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• W1570388980 abstract "Tangier disease, a condition characterized by low levels of high density lipoprotein and cholesterol accumulation in macrophages, is caused by mutations in the ATP-binding cassette transporter ABC1. In cultured macrophages, ABC1 mRNA was induced in an additive fashion by 22(R)-hydroxycholesterol and 9-cis-retinoic acid (9CRA), suggesting induction by nuclear hormone receptors of the liver X receptor (LXR) and retinoid X receptor (RXR) family. We cloned the 5′-end of the human ABC1 transcript from cholesterol-loaded THP1 macrophages. When transfected into RAW macrophages, the upstream promoter was induced 7-fold by 22(R)-hydroxycholesterol, 8-fold by 9CRA, and 37-fold by 9CRA and 22(R)-hydroxycholesterol. Furthermore, promoter activity was increased in a sterol-responsive fashion when cotransfected with LXRα/RXR or LXRβ/RXR. Further experiments identified a direct repeat spaced by four nucleotides (from −70 to −55 base pairs) as a binding site for LXRα/RXR or LXRβ/RXR. Mutations in this element abolished the sterol-mediated activation of the promoter. The results show sterol-dependent transactivation of the ABC1 promoter by LXR/RXR and suggest that small molecule agonists of LXR could be useful drugs to reverse foam cell formation and atherogenesis. Tangier disease, a condition characterized by low levels of high density lipoprotein and cholesterol accumulation in macrophages, is caused by mutations in the ATP-binding cassette transporter ABC1. In cultured macrophages, ABC1 mRNA was induced in an additive fashion by 22(R)-hydroxycholesterol and 9-cis-retinoic acid (9CRA), suggesting induction by nuclear hormone receptors of the liver X receptor (LXR) and retinoid X receptor (RXR) family. We cloned the 5′-end of the human ABC1 transcript from cholesterol-loaded THP1 macrophages. When transfected into RAW macrophages, the upstream promoter was induced 7-fold by 22(R)-hydroxycholesterol, 8-fold by 9CRA, and 37-fold by 9CRA and 22(R)-hydroxycholesterol. Furthermore, promoter activity was increased in a sterol-responsive fashion when cotransfected with LXRα/RXR or LXRβ/RXR. Further experiments identified a direct repeat spaced by four nucleotides (from −70 to −55 base pairs) as a binding site for LXRα/RXR or LXRβ/RXR. Mutations in this element abolished the sterol-mediated activation of the promoter. The results show sterol-dependent transactivation of the ABC1 promoter by LXR/RXR and suggest that small molecule agonists of LXR could be useful drugs to reverse foam cell formation and atherogenesis. high density lipoprotein 9-cis-retinoic acid 22(R)-hydroxycholesterol 25-hydroxycholesterol 7-ketocholesterol liver X receptor retinoid X receptor cotransfected mixture of two receptors putative heterodimer complex steroidogenic factor 1 cholesterol ester transfer protein direct repeat rapid amplification of cDNA ends base pair(s) polymerase chain reaction low density lipoprotein Plasma HDL1-cholesterol levels are inversely related to the incidence of coronary artery disease (1Castelli W.P. Garrison R.J. Wilson P.W. Abbott R.D. Kalousdian S. Kannel W.B. J. Am. Med. Assoc. 1986; 256: 2835-2838Crossref PubMed Scopus (2091) Google Scholar). Two genetic diseases illustrate this phenomenon, the rare Tangier disease and the more common familial HDL deficiency. Tangier disease is characterized by an extremely low concentration of circulating HDL and the accumulation of cholesteryl esters in tonsils, liver, spleen, and intestinal mucosa, mostly in macrophage foam cells (2Serfaty-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). Patients with familial HDL deficiency exhibit a low concentration of HDL particles and an increased risk of coronary artery disease (3Marcil M. Brooks-Wilson A. Clee S.M. Roomp K. Zhang L.H., Yu, L. Collins J.A. van Dam M. Molhuizen H.O. Loubster O. Ouellette B.F. Sensen C.W. Fichter K. Mott S. Denis M. Boucher B. Pimstone S. Genest Jr., J. Kastelein J.J. Hayden M.R. Lancet. 