OSBP-related Protein 8 (ORP8) Suppresses ABCA1 Expression and Cholesterol Efflux from Macrophages

ORP8 is a previously unexplored member of the family of oxysterol-binding protein-related proteins (ORP). We now report the expression pattern, the subcellular distribution, and data on the ligand binding properties and the physiological function of ORP8. ORP8 is localized in the endoplasmic reticulum (ER) via its C-terminal transmembrane span and binds 25-hydroxycholesterol, identifying it as a new ER oxysterol-binding protein. ORP8 is expressed at highest levels in macrophages, liver, spleen, kidney, and brain. Immunohistochemical analysis revealed ORP8 in the shoulder regions of human coronary atherosclerotic lesions, where it is present in CD68(+) macrophages. In advanced lesions the ORP8 mRNA was up-regulated 2.7-fold as compared with healthy coronary artery wall. Silencing of ORP8 by RNA interference in THP-1 macrophages increased the expression of ATP binding cassette transporter A1 (ABCA1) and concomitantly cholesterol efflux to lipid-free apolipoprotein A-I but had no significant effect on ABCG1 expression or cholesterol efflux to spherical high density lipoprotein HDL2. Experiments employing an ABCA1 promoter-luciferase reporter confirmed that ORP8 silencing enhances ABCA1 transcription. The silencing effect was partially attenuated by mutation of the DR4 element in the ABCA1 promoter and synergized with that of the liver X receptor agonist T0901317. Furthermore, inactivation of the E-box in the promoter synergized with ORP8 silencing, suggesting that the suppressive effect of ORP8 involves both the liver X receptor and the E-box functions. Our data identify ORP8 as a negative regulator of ABCA1 expression and macrophage cholesterol efflux. ORP8 may, thus, modulate the development of atherosclerosis. ORP8 is a previously unexplored member of the family of oxysterol-binding protein-related proteins (ORP). We now report the expression pattern, the subcellular distribution, and data on the ligand binding properties and the physiological function of ORP8. ORP8 is localized in the endoplasmic reticulum (ER) via its C-terminal transmembrane span and binds 25-hydroxycholesterol, identifying it as a new ER oxysterol-binding protein. ORP8 is expressed at highest levels in macrophages, liver, spleen, kidney, and brain. Immunohistochemical analysis revealed ORP8 in the shoulder regions of human coronary atherosclerotic lesions, where it is present in CD68(+) macrophages. In advanced lesions the ORP8 mRNA was up-regulated 2.7-fold as compared with healthy coronary artery wall. Silencing of ORP8 by RNA interference in THP-1 macrophages increased the expression of ATP binding cassette transporter A1 (ABCA1) and concomitantly cholesterol efflux to lipid-free apolipoprotein A-I but had no significant effect on ABCG1 expression or cholesterol efflux to spherical high density lipoprotein HDL2. Experiments employing an ABCA1 promoter-luciferase reporter confirmed that ORP8 silencing enhances ABCA1 transcription. The silencing effect was partially attenuated by mutation of the DR4 element in the ABCA1 promoter and synergized with that of the liver X receptor agonist T0901317. Furthermore, inactivation of the E-box in the promoter synergized with ORP8 silencing, suggesting that the suppressive effect of ORP8 involves both the liver X receptor and the E-box functions. Our data identify ORP8 as a negative regulator of ABCA1 expression and macrophage cholesterol efflux. ORP8 may, thus, modulate the development of atherosclerosis. Lipid-laden macrophage foam cells are characteristic constituents of the early atherosclerotic lesion and are present at all stages of lesion development (1Libby P. Theroux P. Circulation. 