Sterol-responsive Element-binding Protein (SREBP) 2 Down-regulates ATP-binding Cassette Transporter A1 in Vascular Endothelial Cells
2004; Elsevier BV; Volume: 279; Issue: 47 Linguagem: Inglês
10.1074/jbc.m407817200
ISSN1083-351X
AutoresLingfang Zeng, Hailing Liao, Yi Liu, Tzong‐Shyuan Lee, Minjia Zhu, Xian Wang, Michael B. Stemerman, Yi Zhu, John Y.‐J. Shyy,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoATP-binding cassette transporter A1 (ABCA1) is a pivotal regulator of cholesterol efflux from cells to apolipoproteins, whereas sterol-responsive element-binding protein 2 (SREBP2) is the key protein regulating cholesterol synthesis and uptake. We investigated the regulation of ABCA1 by SREBP2 in vascular endothelial cells (ECs). Our results showed that sterol depletion activated SREBP2 and increased its target, low density lipoprotein receptor mRNA, with a concurrent decrease in the ABCA1 mRNA. Transient transfection analysis revealed that sterol depletion decreased the ABCA1 promoter activity by 50%, but low density lipoprotein receptor promoter- and the sterol-responsive element-driven luciferase activities were increased. Overexpression of the N terminus of SREBP2 (SREBP2(N)), an active form of SREBP2, also inhibited the ABCA1 promoter activity. Functionally adenovirus-mediated SREBP2(N) expression increased cholesterol accumulation and decreased apoA-I-mediated cholesterol efflux. The conserved E-box motif was responsible for the SREBP2(N)-mediated inhibition since mutation of the E-box increased the basal activity of the ABCA1 promoter and abolished the inhibitory effect of SREBP2(N). Furthermore sterol depletion and SREBP2(N) overexpression induced the binding of SREBP2(N) to both consensus and ABCA1-specific E-box. Chromatin immunoprecipitation assay demonstrated that serum starvation enhanced the association of SREBP2 and the ABCA1 promoter in ECs. To correlate this mechanism pathophysiologically, we found that oscillatory flow caused the activation of SREBP2 and therefore attenuated ABCA1 promoter activity in ECs. Thus, this SREBP-regulated mechanism may control the efflux of cholesterol, which is a newly defined function of SREBP2 in ECs in addition to its role in cholesterol uptake and biosynthesis. ATP-binding cassette transporter A1 (ABCA1) is a pivotal regulator of cholesterol efflux from cells to apolipoproteins, whereas sterol-responsive element-binding protein 2 (SREBP2) is the key protein regulating cholesterol synthesis and uptake. We investigated the regulation of ABCA1 by SREBP2 in vascular endothelial cells (ECs). Our results showed that sterol depletion activated SREBP2 and increased its target, low density lipoprotein receptor mRNA, with a concurrent decrease in the ABCA1 mRNA. Transient transfection analysis revealed that sterol depletion decreased the ABCA1 promoter activity by 50%, but low density lipoprotein receptor promoter- and the sterol-responsive element-driven luciferase activities were increased. Overexpression of the N terminus of SREBP2 (SREBP2(N)), an active form of SREBP2, also inhibited the ABCA1 promoter activity. Functionally adenovirus-mediated SREBP2(N) expression increased cholesterol accumulation and decreased apoA-I-mediated cholesterol efflux. The conserved E-box motif was responsible for the SREBP2(N)-mediated inhibition since mutation of the E-box increased the basal activity of the ABCA1 promoter and abolished the inhibitory effect of SREBP2(N). Furthermore sterol depletion and SREBP2(N) overexpression induced the binding of SREBP2(N) to both consensus and ABCA1-specific E-box. Chromatin immunoprecipitation assay demonstrated that serum starvation enhanced the association of SREBP2 and the ABCA1 promoter in ECs. To correlate this mechanism pathophysiologically, we found that oscillatory flow caused the activation of SREBP2 and therefore attenuated ABCA1 promoter activity in ECs. Thus, this SREBP-regulated mechanism may control the efflux of cholesterol, which is a newly defined function of SREBP2 in ECs in addition to its role in cholesterol uptake and biosynthesis. Epidemiological studies have shown an inverse relationship between levels of high density lipoprotein-cholesterol and risk of coronary artery disease. High density lipoprotein promotes reverse cholesterol transport by facilitating the transfer of cholesterol from peripheral tissues to the liver for disposal (1Tall A.R. J. Clin. Investig. 