Expression of ABCG1, but Not ABCA1, Correlates with Cholesterol Release by Cerebellar Astroglia
2005; Elsevier BV; Volume: 281; Issue: 7 Linguagem: Inglês
10.1074/jbc.m508915200
ISSN1083-351X
AutoresBarbara Karten, Robert B. Campenot, Dennis E. Vance, Jean E. Vance,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoCentral nervous system lipoproteins mediate the exchange of cholesterol between cells and support synaptogenesis and neuronal growth. The primary source of lipoproteins in the brain is astroglia cells that synthesize and secrete apolipoprotein (apo) E in high density lipoprotein-like particles. Small quantities of apoA1, derived from the peripheral circulation, are also present in the brain. In addition to the direct secretion of apoE-containing lipoproteins from astroglia, glia-derived lipoproteins are thought to be formed by cholesterol efflux to extracellular apolipoproteins via ATP-binding cassette (ABC) transporters. We used cultured cerebellar murine astroglia to investigate the relationship among cholesterol availability, apoE secretion, expression of ABCA1 and ABCG1, and cholesterol efflux. In many cell types, cholesterol content, ABCA1 expression, and cholesterol efflux are closely correlated. In contrast, cholesterol enrichment of glia failed to increase ABCA1 expression, although ABCG1 expression and cholesterol efflux to apoA1 were increased. Moreover, the liver X receptor (LXR) agonist TO901317 up-regulated ABCA1 and ABCG1 expression in glia without stimulating cholesterol efflux. Larger lipoproteins were generated when glia were enriched with cholesterol, whereas treatment with the LXR agonist produced smaller particles that were eliminated when the glia were loaded with cholesterol. We also used glia from ApoE–/– mice to distinguish between direct lipoprotein secretion and the extracellular generation of lipoproteins. Our observations indicate that partially lipidated apoE, secreted directly by glia, is likely to be the major extracellular acceptor of cholesterol released from glia in a process mediated by ABCG1. Central nervous system lipoproteins mediate the exchange of cholesterol between cells and support synaptogenesis and neuronal growth. The primary source of lipoproteins in the brain is astroglia cells that synthesize and secrete apolipoprotein (apo) E in high density lipoprotein-like particles. Small quantities of apoA1, derived from the peripheral circulation, are also present in the brain. In addition to the direct secretion of apoE-containing lipoproteins from astroglia, glia-derived lipoproteins are thought to be formed by cholesterol efflux to extracellular apolipoproteins via ATP-binding cassette (ABC) transporters. We used cultured cerebellar murine astroglia to investigate the relationship among cholesterol availability, apoE secretion, expression of ABCA1 and ABCG1, and cholesterol efflux. In many cell types, cholesterol content, ABCA1 expression, and cholesterol efflux are closely correlated. In contrast, cholesterol enrichment of glia failed to increase ABCA1 expression, although ABCG1 expression and cholesterol efflux to apoA1 were increased. Moreover, the liver X receptor (LXR) agonist TO901317 up-regulated ABCA1 and ABCG1 expression in glia without stimulating cholesterol efflux. Larger lipoproteins were generated when glia were enriched with cholesterol, whereas treatment with the LXR agonist produced smaller particles that were eliminated when the glia were loaded with cholesterol. We also used glia from ApoE–/– mice to distinguish between direct lipoprotein secretion and the extracellular generation of lipoproteins. Our observations indicate that partially lipidated apoE, secreted directly by glia, is likely to be the major extracellular acceptor of cholesterol released from glia in a process mediated by ABCG1. The human brain contains 15–20% of total body cholesterol but represents only ∼5% of total body weight (1.Dietschy J.M. Turley S.D. Curr. Opin. Lipidol. 