Sphingomyelin-dependence of cholesterol efflux mediated by ABCG1

• DOI: 10.1194/jlr.m700139-jlr200
• Publication Date: 2007-08-30T01:39:24Z
• Authors: Osamu Sano; Aya Kobayashi; Kohjiro Nagao; Keigo Kumagai; Noriyuki Kioka; Kentaro Hanada; Kazumitsu Ueda; Michinori Matsuo
• Keywords: Efflux; ABCG1

ABCG1, one of the half-type ATP binding cassette (ABC) proteins, mediates efflux cholesterol to HDL and functions in reverse transport from peripheral cells liver. We have shown that ABCG1 not only but also sphingomyelin (SM) phosphatidylcholine. Because SM preferentially associates with cholesterol, we examined whether it plays an important role ABCG1-mediated cholesterol. The mediated by was reduced a mutant CHO-K1 cell line, LY-A, which cellular level is because mutation ceramide transfer protein CERT. In contrast, overexpressing CERT showed increased ABCG1. sensitivity methyl-β-cyclodextrin suggested nonraft domains due disruption raft LY-A cells. These results suggest dependent on distribution plasma membrane. apolipoprotein A-I Chinese hamster ovary liver X receptor phosphatidylcholine scavenger class B type I Cholesterol essential body as component membranes source steroid hormones, excess toxic risk factor for arteriosclerosis. Therefore, removal important. Many proteins been reported be involved homeostasis (1.Takahashi K. Kimura Y. Nagata Yamamoto A. Matsuo M. Ueda ABC proteins: key molecules lipid homeostasis.Med. Mol. Morphol. 2005; 38: 2-12Crossref PubMed Scopus (72) Google Scholar). expression ABCA1 induced via nuclear receptors (LXR) retinoid receptor. expressed ubiquitously (PC) (apoA-I), forms pre-β-HDL (2.Lee J.Y. Parks J.S. ATP-binding transporter AI its formation.Curr. Opin. Lipidol. 16: 19-25Crossref (161) Scholar, 3.Tanaka A.R. Abe-Dohmae S. Ohnishi T. Aoki R. Morinaga G. Okuhira K-i. Ikeda Kano F. Kioka N. et al.Effects mutations first extracellular domain subcellular trafficking binding/hydrolysis.J. Biol. Chem. 2003; 278: 8815-8819Abstract Full Text PDF (110) Mutations are responsible genetic disease, Tangier characterized severe deficiency (4.Brooks-Wilson Marcil Clee S.M. Zhang L.H. Roomp Dam M.van Yu L. Brewer C. Collins J.A. Molhuizen H.O. al.Mutations ABC1 disease familial high-density lipoprotein deficiency.Nat. Genet. 1999; 22: 336-345Crossref (1481) 5.Bodzioch Orso E. Klucken J. Langmann Bottcher Diederich W. Drobnik Barlage Buchler Porsch-Ozcurumez al.The gene encoding 1 mutated disease.Nat. 347-351Crossref (1328) 6.Rust Rosier Funke H. Real Amoura Z. Piette J.C. Deleuze J.F. H.B. Duverger Denefle P. al.Tangier caused 1.Nat. 352-355Crossref (1249) lipid-free apoA-I (7.Wang Lan D. Chen Matsuura Tall transporters G1 G4 mediate lipoproteins.Proc. Natl. Acad. Sci. USA. 2004; 101: 9774-9779Crossref (870) 8.Vaughan A.M. Oram or ABCG4 act sequentially remove generate cholesterol-rich HDL.J. Lipid Res. 2006; 47: 2433-2443Abstract (225) 9.Gelissen I.C. Harris Rye K.A. Quinn Brown A.J. Kockx Cartland Packianathan Kritharides Jessup synergize export apoA-I.Arterioscler. Thromb. Vasc. 26: 534-540Crossref (334) Genetic diseases reported, chow-fed mice lacking accumulation phospholipids neutral lipids including triglyceride lung (10.Kennedy M.A. Barrera G.C. Nakamura Baldan Tarr Fishbein M.C. Frank Francone O.L. Edwards P.A. has critical mediating preventing accumulation.Cell Metab. 1: 121-131Abstract (661) 11.Baldan Vales C.S. Shimotake T.K. Hawgood Deletion transmembrane progressive pulmonary lipidosis.J. 281: 29401-29410Abstract (93) play tissues, macrophages. previously PC well (12.Kobayashi Takanezawa Hirata Shimizu Misasa Arai Efflux sphingomyelin, ABCG1.J. 1791-1802Abstract (154) form ordered microdomains (raft domains, detergent-resistant membranes) assumed influences membrane dynamics, effect change content examined, report found treating rat fibloblasts SMase apoA-I- apoE-dependent (13.Ito J-i. Nagayasu Yokoyama Cholesterol-sphingomyelin interaction apolipoprotein-mediated efflux.J. 2000; 41: 894-904Abstract But, another report, human skin fibroblasts did increase apoA-I-dependent HDL-mediated (14.Mendez Lin Wade D.P. Lawn R.M. Membrane distinct cholesterol/sphingomyelin-rich rafts ABCA1-mediated secretory pathway.J. 2001; 276: 3158-3166Abstract (172) treatment cell, effects could involved. yet clarified. cytosolic PH (phosphatidylinositol 4-phosphate pleckstrin homology) domain, coiled-coil START (steroidogenic acute regulatory protein-related transfer) (15.Hanada Kumagai Yasuda Miura Kawano Fukasawa Nishijima Molecular machinery non-vesicular ceramide.Nature. 