1999; 354: 1341-1346Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). A common explanation for the cardioprotective effect of HDL is the major role it plays in reverse cholesterol transport (4Bruce C. Chouinard Jr., R.A. Tall A.R. Annu. Rev. Nutr. 1998; 18: 297-330Crossref PubMed Scopus (229) Google Scholar). It is commonly accepted that the efflux of cholesterol from cells is caused by two different pathways: the first is passive and promotes efflux from the cell membrane to HDL and the second is energy-dependent and apolipoprotein-mediated (5Rothblat G.H. de la Llera-Moya M. Atger V. Kellner-Weibel G. Williams D.L. Phillips M.C. J. Lipid Res. 1999; 40: 781-796Abstract Full Text Full Text PDF PubMed Google Scholar). The latter was characterized in fibroblasts and macrophages and involves lipid-poor or -free apolipoproteins such as apoA-I, apoA-II, and apo-E (5Rothblat G.H. de la Llera-Moya M. Atger V. Kellner-Weibel G. Williams D.L. Phillips M.C. J. Lipid Res. 1999; 40: 781-796Abstract Full Text Full Text PDF PubMed Google Scholar, 6Yokoyama S. Biochim. Biophys. Acta. 1998; 1392: 1-15Crossref PubMed Scopus (108) Google Scholar, 7Takahashi Y. Smith J.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11358-11363Crossref PubMed Scopus (207) Google Scholar). This active pathway has been reported to be defective in both Tangier disease and familial HDL deficiency (8Remaley A.T. Schumacher U.K. Stonik J.A. Farsi B.D. Nazih H. Brewer Jr., H.B. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1813-1821Crossref PubMed Scopus (191) Google Scholar, 9Marcil M., Yu, L. Krimbou L. Boucher B. Oram J.F. Cohn J.S. Genest Jr., J. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 159-169Crossref PubMed Scopus (90) Google Scholar, 10Francis G.A. Knopp R.H. Oram J.F. J. Clin. Invest. 1995; 96: 78-87Crossref PubMed Scopus (373) Google Scholar). It was recently demonstrated that ABC1 is a key gene in this process (11Lawn 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: R25-31Crossref PubMed Scopus (658) Google Scholar) and that mutations of ABC1 are the major cause of both Tangier disease and familial HDL deficiency (3Marcil M. Brooks-Wilson A. Clee S.M. Roomp K. Zhang L.H., Yu, L. Collins J.A. van Dam M. Molhuizen H.O. Loubster O. Ouellette B.F. Sensen C.W. Fichter K. Mott S. Denis M. Boucher B. Pimstone S. Genest Jr., J. Kastelein J.J. Hayden M.R. Lancet. 1999; 354: 1341-1346Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar, 12Bodzioch M. Orso E. Klucken J. Langmann T. Bottcher A. Diederich W. Drobnik W. Barlage S. Buchler C. Porsch-Ozcurumez 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 (1349) Google Scholar, 13Rust S. Walter M. Funke H. von Eckardstein A. Cullen P. Kroes H.Y. Hordijk R. Geisel J. Kastelein J. Molhuizen H.O. Schreiner M. Mischke A. Hahmann H.W. Assmann G. Nat. Genet. 1998; 20: 96-98Crossref PubMed Scopus (65) Google Scholar, 14Remaley A.T. Rust S. Rosier M. Knapper C. Naudin L. Broccardo C. Peterson K.M. Koch C. Arnould I. Prades C. Duverger N. Funke H. Assman G. Dinger M. Dean M. Chimini G. Santamarina-Fojo S. Fredrickson D.S. Denefle P. Brewer Jr., H.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12685-12690Crossref PubMed Scopus (230) Google Scholar, 15Brooks-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. Genest Jr., J. Hayden M.R. Nat. Genet. 1999; 22: 336-345Crossref PubMed Scopus (1509) Google Scholar, 16Brousseau M.E. Schaefer E.J. Dupuis J. Eustace B. Van Eerdewegh P. Goldkamp A.L. Thurston L.M. FitzGerald M.G. Yasek-McKenna D. O'Neill G. Eberhart G.P. Weiffenbach B. Ordovas J.M. Freeman M.W. Brown Jr., R.H. Gu J.Z. J. Lipid Res. 2000; 41: 433-441Abstract Full Text Full Text PDF PubMed Google Scholar, 17Orso E. Broccardo C. Kaminski W.E. Bottcher A. Liebisch G. Drobnik W. Gotz A. Chambenoit O. Diederich W. Langmann T. Spruss T. Luciani M.F. Rothe G. Lackner K.J. Chimini G. Schmitz G. Nat. Genet. 