1990; 86: 379-384Crossref PubMed Scopus (583) Google Scholar). ATP-binding cassette transporter A1 (ABCA1), 1The abbreviations used are: ABCA1, ATP-binding cassette transporter A1; hABCA1, human ABCA1; SRE, sterol-responsive element; SREBP, sterol-responsive element-binding protein; SREBP2(N), N terminus of SREBP2; EC, endothelial cell; LDL, low density lipoprotein; LDLR, LDL receptor; LXR, liver X receptor; RXR, retinoic X receptor; DR4, direct repeat spaced by four nucleotides; USF, upstream stimulatory factor; HUVEC, human umbilical vein endothelial cell; FBS, fetal bovine serum; SFM, serum-free medium; RT, real time; HA, hemagglutinin; β-gal, β-galactosidase; Ad, adenovirus; ChIP, chromatin immunoprecipitation; SCAP, SREBP-cleavage-activating protein; luc, luciferase.1The abbreviations used are: ABCA1, ATP-binding cassette transporter A1; hABCA1, human ABCA1; SRE, sterol-responsive element; SREBP, sterol-responsive element-binding protein; SREBP2(N), N terminus of SREBP2; EC, endothelial cell; LDL, low density lipoprotein; LDLR, LDL receptor; LXR, liver X receptor; RXR, retinoic X receptor; DR4, direct repeat spaced by four nucleotides; USF, upstream stimulatory factor; HUVEC, human umbilical vein endothelial cell; FBS, fetal bovine serum; SFM, serum-free medium; RT, real time; HA, hemagglutinin; β-gal, β-galactosidase; Ad, adenovirus; ChIP, chromatin immunoprecipitation; SCAP, SREBP-cleavage-activating protein; luc, luciferase. a 254-kDa cytoplasmic membrane protein, is a pivotal regulator of lipid efflux from cells to apolipoproteins (2Tall A.R. Wang N. J. Clin. Investig. 2000; 106: 1205-1207Crossref PubMed Scopus (101) Google Scholar). ABCA1 apparently plays an important role in reverse cholesterol transfer. Mutations in the ABCA1 gene, discovered in patients with Tangier disease, cause impaired efflux of lipids to apoA-I with a near absence of plasma high density lipoprotein (3Brooks-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. Nat. Genet. 1999; 22: 336-345Crossref PubMed Scopus (1481) Google Scholar, 4Bodzioch 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 (1328) Google Scholar, 5Rust 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 (1249) Google Scholar). Study of ABCA1 heterozygotes provides direct evidence that the impaired cholesterol efflux is associated with reduced plasma high density lipoprotein-cholesterol and increased risk of coronary artery disease (6Clee S.M. Kastelein J.J. van Dam M. Marcil M. Roomp K. Zwarts K.Y. Collins J.A. Roelants R. Tamasawa N. Stulc T. Suda T. Ceska R. Boucher B. Rondeau C. DeSouich C. Brooks-Wilson A. Molhuizen H.O. Frohlich J. Genest J. Hayden M.R. J. Clin. Investig. 2000; 106: 1263-1270Crossref PubMed Scopus (282) Google Scholar). Furthermore, under a high cholesterol diet, ABCA1 transgenic mice showed an atheroprotective lipoprotein profile with decreased atherosclerotic lesions, demonstrating the antiatherogenic effect of the ABCA1 transporter in vivo (7Joyce C.W. Amar M.J. Lambert G. Vaisman B.L. Paigen B. Najib-Fruchart J. Hoyt Jr., R.F. Neufeld E.D. Remaley A.T. Fredrickson D.S. Brewer Jr., H.B. Santamarina-Fojo S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 407-412Crossref PubMed Scopus (235) Google Scholar). ABCA1 can be regulated at both the transcriptional and post-transcriptional levels (8Schmitz G. Langmann T. Curr. Opin. Lipidol. 2001; 12: 129-140Crossref PubMed Scopus (179) Google Scholar). The most studied transcriptional regulation of the ABCA1 gene is the binding of liver X receptor (LXR)/retinoic X receptor (RXR) heterodimers to an imperfect direct repeat spaced by four nucleotides (DR4) to up-regulates the gene (9Schwartz K. Lawn R.M. Wade D.P. Biochem. Biophys. Res. Commun. 2000; 274: 794-802Crossref PubMed Scopus (374) Google Scholar). Mutation of the DR4 element strongly reduced the LXR agonist-induced ABCA1 gene activation (9Schwartz K. Lawn R.M. Wade D.P. Biochem. Biophys. Res. Commun. 2000; 274: 794-802Crossref PubMed Scopus (374) Google Scholar, 10Costet P. Luo Y. Wang N. Tall A.R. J. Biol. Chem. 2000; 275: 28240-28245Abstract Full Text Full Text PDF PubMed Scopus (841) Google Scholar). However, significant activity is still retained in DR4-deleted constructs, indicating the existence of regulation other than by LXR/RXR (11Porsch-Ozcurumez M. Langmann T. Heimerl S. Borsukova H. Kaminski W.E. Drobnik W. Honer C. Schumacher C. Schmitz G. J. Biol. Chem. 2001; 276: 12427-12433Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Although a silencing regulatory element in ABCA1 promoter has been mapped to a region containing the E-box motif (12Santamarina-Fojo S. Peterson K. Knapper C. Qiu Y. Freeman L. Cheng J.F. Osorio J. Remaley A. Yang X.P. Haudenschild C. Prades C. Chimini G. Blackmon E. Francois T. Duverger N. Rubin E.M. Rosier M. Denefle P. Fredrickson D.S. Brewer H.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7987-7992Crossref PubMed Scopus (185) Google Scholar), the transcription factors responsive for the negative regulation of the ABCA1 promoter remain elusive. Sterol-responsive element-binding proteins (SREBPs), including SREBP1a, -1c, and -2, modulate the transcription of a number of genes involved in the synthesis and receptor-mediated uptake of cholesterol and fatty acids (13Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (2918) Google Scholar, 14Horton J.D. Shimomura I. Brown M.S. Hammer R.E. Goldstein J.L. Shimano H. J. Clin. Investig. 