2001; 12: 105-112Crossref PubMed Scopus (739) Google Scholar). Cholesterol is a key component of all membranes, including myelin, in the central nervous system (CNS), 4The abbreviations used are: CNS, central nervous system; apo, apolipoprotein; ABC, ATP-binding cassette transporter; DMEM, Dulbecco's modified Eagle's medium; HDL, high density lipoproteins; LDL, low density lipoproteins; LXR, liver X receptor; PBS, phosphate-buffered saline; qPCR, quantitative PCR. and cholesterol is synthesized continuously, albeit at a low rate, in the adult brain (1.Dietschy J.M. Turley S.D. Curr. Opin. Lipidol. 2001; 12: 105-112Crossref PubMed Scopus (739) Google Scholar). All cholesterol in the CNS is synthesized within the CNS rather than being imported from the periphery (2.Turley S.D. Burns D.K. Rosenfeld C.R. Dietschy J.M. J. Lipid Res. 1996; 37: 1953-1961Abstract Full Text PDF PubMed Google Scholar). As a mechanism for maintaining cholesterol homeostasis in the CNS, cholesterol can be converted into the more polar 24-hydroxycholesterol, which is released into the circulation by a subset of neurons (3.Lutjohann D. Breuer O. Ahlborg G. Nennesmo I. Siden A. Diczfalusy U. Bjorkhem I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9799-9804Crossref PubMed Scopus (573) Google Scholar, 4.Lund E.G. Xie C. Kotti T. Turley S.D. Dietschy J.M. Russell D.W. J. Biol. Chem. 2003; 278: 22980-22988Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar). Because cholesterol metabolism and distribution are not uniform across all cell types of the brain, an efficient system is necessary for the transport of cholesterol, and probably other lipids, among cells of the CNS. In the CNS, as in the periphery, cholesterol exchange between cells is mediated by lipoproteins. Lipoproteins in the CNS are derived from glia (astrocytes and microglia), which synthesize and secrete apolipoprotein (apo) E and apoJ (5.LaDu M.J. Gilligan S.M. Lukens J.R. Cabana V.G. Reardon C.A. Van Eldik L.J. Holtzman D.M. J. Neurochem. 1998; 70: 2070-2081Crossref PubMed Scopus (244) Google Scholar, 6.Fagan A.M. Holtzman D.M. Munson G. Mathur T. Schneider D. Chang L.K. Getz G.S. Reardon C.A. Lukens J. Shah J.A. LaDu M.J. J. Biol. Chem. 1999; 274: 30001-30007Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 7.DeMattos R.B. Brendza R.P. Heuser J.E. Kierson M. Cirrito J.R. Fryer J. Sullivan P.M. Fagan A.M. Han X. Holtzman D.M. Neurochem. Int. 2001; 39: 415-425Crossref PubMed Scopus (132) Google Scholar) as well as apoD (8.Patel S.C. Asotra K. Patel Y.C. McConathy W.J. Patel R.C. Suresh S. Neuroreport. 1995; 6: 653-657Crossref PubMed Scopus (74) Google Scholar). Unlike the plasma, in which apoA1 is the most abundant apolipoprotein, apoE is the major apolipoprotein in the CNS (9.Koch S. Donarski N. Goetze K. Kreckel M. Stuerenburg H.J. Buhmann C. Beisiegel U. J. Lipid Res. 2001; 42: 1143-1151Abstract Full Text Full Text PDF PubMed Google Scholar, 10.Pitas R.E. Boyles J.K. Lee S.H. Foss D. Mahley R.W. Biochim. Biophys. Acta. 1987; 917: 148-161Crossref PubMed Scopus (575) Google Scholar). Glia-derived lipoproteins play important roles in the brain by enhancing synaptogenesis and synaptic efficacy (11.Ullian E.M. Sapperstein S.K. Christopherson K.S. Barres B.A. Science. 2001; 291: 657-661Crossref PubMed Scopus (1069) Google Scholar, 12.Mauch D.H. Nagler K. Schumacher S. Goritz C. Muller E.C. Otto A. Pfrieger F.W. Science. 2001; 294: 1354-1357Crossref PubMed Scopus (1271) Google Scholar, 13.Nagler K. Mauch D.H. Pfrieger F.W. J. Physiol. 2001; 533: 665-679Crossref PubMed Scopus (156) Google Scholar) and by promoting axonal growth (14.Hayashi H. Campenot R.B. Vance D.E. Vance J.E. J. Biol. Chem. 2004; 279: 14009-14015Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Glial lipoproteins have been proposed to be taken up by axons of neighboring neurons so that the lipids, particularly cholesterol, can be used in membranes during axon repair or remodeling (15.Mahley R.W. Science. 