27-Hydroxycholesterol Is an Endogenous Ligand for Liver X Receptor in Cholesterol-loaded Cells

The nuclear receptors liver X receptor α (LXRα) (NR1H3) and LXRβ (NR1H2) are important regulators of genes involved in lipid metabolism, including ABCA1,ABCG1, and sterol regulatory element-binding protein-1c (SREBP-1c). Although it has been demonstrated that oxysterols are LXR ligands, little is known about the identity of the physiological activators of these receptors. Here we confirm earlier studies demonstrating a dose-dependent induction of ABCA1 and ABCG1 in human monocyte-derived macrophages by cholesterol loading. In addition, we show that formation of 27-hydroxycholesterol and cholestenoic acid, products of CYP27 action on cholesterol, is dependent on the dose of cholesterol used to load the cells. Other proposed LXR ligands, including 20(S)-hydroxycholesterol, 22(R)-hydroxycholesterol, and 24(S),25-epoxycholesterol, could not be detected under these conditions. A role for CYP27 in regulation of cholesterol-induced genes was demonstrated by the following findings. 1) Introduction of CYP27 into HEK-293 cells conferred an induction of ABCG1 and SREBP-1c; 2) upon cholesterol loading, CYP27-expressing cells induce these genes to a greater extent than in control cells; 3) in CYP27-deficient human skin fibroblasts, the induction of ABCA1 in response to cholesterol loading was ablated; and 4) in a coactivator association assay, 27-hydroxycholesterol functionally activated LXR. We conclude that 27-hydroxylation of cholesterol is an important pathway for LXR activation in response to cholesterol overload. The nuclear receptors liver X receptor α (LXRα) (NR1H3) and LXRβ (NR1H2) are important regulators of genes involved in lipid metabolism, including ABCA1,ABCG1, and sterol regulatory element-binding protein-1c (SREBP-1c). Although it has been demonstrated that oxysterols are LXR ligands, little is known about the identity of the physiological activators of these receptors. Here we confirm earlier studies demonstrating a dose-dependent induction of ABCA1 and ABCG1 in human monocyte-derived macrophages by cholesterol loading. In addition, we show that formation of 27-hydroxycholesterol and cholestenoic acid, products of CYP27 action on cholesterol, is dependent on the dose of cholesterol used to load the cells. Other proposed LXR ligands, including 20(S)-hydroxycholesterol, 22(R)-hydroxycholesterol, and 24(S),25-epoxycholesterol, could not be detected under these conditions. A role for CYP27 in regulation of cholesterol-induced genes was demonstrated by the following findings. 1) Introduction of CYP27 into HEK-293 cells conferred an induction of ABCG1 and SREBP-1c; 2) upon cholesterol loading, CYP27-expressing cells induce these genes to a greater extent than in control cells; 3) in CYP27-deficient human skin fibroblasts, the induction of ABCA1 in response to cholesterol loading was ablated; and 4) in a coactivator association assay, 27-hydroxycholesterol functionally activated LXR. We conclude that 27-hydroxylation of cholesterol is an important pathway for LXR activation in response to cholesterol overload. liver X receptor cerebrotendinous xanthomatosis gas chromatography mass spectrometry trimethylsilyl steroidogenic acute regulatory protein sterol regulatory element sterol regulatory element-binding protein internal ribosomal entry site cytochrome P450 cytomegalovirus reverse transcription polymerase chain reaction 23-kDa highly basic protein stearoyl-CoA desaturase steroid receptor coactivator-1 human embryonic kidney low density lipoprotein 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid Smith-Lemli-Opitz syndrome The nuclear receptors LXRα1 (NR1H3) and LXRβ (NR1H2) have recently been shown to be important regulators of cholesterol and fatty acid metabolism (reviewed in Ref. 1Repa J.J. Mangelsdorf D.J. Curr. Opin. Biotechnol. 