1998; 101: 2331-2339Crossref PubMed Google Scholar, 15Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11041-11048Crossref PubMed Scopus (1085) Google Scholar). Results to date support the notion that SREBP1 primarily activates the fatty acid triglyceride and phospholipid pathways, whereas SREBP2 is the prominent isoform for cholesterol synthesis and uptake (13Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (2918) Google Scholar, 14Horton J.D. Shimomura I. Brown M.S. Hammer R.E. Goldstein J.L. Shimano H. J. Clin. Investig. 1998; 101: 2331-2339Crossref PubMed Google Scholar, 16Shimano H. Horton J.D. Hammer R.E. Shimomura I. Brown M.S. Goldstein J.L. J. Clin. Investig. 1996; 98: 1575-1584Crossref PubMed Scopus (693) Google Scholar). Containing a basic-helix-loop-helix-leucine zipper motif, the N terminus of SREBPs (SREBP(N)) mediates dimerization, nuclear entry, and DNA binding. In sterol-depleted cells, SREBPs are cleaved by proteases in Golgi, releasing the N termini, which translocate into the nucleus and bind to SREs in the enhancers of multiple genes encoding enzymes and proteins involved in cholesterol biosynthesis, unsaturated fatty acid biosynthesis, triglyceride biosynthesis, and lipid uptake (for reviews, see Refs. 15Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11041-11048Crossref PubMed Scopus (1085) Google Scholar, 16Shimano H. Horton J.D. Hammer R.E. Shimomura I. Brown M.S. Goldstein J.L. J. Clin. Investig. 1996; 98: 1575-1584Crossref PubMed Scopus (693) Google Scholar, 17Horton J.D. Shimomura I. Curr. Opin. Lipidol. 1999; 10: 143-150Crossref PubMed Scopus (268) Google Scholar). In addition to the SRE motif, SREBPs recognize several other sequences, including E-box in the promoters of certain genes (15Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11041-11048Crossref PubMed Scopus (1085) Google Scholar, 18Amemiya-Kudo M. Shimano H. Hasty A.H. Yahagi N. Yoshikawa T. Matsuzaka T. Okazaki H. Tamura Y. Iizuka Y. Ohashi K. Osuga J. Harada K. Gotoda T. Sato R. Kimura S. Ishibashi S. Yamada N. J. Lipid Res. 2002; 43: 1220-1235Abstract Full Text Full Text PDF PubMed Google Scholar). The vascular endothelium forms a barrier between the vessel wall and lipoproteins in the circulation. It plays an important role in maintaining vascular integrity. The disturbance or injury of endothelium can lead to cardiovascular impairments such as atherosclerosis. ABCA1 is expressed in vascular endothelial cells (ECs) and transcriptionally up-regulated by LDL and cholesterol, suggesting that ABCA1 in ECs plays an important role in cholesterol homeostasis in the vessel wall (19Liao H. Langmann T. Schmitz G. Zhu Y. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 127-132Crossref PubMed Scopus (52) Google Scholar). However, the regulation of ABCA1 and its role in lipid trafficking in ECs remain largely unknown. We previously reported that shear stress activated SREBPs and hence up-regulated their targeting genes in ECs (20Liu Y. Chen B.P. Lu M. Zhu Y. Stemerman M.B. Chien S. Shyy J.Y. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 76-81Crossref PubMed Scopus (125) Google Scholar, 21Lin T. Zeng L. Liu Y. DeFea K. Schwartz M.A. Chien S. Shyy J.Y. Circ. Res. 2003; 92: 1296-1304Crossref PubMed Scopus (90) Google Scholar). Given the important role of SREBP2 and ABCA1 in cholesterol homeostasis and the significance of cholesterol traffic in the vessel wall, we investigated the regulation of ABCA1 by SREBP2 in ECs. Our results showed that, by binding to the E-box, SREBP2 could inhibit ABCA1 transcription. Importantly oscillatory flow caused the activation of SREBP2 and inhibition of ABCA1 promoter activity in ECs. Thus, this SREBP-regulated mechanism controlling cholesterol efflux is a newly defined function of SREBP2 in the vascular wall. Reagents—Cholesterol was purchased from Avanti, Inc. (Alabaster, AL). [γ-32P]ATP, [α-32P]dCTP, and [3H]cholesterol were from MP Biomedicals (Irvine, CA). The DECApriming II DNA labeling kit was from Ambion (Austin, TX). All of the DNA-modifying enzymes and PCR enzymes were obtained from Promega Corp. (Madison, WI). LXR agonist TO901317 was purchased from Cayman Chemical (Ann Arbor, MI). Human apoA-I was from Biodesign International (Saco, ME). Anti-ABCA1 antibody was purchased from Novus Biologicals (Littleton, CO), and antibodies against SREBP2, upstream stimulatory factor 1 (USF1), USF2, c-Myc, Max, HA tag, and α-tubulin were from Santa Cruz Biotechnology (Santa Cruz, CA). LDL was isolated from non-frozen human plasma as described previously (19Liao H. Langmann T. Schmitz G. Zhu Y. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 127-132Crossref PubMed Scopus (52) Google Scholar). Recombinant human fibroblast growth factor was a generous gift from Dr. J. A. Thompson at the University of Alabama. All other reagents, purchased from Invitrogen or Sigma, were all of tissue culture or molecular biology grade. Cell Culture—Human umbilical vein endothelial cells (HUVECs) were isolated and maintained as described previously (22Zhu Y. Lin J.H. Liao H.L. Friedli O.J. Verna L. Marten N.W. Straus D.S. Stemerman M.B. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 473-480Crossref PubMed Scopus (72) Google Scholar). Briefly HUVECs were maintained in medium 199 supplemented with 20% fetal bovine serum (FBS), 5 ng/ml fibroblast growth factor, and 90 μg/ml heparin (EC medium). In experiments involving serum-free treatment, human endothelial serum-free medium (basal growth medium, Invitrogen) supplemented with 5 ng/ml fibroblast growth factor (SFM), was used. Bovine aortic endothelial cells were cultured under standard culture conditions. All experiments were performed with HUVECs up to passage three, and all cells were cultured to confluence before treatment. Northern Blotting, Quantitative Real Time (RT)-PCR, and Western Blotting—Total RNA isolation and Northern blotting for hABCA1, LDL receptor (LDLR), and von Willebrand factor mRNA was carried out according to standard protocols. The cDNA probes for LDLR and hABCA1 were generated via RT-PCR, and the probes were labeled with [α-32P]dCTP by DECApriming (Ambion). For quantitative RT-PCR, total RNA was converted into cDNA by using reverse transcriptase with oligo(dT) as the primer. The obtained cDNAs were then used as the templates for quantitative RT-PCR with the use of Brilliant SYBR Green QPCR Master Mix (Stratagene, La Jolla, CA). The relative amount of ABCA1 mRNAs was calculated using the comparative method with the β-actin mRNA as internal control. The nucleotide sequences of the primers were: hABCA1, 5′-GCTGCCTCCTCCACAA-AGAAAAC-3′ and 5′-GCTTTGCTGACCCGCTCCTGGATC-3′; β-actin, 5′-TGACCGGGTCACCCACACTGTGCCCATCTA-3′ and 5′-CTAGAA-GCATTTGCGGTGGACGATGGAGGG-3′. HUVECs were solubilized in a buffer containing detergent, and cellular proteins were separated by SDS-PAGE. The Western blotting analysis was performed with antibodies against ABCA1, SREBP2, SREBP1, USF1, USF2, c-Myc, Max, HA, or α-tubulin. Plasmid Construction and Transient Transfection—Plasmid pABCA1(E-boxmut)-luc was generated using the QuikChange site-directed mutagenesis kit (Stratagene) and pABCA1(–928)-luc construct (10Costet P. Luo Y. Wang N. Tall A.R. J. Biol. Chem. 2000; 275: 28240-28245Abstract Full Text Full Text PDF PubMed Scopus (841) Google Scholar) as template. The mutagenic primer for E-box mutation was: 5′-G-GGCCCCGGCTCCACGgaCTTTCTGCTGAG-3′. The sequences underlined indicate the E-box motif, and the bases in small letters are the designated mutations. Mutagenesis was performed according to the manufacturer's protocol. pCMV5-HA-SREBP2(N), the expression plasmid of the N terminus of human SREBP2, was generated as described previously (21Lin T. Zeng L. Liu Y. DeFea K. Schwartz M.A. Chien S. Shyy J.Y. Circ. Res. 2003; 92: 1296-1304Crossref PubMed Scopus (90) Google Scholar). For the promoter activation study, the reporter plasmids of the hABCA1 promoter, 4XSRE-luc, LDLR-luc, and SCAP-C (20Liu Y. Chen B.P. Lu M. Zhu Y. Stemerman M.B. Chien S. Shyy J.Y. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 76-81Crossref PubMed Scopus (125) Google Scholar), were transiently transfected into HUVECs with the use of the Targefect F2 (Targeting Systems, San Diego, CA). pRSV-β-gal was co-transfected as a transfection control. Adenovirus Construction and Infection—Recombinant adenovirus encoding Ad-HA-SREBP2(N) was created, amplified, and titered as reported previously (23Zeng L. Lu M. Mori K. Luo S. Lee A.S. Zhu Y. Shyy J.Y. EMBO J. 2004; 23: 950-958Crossref PubMed Scopus (162) Google Scholar). For adenovirus infection, the virus mixture with multiplicity of infection as indicated was added to confluent HUVECs in culture and incubated for 12 h. Ad-β-gal was used as an infection control. The infected cells were then incubated in fresh growth medium for 24 h followed by RNA or protein extraction. Assessment of Cholesterol Efflux—The cholesterol efflux was assessed as described previously (19Liao H. Langmann T. Schmitz G. Zhu Y. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 127-132Crossref PubMed Scopus (52) Google Scholar) with modification. Briefly HUVECs in 12-well plates were infected with Ad-HA-SREBP2(N) or control virus for 12 h. The cells were then treated with LXR agonist TO901317 (10 μm) in EC medium or SFM for 18 h and labeled with [3H]cholesterol (0.2 μCi/ml) for 6 h. After washing with phosphate-buffered saline-bovine serum albumin, the cells were incubated with fresh SFM with or without apoA-I (10 μg/ml) for 2 h. The aliquots of medium and cell lysates were assayed by liquid scintillation. Electrophoretic Mobility Shift Assay—Confluent HUVECs were treated with EC medium or SFM for 6 h or infected with Ad-HA-SREBP2(N) for 24 h. The cells were then lysed, and nuclear extracts were prepared. Double strand oligonucleotides containing the divergent E-box sequence in the ABCA1 promoter (5′-GGGCCCCGGCTCCACG-AGCTTTCTGCTGAG-3′) or consensus E-box oligos (Santa Cruz Biotechnology) were end-labeled with [γ-32P]ATP. Electrophoretic mobility shift assays were performed as described previously (24Zhu Y. Liao H.L. Wang N. Yuan Y. Ma K.S. Verna L. Stemerman M.B. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 2465-2470Crossref PubMed Scopus (54) Google Scholar). To test the specificity of binding, a 100-fold molar excess of unlabeled ABCA1-E-box or irrelevant AP-1 and SP-1 probes were used for competition experiments. In supershift experiments, the nuclear extracts were incubated with anti-HA antibody for 3 h on ice before the addition of the labeled probes. Chromatin Immunoprecipitation (ChIP) Assay—The ChIP assays were performed as described previously (23Zeng L. Lu M. Mori K. Luo S. Lee A.S. Zhu Y. Shyy J.Y. EMBO J. 2004; 23: 950-958Crossref PubMed Scopus (162) Google Scholar). In brief, cells were fixed with 1% formaldehyde and quenched prior to harvest and sonication. Goat anti-SREBP2 or normal goat IgG and single strand salmon sperm DNA saturated with Protein A-Sepharose 4B beads were added to sheared samples for immunoprecipitation. Immunoprecipitates were pelleted by centrifugation, and the supernatant of the control group was collected as an input control. The immunoprecipitates were eluted from Sepharose 4B beads, and proteinase K solution was added and incubated at 60 °C for 8 h. DNA was extracted, purified, and then used to amplify target sequences by PCR. The ABCA1 promoter containing the E-box element was amplified by use of primer set 5′-CTGCACCGAGC-GCAGAGGTTA-3′ and 5′-CAACTCCCTAGATGTGTCGTG-3′. Flow Experiments—The flow experiments were performed as described previously (25Chappell D.C. Varner S.E. Nerem R.M. Medford R.M. Alexander R.W. Circ. Res. 1998; 82: 532-539Crossref PubMed Scopus (464) Google Scholar). In brief, ECs were seeded on glass until confluence. Cells were then subjected to a parallel plate flow channel to impose an oscillatory flow (mean shear stress, 0 dyne/cm2; amplitude of pulsatility, ±3 dyne/cm2; 1 Hz) or a steady laminar flow (12 dyne/cm2) for 12 h. The flow system was kept at 37 °C and ventilated with 95% humidified air with 5% CO2. Statistics—Quantitative data were expressed as mean ± S.D. Statistical significance of the data was evaluated by analysis of variance or Student's t test. p values less than 0.05 were considered significant. For nonquantitative data, results were representative from at least three independent experiments. The Reciprocal Response of ABCA1 and SREBP2 Transcripts to Serum Deprivation—To study the regulation of ABCA1 in ECs under serum-free condition, we cultured confluent HUVECs in SFM in the presence or absence of LDL for 24 h. Under this condition, HUVECs had normal morphology, and no cell death was detected. As shown in Fig. 1A, SFM greatly decreased ABCA1 mRNA in HUVECs, and the inclusion of LDL in SFM reversed such an inhibition. In control experiments, cells in EC medium containing 20% FBS were exposed to LDL or cholesterol. LDL and cholesterol increased the level of ABCA1 mRNA. The level of mRNA encoding LDLR, an SREBP2 target gene, was much lower in cells in FBS than those in SFM. Supplementing SFM with LDL inhibited the expression of LDLR mRNA. Consistent with the SFM-up-regulated LDLR, SREBP2 was activated as revealed by the increased SREBP2 cleavage at 6 h after exposure to SFM and the subsequent increase in both precursor and cleavage forms of SREBP2 (Fig. 1B). Furthermore results from transient transfection assays showed that SFM decreased the luciferase reporter driven by the ABCA1 promoter by 49 ± 6% compared with cells under 20% FBS (Fig. 1C). However, SFM increased the luciferase reporter driven by the LDLR promoter or SRE by 5–6 times. Thus, ABCA1 and SREBP2 transcripts in ECs responded reciprocally to SFM. SREBP2 Inhibits the Expression of ABCA1—To ascertain whether SREBP2 up-regulation inhibits the expression of ABCA1, we compared the level of ABCA1 protein in HUVECs infected with recombinant Ad-HA-SREBP2(N) encoding the N terminus of SREBP2 with those infected with Ad-β-gal. Western blotting analysis revealed that ABCA1 in HUVECs was decreased by SREBP2(N) overexpression. This inhibitory effect was similar to that by SFM (Fig. 2A). Because ABCA1 is a pivotal regulator of cholesterol efflux from cells to apolipoproteins such as apoA-I, we explored cholesterol efflux to apoA-I as a functional consequence of the SREBP2-suppressed ABCA1. Compared with the Ad-β-gal-infected controls, the infection of Ad-HA-SREBP2(N) increased the [3H]cholesterol uptake and/or accumulation by 20 ± 8% (Fig. 2B). Furthermore cholesterol efflux in the presence of apoA-I was significantly decreased in Ad-HA-SREBP2(N)-infected cells (Fig. 2C). As an LXR target gene, ABCA1 is up-regulated by TO901317, a selective LXR agonist (26Schultz J.R. Tu H. Luk A. Repa J.J. Medina J.C. Li L. Schwendner S. Wang S. Thoolen M. Mangelsdorf D.J. Lustig K.D. Shan B. Genes Dev. 2000; 14: 2831-2838Crossref PubMed Scopus (1373) Google Scholar, 27Repa 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 (1394) Google Scholar). Treating Ad-β-gal-infected HU-VECs with TO901317 promoted the apoA-I-mediated cholesterol efflux when the cholesterol accumulation was unchanged. SREBP2 Down-regulates the ABCA1 Transcription via E-box Motif—To study whether the inhibition of ABCA1 by serum starvation or SREBP2 is at the level