1988; 240: 622-630Crossref PubMed Scopus (3395) Google Scholar). The importance of cholesterol homeostasis in the CNS is underscored by the link between disturbances in CNS cholesterol metabolism and several neurodegenerative diseases such as Niemann-Pick disease type C and Alzheimer's disease (reviewed in Ref. 16.Vance J.E. Hayashi H. Karten B. Semin. Cell Dev. Biol. 2005; 16: 193-212Crossref PubMed Scopus (160) Google Scholar). Moreover, imbalances in cholesterol homeostasis in the CNS have been proposed to cause synaptic dysfunction (17.Koudinov A.R. Koudinova N.V. J. Neurol. Sci. 2005; 229–230: 233-240Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Glial lipoproteins are thought to be formed by two poorly characterized processes: the direct secretion of lipidated apolipoproteins, and the efflux of lipids from glial cells to lipid-free or lipid-poor extracellular apolipoproteins. Lipoprotein particles in the brain and cerebrospinal fluid are the size and density of plasma high density lipoproteins (HDLs) (reviewed in Refs. 9.Koch S. Donarski N. Goetze K. Kreckel M. Stuerenburg H.J. Buhmann C. Beisiegel U. J. Lipid Res. 2001; 42: 1143-1151Abstract Full Text Full Text PDF PubMed Google Scholar and 18.LaDu M.J. Shah J.A. Reardon C.A. Getz G.S. Bu G. Hu J. Guo L. van Eldick L.J. J. Biol. Chem. 2000; 275: 33974-33980Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Whereas all apoE in the CNS is derived from cells within the CNS, some plasma apoA1 crosses the blood-brain barrier by an unknown mechanism (19.Pitas R.E. Boyles J.K. Lee S.H. Hui D. Weisgraber K.H. J. Biol. Chem. 1987; 262: 14352-14360Abstract Full Text PDF PubMed Google Scholar) and is incorporated into CNS lipoproteins. ApoA1 is also synthesized by endothelial cells of the blood-brain barrier (20.Mockel B. Zinke H. Flach R. Weiss B. Weiler-Guttler H. Gassen H.G. J. Neurochem. 1994; 62: 788-798Crossref PubMed Scopus (69) Google Scholar, 21.Panzenboeck 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 (163) Google Scholar). Nascent discoidal lipoproteins are transformed into spherical particles in cerebrospinal fluid by the action of lecithin: cholesterol acyltransferase (5.LaDu M.J. Gilligan S.M. Lukens J.R. Cabana V.G. Reardon C.A. Van Eldik L.J. Holtzman D.M. J. Neurochem. 1998; 70: 2070-2081Crossref PubMed Scopus (244) Google Scholar). Lipid efflux from cells to apolipoproteins is thought to be mediated by members of the ATP-binding cassette (ABC) transporter family. During HDL formation in the plasma ABCA1 is required for transferring phospholipids and/or cholesterol to lipid-free, or lipid-poor, apoA1 (reviewed in Ref. 22.Oram J.F. Lawn R.M. J. Lipid Res. 2001; 42: 1173-1179Abstract Full Text Full Text PDF PubMed Google Scholar), but the role of ABC transporters in brain lipid metabolism is incompletely understood. The expression of ABCA1 in several cell types increases in response to an increased cellular concentration of cholesterol and/or oxysterols through activation of the transcription factor, liver X receptor (LXR) (reviewed in Ref. 22.Oram J.F. Lawn R.M. J. Lipid Res. 2001; 42: 1173-1179Abstract Full Text Full Text PDF PubMed Google Scholar). Other ABC transporters, such as the half-transporter ABCG1, have also been proposed to play a role in the generation of plasma lipoproteins (reviewed in Refs. 23.Schmitz G. Langmann T. Heimerl S. J. Lipid Res. 2001; 42: 1513-1520Abstract Full Text Full Text PDF PubMed Google Scholar and 24.Ito T. Drug News Perspect. 2003; 16: 490-492Crossref PubMed Scopus (18) Google Scholar). Recent studies on the mechanism of formation of plasma HDL indicate that ABCA1 mediates an initial lipidation of lipid-poor or lipid-free apoA1, whereas ABCG1 is responsible for the further lipidation of particles that have been partially lipidated by ABCA1 (25.Wang N. Lan D. Chen W. Matsuura F. Tall A.R. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9774-9779Crossref PubMed Scopus (889) Google Scholar, 26.Nakamura K. Kennedy M.A. Baldan A. Bojanic D.D. Lyons K. Edwards P.A. J. Biol. Chem. 2004; 279: 45980-45989Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 27.Kennedy M.A. Barrera G.C. Nakamura K. Baldan A. Tarr P. Fishbein M.C. Frank J. Francone O.L. Edwards P.A. Cell Metab. 