1999; 10: 557-563Crossref PubMed Scopus (164) Google Scholar). Originally identified as orphan receptors (1Repa J.J. Mangelsdorf D.J. Curr. Opin. Biotechnol. 1999; 10: 557-563Crossref PubMed Scopus (164) Google Scholar), LXR isoforms were subsequently shown to bind and be activated by certain oxysterols, oxygenated derivatives of cholesterol (2Janowski B.A. Willy P.J. Devi T.R. Falck J.R. Mangelsdorf D.J. Nature. 1996; 383: 728-731Crossref PubMed Scopus (1441) Google Scholar, 3Lehmann 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 (1029) Google Scholar, 4Forman B.M. Ruan B. Chen J. Schroepfer Jr., G.J. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10588-10593Crossref PubMed Scopus (249) Google Scholar). Among candidates proposed, 24(S),25-epoxycholesterol is one of the most potent activators (5Spencer T.A. Li D. Russel J.S. Collins J.L. Bledsoe R.K. Consler T.G. Moore L.B. Galardi C.M. McKee D.D. Moore J.T. Watson M.A. Parks D.J. Lambert M.H. Willson T.M. J. Med. Chem. 2001; 44: 886-897Crossref PubMed Scopus (108) Google Scholar). At least in certain cell culture systems, this compound is formed via a shunt in the cholesterol biosynthetic pathway (6Spencer T.A. Acc. Chem. Res. 1994; 27: 83-90Crossref Scopus (59) Google Scholar). Other proposed activators include 20(S)-hydroxycholesterol and 22(R)-hydroxycholesterol (2Janowski B.A. Willy P.J. Devi T.R. Falck J.R. Mangelsdorf D.J. Nature. 1996; 383: 728-731Crossref PubMed Scopus (1441) Google Scholar). These compounds are formed as transient intermediates in steroid hormone synthesis; thus, their distribution is limited.The first demonstration of the importance of LXRα for maintenance of cholesterol homeostasis came with results from studies using mice deficient in that receptor (7Peet D.J. Turley S.D. Ma W. Janowski B.A. Lobaccaro J.M. Hammer R.E. Mangelsdorf D.J. Cell. 1998; 93: 693-704Abstract Full Text Full Text PDF PubMed Scopus (1230) Google Scholar). Upon feeding a high cholesterol diet, the livers of the LXRα −/− mice become engorged with cholesterol, which was shown to be caused by a failure to up-regulate CYP7A1 and bile acid synthesis as a response to cholesterol overload. Subsequently, several genes of importance for cholesterol and fatty acid metabolism have been shown to be responsive to LXR. Included in that list is a group of genes involved in reverse cholesterol transport (ABCA1, 2Especially in older literature, 27-hydroxycholesterol may be called 26-hydroxycholesterol or 25(R),26-hydroxycholesterol. ABCA1 is the same as ABC1, and ABCG1 is also called ABC8. 2Especially in older literature, 27-hydroxycholesterol may be called 26-hydroxycholesterol or 25(R),26-hydroxycholesterol. ABCA1 is the same as ABC1, and ABCG1 is also called ABC8.ABCG1,2ApoE, and CETP) (8Luo Y. Tall A.R. J. Clin. 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Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 507-512Crossref PubMed Scopus (567) Google Scholar), and LXR has indeed been proposed to be a coordinator of HDL cholesterol catabolism and bile acid synthesis (8Luo Y. Tall A.R. J. Clin. Invest. 