of transcription and to determine the involved transcriptional element within the ABCA1 promoter, we performed transient transfection assays using ABCA1 promoter-driven reporter constructs. Fig. 3A shows that overexpression of SREBP2(N) by the co-transfected pCMV5-HA-SREBP2(N) decreased the activities of promoter constructs pABCA1(–928)-luc and pABCA1(–156)-luc by 50% compared with controls transfected with vector plasmid pCMV5. Further deletion of a segment between –156 and –116 containing the E-box site, a silencer of the ABCA1 promoter (28Langmann T. Porsch-Ozcurumez M. Heimerl S. Probst M. Moehle C. Taher M. Borsukova H. Kielar D. Kaminski W.E. Dittrich-Wengenroth E. Schmitz G. J. Biol. Chem. 2002; 277: 14443-14450Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 29Yang X.P. Freeman L.A. Knapper C.L. Amar M.J. Remaley A. Brewer Jr., H.B. Santamarina-Fojo S. J. Lipid Res. 2002; 43: 297-306Abstract Full Text Full Text PDF PubMed Google Scholar), abolished the inhibitory effect of SREBP2(N). Indeed mutation of the E-box in pABCA1(–928 E-boxmut)-luc not only increased the basal activity but also reversed the inhibition of ABCA1 promoter by SREBP2(N). Furthermore we studied the effect of serum starvation on the ABCA1 promoter by incubating the various transfected HUVECs with SFM or 20% FBS. As shown in Fig. 3B, SFM and SREBP2(N) overexpression exhibited a similar pattern of regulation on ABCA1 promoter constructs. SREBP2(N) Binds to the E-box of the ABCA1 Promoter— Given the possibility that E-box is an SREBP-responsive element, which down-regulates the ABCA1 expression, we studied the binding of SRBEP2(N) to E-box by electrophoretic mobility shift assay. As shown in Fig. 4A, SREBP2(N) overexpression in ECs because of Ad-HA-SREBP2(N) infection increased the binding of nuclear extracts to the ABCA1-specific E-box compared with control cells infected with Ad-β-gal. The supershifted band that resulted from the inclusion of anti-HA antibody revealed the specific binding of SREBP2 to the ABCA1-E-box motif. Furthermore SFM treatment for 6 h increased the bindings of nuclear extracts to both consensus and ABCA1-specific E-box (Fig. 4B). Addition of LDL or cholesterol to SFM attenuated such a binding. We also performed ChIP assay to ascertain whether SREBP2(N) binds to the ABCA1 promoter in vivo. As shown in Fig. 4C, although SREBP2(N) associated with the ABCA1 promoter in cells in 20% FBS, SFM greatly enhanced the association. Thus, the inhibition of ABCA1 transcription by SREBP2(N) is most likely through the binding of SREBP2 to the E-box site in the ABCA1 promoter. Serum Starvation Does Not Increase Other E-box-related Transcription Factors—In addition to SREBPs, E-box element also binds to several other nuclear proteins, including c-Myc, Max, and c-Myc-related regulatory factors such as USF1 and USF2. To explore the possible regulation of these E-box-binding proteins, the induction of Myc, Max, USF1, and USF2 by SFM was detected in the nuclear extract. Western blotting showed that SFM increased SREBP1 and SREBP2. However, levels of nuclear c-Myc and USF2 were decreased, and those of Max and USF1 remained unchanged (Fig. 5). This result suggested that Myc, Max, or USFs were unlikely to be involved in the binding of the E-box of ABCA1 promoter, leading to the suppression of the ABCA1 in response to SFM. Flow Inhibits ABCA1 Promoter Activation in ECs—We previously reported that steady laminar flow caused a transient activation of the SREBP-regulated genes, whereas disturbed flow patterns caused a sustained activation of SREBPs and their targeting genes in ECs (20Liu Y. Chen B.P. Lu M. Zhu Y. Stemerman M.B. Chien S. Shyy J.Y. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 76-81Crossref PubMed Scopus (125) Google Scholar). To determine whether the oscillatory flow-activated SREBP2, like serum depletion, can down-regulate ABCA1, HUVECs were subjected to an oscillatory flow (0 ± 3 dyne/cm2), and the levels of ABCA1 mRNA were determined by quantitative RT-PCR. As shown in Fig. 6A, oscillatory flow indeed decreased the level of ABCA1 mRNA compared with static controls or cells subjected to laminar flow. Furthermore ECs were transiently transfected with pABCA1(–156)-luc, and the transfected cells were then subjected to different patterns of flow for luciferase induction assays. As shown in Fig. 6B, oscillatory flow decreased the ABCA1 promoter activity compared with static controls or laminar flow. The oscillatory flow-decreased ABCA1 promoter activity was reversed if E-box site was mutated or by co-transfection with SCAP-C, which encodes a truncated C terminus of SCAP and has been shown to block SREBP translocation from the endoplasmic reticulum to the Golgi (Fig. 6C) (30DeBose-Boyd R.A. Brown M.S. Li W.P. Nohturfft A. Goldstein J.L. Espenshade P.J. Cell. 1999; 99: 703-712Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). These data suggest that oscillatory flow down-regulates ABCA1 in ECs, which is mediated through the up-regulation of SREBP2. In the present study we reported that the binding of SREBP2(N) to E-box motif within the hABCA1 promoter is responsible for the repressive effect of serum deprivation and oscillatory flow on the expression of ABCA1 in ECs. Evidence supporting such a thesis includes the following. 