2000; 24: 192-196Crossref PubMed Scopus (431) Google Scholar). ABC1 (ABCA1) belongs to the large ATP-binding cassette transporter family. These transmembrane proteins transport many diverse substrates across membranes because of their channel-like topology (18Croop J.M. Methods Enzymol. 1998; 292: 101-116Crossref PubMed Scopus (53) Google Scholar, 19Broccardo C. Luciani M. Chimini G. Biochim. Biophys. Acta. 1999; 1461: 395-404Crossref PubMed Scopus (90) Google Scholar). The human ABC1 gene was assigned to chromosome 9q31, spanning a minimum of 70 kilobases and containing at least 49 exons (14Remaley A.T. Rust S. Rosier M. Knapper C. Naudin L. Broccardo C. Peterson K.M. Koch C. Arnould I. Prades C. Duverger N. Funke H. Assman G. Dinger M. Dean M. Chimini G. Santamarina-Fojo S. Fredrickson D.S. Denefle P. Brewer Jr., H.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12685-12690Crossref PubMed Scopus (230) Google Scholar, 16Brousseau M.E. Schaefer E.J. Dupuis J. Eustace B. Van Eerdewegh P. Goldkamp A.L. Thurston L.M. FitzGerald M.G. Yasek-McKenna D. O'Neill G. Eberhart G.P. Weiffenbach B. Ordovas J.M. Freeman M.W. Brown Jr., R.H. Gu J.Z. J. Lipid Res. 2000; 41: 433-441Abstract Full Text Full Text PDF PubMed Google Scholar, 20Luciani M.F. Denizot F. Savary S. Mattei M.G. Chimini G. Genomics. 1994; 21: 150-159Crossref PubMed Scopus (231) Google Scholar). Whereas its expression is ubiquitous, the highest levels of human or murine mRNAs were found in placenta, fetal tissues, liver, lung, and adrenal glands (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 (429) Google Scholar, 22Klucken J. Buchler C. Orso E. Kaminski W.E. Porsch-Ozcurumez M. Liebisch G. Kapinsky M. Diederich W. Drobnik W. Dean M. Allikmets R. Schmitz G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 817-822Crossref PubMed Scopus (474) Google Scholar). The predicted human protein contains 2201 amino acids (220-kDa protein) (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 (429) Google Scholar). The expression of hABC1 is induced by cholesterol loading of human macrophages. Both the protein and the mRNA are up-regulated in the presence of acetylated LDL, and down-regulated by cholesterol unloading via HDL3 (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 (429) Google Scholar). Whereas the cholesterol-mediated regulation of genes involved in cholesterol uptake or biosynthesis via sterol regulatory element binding protein (SREBP) pathways is well understood (23Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (3029) Google Scholar), much less is known about direct mechanisms of sterol-mediated up-regulation of gene expression. Two families of nuclear receptors are known to be activated by oxysterols and to mediate a positive response by binding to specific DNA elements, the liver X receptor (LXR) and steroidogenic factor 1 (SF1) (24Peet 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 (1252) Google Scholar, 25Luo Y. Tall A.R. J. Clin. Invest. 2000; 105: 513-520Crossref PubMed Scopus (309) Google Scholar, 26Christenson L.K. McAllister J.M. Martin K.O. Javitt N.B. Osborne T.F. Strauss III, J.F. J. Biol. Chem. 1998; 273: 30729-30735Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 27Lala D.S. Syka P.M. Lazarchik S.B. Mangelsdorf D.J. Parker K.L. Heyman R.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4895-4900Crossref PubMed Scopus (170) Google Scholar). SF1 acts as a monomer and has been implicated in the regulation of steroidogenic acute regulatory protein gene expression (StAR) activity (26Christenson L.K. McAllister J.M. Martin K.O. Javitt N.B. Osborne T.F. Strauss III, J.F. J. Biol. Chem. 1998; 273: 30729-30735Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Recently, two different genes involved in the reverse cholesterol transport pathways, cholesterol 7α-hydoxylase (24Peet 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 (1252) Google Scholar) and cholesterol ester transfer protein (CETP) (25Luo Y. Tall A.R. J. Clin. Invest. 