2000; 105: 513-520Crossref PubMed Scopus (304) Google Scholar). Also regulated by LXR is SREBP-1c (15Repa J.J. Liang G. Ou J. Bashmakov Y. Lobaccaro J.M. Shimomura I. Shan B. Brown M.S. Goldstein J.L. Mangelsdorf D.J. Genes Dev. 2000; 14: 2819-2830Crossref PubMed Scopus (1401) Google Scholar, 16Yoshikawa T. Shimano H. Amemiya-Kudo M. Yahagi N. Hasty A.H. Matsuzaka T. Okazaki H. Tamura Y. Iizuka Y. Ohashi K. Osuga J.I. Harada K. Gotoda T. Kimura S. Ishibashi S. Yamada N. Mol. Cell. Biol. 2001; 21: 2991-3000Crossref PubMed Scopus (431) Google Scholar), a master regulator of fatty acid synthesis.Among LXR-responsive genes, ABCA1 is of particular interest since the recent discovery that mutations in this gene are the underlying cause of Tangier disease (17Brooks-Wilson A. Marcil M. Clee S.M. Zhang L.H. Roomp K. van Dam M., Yu, L. Brewer C. Collins J.A. Molhuizen H.O. Loubser O. Ouelette B.F. Fichter K. Ashbourne-Excoffon K.J. Sensen C.W. Scherer S. Mott S. Denis M. Martindale D. Frohlich J. Morgan K. Koop B. Pimstone S. Kastelein J.J. Hayden M.R. et al.Nat. Genet. 1999; 22: 336-345Crossref PubMed Scopus (1493) Google Scholar, 18Bodzioch M. Orsó E. Klucken J. Langmann T. Bottcher A. Diederich W. Drobnik W. Barlage S. Buchler C. Porsch-Özcürümez M. Kaminski W.E. Hahmann H.W. Oette K. Rothe G. Aslanidis C. Lackner K.J. Schmitz G. Nat. Genet. 1999; 22: 347-351Crossref PubMed Scopus (1337) Google Scholar, 19Rust S. Rosier M. Funke H. Real J. Amoura Z. Piette J.C. Deleuze J.F. Brewer H.B. Duverger N. Denefle P. Assmann G. Nat. Genet. 1999; 22: 352-355Crossref PubMed Scopus (1258) Google Scholar, 20Lawn R.M. Wade D.P. Garvin M.R. Wang X. Schwartz K. Porter J.G. Seilhamer J.J. Vaughan A.M. Oram J.F. J. Clin. Invest. 1999; 104: R25-R31Crossref PubMed Scopus (649) Google Scholar). This condition is characterized by defective formation of nascent HDL and extremely low levels of circulating apoA-I-containing lipoproteins. Before the realization that ABCA1 is an LXR-responsive gene, it had been shown that loading of human monocyte-derived macrophages with cholesterol would induce the expression of that gene and, conversely, that cholesterol unloading would decrease the expression (21Langmann T. Klucken J. Reil M. Liebisch G. Luciani M.F. Chimini G. Kaminski W.E. Schmitz G. Biochem. Biophys. Res. Commun. 1999; 257: 29-33Crossref PubMed Scopus (425) Google Scholar). These cells do not synthesize steroid hormones and therefore not 20(S)- or 22(R)-hydroxycholesterol. We also found it counterintuitive that overload of cholesterol, well known to down-regulate early steps in cholesterol biosynthesis, would induce formation of intermediates in this pathway, including 24(S),25-epoxycholesterol, and hypothesized that some other derivative of cholesterol would mediate the activation of LXR in cholesterol-loaded cells.Sterol 27-hydroxylase, encoded by the CYP27 gene, is an enzyme with multiple functions (recently reviewed in Ref. 22Javitt N.B. Biochim. Biophys. Acta. 2000; 1529: 136-141Crossref PubMed Scopus (16) Google Scholar). In the liver, its primary function is to hydroxylate bile acid intermediates such as cholestane-3α,7α,12α,-triol, thereby initiating side chain degradation. In extrahepatic tissues and in liver, the enzyme hydroxylates cholesterol to yield 27-hydroxycholesterol2and cholestenoic acid. 27-Hydroxycholesterol is the most abundant hydroxycholesterol in human circulation, with average concentrations of about 0.4 µm in normal subjects (23Dzeletovic S. Breuer O. Lund E. Diczfalusy U. Anal. Biochem. 1995; 225: 73-80Crossref PubMed Scopus (471) Google Scholar). The absence of a functional sterol 27-hydroxylase is the cause of the rare inherited metabolic disorder cerebrotendinous xanthomatosis (CTX) (24Oftebro H. Björkhem I. Skrede S. Schreiner A. Pedersen J.I. J. Clin. Invest. 