2005; 1: 121-131Abstract Full Text Full Text PDF PubMed Scopus (688) Google Scholar). Because both ABCA1 and ABCG1 are expressed in the brain, a key determinant of the lipoprotein population in the CNS is likely to be the lipidation of apoE (synthesized by glia) and apoA1 (derived from the plasma) mediated by one or more of these transporters. Recently, the secretion and lipidation of apoE by astrocytes were shown to depend, at least in part, on ABCA1 (28.Hirsch-Reinshagen V. Zhou S. Burgess B.L. Bernier L. McIsaac S.A. Chan J.Y. Tansley G.H. Cohn J.S. Hayden M.R. Wellington C.L. J. Biol. Chem. 2004; 279: 41197-41207Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 29.Wahrle S.E. Jiang H. Parsadanian M. Legleiter J. Han X. Fryer J.D. Kowalewski T. Holtzman D.M. J. Biol. Chem. 2004; 279: 40987-40993Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). However, these studies revealed that astrocytes and microglia also operate pathways for the release of cholesterol to apoE that are independent of ABCA1 (28.Hirsch-Reinshagen V. Zhou S. Burgess B.L. Bernier L. McIsaac S.A. Chan J.Y. Tansley G.H. Cohn J.S. Hayden M.R. Wellington C.L. J. Biol. Chem. 2004; 279: 41197-41207Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar). The currently emerging model for the formation of plasma HDL is that apoA1 and apoE bind to ABCA1 (30.Oram J.F. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 720-727Crossref PubMed Scopus (214) Google Scholar, 31.Lee J.Y. Parks J.S. Curr. Opin. Lipidol. 2005; 16: 19-25Crossref PubMed Scopus (165) Google Scholar, 32.Remaley A.T. Stonik J.A. Demosky S.J. Neufeld E.B. Bocharov A.V. Vishnyakova T.G. Eggerman T.L. Patterson A.P. Duverger N.J. Santamarina-Fojo S. Brewer Jr., H.B. Biochem. Biophys. Res. Commun. 2001; 280: 818-823Crossref PubMed Scopus (279) Google Scholar, 33.Krimbou L. Denis M. Haidar B. Carrier M. Marcil M. Genest Jr., J. J. Lipid Res. 2004; 45: 839-848Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), most likely via a direct protein-protein interaction that results in the transfer of phospholipids and cholesterol from cells to the acceptor apolipoprotein. Once apoE associates with lipids, however, the ability of ABCA1 to interact with apoE is reduced (33.Krimbou L. Denis M. Haidar B. Carrier M. Marcil M. Genest Jr., J. J. Lipid Res. 2004; 45: 839-848Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). These findings support the idea that ABCA1 is responsible for the initial lipidation of apoA1/apoE, whereas another mechanism, perhaps involving ABCG1, provides additional lipids for complete lipidation of the particle. To gain a better understanding of the mechanisms involved in lipoprotein formation in the CNS, we have investigated the regulation of expression of three key components of lipoprotein formation by cerebellar astroglia, apoE, ABCA1, and ABCG1, in relation to cholesterol release. Our data demonstrate that the expression of apoE, ABCA1, and ABCG1 in astroglia is differentially regulated by cholesterol and that cholesterol efflux from cerebellar astroglia, in contrast to peripheral cells, does not correlate with the expression of ABCA1. Materials—Dulbecco's modified Eagle's medium (DMEM) and phospholipase C (from Clostridium welchii) were purchased from Sigma. DNase I was from Cedarlane (Hornby, Ontario, Canada). All other materials for cell culture and PCR were from Invitrogen. Cholesterol was purchased from Sigma. Supplies for polyacrylamide gel electrophoresis and immunoblotting were from Bio-Rad (Mississauga, Ontario, Canada). The LXR agonist TO901317 was from Key Organics (Camelford, UK). The inhibitor of acyl-CoA:cholesterol acyltransferase Sandoz 58-035 was purchased from Sigma. Nile Red was from Invitrogen Molecular Probes (Burlington, Ontario, Canada). [1-14C]Acetic acid (57 mCi/mmol) was from Amersham Biosciences. Silica gel G60 thin-layer chromatography plates were from Merck (Darmstadt, Germany). A rabbit anti-human ABCA1 antibody, that recognizes mouse ABCA1, was from Novus Biologicals (Littleton, CO) and the goat anti-human apoE antibody that recognizes mouse apoE was from Biodesign (Saco, ME). A rabbit polyclonal anti-dog calnexin antibody was obtained from Stressgen (Victoria, British Columbia, Canada). Human apoA1 and human low density lipoproteins (LDLs) were