2000; 105: 513-520Crossref PubMed Scopus (309) Google Scholar), have been shown to be up-regulated by the heterodimer LXR·RXR. This suggests the hypothesis that LXRs might coordinate different steps of reverse cholesterol transport (25Luo Y. Tall A.R. J. Clin. Invest. 2000; 105: 513-520Crossref PubMed Scopus (309) Google Scholar). LXRα (NR1H3) and LXRβ (NR1H2) heterodimerize with their partner RXR. The resulting complex up-regulates genes through binding sites typically composed of direct repeats (DR) of the motif AGGTCA, spaced by 4 nucleotides (LXRα and LXRβ) or 1 nucleotide (LXRβ) (28Willy P.J. Umesono K. Ong E.S. Evans R.M. Heyman R.A. Mangelsdorf D.J. Genes Dev. 1995; 9: 1033-1045Crossref PubMed Scopus (923) Google Scholar, 29Teboul M. Enmark E. Li Q. Wikstrom A.C. Pelto-Huikko M. Gustafsson J.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2096-2100Crossref PubMed Scopus (200) Google Scholar, 30Feltkamp D. Wiebel F.F. Alberti S. Gustafsson J.A. J. Biol. Chem. 1999; 274: 10421-10429Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). The dimer can be activated by both the ligands of RXR (retinoids) and LXR (oxysterols) separately or together (29Teboul M. Enmark E. Li Q. Wikstrom A.C. Pelto-Huikko M. Gustafsson J.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2096-2100Crossref PubMed Scopus (200) Google Scholar, 31Song C. Kokontis J.M. Hiipakka R.A. Liao S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10809-10813Crossref PubMed Scopus (210) Google Scholar, 32Janowski B.A. Willy P.J. Devi T.R. Falck J.R. Mangelsdorf D.J. Nature. 1996; 383: 728-731Crossref PubMed Scopus (1477) Google Scholar). Here we report the sequence of the hABC1 promoter and show that this promoter is active in macrophages and that its sterol-mediated activation depends on the binding of LXR/RXRα to a DR4 element. 5′-RACE PCR was performed with the SMART RACE cDNA kit (CLONTECH, Palo Alto, CA) using 1 μg of poly(A)+ mRNA from HepG2 and THP-1 cells that were differentiated into macrophage with phorbol 12-myristate 13-acetate and exposed to acetylated LDL (25 μg/ml) for 48 h. After reverse transcription (M-MLV reverse transcriptase, Life Technologies Inc.), a first PCR (hot start, 94 °C 30 s, 65 °C 30 s, 72 °C 3 min, 25 cycles, and then 72 °C 10 min) was performed using the reverse primer 5′-CCCCCTCCCTCGGGATGCCCGCAGACAA-3′. A second PCR (hot start, 94 °C 30 s, 55 °C 30 s, 72 °C 3 min, 25 cycles, and then 72 °C 10 min) was done on 2.5 μl of the 50×-diluted first PCR sample with the nested primer, 5′-GCCTCCGAGCATCTGAGAACAGGC-3′. The forward primers were provided by CLONTECH. The screening of the human RPC.11 BAC clone library was performed (Research Genetics, Inc., Huntsville, AL) with a 68-mer oligonucleotide probe corresponding to nucleotides 11–79 of the publishedhABC1 sequence. Two BAC clones were recovered that were positive by PCR for exon 1 (BAC553F19) and exon 3 (BAC 522C12). After digestion by PstI, a Southern blot was performed using the32P-radiolabeled probes generated by PCR with the previously cited exons. Positive bands were cloned in pBluescript KS(+) (Stratagene, La Jolla, California). A colony hybridization (probes used for Southern blot) (33Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) allowed us to isolate positive clones for thehABC1 promoter (5 kilobases) and intron 2. Sequencing performed on both strands showed that we also cloned intron 2 from BAC 522C12. The sequences of these introns are contained in the sequence of human genomic clone RP11–1M10, which also contains exons 1, 2, and 3 (see Fig. 2 a). The cell lines were purchased from ATCC (Manassas, VA). The murine RAW 264.7, African green monkey CV-1, and human 293 or HepG2 cell lines were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 100 units/ml penicillin, and 100 mg/ml streptomycin. THP-1 cells were maintained in RPMI 1640 containing l-glutamine, 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin supplemented with 0.5 μmβ-mercaptoethanol. Confluent cells were differentiated with 0.2 μm phorbol 12-myristate 13-acetate (Sigma) in ethanol over 72 h. Thioglycolate-elicited peritoneal macrophages were isolated from C57 Bl/6 mice as described previously (34Shipley J.M. Wesselschmidt R.L. Kobayashi D.K. Ley T.J. Shapiro S.