426: 803-809Crossref (807) 16.Hanada Discovery molecular endoplasmic reticulum-to-Golgi ceramide.Mol. Cell. Biochem. 286: 23-31Crossref (64) transfers reticulum, where synthesized, Golgi, used synthesize (17.Kumagai Okemoto Kobayashi Hanada intermembrane various species ceramides.J. 280: 6488-6495Abstract (170) This impaired (CHO) misssense (18.Hanada Hara Yamaji Umeda Mammalian mutants resistant sphingomyelin-directed cytolysin. biochemical evidence complex formation LCB1 LCB2 serine palmitoyltransferase.J. 1998; 273: 33787-33794Abstract (164) line allowed us investigate reduction without accumulation. present study, changed using missense mutation, knockdown, overexpression, demonstrated correlated level. rabbit polyclonal anti-ABCG1 antibody mouse monoclonal anti-FLAG were purchased Santa Cruz. anti-vinculin (from Bacillus cereus) obtained Sigma. acquired Calbiochem. Other chemicals Sigma, Amersham Biosciences, Wako Pure Chemical Industries, Nacalai Tesque. CHO grown Ham's F-12 medium supplemented 10% (v/v) FBS 5% CO2 at 37°C. HeLaS3 Dulbecco's modified Eagle's (DMEM) When cultured sphingolipid-deficient medium, washed serum-free incubated Nutridoma-BO [Ham's (for cells) DMEM containing 1% Nutridoma-SP (Roche Biochemicals), 0.1% FBS, 10 μM sodium oleate-BSA complex, μg/ml gentamicin] given period (19.Hanada Kiso Hasegawa Fujita Ogawa Akamatsu Sphingolipids growth Restoration defective sphingoid base biosynthesis exogenous sphingolipids.J. 1992; 267: 23527-23533Abstract transfected pcDNA3.1Hygro(+)/human Scholar) inserted into NotI site pcDNA3.1/Hygro(+) (Invitrogen), pcDNA3.1(+)/FLAG-CERT (20.Kawano Efficient reticulum Golgi apparatus requires VAMP-associated protein-interacting FFAT motif CERT.J. 30279-30288Abstract (221) Scholar), LipofectAMINE (Invitrogen) according manufacturer's instructions. Forward (5′-GATCCCGCGAGAGTATCCTAAATTTTTCAAGAGAAAATTTAGGATACTCTCGCTTTTTTGGAAA-3′) (5′-AGCTTTTCCAAAAAAGCGAGAGTATCCTAAATTTTCTCTTGAAAAATTTAGGATACTCTCGCGG-3′) oligo sequences synthesized BamHI-HindIII pSilencer3.0-H1 (Ambion) vector silencing (1719-GCGAGAGTATCCTAAATTT-1737) expected silence LipofectAMINE2000. Cells subcultured 6-well plates density 1.2 × 106 cells/well. After incubation indicated period, fresh 0.02% BSA 20 HDL. amount determined after 12 h 24 described (21.Abe-Dohmae Suzuki Wada Aburatani Vance D.E. Characterization generation cAMP murine macrophage line.Biochemistry. 39: 11092-11099Crossref (100) Hojjati Jiang (22.Hojjati M.R. X-C. Rapid, specific, sensitive measurements phosphatidylcholine.J. 673-676Abstract (91) hydrolyzed bacterial SMase, followed treatments alkaline phosphatase, choline oxidase, peroxidase, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, 4-aminoantipyrine. absorbance 595 nm blue dye generated measured spectrophotometer (BIO-RAD). added contained SM, calculated subtracting amounts those medium. twice PBS, extracted n-hexane/2-propanol (3:2). above. PBS lysed lysis buffer (50 mM Tris-Cl (pH 7.5), 150 NaCl, Triton X-100) protease inhibitors [100 (p-amidinophenyl)methanesulfonyl fluoride, 2 leupeptin, aprotinin]. Samples electrophoresed SDS-polyacrylamide gel immunodetected FLAG antibody. glass coverslips fixed 4% paraformaldehyde PBS+ (phosphate-buffered saline 0.1 g/l CaCl2 MgCl26H2O), permeabilized 0.4% X-100 5 min. To diminish nonspecific antibodies, goat serum PBS+. diluted 1:500 serum, then fluorescent Alexa488-conjugated anti-rabbit IgG (Molecular Probes) h. directly viewed 63× Plan-Neofluar oil immersion objective Zeiss confocal microscope (LSM5 Pascal). 