1980; 65: 1418-1430Crossref PubMed Scopus (157) Google Scholar, 25Cali J.J. Hsieh C.-L. Francke U. Russell D.W. J. Biol. Chem. 1991; 266: 7779-7783Abstract Full Text PDF PubMed Google Scholar). CTX patients have a complex phenotype, partly reflecting the importance of CYP27 for bile acid synthesis (26Björkhem I. Boberg K.M. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Basis of Inherited Disease. McGraw-Hill Inc., New York1995: 2073-2099Google Scholar). Thus, high levels of toxic bile acid intermediates circulate in these patients. There are, however, other features of CTX that are not readily explained by the bile acid biosynthetic defect but are compatible with the notion of a defective formation of endogenous LXR ligands. For example, intracellular cholesterol is elevated in CTX patients, and premature atherosclerosis is relatively common despite low to normal LDL cholesterol levels. A single study (27Shore V. Salen G. Cheng F.W. Forte T. Shefer S. Tint G.S. Lindgren F.T. J. Clin. Invest. 1981; 68: 1295-1304Crossref PubMed Scopus (33) Google Scholar), which has not been confirmed (28Fujiyama J. Kuriyama M. Arima S. Shibata Y. Nagata K. Takenaga S. Tanaka H. Osame M. Clin. Chim. Acta. 1991; 200: 1-11Crossref PubMed Scopus (57) Google Scholar), reported very low HDL levels in CTX patients. This would be expected if the absence of CYP27 caused a low intracellular LXR tone leading to low ABCA1-mediated high density lipoprotein formation.To determine the identity of the LXR ligands formed as a response to cholesterol overload, we treated human primary monocyte-derived macrophages with acetylated LDL and determined oxysterol formation. A dose dependence between cholesterol loading, formation of 27-hydroxycholesterol and cholestenoic acid, and activation of LXR-responsive genes was observed. In a coactivator association experiment, 27-hydroxycholesterol was shown to activate LXR. We were then able to show that transient or stable transfection of CYP27 into HEK293 cells confers activation of LXR-responsive genes. Finally, in primary fibroblasts derived from a patient deficient in CYP27 activity, LXR activation in response to cholesterol loading was impaired. We conclude that intracellular 27-hydroxylation of cholesterol is an important pathway for the generation of LXR agonists upon cholesterol loading.EXPERIMENTAL PROCEDURESMaterialsAll of the DNA-modifying enzymes were purchased from Life Technologies, Inc. Vectors and cells (293-Tet-Off) for the generation of tetracycline-regulated stably transfected cells were obtained fromCLONTECH (Palo Alto, CA). The expression vector pcDNA3.1 was obtained from Invitrogen (Carlsbad, CA). Human low density lipoprotein was obtained by sequential ultracentrifugation as described (29Basu S.K. Goldstein J.L. Anderson G.W. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 3178-3182Crossref PubMed Scopus (822) Google Scholar). Acetylation of LDL was performed according to the method of Goldstein et al. (30Goldstein J.L. Ho Y.K. Basu S.K. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 333-337Crossref PubMed Scopus (1926) Google Scholar). Steroids were obtained from Sigma, Steraloids, (Newport, RI), or Research Plus (Bayonne, NJ). Cholestenoic acid was kindly provided by Dr. Norman B. Javitt (New York University, New York, NY).Cell CultureHuman skin fibroblasts from a CTX patient were kindly provided by Dr. Ingemar Björkhem (Huddinge University Hospital, Huddinge, Sweden). This patient has been extensively characterized, and the mutation has been identified (R441W). 