2005; 111: 3481-3488Crossref PubMed Scopus (1269) Google Scholar). Macrophages also play a central role in the inflammatory signaling within the developing plaque (2Hansson G.K. Libby P. Nat. Rev. Immunol. 2006; 6: 508-519Crossref PubMed Scopus (1789) Google Scholar), and hydrolytic enzymes secreted by macrophages and other inflammatory cells influence plaque structure and stability (3Lindstedt K.A. Kovanen P.T. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 2205-2206Crossref PubMed Scopus (15) Google Scholar). In addition to cholesterol, 27-carbon oxygenated derivatives of cholesterol, referred to as oxysterols (4Björkhem I. Diczfalusy U. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 734-742Crossref PubMed Scopus (255) Google Scholar), are enriched in atherosclerotic plaques in both humans and in animal models (5Brown A.J. Jessup W. Atherosclerosis. 1999; 142: 1-28Abstract Full Text Full Text PDF PubMed Scopus (765) Google Scholar, 6Olkkonen V.M. Lehto M. Ann. Med. 2004; 36: 562-572Crossref PubMed Scopus (75) Google Scholar). Oxysterols have cytotoxic, pro-apoptotic, and pro-inflammatory effects and facilitate the differentiation of monocytes into macrophages. They have, therefore, been suggested to adversely affect lesion development and stability (6Olkkonen V.M. Lehto M. Ann. Med. 2004; 36: 562-572Crossref PubMed Scopus (75) Google Scholar, 7Colles S.M. Maxson J.M. Carlson S.G. Chisolm G.M. Trends Cardiovasc. Med. 2001; 11: 131-138Crossref PubMed Scopus (167) Google Scholar). The central apparatus for the transcriptional regulation of sterol metabolism consists of the sterol regulatory element-binding proteins (8Eberle D. Hegarty B. Bossard P. Ferre P. Foufelle F. Biochimie (Paris). 2004; 86: 839-848Crossref PubMed Scopus (1019) Google Scholar, 9Goldstein J.L. DeBose-Boyd R.A. Brown M.S. Cell. 2006; 124: 35-46Abstract Full Text Full Text PDF PubMed Scopus (1237) Google Scholar) and the liver X receptors (LXRs) 2The abbreviations used are:LXRliver X receptorABCATP binding cassette transporterapoA-Iapolipoprotein A-IERendoplasmic reticulumGSTglutathione S-transferaseHDLhigh density lipoproteinOHChydroxycholesterolOSBPoxysterol-binding proteinORPOSBP-related proteinsiRNAshort interfering double-stranded RNA (10Li A.C. Glass C.K. J. Lipid Res. 2004; 45: 2161-2173Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 11Tontonoz P. Mangelsdorf D.J. Mol. Endocrinol. 2003; 17: 985-993Crossref PubMed Scopus (530) Google Scholar, 12Zelcer N. Tontonoz P. J. Clin. Investig. 2006; 116: 607-614Crossref PubMed Scopus (768) Google Scholar), both of which are responsive to oxysterols. The Insig proteins that control the intracellular transport and proteolytic activation of sterol regulatory element-binding protein (SREBP) act as 25-hydroxycholesterol receptors in the endoplasmic reticulum (ER) and mediate oxysterol regulation of SREBP maturation (13Radhakrishnan A. Ikeda Y. Kwon H.J. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 6511-6518Crossref PubMed Scopus (436) Google Scholar). The LXRs are activated by oxysterol ligands such as 22(R)-, 24(S)-, and 27-hydroxycholesterol and 24(S), 25-epoxycholesterol (14Fu X. Menke J.G. Chen Y. Zhou G. MacNaul K.L. Wright S.D. Sparrow C.P. Lund E.G. J. Biol. Chem. 2001; 276: 38378-38387Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar, 15Janowski B.A. Willy P.J. Devi T.R. Falck J.R. Mangelsdorf D.J. Nature. 