 2 µM sodium taurocholate. TG substrate contained 25 nmol triolein/assay, 40,000 cpm/nmol of [9,10-3H(N)]triolein (NEN Life Science Products, Boston, MA), 15 µg mixed micelles of PC and PI (3:1, w:w), and 4 µM sodium taurocholate. DG substrate contained 40 nmol 1,2-diolein/assay and 120,000 cpm/nmol of dioleyl-rac-glycerol[oleoyl-1-14C] (American Radiolabeled Chemicals, St. Louis, MO), 15 µg mixed micelles of PC and PI (3:1, w:w), and 4 µM sodium taurocholate. After evaporation, substrates were sonicated (Labsonic, B. Braun, Melsungen, Germany) in 100 mM potassium phosphate buffer (pH 7.0) containing 4 µM sodium taurocholate and 2.5% fatty acid-free BSA exactly as described by Holm et al. (31Holm C. Osterlund T. Hormone-sensitive lipase and neutral cholesteryl ester lipase.in: Doolittle M.H. Reue K. Lipase and Phospholipase Protocols. Humana Press, Totowa, NJ1999: 109-121Google Scholar) One hundred µg of cell extracts (lysates or subfractions) and 100 µl substrate were incubated in a water bath at 37°C for 1 h. The reaction was terminated by the addition of 3.25 ml of methanol/ chloroform/heptane (10:9:7) and 1 ml of 100 mM potassium carbonate, 100 mM boric acid, pH 10.5. After centrifugation (800 g, 20 min), the radioactivity in 1 ml of the upper phase was determined by liquid scintillation counting, and the release of FFA was calculated. For lysate preparation, cells (COS-7 or MPM) were washed two times with PBS and sonicated twice for 10 s in lysis buffer (100 mM potassium phosphate, 250 mM sucrose, 1 mM EDTA, 1 mM dithiothreitol, 20 µg/ml leupeptin, 2 µg/ml antipain, 1 µg/ml pepstatin, pH 7.0). Nuclei and cell debris were removed by centrifugation at 1,000 g for 5 min (4°C). Cytosolic and membrane fractions were prepared by centrifugation at 100,000 g for 1 h (4°C). For determination of CE hydrolase activities in tissues of WT, HSL−/−, and KIAA1363−/− mice, tissues were surgically removed and washed in PBS containing 1 mM EDTA. Homogenization was performed in lysis buffer on ice using a Precellys24 homogenizer (Bertin, Montigny-le-Bretonneux, France). Lysates were centrifuged for 30 min at 20,000 g (4°C). Radioactivity corresponding to the release of FFA in 100 µg of the lipid-free infranatant was determined by liquid scintillation counting. Protein concentrations were determined using a Bradford assay (Bio-Rad Laboratories, Vienna, Austria). Cytoplasmic extracts of COS-7 cells were prepared as described above. A total of 50 µg of cellular protein were incubated with 1 nmol of the specific fluorescently labeled probes 7-nitrobenz- 2-oxa-1,3-diazole (NBD) cholesteryl phosphonate (CP) and enantiomeric TG analogs (NBD-sn1-TGP and NBD-sn3-TGP) (32Schmidinger H. Birner-Gruenberger R. Riesenhuber G. Saf R. Susani-Etzerodt H. Hermetter A. Novel fluorescent phosphonic acid esters for discrimination of lipases and esterases.Chembiochem. 2005; 6: 1776-1781Crossref PubMed Scopus (47) Google Scholar), and 1 mM Triton X-100 (Hoffmann La Roche, Basel, Switzerland) at 37°C for 2 h under shaking as described (33Birner-Gruenberger R. Susani-Etzerodt H. Waldhuber M. Riesenhuber G. Schmidinger H. Rechberger G. Kollroser M. Strauss J.G. Lass A. Zimmermann R. et al.The lipolytic proteome of mouse adipose tissue.Mol. Cell. Proteomics. 2005; 4: 1710-1717Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Total protein was precipitated with 10% trichloroacetic acid for 1 h on ice, washed with acetone, and subjected to 10% SDS-PAGE. Gels were treated with 10% ethanol and 7% acetic acid, and fluorescent signals were detected on a BioRad FX Pro laser scanner (excitation 488 nm, emission 530 nm) (BioRad Laboratories, Hercules, CA). Lipids from MPM were extracted with 2 ml hexane:isopropanol (3:2, v:v) for 1 h at 4°C. Lipid extracts were dried under nitrogen, redissolved in 100 µl 1% Triton X-100 in chloroform, dried under nitrogen, and resuspended in 100 µl distilled water for 15 min at 37°C. Aliquots (30 µl) were used for enzymatic determinations of TG (DiaSys, Holzheim, Germany), TC (Greiner Diagnostics, Bahlingen, Germany), and FC (DiaSys, Holzheim, Germany) concentrations. Proteins of extracted cells were dissolved in 2 ml of 300 mM NaOH for 1 h at room temperature, and protein content was quantitated using a Bradford assay (Bio-Rad Laboratories, Vienna, Austria). The amount of 50 µg of freshly prepared acLDL (not older than one week) was enriched with 1 µCi [1, 2 3H]cholesterol (Hartmann Analytik, Braunschweig, Germany). MPM from WT, HSL−/−, and KIAA1363−/− mice were seeded in 12-well plates and incubated with 50 µg 3H-cholesterol-enriched acLDL for 24 h. Afterwards, cells were equilibrated overnight in DMEM containing 0.2% fatty acid-free BSA (Sigma, Vienna, Austria). After two washing steps with PBS/0.2% fatty acid-free BSA, cells were incubated with 100 µg/ml human HDL3 or 15 µg/ml purified human apoA-I (B

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