3I. Björkhem, personal communication. This mutation has also been described and characterized in a number of Japanese patients (31Wakamatsu N. Hayashi M. Kawai H. Kondo H. Gotoda Y. Nishida Y. Kondo R. Tsuji S. Matsumoto T. J. Neurol. Neurosurg. Psychiatry. 1999; 67: 195-198Crossref PubMed Scopus (19) Google Scholar). Control human skin fibroblasts were obtained from the ATCC (catalog no. CRL-2201). Experiments with both normal and CTX fibroblasts were performed between passages 14 and 19. Fibroblasts were cultured in medium A (Dulbecco's modified Eagle's medium (high glucose), supplemented with 25 mm HEPES, 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin sulfate).Human embryonic kidney 293 cells (HEK-293; ATCC catalog no. CRL-1573) were cultured in medium B (Dulbecco's modified Eagle's medium (low glucose), supplemented with 25 mm HEPES, 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin sulfate). Tet-293-based cell lines were cultured in medium C (medium A supplemented with 100 µg/ml hygromycin and 100 µg/ml G418) supplemented with 2 µg/ml doxycycline as specifically stated. Transfection of 293 and Tet-293 cells was accomplished using LipofectAmine (Life Technologies, Inc.) according to the instructions given by the manufacturer. Human primary monocytes were prepared as described (32Wright S.D. Silverstein S.C. J. Exp. Med. 1982; 156: 1149-1164Crossref PubMed Scopus (293) Google Scholar) and differentiated to macrophages by culturing for 7 days in Teflon jars in medium D (RPMI 1640 medium supplemented with 12% human serum, 100 units/ml penicillin, and 100 µg/ml streptomycin sulfate) (33Hailman E. Vasselon T. Kelley M. Busse L.A. Hu M.C. Lichenstein H.S. Detmers P.A. Wright S.D. J. Immunol. 1996; 156: 4384-4390PubMed Google Scholar). They were then plated at a density of 1 million cells/well (12-well plate) in medium D. Loading with acetylated LDL at concentrations specified elsewhere was performed for 72 h before harvesting cells and media for analysis with GC-MS and quantitative real time PCR. All determinations of cell protein were done after dissolution in 0.1 n NaOH using the BCA kit from Pierce according to the instructions by the manufacturer.All cells were cultured in a humidified atmosphere consisting of 95% air and 5% carbon dioxide.Co-activator Association AssaysA homogeneous time-resolved fluorescence assay was used to examine the ligand-dependent interaction of LXRα or LXRβ with the co-activator steroid receptor coactivator-1 (SRC-1) as we have previously described (34Zhou G. Cummings R. Li Y. Mitra S. Wilkinson H.A. Elbrecht A. Hermes J.D. Schaeffer J.M. Smith R.G. Moller D.E. Mol. Endocrinol. 1998; 12: 1594-1604Crossref PubMed Scopus (160) Google Scholar). Briefly, 198 µl of reaction mixture (50 mm HEPES, 125 mm KF, 0.125% (w/v) CHAPS, 0.05% casein, 5 nm GST-LXR LBD, 2 nmanti-GST-(Eu)K, 10 nm biotin-SRC-1-(568–780), 20 nm SA/XL665) were added to each well. The testing compounds in Me2SO were added to the appropriate wells. The reaction was incubated overnight at 4 °C, followed by measurement of fluorescence reading on a Discovery instrument (Packard). The results were expressed as the ratio, multiplied by a factor of 104, of the emission intensity at 665 nm to that at 620 nm.RNA IsolationTotal RNA was prepared using Trizol reagent (Life Technologies) according to the instructions given by the manufacturer, with the exception that 1-bromo-3-chloropropane (Sigma) was substituted for chloroform. The RNA was treated with DNase (Ambion) before analysis by real time quantitative RT-PCR.Real Time Quantitative RT-PCR (TaqMan)Real time quantitative PCR analysis (35Heid C.A. Stevens J. Livak K.J. Williams P.M. Genome Res. 1996; 6: 986-994Crossref PubMed Scopus (4970) Google Scholar) was used to determine the relative levels of ABCA1, ABCG1, SREBP-1c, CYP27, SCD, LXRα, and LXRβ mRNA. Reverse transcription and PCRs were performed according to the manufacturer's instructions (Applied Biosystems; TaqMan Gold RT-PCR protocol and TaqMan Universal PCR Master Mix). Sequence-specific amplification was detected with an increasing fluorescent signal of FAM (reporter dye) during the amplification cycle. Amplification of the mRNA for the human 23-kDa highly basic protein (HBP), also called ribosomal protein L13a, was performed in the same reaction on all samples tested as an internal control for variations in RNA amounts. Levels of the different mRNAs were subsequently normalized to HBP mRNA levels.Primers and Fluorogenic ProbesOligonucleotide primers and TaqMan probes were designed using Primer Express software (Applied Biosystems) and were synthesized by Applied Biosystems. See Table I for sequences of probes and primers.Table ISequences of primers and probes for real time quantitative RT-PCRGeneForward primerReverse primerProbeAccession no.ABCA1TGTCCAGTCCAGTAATGGTTCTGTAAGCGAGATATGGTCCGGATT6FAM-ACACCTGGAGAGAAGCTTTCAACGAGACTAACC-TAMRANM_005502ABCG1TGCAATCTTGTGCCATATTTGACCAGCCGACTGTTCTGATCA6FAM-TACCACAACCCAGCAGATTTTGTCATGGA-TAMRAXM_016998LXRαAGAGGAGGAACAGGCTCATGCAAAGGAGCGCCGGTTACACT6FAM-ATGATCGAGAAGCTCGTCGCTGCC-TAMRAU22662LXRβGGAGCTGGCCATCATCTCAGTCTCTAGCAGCATGATCTCGATAGT6FAM-CAGGAGATCGTGGACTTCGCTAAGCAA-TAMRANM_007121CYP27AGATGCAGCTACTCCTCGCAAAGGCCCACTTTCTTATTGGGA6FAM-AAGGTGGTCCTGGCCCCGGA-TAMRAM62401SREBP-1cGGTAGGGCCAACGGCCTCTGTCTTGGTTGTTGATAAGCTGAA6FAM-ATCGCGGAGCCATGGATTGCACT-TAMRAU00968,AH004383SCDAGGCAGCCTCCTTTGTGTGTATTGGTTCAGTGACCCTGAGCAT6FAM-AGGCAGCTCCCTCCTGCACACAGA-TAMRAXM_005719HBPGCTGGAAGTACCAGGCAGTGAACCGGTAGTGGATCTTGGCTTTVIC-TCTTTCCTCTTCTCCTCCAGGGTGGCT-TAMRANM_012423Reference is given to the GenBank™ accession numbers of the DNA sequences from which the primers and probes were designed. Open table in a new tab cDNA CloningA full-length expressible cDNA encoding human CYP27 was obtained by single-tube RT-PCR using RNA from human primary monocyte-derived macrophages. A Titan RT-PCR kit (Roche Molecular Biochemicals) was used according to the instructions supplied by the manufacturer. Primers used were AAAGCGGCCGCAGGCGCGCGAGCACAACCCAT (forward primer, includes a NotI restriction site) and AAAGAATTCGAGCCTCTAGGACAAGCCCCAGCAA (reverse primer, includes anEcoRI restriction site). The following program was used for the Perkin-Elmer 9600 thermocycler: reverse transcription at 50 °C for 30 min, denaturing at 94 °C for 2 min, 10 cycles of denaturing at 94 °C for 30 s, annealing at 62 °C for 30 s, elongation at 68 °C for 90 s, and 30 more cycles with the elongation time increased by 5 s/cycle, concluded with a final elongation step at 68 °C for 7 min. After purification from agarose gel, the amplified fragment was cloned into the expression vector pcDNA3.1-His5 using the Topo TA cloning kit from Invitrogen, yielding plasmid pcDNA3.1-hCYP27. A full-length cDNA encoding the human StAR protein was obtained by RT-PCR and cloned into pcDNA3.1-His5 in the same manner, using human adrenal gland mRNA (CLONTECH). The primers used were CCACCACTGCCACATTTGCCAGGAA (forward primer) and CAGTTGGGAACAGCAGGCTGGTCTT (reverse primer), and the temperature program was identical to that used for amplification of CYP27. The resulting plasmid was designated as pcDNA3.1-hStAR. The endogenous stop codon was included in the reverse primer for both CYP27 and StAR to prevent incorporation of a His tag in the expressed protein, which may have interfered with activity.Stable Cell LinesA Tetracycline-suppressible HEK293 Cell Line Expressing Human CYP27The insert from pcDNA3.1-hCYP27 was subcloned into the plasmid pTRE2 (CLONTECH) using standard molecular biology techniques (36Molecular Cloning: A Laboratory Manual.in: Sambrook J.F. Russell D.W. Cold Spring Harbor Laboratory Press. 