24- 96-well 5.0 0.4 104 cells/well, respectively. h, amphotericin (MβCD) 3-(4,5-dimethyl-thiazoyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) MTT solution removed. Formazan produced dissolved dimethyl sulfoxide, 570 Values presented means ± SD. Statistical significance Student's t-test. A value P < 0.05 considered statistically significant. HEK293 HDL- BSA-dependent manner examine possibility named CERT, stable transformant cDNA LY-A/CERT line. FBS-containing similar (Fig. 1A , B). However, when (16 3.2 μg/mg protein) about 64% (25 2.3 1C). total these under conditions 1D). transiently plasmid transfection, observed Western blotting cells, 2) (data shown). Immunostaining revealed mainly localized 3A B, E, F), localization affected culture Thus, affect cells.Fig. 2.Expression preincubated 40 mock-transfected At 18 lysates prepared. Cell (10 μg separated polyacrylamide electrophoresis, detected Nonspecific bands asterisk.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3.Subcellular wild-type (A, E), (B, (C, D, G, H) (A–C, E–G) plus FLAG-CERT (D, H). reacted IgG-Alexa488. Immunostained images A–D, differential interference contrast E–H.View (PPT) function analyzed presence 4). (0.49 0.15 cholesterol) took up passive diffusion, became 0.58 0.022 0.29 0.075 μg/ml, respectively, incubation. expressing further 0.95 0.066 0.43 0.099 (1.1 0.39 1.1 0.22 protein, respectively) (1.2 0.18 1.3 0.45 compared 0.28 0.60 0.080 (0.41 0.10 0.37 0.20 they 5), slightly (0.32 0.86 0.059 (0.27 0.19 0.65 0.13 respectively), whereas (1.6 0.79 1.7 0.21 (0.47 0.25 respectively). HDL-2 instead HDL, same Under conditions, neither nor significantly contents free esterified shown).Fig. 5.Efflux 6 (A) (B) during analyzed. average values three nine experiments ** 0.01; # 0.05; ## 0.01.View verify result knocked down RNA co-transfecting knockdown FLAG-tagged parallel experiment (see supplementary Fig. I). Expression suppressed 60–90% approximately 60% 6). Knockdown itself decrease impairs Next, overexpression 7). (0.84 0.30 (0.35 0.12 Co-expression (1.8 0.35 1.6 0.23 Overexpression although 29% IIA). IIB). shown) 3C, Similar III). stimulates As above, One might kills surface IVA), contain numbers membrane, consistent previous study (23.Fukasawa Itabe Takano Reduction affects enhances methyl-beta -cyclodextrin.J. 275: 34028-34034Abstract (94) Then, MβCD large difference between IVB), agreement CHO-K1, all MβCD, highly changed, more accessible reduced. this Although efflux. produces ceramide, serves modulator functions, can lowered CERT-knockdown reduced, CERT-overexpressing increased. clearly medium; however, significantly. It probably reflected less than mass effluxed, easily detectable. Furthermore, little observed, affected. confirmed interference. Moreover, brought content, stimulated (SR-BI) (24.Ji Jian B. Wang Sun Moya M.L. Phillips Rothblat G.H. Swaney J.B. Scavenger BI promotes high lipoprotein-mediated 1997; 272: 20982-20985Abstract (629) SR-BI expression. content. There several possible explanations above results. First, regulated level, distributes perinuclear (9.Gelissen intracellular macrophages, LXR activation (25.Neufeld E.B. Remaley A.T. Demosky S.J. Stonik Cooney Comly Dwyer N.K. Blanchette-Mackie Santamarina-Fojo al.Cellular transporter.J. 27584-27590Abstract (279) study. second activity would happen if interacts unlikely, FBS. third either, examined. surface. does number fourth recognizes both substrates transport. If case, molar ratios released constant apparently varied 1.5 8, suggesting simple co-transport Finally, fifth components promoted macrophages 26.Drobnik Borsukova Pfeiffer Liebisch Schutz G.J. Schindler Schmitz Apo AI/ABCA1-dependent HDL3-mediated compositionally cholesterol-based microdomains.Traffic. 2002; 3: 268-278Crossref (140) Indeed, becomes changed. data reorganizes generates loosely packed may facilitate HDL-dependent al. there no concentration (0.5 0.0 nmol/mg causes changes some specific cannot ruled out entirely. Further localizes necessary. removed Lubrol rafts, additionally redistributes removable differ (27.Vaughan lipid-depleted apolipoproteins.J. 30150-30157Abstract (219) (28.Nagao Takahashi Enhanced ApoA-I-dependent sphingomyelin-deficient cells.J. 2007; 282: 14868-14874Abstract (59) findings mechanisms behind summary, correlates SM. sphingomylin work supported Grant-in-aid Scientific Research Creative 15GS0301 Ministry Education, Culture, Sports, Science, Technology, Japan, grants Bio-oriented Technology Advancement Institution Pharmaceutical Medical Devices Agency. .pdf (.72 MB) Help pdf files