isolated from plasma of healthy volunteers and were a generous gift from Dr. G. Francis (University of Alberta). Cell Culture—Primary cerebellar astroglia were cultured as described previously (34.Karten B. Hayashi H. Francis G.A. Campenot R.B. Vance D.E. Vance J.E. Biochem. J. 2005; 387: 779-788Crossref PubMed Scopus (36) Google Scholar). Briefly, cerebella were dissected from 1- to 2-day-old Balb/cCr/AltBM or B6.129P2-Apoetm1Unc mice (stock 002052, Jackson Laboratories, Bar Harbor, ME). Meninges and surface blood vessels were removed and discarded, and then the tissue was finely chopped, briefly digested with trypsin and DNase I, and dissociated by gentle trituration through a Pasteur pipette. Cells were plated in DMEM containing 10% fetal bovine serum at a density of one cerebellum per 25-mm2 flask. Upon reaching confluence (∼7 days after plating) the cells were washed three times with PBS, trypsinized, and replated at a density of 1:3. All experiments were performed with confluent cells. Under these conditions, 90–95% of cells were astroglia as assessed by immunoreactivity of glial fibrillary acidic protein (34.Karten B. Hayashi H. Francis G.A. Campenot R.B. Vance D.E. Vance J.E. Biochem. J. 2005; 387: 779-788Crossref PubMed Scopus (36) Google Scholar). All procedures were approved by the Health Sciences Animal Welfare Committee of the University of Alberta. Immunoblotting—Glial cells were scraped into PBS then pelleted by centrifugation at 16,500 × g for 2 min. Cell pellets were resuspended in buffer containing 10 mm Tris/HCl (pH 7.4), 1 mm phenylmethylsulfonyl fluoride, and a protease inhibitor mixture (Complete Mini, Roche Diagnostics, Mannheim, Germany) and sonicated for 3 s. For immunoblotting of ABCA1, proteins were resolved on 7% polyacrylamide gels containing 0.1% SDS under reducing conditions, and then transferred to polyvinylidene difluoride membranes. The membranes were probed with rabbit anti-human ABCA1 antibody (dilution 1:1,000). Immunoreactive proteins were detected by reaction with peroxidase-conjugated goat anti-rabbit IgG (dilution 1:10,000) and visualized with ECL reagent (ECL Western blotting System, Amersham Biosciences). Immunoblotting for apoE was performed after separation of proteins on 12% polyacrylamide gels containing 0.1% SDS. The membranes were probed with goat anti-human apoE antibody (dilution 1:2,500), and immunoreactive proteins were detected by reaction with peroxidase-conjugated donkey anti-goat IgG (dilution 1:10,000) and ECL reagent. Isolation of Glia-conditioned Medium—Confluent glia were incubated with or without LDL (0.1 mg of protein/ml) in serum-free DMEM for 24 h, then washed three times with PBS. Serum-free DMEM, containing or lacking 10 μg/ml apoA1, was added, and the cells were incubated for a further 3 days. Medium was collected and centrifuged for 10 min at 1000 × g to remove cell debris. The supernatant (glia-conditioned medium) was used for immunoblotting, cholesterol analysis by gas-liquid chromatography, and fast protein liquid chromatography over a gel-filtration column, as indicated. Efflux of Endogenously Synthesized Cholesterol—Upon reaching ∼60% confluency, glial cells were incubated with 1 μCi/ml [14C]sodium acetate in DMEM containing 5% fetal bovine serum for ∼3 days until confluency was attained. The cellular concentration of cholesterol was then raised by incubation of glia for 24 h in serum-free DMEM with or without 30 μg/ml cholesterol (added from a 10 mg/ml stock solution in ethanol) in the absence of radiolabel to minimize the presence of radiolabeled cholesterol precursors. In some experiments, glia were incubated with the LXR agonist TO901317 (2 μm) for 24 h. The cells were washed three times with PBS then incubated in serum-free DMEM, with or without 10 μg/ml apoA1, for 24 h. Medium was collected and centrifuged for 10 min at 1000 × g to remove cell debris. Cells were washed three times with PBS, and cellular lipids were extracted into hexane/isopropanol (3:2, v/v). Cellular protein was dissolved in 0.3 n NaOH and analyzed using the BCA protein assay (Pierce). Lipid Analyses—Medium from