3rd Ed. Cold Spring Harbor, NY2000Google Scholar), yielding plasmid pTRE2-hCYP27. This plasmid encodes human sterol 27-hydroxylase under the control of a tetracycline response element and a minimal CMV promoter. This plasmid and plasmid pTK-hyg (CLONTECH), which expresses a hygromycin resistance gene, were cotransfected into 293-Tet-Off cells (CLONTECH), a stable cell line enabling the suppression by tetracycline or doxycycline of genes under control of a tetracycline response element. LipofectAmine (Life Technologies) was used as a transfection reagent according to the instructions given by the manufacturer. After 3 weeks of selection in medium C supplemented with 2 µg/ml doxycycline, several clones were isolated. The clones were screened for CYP27 expression by plating on 60-mm dishes at a density of 1 million cells/dish and cultured in medium C for 48 h in the presence or absence of 2 µg/ml doxycycline. RNA was prepared and analyzed by quantitative real time RT-PCR for induction of CYP27 in the absence of doxycycline, and medium was collected and analyzed by GC-MS for accumulation of 27-hydroxycholesterol and cholestenoic acid. One clone, designated XF-4, showed a 3-fold induction of CYP27 mRNA expression in the absence of doxycycline. Medium concentrations of 27-hydroxycholesterol and cholestenoic acid were 72 and 10 nmol/liter, respectively, in the suppressed state and 90 and 140 nmol/liter, respectively, in the induced state after a 48-h incubation in the presence of 10% fetal bovine serum.An HEK293 Cell Line Expressing Human CYP27 and Human StAR Constitutively and a Control Cell LineThe inserts from pcDNA3.1-hCYP27 and pcDNA3.1-hStAR were subcloned into the plasmid pIRES (CLONTECH) using standard molecular biology techniques. The resulting plasmid, p27ST, encodes the human CYP27 and the human StAR, separated by an IRES, under the control of a CMV promoter. The IRES functions as a ribosomal start site; therefore, translation can be initiated at the beginning of the mRNA transcript as well as following the IRES. This feature allows for the simultaneous functional expression of both of the two proteins when transfected into a suitable cell line. The plasmid p27ST was used to transfect 293-Tet-Off cells using LipofectAmine as described above. After 3 weeks of selection in medium C supplemented with 2 µg/ml doxycycline, several clones were isolated and propagated. The different isolated cell lines were plated on 60-mm dishes at a density of 1 million cells/dish and cultured for 24 h in medium C, and the medium was collected and analyzed by GC-MS for the presence of cholestenoic acid and 27-hydroxycholesterol. One cell line, designated XF-13, that did not have detectable 27-hydroxylase activity was saved and propagated as a control line. Another cell line, designated XF-12, demonstrated a constitutive sterol 27-hydroxylase activity, and after culturing for 24 h in medium C, the final concentration of cholestenoic acid was 220 nmol/liter (27-hydroxycholesterol not determined). This cell line was saved, propagated, and used in further experiments.Cholesterol Loading in CTX and Normal Skin FibroblastsCTX and normal skin fibroblasts were cultured and cholesterol-loaded as described by Francis et al. (37Francis G.A. Knopp R.H. Oram J.F. J. Clin. Invest. 