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3942-3946Crossref PubMed Scopus (412) Google Scholar). Transfections were performed in 24-well plates with LipofectAMINE reagent (transactivation experiments in CV-1 and 293 cells, see Figs. 4and 5) or LipofectAMINE-Plus reagent (basal activation experiments in RAW 264.7, see Figs. 4, 6, and 8) according to the manufacturer's instructions (Life Technologies Inc.). For basal activation experiments, a total of 0.15 μg of reporter DNA and 0.05 μg of PRL-CMV (Renilla, Promega) per well were used. For transactivation studies, we used 0.025 μg/well PRL-CMV, 0.2 μg of reporter DNA, and 0.1 μg of each receptor (CMX-hRXRα, CMX-hLXRα, CMV-mLXRβ). pcDNA3.1 plasmid was included to obtain a final quantity of 0.45 μg of total DNA per well. The transfected cells were cultured in lipoprotein-deficient serum medium in the presence of 4 μg/ml (see Figs. 4, 6, and 8) or 2 μg/ml (transactivation experiments, see Fig. 5) of 22(R)-hydroxycholesterol (22(R)-Hch), 25-hydroxycholesterol (25-Hch), or 7-ketocholesterol (7-Kch), 10 μm9-cis-retinoic acid (9CRA, Sigma) or ethanol alone for 24 h. The luciferase activities were measured using the Promega dual luciferase assay system. A reporter plasmid used to analyze the activity of the hABC1 promoter was constructed by subcloning a 1029-bp PCR fragment of the hABC1 promoter (from −928 to +101 bp) into the pGL3-Luc basic vector (Promega). A shorter promoter (from −469 to +101 bp) was generated by digestion of this plasmid withSacI. Deletions (see Fig. 7) were performed by enzymatic digestion (ApaI, from −156 to +101 bp) or PCR. The sequence of the PCR fragments were verified. Where shown, error barsrepresent S.D. Non-parametric Mann Whitney tests were performed to obtain p values.Figure 5Expression of LXR α and LXR β in various cell lines.Sterol transactivation by LXR/RXR of the hABC1 promoter is shown. a, cells were isolated and cultured as described under “Material and Methods.” A Northern blot was performed with 35 μg of total RNA for each cell line. Hybridizations were performed using probes of similar specific activities for hLXRα, mLXRβ, and mouse glycerol-3-phosphate dehydrogenase (G3PDH) as an internal standard. h, human; m, mouse.b, 293 cells were transfected with the hABC1promoter (from −469 to +101 bp) or a construct containing three copies of a sterol-responsive element of the CETP promoter (c) (25Luo Y. Tall A.R. J. Clin. Invest. 2000; 105: 513-520Crossref PubMed Scopus (309) Google Scholar). These constructs were cotransfected with theRenilla luciferase reporter gene and hLXRα, mLXRβ, and/or hRXRα as designated. The cells were treated 24 h with vehicle alone or 22(R)-Hch (5 μm) and/or 9CRA (10 μm) in fetal bovine serum medium with 10% lipoprotein-deficient serum. The results represent 2 independent experiments in duplicate for the transfection using thehABC1 promoter and 1–2 experiments in duplicates for the transfection using the hCETP promoter. d, CV-1 cells were transfected and treated according to the protocols described in b. 2–3 independent experiments in duplicates were performed. Bars indicate mean ± S.D. Significance of treatment versus ethanol is indicated by **,p < 0.01; *, p < 0.05.View Large Image Figure ViewerDownload (PPT)Figure 6Deletional analysis of hABC1promoter. Deletions were performed by enzymatic digestion or PCR amplification of the hABC1 promoter. The results represent two independent experiments of duplicates. Barsindicate mean ± S.D. Significance of treatment versusethanol is indicated by ***, p < 0.001; *,p < 0.05.View Large Image Figure ViewerDownload (PPT)Figure 8Electrophoretic mobility shift assay of humanABC1 promoter fragment. Oligonucleotides containing the DR4 element (see Fig. 6) that was identified as a potential binding site for LXR/RXR or a mutated version (mut. DR4) were 32P-radiolabeled. Competitors (lanes 2 and 3) correspond to cold wild-type or mutant oligonucleotides. After incubation with these oligonucleotides and nuclear extracts from 293 cells transfected with LXRα/hRXRα (B) or LXRβ/hRXRα (A), some samples were incubated with polyclonal antibodies for LXRα/β, LXRα, RXRα, or RORα (negative control) or with a monoclonal antibody targeting LXRβ. Three different experiments were performed with similar results. The arrow indicates the position of the retarded complex.View Large Image Figure ViewerDownload (PPT)Figure 7Mutational analysis of hABC1promoter. Raw 264.7 cells were transfected with wild-type (WT) hABC1 promoter (from −928 to +101 bp) or its mutated version. The mutations are presented in Fig. 7. The results represent two independent experiments of duplicates. Barsindicate mean ± S.D. Significance of treatment versusethanol is indicated by ***, p < 0.001; *,p < 0.05.View Large Image Figure ViewerDownload (PPT) Total RNA was isolated with RNAzol B reagent (TEL-TEST, Inc., Friendwood, TX). Northern blots were performed as described previously (33Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). A human ABC1 probe corresponding to exons 2–8 of the published sequence was synthesized by reverse transcriptase-PCR using the forward primer, 5′-AGGTGGCCTGGCCTCTATTTATCTTC-3′ and the reverse primer, 5′-GCCTCCGAGCATCTGAGAACAGGC-3′. LXR probes were synthesized from humanLXRα and mouse LXRβ sequences (28Willy P.J. Umesono K. Ong E.S. Evans R.M. Heyman R.A. Mangelsdorf D.J. Genes Dev. 1995; 9: 1033-1045Crossref PubMed Scopus (923) Google Scholar, 35Seol W. Choi H.S. Moore D.D. Mol. Endocrinol. 1995; 9: 72-85Crossref PubMed Google Scholar). A mouse glycerol-3-phosphate dehydrogenase probe was used as an internal standard (reverse transcriptase-PCR synthesized fragment: forward primer, 5′-ACCACAGTCCATGCCATCAC-3′ and reverse primer, 5′-TCCACCACCCTGTTGCTGTA-3′). Signals were quantitated with phosphor imaging. Non-parametric Mann Whitney tests were performed to obtainp values. Electrophoretic mobility shift assays were performed as described previously (25Luo Y. Tall A.R. J. Clin. Invest. 2000; 105: 513-520Crossref PubMed Scopus (309) Google Scholar). Nuclear extracts were prepared from 293 cells cotransfected with LXRα and hRXRα or LXRβ and hRXR. Double-stranded oligonucleotides containing the DR4 element or its mutated version (see Fig. 7) were synthesized with overhangs and used at a final concentration of 0.1 pm (hABC1DR4) or 0.5 μm (competitors). An oligonucleotide corresponding to a canonical half-site sequence (AGGTCA) was added to each sample to reduce the background (1 μm). Polyclonal antibodies against peptides from LXRα (P20, sc-1202X), LXRα/β (C19, sc-2101X), RXRα (D20, sc-553X), and RORα (K-20, sc-6063X) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). To investigate whether the endogenousABC1 gene can be activated by oxysterols and/or retinoic acid in macrophages, we performed Northern blot analysis of total RNA from human THP-1 macrophages. Fig. 1shows a significant increase of ABC1 mRNA in cells treated with 22(R)-Hch (2-fold induction, p < 0.05) or 9CRA (2-fold, p < 0.05). An additive effect was obtained with combined treatment (4-fold, p < 0.05 when compared with separate treatments). These responses suggest possible activation of transcription by LXR/RXR (32Janowski B.A. Willy P.J. Devi T.R. Falck J.R. Mangelsdorf D.J. Nature. 1996; 383: 728-731Crossref PubMed Scopus (1477) Google Scholar). To identify the promoter of the human ABC1 gene, we performed 5′-RACE PCR using poly(A)+ mRNA from cholesterol-loaded THP-1 macrophages and HepG2 cells (Fig.2 b). In macrophages this revealed a single major transcript (transcript A) consisting of a first exon of 217 bp followed by a second exon of 160 bp, 73% identical to mouse exon 1 (GenBank™/EBI accession number X75926). This exon is then followed by the published human exons 2, 3, and 4 (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 (429) Google Scholar). In HepG2 cells, 5′-RACE