radiolabeling experiments was extracted twice with hexane/isopropanol (3:2, v/v). Lipid extracts from medium and cells were separated by thin-layer chromatography in heptane/di-isopropyl ether/acetic acid, 65:35:4 (v/v). The band corresponding to unesterified cholesterol was scraped, and radioactivity was measured. Lipids in the medium from experiments without radioactivity were extracted twice with hexane. The lipid extract was dried under a stream of nitrogen then silylated with N,O-bis[trimethylsilyl]trifluoroacetamide/1% trimethylsilane in acetone and analyzed by gas-liquid chromatography using 5-α-cholestane as internal standard. Cellular lipids were incubated with phospholipase C to hydrolyze the phospholipids, extracted into hexane/diethyl ether (2:1), silylated with N,O-bis[trimethylsilyl]trifluoroacetamide/1% trimethylsilane in acetone, and analyzed by gas-liquid chromatography. Visualization of Neutral Lipids with Nile Red—Glia were grown to confluency in DMEM containing 10% fetal bovine serum. The cells were then incubated in serum-free DMEM with or without 30 μg/ml cholesterol for 24 h, washed three times with PBS, and fixed for 15 min in 3% (w/v) paraformaldehyde in PBS. The cells were permeabilized by treatment for 20 min with 50 μg/ml saponin in PBS and then stained with Nile Red (0.1 μg/ml in PBS) for 10 min. The cells were washed and examined in a Leica DM IRE2 digital microscope (Leica Microsystems, Wetzlar, Germany) equipped with an N PLAN L 20×/0.40 objective and a Leica ebq100 fluorescence lamp. Lipoprotein Size—The size of glia-derived lipoprotein particles was evaluated by fast protein liquid chromatography. Glia-conditioned medium was concentrated 50-fold using an Amicon Ultra Filter (30-kDa molecular mass cut-off) and applied to a Superose 6 gel filtration column (Amersham Biosciences) attached to a Beckman Systems Gold or Nouveau Gold apparatus. The cholesterol content of the eluate was monitored by an in-line detection assay (Infinity Cholesterol Reagent, Sigma). RNA Isolation and Real-time qPCR—Total RNA was isolated from cultured glia by extraction with TRIzol (Invitrogen). cDNA was synthesized from 1.5 μg of total RNA using oligo(dT)12–18 random primers and Superscript II reverse transcriptase (Invitrogen) according to the manufacturer's instructions. Real-time qPCR was performed using Platinum® Quantitation PCR supermix (Invitrogen), SYBR Green I (Molecular Probes), and intron-spanning, gene-specific oligonucleotides (250 nm of each primer) in a total volume of 25 μl. Transcripts were detected by real-time qPCR with a Rotor-Gene 3000 instrument (Montreal Biotech, Montreal, Quebec, Canada). Data were analyzed using the Rotor-Gene 6.0.19 program. A standard curve was used to calculate mRNA level relative to that of a control gene, cyclophilin. The specificity of products was confirmed by agarose gel electrophoresis and sequence analysis. All primers were synthesized at the DNA Core Facility of the University of Alberta with sequences as follows: cyclophilin, 5′-TCC AAA GAC AGC AGA AAA CTT TCG (sense), 5′-TCT TCT TGC TGG TCT TGC CAT TCC (antisense); ABCA1, 5′-TTG GAT GGA TTA GAT TGG AC (sense), 5′-ATG CCT GTG AAC ACG ATG (antisense); ABCG1, 5′-TGA CAC ATC TGC GAA TCA C (sense), 5′-AGG GGA AAG GTC AGA ACA (antisense). Determination of the Cellular Distribution of ABCA1—The partitioning of ABCA1 between the cell surface and intracellular membranes was assessed by biotinylation of surface proteins. Confluent glia were incubated with 30 μg/ml cholesterol or 2 μm TO901317 in serum-free DMEM for 24 h. Surface proteins were biotinylated using the Cell Surface Protein Biotinylation and Purification Kit from Pierce. Briefly, surface proteins were biotinylated then cells were harvested and lysed. Biotinylated proteins were isolated by passage of the cell lysate over a streptavidin column. Proteins that bound to the streptavidin column (i.e. biotinylated surface proteins) and proteins that did not bind to the streptavidin column (i.e. non-plasma membrane proteins) were separated by denaturing gel electrophoresis and analyzed for ABCA1 by