1995; 96: 78-87Crossref PubMed Scopus (370) Google Scholar). Briefly, cells were set up in medium A and were allowed to reach confluence before loading with cholesterol or treatment with LXR agonist. For determination of induction of gene expression by cholesterol loading, cells were washed twice with phosphate-buffered saline, and the medium was changed to medium E (like medium A but without serum) supplemented with 0.2% fatty acid-free bovine serum albumin and 30 µg/ml cholesterol or vehicle (ethanol) control. After 72 h of incubation, cells were harvested to determine loading efficiency by GC-MS and gene expression by real time PCR as described above. For determination of induction of gene expression by the LXR agonist 24(S),25-epoxycholesterol, cells were incubated with that compound (10 µm) or vehicle (ethanol) in medium A for 24 h. After incubation, cells were harvested to determine gene expression by real time PCR as described above.Cholesterol Loading in HEK293 Cells Constitutively Expressing CYP27 and StARThe stable cell line XF-12 and a control cell line XF-13 were cultured in medium C to reach 80% confluence. Cholesterol loading was carried out in medium F (like medium C but without serum) supplemented with 30 µg/ml cholesterol or vehicle (ethanol) control. After 24 h of incubation, loading efficiency and gene expression were determined as described above.Gas Chromatography-Mass SpectrometryGC-MS was performed using a ThermoQuest GCQ instrument equipped with an RTX-5MS column (30 m × 0.25 mm inner diameter, 0.25-µm phase thickness; Restek Corp., Bellefonte, PA). The gas chromatography program was 180 °C for 1 min, followed by a temperature gradient of 20 °C/min to 290 °C and a final elution at 290 °C for 20 min The injector was operated in the split mode (1:10 split), and the temperature was kept at 275 °C. Helium was used as the carrier gas at a constant flow rate of 1 ml/min. The instrument was operated in the electron ionization mode with the electron energy set to 70 eV. The ion trap was scanned between m/z 50 and 550 at the rate of 1 scan/1.5 s except when used for quantitation of cholesterol, when the ion trap was used for the selected ion monitoring ofm/z 458 and 464.Determination of 27-Hydroxycholesterol and Cholestenoic AcidTo aliquots of cell media, defined amounts (500–1000 ng of each) of 22(S)-hydroxycholesterol and cholenic acid (internal standards for 27-hydroxycholesterol and cholestenoic acid, respectively) were added. Medium (2–3 ml) was extracted with 3 volumes of chloroform/methanol (2:1, v/v), and the organic phase was collected and separated into a neutral fraction containing neutral oxysterols and an acidic fraction containing cholenic acid and cholestenoic acid (38Kaluzny M.A. Duncan L.A. Merritt M.V. Epps D.E. J. Lipid Res. 1985; 26: 135-140Abstract Full Text PDF PubMed Google Scholar). The neutral fraction was further purified using Isolute Silica columns as described (23Dzeletovic S. Breuer O. Lund E. Diczfalusy U. Anal. Biochem. 1995; 225: 73-80Crossref PubMed Scopus (471) Google Scholar), with the exception that 6 instead of 8 ml of 0.5% isopropyl alcohol in hexane was used to elute cholesterol. 27-Hydroxycholesterol and other neutral oxysterols were then eluted with 2 ml of 30% isopropyl alcohol in hexane. Prior to analysis by GC-MS, purified neutral fractions were converted to TMS ethers using Sigma Sil-A (60 °C