PCR revealed three different transcripts (Fig.2 b). Transcript B represents a truncated version of exon 2 found in THP-1 cells (only the last 29 bp) followed by the published exons 2, 3, and 4 (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 (429) Google Scholar). Transcript C contains one exon of 372 bp upstream of the published exon 2, which is different from the exons found in THP1 cells. Transcript D has the same 5′ structure as transcript C but lacks the published exon 3. A BLAST search of the GenBank™/EBI Data Bank (htgs) revealed 100% homology of these exons (Fig. 2 a, exons 1–3) with fragments of the human genomic clone RP11–1M10 (working draft sequence, GenBank™/EBI accession numberAC012230). A comparison of the sequences from the published exon 2, the 5′-RACE PCR product, and RP11–1M10 revealed a C instead of a T at position +15 and a G instead of an A at position +17. Conceptual translation of the transcripts revealed two new start codons in frame with the previously published ATG located in exon 5 (14Remaley A.T. Rust S. Rosier M. Knapper C. Naudin L. Broccardo C. Peterson K.M. Koch C. Arnould I. Prades C. Duverger N. Funke H. Assman G. Dinger M. Dean M. Chimini G. Santamarina-Fojo S. Fredrickson D.S. Denefle P. Brewer Jr., H.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12685-12690Crossref PubMed Scopus (230) Google Scholar) (Fig.2, a and c). In the case of the transcript characteristic of THP1 cells, a new ATG located in exon 2 resulted in an extra 60 amino acids at the amino terminus. In the case of HepG2 cells, a new start codon at the 3′-end of exon 3 may be functional in transcript C and also transcript D, which lacks the previously published start codon. This results in an extra 39-amino acid fragment for transcript C. A comparison of the putative amino-terminal amino acid sequences of ABC1 (transcripts A, B) with nucleotide data bases revealed strong homology to the amino-terminal sequences of two members of the ABC1 family (57% identity with ABCR and 45% identity with ABC3 (Fig. 1c). This strongly suggests that the amino-terminal sequence of hABC1 is authentic. The promoter region upstream of exon 1 was responsive to sterols when transfected into cells (see below), whereas the 2.3-kilobase region upstream of transcript B was not responsive (data not shown). Thus, we focused our attention on the former region. Fig. 3 presents a partial sequence of genomic DNA with a fragment of exon 1 cloned from the human RPCI.11 BAC library. A potential TATA box is present at −32 bp and an Sp1 site at −101 bp. An analysis of this sequence revealed several potential transcription factor binding sites. To investigate the function of the potentialhABC1 promoter, we transfected the macrophage-like RAW 264.7 cell line with a promoter-luciferase construct (Fig.4 a). To test for activation by sterols we used 22(R)-Hch, a potent activator of LXR·RXR (36Lehmann 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 (1048) Google Scholar) but a poor activator of SF1 (27Lala D.S. Syka P.M. Lazarchik S.B. Mangelsdorf D.J. Parker K.L. Heyman R.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4895-4900Crossref PubMed Scopus (170) Google Scholar) and 9CRA, to activate endogenous RXR. Compared with basal conditions, transfected cells treated with 22(R)-Hch or 9CRA exhibited 7- and 8-fold higher promoter activity, respectively (p < 0.001) (Fig.4 a). When both compounds were added together, there was a synergistic 37-fold induction (p < 0.001). A similar response was obtained with promoter fragments containing 928 bp (Fig.4 a) or 469 bp (data not shown) of upstream sequence. Next we compared the response of the ABC1 promoter to different sterols (Fig. 4 b). We treated the transfected cells with 25-Hch, which" @default.
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• W1570388980 title "Sterol-dependent Transactivation of theABC1 Promoter by the Liver X Receptor/Retinoid X Receptor" @default.
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