1) The adenovirus-mediated overexpression of SREBP2(N) could bind to the E-box motif of the ABCA1 promoter, hence inhibiting its activity, and the mutation of the E-box abolished the inhibitory effect of SREBP2(N). 2) Electrophoretic mobility shift and ChIP assays showed the association of SREBP2 and the ABCA1 promoter in ECs. 3) Functionally the SREBP2-down-regulated ABCA1 increased cholesterol accumulation and decreased the apoA-I-mediated cholesterol efflux. 4) Pathophysiologically disturbed flow patterns caused sustained activation of SREBP2 (21Lin T. Zeng L. Liu Y. DeFea K. Schwartz M.A. Chien S. Shyy J.Y. Circ. Res. 2003; 92: 1296-1304Crossref PubMed Scopus (90) Google Scholar) with consequent decrease in ABCA1 expression in ECs. Thus, this SREBP-regulated mechanism controls the efflux of cholesterol, which is a newly defined function of SREBP2 in the vascular wall. Apparently ABCA1 plays an important role in reverse cholesterol transfer and atherogenic protection (2Tall A.R. Wang N. J. Clin. Investig. 2000; 106: 1205-1207Crossref PubMed Scopus (101) Google Scholar). Most in vitro experiments involving ABCA1 function and regulation have been performed with the use of cultured macrophages, fibroblasts, and hepatocytes. Because it is exposed to the lipoproteins in the circulation, the vascular endothelium plays an important role in maintaining vascular integrity. We and others reported that ABCA1 is expressed in human aortic ECs, HUVECs, porcine brain capillary ECs, and rat liver ECs (19Liao H. Langmann T. Schmitz G. Zhu Y. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 127-132Crossref PubMed Scopus (52) Google Scholar, 31Panzenboeck U. Balazs Z. Sovic A. Hrzenjak A. Levak-Frank S. Wintersperger A. Malle E. Sattler W. J. Biol. Chem. 2002; 277: 42781-42789Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 32Hoekstra M. Kruijt J.K. Van Eck M. Van Berkel T.J. J. Biol. Chem. 2003; 278: 25448-25453Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Although the dysfunction of lipid metabolism and the ensuing oxidation and deposition in vascular cells impose important pathophysiological consequences, there have been only a few documented reports on ABCA1 regulation in ECs. The most studied transcriptional regulation of ABCA1 is the binding of the LXR/RXR heterodimer to the DR4 site of the ABCA1 promoter to up-regulate the expression of the ABCA1 gene (9Schwartz K. Lawn R.M. Wade D.P. Biochem. Biophys. Res. Commun. 2000; 274: 794-802Crossref PubMed Scopus (374) Google Scholar). Similar regulation has also been observed in ECs (19Liao H. Langmann T. Schmitz G. Zhu Y. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 127-132Crossref PubMed Scopus (52) Google Scholar). The E-box of the ABCA1 promoter has been reported to be a silencing regulatory element (12Santamarina-Fojo S. Peterson K. Knapper C. Qiu Y. Freeman L. Cheng J.F. Osorio J. Remaley A. Yang X.P. Haudenschild C. Prades C. Chimini G. Blackmon E. Francois T. Duverger N. Rubin E.M. Rosier M. Denefle P. Fredrickson D.S. Brewer H.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7987-7992Crossref PubMed Scopus (185) Google Scholar, 29Yang X.P. Freeman L.A. Knapper C.L. Amar M.J. Remaley A. Brewer Jr., H.B. Santamarina-Fojo S. J. Lipid Res. 2002; 43: 297-306Abstract Full Text Full Text PDF PubMed Google Scholar) that binds to various transcription factors such as USF1, USF2, and Fra2 for the repression of the human ABCA1 promoter in murine RAW cells. However, overexpression of USF1 and USF2 enhanced activity of the wild-type proximal hABCA1 promoter. In addition, the bindings of USFs were not modulated by any known activators of ABCA1. In this report, we found that serum starvation activated SREBP2(N) bound to both consensus and the ABCA1-specific E-box. The mutation of the E-box abolished the inhibitory effect of SREBP2 in the transient transfection assay. Under the same condition, the levels of USF-1 showed little change, and USF-2 was down-regulated. Thus, SREBP-2, but not USFs, was most likely to be involved in the negative regulation of ABCA1 promoter activity in ECs. Since USFs can activate or inhibit transcription by competing with other transcriptional factors, repressors, or activators for the E-box, the possibility that SREBPs replace USFs bound to the E-box under serum-free condition could not be excluded. In sterol-depleted cells, SREBP(N) is released from membranes and then translocates into the nucleus where it binds to SREs in the promoters of multiple genes encoding enzymes for cholesterol biosynthesis, unsaturated fatty acid biosynthesis, triglyceride biosynthesis, and lipid uptake (for reviews, see Refs. 15Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11041-11048Crossref PubMed Scopus (1085) Google Scholar, 16Shimano H. Horton J.D. Hammer R.E. Shimomura I. Brown M.S. Goldstein J.L. J. Clin. Investig. 