1996; 383: 728-731Crossref PubMed Scopus (1468) Google Scholar, 16Lehmann J.M. Kliewer S.A. Moore L.B. Smith-Oliver T.A. Oliver B.B. Su J.L. Sundseth S.S. Winegar D.A. Blanchard D.E. Spencer T.A. Willson T.M. J. Biol. Chem. 1997; 272: 3137-3140Abstract Full Text Full Text PDF PubMed Scopus (1043) Google Scholar, 17Rowe A.H. Argmann C.A. Edwards J.Y. Sawyez C.G. Morand O.H. Hegele R.A. Huff M.W. Circ. Res. 2003; 93: 717-725Crossref PubMed Scopus (82) Google Scholar, 18Szanto A. Benko S. Szatmari I. Balint B.L. Furtos I. Ruhl R. Molnar S. Csiba L. Garuti R. Calandra S. Larsson H. Diczfalusy U. Nagy L. Mol. Cell. Biol. 2004; 24: 8154-8166Crossref PubMed Scopus (104) Google Scholar, 19Chen W. Chen G. Head D.L. Mangelsdorf D.J. Russell D.W. Cell Metab. 2007; 5: 73-79Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 20Wong J. Quinn C.M. Brown A.J. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 2365-2371Crossref PubMed Scopus (131) Google Scholar). Together with the retinoid X receptor, the LXR form obligate heterodimers which facilitate via recruitment of coactivators, the transcription of specific target genes. Genes regulated by LXR are involved in sterol absorption in the intestine, the reverse cholesterol transport process, bile acid synthesis, biliary neutral sterol secretion, hepatic lipogenesis, and synthesis of nascent high density lipoproteins (10Li A.C. Glass C.K. J. Lipid Res. 2004; 45: 2161-2173Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 11Tontonoz P. Mangelsdorf D.J. Mol. Endocrinol. 2003; 17: 985-993Crossref PubMed Scopus (530) Google Scholar). Several ATP binding cassette (ABC) transporters are subject to transcriptional control by LXR (11Tontonoz P. Mangelsdorf D.J. Mol. Endocrinol. 2003; 17: 985-993Crossref PubMed Scopus (530) Google Scholar). ABCA1 mediates phospholipid and cholesterol efflux to lipid-poor apolipoprotein A-I, whereas ABCG1 acts in cholesterol efflux to spherical high density lipoprotein particles (21Oram J.F. Vaughan A.M. Circ. Res. 2006; 99: 1031-1043Crossref PubMed Scopus (327) Google Scholar, 22Yokoyama S. Curr. Opin. Lipidol. 2005; 16: 269-279Crossref PubMed Scopus (54) Google Scholar). The removal of excess cholesterol from macrophages is regarded as an important anti-atherogenic process (1Libby P. Theroux P. Circulation. 2005; 111: 3481-3488Crossref PubMed Scopus (1269) Google Scholar). Consistent with this, animal model and human genetic/epidemiologic studies support an atheroprotective function of ABCA1 (23Aiello R.J. Brees D. Bourassa P.A. Royer L. Lindsey S. Coskran T. Haghpassand M. Francone O.L. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 630-637Crossref PubMed Scopus (348) Google Scholar, 24Singaraja R.R. Brunham L.R. Visscher H. Kastelein J.J. Hayden M.R. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1322-1332Crossref PubMed Scopus (226) Google Scholar, 25Van Eck M. Bos I.S. Kaminski W.E. Orso E. Rothe G. Twisk J. Bottcher A. Van Amersfoort E.S. Christiansen-Weber T.A. Fung-Leung W.P. Van Berkel T.J. Schmitz G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6298-6303Crossref PubMed Scopus (322) Google Scholar, 26Van Eck M. Singaraja R.R. Ye D. Hildebrand R.B. James E.R. Hayden M.R. Van Berkel T.J. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 929-934Crossref PubMed Scopus (150) Google Scholar, 27Frikke-Schmidt R. Nordestgaard B.G. Schnohr P. Steffensen R. Tybjaerg-Hansen A. J. Am. Coll. Cardiol. 2005; 46: 1516-1520Crossref PubMed Scopus (59) Google Scholar), whereas the role of ABCG1 in atherogenesis is more controversial (28Baldan A. Pei L. Lee R. Tarr P. Tangirala R.K. Weinstein M.M. Frank J. Li A.C. Tontonoz P. Edwards P.A. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 2301-2307Crossref PubMed Scopus (151) Google Scholar, 29Out R. Hoekstra M. Hildebrand R.B. Kruit J.K. Meurs I. Li Z. Kuipers F. Van Berkel T.J. Van Eck M. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 2295-2300Crossref PubMed Scopus (174) Google Scholar, 30Ranalletta M. Wang N. Han S. Yvan-Charvet L. Welch C. Tall A.R. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 2308-2315Crossref PubMed Scopus (149) Google Scholar). liver X receptor ATP binding cassette transporter apolipoprotein A-I endoplasmic reticulum glutathione S-transferase high density lipoprotein hydroxycholesterol oxysterol-binding protein OSBP-related protein short interfering double-stranded RNA A third protein family that can function as oxysterol sensors consists of oxysterol-binding protein (OSBP) and its homologues. OSBP is a cytoplasmic protein with affinity for several oxysterols (31Dawson P.A. Ridgway N.D. Slaughter C.A. Brown M.S. Goldstein J.L. J. Biol. Chem. 1989; 264: 16798-16803Abstract Full Text PDF PubMed Google Scholar, 32Dawson P.A. Van der Westhuyzen D.R. Goldstein J.L. Brown M.S. J. Biol. Chem. 1989; 264: 9046-9052Abstract Full Text PDF PubMed Google Scholar, 33Taylor F.R. Saucier S.E. Shown E.P. Parish E.J. Kandutsch A.A. J. Biol. Chem. 1984; 259: 12382-12387Abstract Full Text PDF PubMed Google Scholar). It plays a role in the transport of ceramide from the ER to the Golgi apparatus for sphingomyelin synthesis (34Perry R.J. Ridgway N.D. Mol. Biol. Cell. 2006; 17: 2604-2616Crossref PubMed Scopus (198) Google Scholar) and acts as a sterol-dependent scaffold that regulates the activity of extracellular signal-regulated kinases (35Wang P.Y. Weng J. Anderson R.G. Science. 2005; 307: 1472-1476Crossref PubMed Scopus (245) Google Scholar). Proteins displaying sequence homology to the C-terminal sterol binding domain of OSBP are present in most eukaryotic organisms (36Lehto M. Olkkonen V.M. Biochim. Biophys. Acta. 2003; 1631: 1-11Crossref PubMed Scopus (97) Google Scholar, 37Olkkonen V.M. Curr. Opin. Lipidol. 2004; 15: 321-327Crossref PubMed Scopus (42) Google Scholar). In humans the gene/protein family consists of 12 members (38Jaworski C.J. Moreira E. Li A. Lee R. Rodriguez I.R. Genomics. 2001; 78: 185-196Crossref PubMed Scopus (101) Google Scholar, 39Lehto M. Laitinen S. Chinetti G. Johansson M. Ehnholm C. Staels B. Ikonen E. Olkkonen V.M. J. Lipid Res. 2001; 42: 1203-1213Abstract Full Text Full Text PDF PubMed Google Scholar). The mammalian OSBP-related proteins (ORP) have been implicated as sterol sensors that regulate a number of cellular functions ranging from sterol and neutral lipid metabolism to vesicle transport and cell signaling (40Olkkonen V.M. Johansson M. Suchanek M. Yan D. Hynynen R. Ehnholm C. Jauhiainen M. Thiele C. Lehto M. Biochem. Soc. Trans. 2006; 34: 389-391Crossref PubMed Scopus (46) Google Scholar). However, their connections with the transcriptional control of cellular sterol homeostasis are relatively unexplored. ORP8 is a previously unexplored member of the ORP family. We now report the expression pattern, the subcellular distribution, and data on the ligand binding properties and the physiological function of ORP8. Our results suggest that ORP8 acts as a sterol sensor that affects the reverse cholesterol transport process via modulation of ABCA1 expression and macrophage cholesterol efflux. Antibodies and Other Reagents—A glutathione S-transferase (GST) fusion protein carrying amino acid residues 1-60 of human ORP8 was expressed in Escherichia coli BL21(DE3), purified by affinity chromatography on glutathione-Sepharose 4B (GE Healthcare), and used for immunization of New Zeal- and White rabbits according to a standard protocol. The ORP8 antibodies were bound on a glutathione-Sepharose 4B column carrying covalently coupled GST-ORP8 (1Libby P. Theroux P. Circulation. 2005; 111: 3481-3488Crossref PubMed Scopus (1269) Google Scholar, 2Hansson G.K. Libby P. Nat. Rev. Immunol. 2006; 6: 508-519Crossref PubMed Scopus (1789) Google Scholar, 3Lindstedt K.A. Kovanen P.T. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 2205-2206Crossref PubMed Scopus (15) Google Scholar, 4Björkhem I. Diczfalusy U. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 734-742Crossref PubMed Scopus (255) Google Scholar, 5Brown A.J. Jessup W. Atherosclerosis. 1999; 142: 1-28Abstract Full Text Full Text PDF PubMed Scopus (765) Google Scholar, 6Olkkonen V.M. Lehto M. Ann. Med. 2004; 36: 562-572Crossref PubMed Scopus (75) Google Scholar, 7Colles S.M. Maxson J.M. Carlson S.G. Chisolm G.M. Trends Cardiovasc. Med. 2001; 11: 131-138Crossref PubMed Scopus (167) Google Scholar, 8Eberle D. Hegarty B. Bossard P. Ferre P. Foufelle F. Biochimie (Paris). 2004; 86: 839-848Crossref PubMed Scopus (1019) Google Scholar, 9Goldstein J.L. DeBose-Boyd R.A. Brown M.S. Cell. 2006; 124: 35-46Abstract Full Text Full Text PDF PubMed Scopus (1237) Google Scholar, 10Li A.C. Glass C.K. J. Lipid Res. 2004; 45: 2161-2173Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 11Tontonoz P. Mangelsdorf D.J. Mol. Endocrinol. 2003; 17: 985-993Crossref PubMed Scopus (530) Google Scholar, 12Zelcer N. Tontonoz P. J. Clin. Investig. 2006; 116: 607-614Crossref PubMed Scopus (768) Google Scholar, 13Radhakrishnan A. Ikeda Y. Kwon H.J. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 6511-6518Crossref PubMed Scopus (436) Google Scholar, 14Fu X. Menke J.G. Chen Y. Zhou G. MacNaul K.L. Wright S.D. Sparrow C.P. Lund E.G. J. Biol. Chem. 2001; 276: 38378-38387Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar, 15Janowski B.A. Willy P.J. Devi T.R. Falck J.R. Mangelsdorf D.J. Nature. 1996; 383: 728-731Crossref PubMed Scopus (1468) Google Scholar, 16Lehmann J.M. Kliewer S.A. Moore L.B. Smith-Oliver T.A. Oliver B.B. Su J.L. Sundseth S.S. Winegar D.A. Blanchard D.E. Spencer T.A. Willson T.M. J. Biol. Chem. 1997; 272: 3137-3140Abstract Full Text Full Text PDF PubMed Scopus (1043) Google Scholar, 17Rowe A.H. Argmann C.A. Edwards J.Y. Sawyez C.G. Morand O.H. Hegele R.A. Huff M.W. Circ. Res. 2003; 93: 717-725Crossref PubMed Scopus (82) Google Scholar, 18Szanto A. Benko S. Szatmari I. Balint B.L. Furtos I. Ruhl R. Molnar S. Csiba L. Garuti R. Calandra S. Larsson H. Diczfalusy U. Nagy L. Mol. Cell. Biol. 2004; 24: 8154-8166Crossref PubMed Scopus (104) Google Scholar, 19Chen W. Chen G. Head D.L. Mangelsdorf D.J. Russell D.W. Cell Metab. 2007; 5: 73-79Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 20Wong J. Quinn C.M. Brown A.J. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 2365-2371Crossref PubMed Scopus (131) Google Scholar, 21Oram J.F. Vaughan A.M. Circ. Res. 2006; 99: 1031-1043Crossref PubMed Scopus (327) Google Scholar, 22Yokoyama S. Curr. Opin. Lipidol. 2005; 16: 269-279Crossref PubMed Scopus (54) Google Scholar, 23Aiello R.J. Brees D. Bourassa P.A. Royer L. Lindsey S. Coskran T. Haghpassand M. Francone O.L. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 630-637Crossref PubMed Scopus (348) Google Scholar, 24Singaraja R.R. Brunham L.R. Visscher H. Kastelein J.J. Hayden M.R. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1322-1332Crossref PubMed Scopus (226) Google Scholar, 25Van Eck M. Bos I.S. Kaminski W.E. Orso E. Rothe G. Twisk J. Bottcher A. Van Amersfoort E.S. Christiansen-Weber T.A. Fung-Leung W.P. Van Berkel T.J. Schmitz G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6298-6303Crossref PubMed Scopus (322) Google Scholar, 26Van Eck M. Singaraja R.R. Ye D. Hildebrand R.B. James E.R. Hayden M.R. Van Berkel T.J. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 929-934Crossref PubMed Scopus (150) Google Scholar, 27Frikke-Schmidt R. Nordestgaard B.G. Schnohr P. Steffensen R. Tybjaerg-Hansen A. J. Am. Coll. Cardiol. 2005; 46: 1516-1520Crossref PubMed Scopus (59) Google Scholar, 28Baldan A. Pei L. Lee R. Tarr P. Tangirala R.K. Weinstein M.M. Frank J. Li A.C. Tontonoz P. Edwards P.A. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 2301-2307Crossref PubMed Scopus (151) Google Scholar, 29Out R. Hoekstra M. Hildebrand R.B. Kruit J.K. Meurs I. Li Z. Kuipers F. Van Berkel T.J. Van Eck M. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 2295-2300Crossref PubMed Scopus (174) Google Scholar, 30Ranalletta M. Wang N. Han S. Yvan-Charvet L. Welch C. Tall A.R. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 2308-2315Crossref PubMed Scopus (149) Google Scholar, 31Dawson P.A. Ridgway N.D. Slaughter C.A. Brown M.S. Goldstein J.L. J. Biol. Chem. 1989; 264: 16798-16803Abstract Full Text PDF PubMed Google Scholar, 32Dawson P.A. Van der Westhuyzen D.R. Goldstein J.L. Brown M.S. J. Biol. Chem. 1989; 264: 9046-9052Abstract Full Text PDF PubMed Google Scholar, 33Taylor F.R. Saucier S.E. Shown E.P. Parish E.J. Kandutsch A.A. J. Biol. Chem. 1984; 259: 12382-12387Abstract Full Text PDF PubMed Google Scholar, 34Perry R.J. Ridgway N.D. Mol. Biol. Cell. 2006; 17: 2604-2616Crossref PubMed Scopus (198) Google Scholar, 35Wang P.Y. Weng J. Anderson R.G. Science. 2005; 307: 1472-1476Crossref PubMed Scopus (245) Google Scholar, 36Lehto M. Olkkonen V.M. Biochim. Biophys. Acta. 2003; 1631: 1-11Crossref PubMed Scopus (97) Google Scholar, 37Olkkonen V.M. Curr. Opin. Lipidol. 2004; 15: 321-327Crossref PubMed Scopus (42) Google Scholar, 38Jaworski C.J. Moreira E. Li A. Lee R. Rodriguez I.R. Genomics. 2001; 78: 185-196Crossref PubMed Scopus (101) Google Scholar, 39Lehto M. Laitinen S. Chinetti G. Johansson M. Ehnholm C. Staels B. Ikonen E. Olkkonen V.M. J. Lipid Res. 2001; 42: 1203-1213Abstract Full Text Full Text PDF PubMed Google Scholar, 40Olkkonen V.M. Johansson M. Suchanek M. Yan D. Hynynen R. Ehnholm C. Jauhiainen M. Thiele C. Lehto M. Biochem. Soc. Trans. 2006; 34: 389-391Crossref PubMed Scopus (46) Google Scholar, 41Havel R.J. Eder H.A. Bragdon J.H. J. Clin. Investig. 1955; 34: 1345-1353Crossref PubMed Scopus (6487) Google Scholar, 42Mäyränpää M.I. Heikkilä H.M. Lindstedt K.A. Walls A.F. Kovanen P.T. Coron. Artery Dis. 2006; 17: 611-621Crossref PubMed Scopus (68) Google Scholar, 43Taylor F.R. Kandutsch A.A. Methods Enzymol. 1985; 110: 9-19Crossref PubMed Scopus (16) Google Scholar, 44Pfaffl M.W. Nucleic Acids Res. 2001; 29: e45Crossref PubMed Scopus (25597) Google Scholar, 45Costet P. Luo Y. Wang N. Tall A.R. J. Biol. Chem. 2000; 275: 28240-28245Abstract Full Text Full Text PDF PubMed Scopus (851) Google Scholar, 46Wong J. Quinn C.M. Brown A.J. Biochem. J. 2006; 400: 485-491Crossref PubMed Scopus (116) Google Scholar, 47Schmitz G. Langmann T. Biochim. Biophys. Acta. 2005; 1735: 1-19Crossref PubMed Scopus (183) Google Scholar, 48Im Y.J. Raychaudhuri S. Prinz W.A. Hurley J.H. Nature. 2005; 437: 154-158Crossref PubMed Scopus (339) Google Scholar, 49Moreira E.F. Jaworski C. Li A. Rodriguez I.R. J. Biol. Chem. 2001; 276: 18570-18578Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 50Wang C. JeBailey L. Ridgway N.D. Biochem. J. 2002; 361: 461-472Crossref PubMed Scopus (98) Google Scholar, 51Suchanek M. Hynynen R. Wohlfahrt G. Lehto M. Johansson M. Saarinen H. Radzikowska A. Thiele C. Olkkonen V.M. Biochem. J. 2007; 405: 473-480Crossref PubMed Scopus (120) Google Scholar, 52Serfaty-Lacrosniere C. Civeira F. Lanzberg A. Isaia P. Berg J. Janus E.D. Smith Jr., M.P. Pritchard P.H. Frohlich J. Lees R.S. Barnard G.F. Ordovas J.M. Schaefer E.J. Atherosclerosis. 1994; 107: 85-98Abstract Full Text PDF PubMed Scopus (216) Google Scholar, 53Björkhem I. J. Clin. Investig. 2002; 110: 725-730Crossref PubMed Scopus (216) Google Scholar, 54Beyea M.M. Heslop C.L. Sawyez C.G. Edwards J.Y. Markle J.G. Hegele R.A. Huff M.W. J. Biol. Chem. 2007; 282: 5207-5216Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 55Fujiyoshi M. Ohtsuki S. Hori S. Tachikawa M. Terasaki T. J. Neurochem. 