immunoblotting. Calnexin was used as a negative control to demonstrate the lack of contamination of the cell-surface fraction with intracellular proteins. Statistical Analyses—Statistical significance of differences (p < 0.05) was determined by the Student's t test. Cholesterol Loading of Astroglia Increases Cholesterol Efflux to Exogenous ApoA1—Astroglia release cholesterol in the form of apoE-containing lipoproteins in the absence of any exogenously added apolipoprotein acceptor. Astroglia can also release cholesterol to extracellular acceptors such as apoA1 in a process mediated by ABC transporters (28.Hirsch-Reinshagen V. Zhou S. Burgess B.L. Bernier L. McIsaac S.A. Chan J.Y. Tansley G.H. Cohn J.S. Hayden M.R. Wellington C.L. J. Biol. Chem. 2004; 279: 41197-41207Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 29.Wahrle S.E. Jiang H. Parsadanian M. Legleiter J. Han X. Fryer J.D. Kowalewski T. Holtzman D.M. J. Biol. Chem. 2004; 279: 40987-40993Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). To examine cholesterol efflux mediated by ABC transporters, we measured cholesterol output into the medium of cerebellar glial cells in the absence and presence of apoA1. We also investigated cholesterol efflux from glia in which the intracellular cholesterol content had been raised by pre-treatment with cholesterol, because cholesterol loading of fibroblasts is known to increase ABCA1 expression (35.Langmann 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, 36.Klucken 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). Using this protocol for enrichment of glia with cholesterol, the cholesterol content of the glia increased from 39.9 ± 7.2 μg/mg of protein to 115.5 ± 6.2 μg/mg of protein. As shown in Fig. 1A, the efflux of cholesterol synthesized endogenously from [14C]acetate was not increased by the addition of apoA1 alone, or by cholesterol loading alone. However, when glia were enriched with cholesterol, apoA1 promoted [14C]cholesterol efflux. We also measured the mass of cholesterol in the culture medium, because the amount of radiolabeled cholesterol in the medium does not necessarily reflect cholesterol mass. In these experiments, as an alternative method for increasing the cholesterol content, glia were preincubated for 24 h in serum-free medium containing low density lipoproteins (LDL), which increased the cholesterol content of the glia from 39.9 ± 7.2 μg/mg of protein to 57.3 ± 6.0 μg/mg of protein. The cholesterol-enriched cells were incubated for 3 days in serum- and LDL-free medium, containing or lacking apoA1, and the amount of cholesterol in cells and medium was determined (Fig. 1B). A longer conditioning period, compared with that used for the experiments in Fig. 1A, was necessary to attain sufficient cholesterol concentrations in the medium for analysis by gas-liquid chromatography. In agreement with the data shown in Fig. 1A, the release of cholesterol was enhanced only when the cholesterol content of the cells was increased and, in addition, apoA1 was added exogenously (Fig. 1B). The amount of apoE in the medium was not changed by these treatments (Fig. 1C). Cholesterol Efflux to Exogenous ApoA1 Is Not Increased by the LXR Agonist TO901317—LXR agonists increase the expression of ABCA1 and other ABC transporters in fibroblasts and macrophages (37.Schwartz K. Lawn R.M. Wade D.P. Biochem. Biophys. Res. 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Surprisingly, treatment of glia with TO901317 did not increase the efflux of either radiolabeled cholesterol (Fig. 2A), or cholesterol mass (Fig. 2B). Nor did a combination of apoA1 and TO90137 increase cholesterol efflux (Fig. 2, A and B). Fig. 2C shows that apoE secretion was independent of the presence of apoA1, whereas the LXR agonist (2 μm) did increase apoE secretion. Treatment of glia with a higher concentration of TO901317 (10 μm) also failed to stimulate cholesterol efflux but increased apoE secretion to a greater extent than did 2 μm TO90137 (not shown). Thus, in cerebellar glia, in contrast to other types of cells, the LXR agonist TO
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