1996; 98: 1575-1584Crossref PubMed Scopus (693) Google Scholar, 17Horton J.D. Shimomura I. Curr. Opin. Lipidol. 1999; 10: 143-150Crossref PubMed Scopus (268) Google Scholar). SREBPs can bind both the SRE sequence and E-box motif in the promoters of several genes (15Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11041-11048Crossref PubMed Scopus (1085) Google Scholar). SREBP1 primarily activates the fatty acid triglyceride and phospholipid pathways, whereas SREBP2 is responsible for cholesterol synthesis and uptake. Since the major function of ABCA1 is to mediate intracellular free cholesterol efflux to apolipoproteins, we therefore focused on the regulation of ABCA1 by SREBP2. It was reported that LDL incubation decreases the expression of SREBP2 and its target genes in cultured ECs (33Rodriguez C. Raposo B. Martinez-Gonzalez J. Llorente-Cortes V. Vilahur G. Badimon L. Cardiovasc. Res. 2003; 58: 178-185Crossref PubMed Scopus (29) Google Scholar). We found that sterol depletion activated SREBP2 with the concurrent increase in LDLR and decrease in ABCA1 at the transcription level. This regulation was mimicked by the SREBP2(N) overexpression, which inhibited ABCA1 at the protein level with ensuing increase in cholesterol accumulation and decrease in apoA-I-mediated cholesterol efflux. In peripheral cells, intracellular cholesterol homeostasis is exquisitely regulated and depends on the balance between cholesterol synthesis, degradation, cholesterol ester formation, influx, and efflux (34Hayden M.R. Clee S.M. Brooks-Wilson A. Genest J. Attie A. Kastelein J.J. Curr. Opin. Lipidol. 2000; 11: 117-122Crossref PubMed Scopus (108) Google Scholar, 35Fielding C.J. Fielding P.E. J. Lipid Res. 1997; 38: 1503-1521Abstract Full Text PDF PubMed Google Scholar). In principle, the sterol deprivation-activated SREBPs serve as transcriptional factors for lipid/cholesterol synthesis, uptake, and storage. But whether SREBPs also regulate genes governing cholesterol efflux is not clear. It was postulated that caveolin functions as a regulator of cellular free cholesterol homeostasis in quiescent peripheral cells in which caveolin mediated free cholesterol efflux. Previous study by others showed that SREBPs inhibited the expression of the caveolin gene, which is contradictory to its stimulating effect on other promoters (36Bist A. Fielding P.E. Fielding C.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10693-10698Crossref PubMed Scopus (230) Google Scholar). Our result indicates that SREBP2 binds directly to the E-box of the ABCA1 promoter. Whether SREBP2 forms a complex with other transcriptional factors or the binding of SREBP2 recruits other repressors deserves further studies. Recently we found that endoplasmic reticulum stress-induced activating transcription factor 6 suppressed the SREBP-mediated transcription in glucose-deprived HepG2 cells. The attenuated transcriptional activity of SREBP was due to the recruitment of a repressor, histone deacetylase 1, to the ATF6-SREBP complex (23Zeng L. Lu M. Mori K. Luo S. Lee A.S. Zhu Y. Shyy J.Y. EMBO J. 2004; 23: 950-958Crossref PubMed Scopus (162) Google Scholar, 24Zhu Y. Liao H.L. Wang N. Yuan Y. Ma K.S. Verna L. Stemerman M.B. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 2465-2470Crossref PubMed Scopus (54) Google Scholar). Nevertheless ABCA1 down-regulation by SREBP2 is another example of the coordinated regulation of cholesterol synthesis, influx, and efflux. In vitro study suggested that SREBPs in peripheral cells were suppressed by high levels of serum or cholesterol (17Horton J.D. Shimomura I. Curr. Opin. Lipidol. 1999; 10: 143-150Crossref PubMed Scopus (268) Google Scholar). The physiological implications of the SREBP2 activation and ABCA1 down-regulation in ECs by SFM would be plausible since endothelium is exposed to full blood in vivo. We observed that oscillatory flow activated SREBPs and up-regulated their targeting genes in ECs in the presence of high levels of LDL and 25-hydroxycholesterol, an agent that is commonly used to suppress SREBP activation (20Liu Y. Chen B.P. Lu M. Zhu Y. Stemerman M.B. Chien S. Shyy J.Y. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 76-81Crossref PubMed Scopus (125) Google Scholar, 21Lin T. Zeng L. Liu Y. DeFea K. Schwartz M.A. Chien S. Shyy J.Y. Circ. Res. 2003; 92: 1296-1304Crossref PubMed Scopus (90) Google Scholar). Consistently we demonstrated in this study that oscillatory flow inhibited the expression of ABCA1 and the ABCA1 promoter activities in ECs. The block of SREBP activation by SCAP-C was able to reverse such an inhibitory effect, indicating the involvement of SREBPs. Thus, the pathophysiological implication for this result is that disturbed flow patterns, causing sustained SREBP2 activation and ABCA1 suppression, may impair the lipid/cholesterol homeostasis in the curves or branches of the arterial tree, promoting atherogenesis.
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