2007; 100: 968-978Crossref PubMed Scopus (47) Google Scholar, 56Khovidhunkit W. Moser A.H. Shigenaga J.K. Grunfeld C. Feingold K.R. J. Lipid Res. 2003; 44: 1728-1736Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 57Costet P. Lalanne F. Gerbod-Giannone M.C. Molina J.R. Fu X. Lund E.G. Gudas L.J. Tall A.R. Mol. Cell. Biol. 2003; 23: 7756-7766Crossref PubMed Scopus (118) Google Scholar, 58Kaplan R. Gan X. Menke J.G. Wright S.D. Cai T.Q. J. Lipid Res. 2002; 43: 952-959Abstract Full Text Full Text PDF PubMed Google Scholar, 59Hu X. Li S. Wu J. Xia C. Lala D.S. Mol. Endocrinol. 2003; 17: 1019-1026Crossref PubMed Scopus (113) Google Scholar, 60Wagner B.L. Valledor A.F. Shao G. Daige C.L. Bischoff E.D. Petrowski M. Jepsen K. Baek S.H. Heyman R.A. Rosenfeld M.G. Schulman I.G. Glass C.K. Mol. Cell. Biol. 2003; 23: 5780-5789Crossref PubMed Scopus (192) Google Scholar) and eluted with 0.2 m glycine, pH 2.8, neutralized, dialyzed against phosphate-buffered saline, 20% glycerol, and stored at -20 °C. Rabbit antibodies against calnexin were a kind gift from Prof. Ralf Pettersson (Ludwig Institute for Cancer Research, Stockholm, Sweden), monoclonal anti-β-actin from the Developmental Studies Hybridoma Bank (University of Iowa), anti-protein disulfide isomerase from Stressgen (San Diego, CA), anti-ABCA1 from Novus Biologicals (Littleton, CO), and anti-CD68 from Dako (Glostrup, Denmark). Lipid-free human apoA-I was kindly provided by Dr. Peter Lerch (Swiss Red Cross, Bern, Switzerland). HDL2 was purified from human plasma by ultracentrifugation (41Havel R.J. Eder H.A. Bragdon J.H. J. Clin. Investig. 1955; 34: 1345-1353Crossref PubMed Scopus (6487) Google Scholar). [3H]25OHC (20 Ci/mmol) was kindly provided by Dr. Christoph Thiele (Max-Planck-Institute of Molecular Cell Biology and Genetics, Dresden, Germany), and [3H]24(S)OHC (40-60 Ci/mmol) was provided by Prof. Ingemar Björkhem (Karolinska Institute, Huddinge, Sweden). 7-[3H]Ketocholesterol (65 Ci/mmol) was purchased from American Radiolabeled Chemicals (St. Louis, MO), and unlabeled oxysterols were from Sigma-Aldrich. cDNA Constructs—Full-length human ORP8 cDNA (accession number NM_001003712) was inserted into the XbaI site of pcDNA4HisMaxC (Invitrogen) to obtain a construct fused with an N-terminal Xpress epitope tag. A truncated cDNA encoding ORP8 that lacks the 19-amino acid C-terminal transmembrane span (ORP8ΔC) was engineered by PCR. Furthermore, a GST fusion of ORP8 ligand binding domain (ORP8-(242-828)) was created in pGEX-1λT (GE Healthcare) for protein production in E. coli. Sequence changes were verified by sequencing with a cycle-sequencing kit (BIGDYE, Applied Biosystems, Foster City, CA) and an automated ABI3730 sequencer (Applied Biosystems). Western Blotting—Protein samples for SDS-PAGE were prepared by homogenizing cultured cells or mouse tissues in 250 mm Tris-HCl, pH 6.8, 8% SDS, protease inhibitor mixture (Roche Diagnostics). The crude extracts were cleared by centrifugation at 16,000 × g for 3 min, and the protein concentration of the supernatant was determined by the DC assay (Bio-Rad). The proteins were electrophoresed on Laemmli gels and electrotransferred to Hybond-C Extra nitrocellulose (GE Healthcare). Nonspecific binding of antibodies was blocked with, and all antibody incubations were carried out in 5% fat-free powdered milk in 10 mm Tris-HCl, pH 7.4, 150 mm NaCl, and 0.05% Tween 20. The bound primary antibodies were visualized with horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse IgG (Bio-Rad) and the enhanced chemiluminescence system ECL (GE Healthcare). Cell Culture—The human monocytic THP-1 cells were cultured in RPMI1640 (Sigma-Aldrich), 10% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin and differentiated into macrophages by 72 h incubation in the presence of phorbol 12-myristate 13-acetate at 100 ng/ml. HEK293 cells were cultured in Eagle's minimal essential medium with Earle's salts (Sigma-Aldrich), 10% fetal calf serum, nonessential amino acid supplement, 10 mm HEPES, pH 7.4, and the above antibiotics. Immunofluorescence Microscopy—THP-1 macrophages or HEK293 cells transfected with Xpress epitope-tagged ORP8 or ORP8ΔC cDNA using Lipofectamine 2000 (Invitrogen) were fixed with 4% paraformaldehyde, 250 mm HEPES, pH 7.4, for 30 min, permeabilized for 30 min with 0.1% Saponin in phosphate-buffered saline, and processed for indirect immunofluorescence microscopy using primary antibodies and Alexa Fluor secondary antibody conjugates (Invitrogen). The specimens were analyzed with a TCS SP1 laser scanning confocal microscope (Leica, Wetzlar, Germany). Immunohistochemical Analysis of Human Tissue Specimens—Formalin-fixed, paraffin-embedded 5-μm-thick human coronary artery sections were stained for ORP8, smooth muscle cell α-actin, CD31, and CD68 as described (42Mäyränpää M.I. Heikkilä H.M. Lindstedt K.A. Walls A.F. Kovanen P.T. Coron. Artery Dis. 2006; 17: 611-621Crossref PubMed Scopus (68) Google Scholar). Specificity of the ORP8 immunostaining was verified by simultaneous staining of adjacent coronary sections with antibody aliquots preincubated overnight at +4 °C with GST-ORP8 (1Libby P. Theroux P. Circulation. 2005; 111: 3481-3488Crossref PubMed Scopus (1269) Google Scholar, 2Hansson G.K. Libby P. Nat. Rev. Immunol. 2006; 6: 508-519Crossref PubMed Scopus (1789) Google Scholar, 3Lindstedt K.A. Kovanen P.T. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 2205-2206Crossref PubMed Scopus (15) Google Scholar, 4Björkhem I. Diczfalusy U. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 734-742Crossref PubMed Scopus (255) Google Scholar, 5Brown A.J. Jessup W. Atherosclerosis. 1999; 142: 1-28Abstract Full Text Full Text PDF PubMed Scopus (765) Google Scholar, 6Olkkonen V.M. Lehto M. Ann. Med. 2004; 36: 562-572Crossref PubMed Scopus (75) Google Scholar, 7Colles S.M. Maxson J.M. Carlson S.G. Chisolm G.M. Trends Cardiovasc. Med. 2001; 11: 131-138Crossref PubMed Scopus (167) Google Scholar, 8Eberle D. Hegarty B. Bossard P. Ferre P. Foufelle F. Biochimie (Paris). 2004; 86: 839-848Crossref PubMed Scopus (1019) Google Scholar, 9Goldstein J.L. DeBose-Boyd R.A. Brown M.S. Cell. 2006; 124: 35-46Abstract Full Text Full Text PDF PubMed Scopus (1237) Google Scholar, 10Li A.C. Glass C.K. J. Lipid Res. 2004; 45: 2161-2173Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 11Tontonoz P. Mangelsdorf D.J. Mol. Endocrinol. 2003; 17: 985-993Crossref PubMed Scopus (530) Google Scholar, 12Zelcer N. Tontonoz P. J. Clin. Investig. 2006; 116: 607-614Crossref PubMed Scopus (768) Google Scholar, 13Radhakrishnan A. Ikeda Y. Kwon H.J. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 6511-6518Crossref PubMed Scopus (436) Google Scholar, 14Fu X. Menke J.G. Chen Y. Zhou G. MacNaul K.L. Wright S.D. Sparrow C.P. Lund E.G. J. Biol. Chem. 2001; 276: 38378-38387Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar, 15Janowski B.A. Willy P.J. Devi T.R. Falck J.R. Mangelsdorf D.J. Nature. 1996; 383: 728-731Crossref PubMed Scopus (1468) Google Scholar, 16Lehmann J.M. Kliewer S.A. Moore L.B. Smith-Oliver T.A. Oliver B.B. Su J.L. Sundseth S.S. Winegar D.A. Blanchard D.E. Spencer T.A. Willson T.M. J. Biol. Chem. 1997; 272: 3137-3140Abstract Full Text Full Text PDF PubMed Scopus (1043) Google Scholar, 17Rowe A.H. Argmann C.A. Edwards J.Y. Sawyez C.G. Morand O.H. Hegele R.A. Huff M.W. Circ. Res