Serum amyloid A generates high density lipoprotein with cellular lipid in an ABCA1- or ABCA7-dependent manner
2006; Elsevier BV; Volume: 47; Issue: 7 Linguagem: Inglês
10.1194/jlr.m600145-jlr200
ISSN1539-7262
AutoresSumiko Abe-Dohmae, Koichi H. Kato, Yoshitaka Kumon, Wei Hu, Hideaki Ishigami, Noriyuki Iwamoto, Mitsuyo Okazaki, Chen-Ai Wu, Maki Tsujita, Kazumitsu Ueda, Shinji Yokoyama,
Tópico(s)Lipid metabolism and biosynthesis
ResumoSerum amyloid A (SAA) is an amphiphilic helical protein that is found associated with plasma HDL in various pathological conditions, such as acute or chronic inflammation. Cellular lipid release and generation of HDL by this protein were investigated, in comparison with the reactions by apolipoprotein A-I (apoA-I) and several types of cells that appear with various specific profiles of cholesterol and phospholipid release. SAA mediated cellular lipid release from these cells with the same profile as apoA-I. Upregulation of cellular ABCA1 protein by liver X receptor/retinoid X receptor agonists resulted in an increase of cellular lipid release by apoA-I and SAA. SAA reacted with the HEK293-derived clones that stably express human ABCA1 (293/2c) or ABCA7 (293/6c) to generate cholesterol-containing HDL in a similar manner to apoA-I. Dibutyryl cyclic AMP and phorbol 12-myristate 13-acetate, which differentiate apoA-I-mediated cellular lipid release between 293/2c and 293/6c, also exhibited the same differential effects on the SAA-mediated reactions. No evidence was found for the ABCA1/ABCA7-independent lipid release by SAA. Characterization of physicochemical properties of the HDL revealed that SAA-generated HDL particles had higher density, larger diameter, and slower electrophoretic mobility than those generated by apoA-I. These results demonstrate that SAA generates cholesterol-containing HDL directly with cellular lipid and that the reaction is mediated by ABCA1 and ABCA7. Serum amyloid A (SAA) is an amphiphilic helical protein that is found associated with plasma HDL in various pathological conditions, such as acute or chronic inflammation. Cellular lipid release and generation of HDL by this protein were investigated, in comparison with the reactions by apolipoprotein A-I (apoA-I) and several types of cells that appear with various specific profiles of cholesterol and phospholipid release. SAA mediated cellular lipid release from these cells with the same profile as apoA-I. Upregulation of cellular ABCA1 protein by liver X receptor/retinoid X receptor agonists resulted in an increase of cellular lipid release by apoA-I and SAA. SAA reacted with the HEK293-derived clones that stably express human ABCA1 (293/2c) or ABCA7 (293/6c) to generate cholesterol-containing HDL in a similar manner to apoA-I. Dibutyryl cyclic AMP and phorbol 12-myristate 13-acetate, which differentiate apoA-I-mediated cellular lipid release between 293/2c and 293/6c, also exhibited the same differential effects on the SAA-mediated reactions. No evidence was found for the ABCA1/ABCA7-independent lipid release by SAA. Characterization of physicochemical properties of the HDL revealed that SAA-generated HDL particles had higher density, larger diameter, and slower electrophoretic mobility than those generated by apoA-I. These results demonstrate that SAA generates cholesterol-containing HDL directly with cellular lipid and that the reaction is mediated by ABCA1 and ABCA7. Serum amyloid A (SAA) is a protein family that comprises acute-phase and constitutive members, both of which are synthesized mainly in the liver and the former is in reaction to the inflammatory status (1Uhlar C.M. Whitehead A.S. Serum amyloid A, the major vertebrate acute-phase reactant.Eur. J. Biochem. 1999; 265: 501-523Crossref PubMed Scopus (877) Google Scholar). Although constitutive SAA (e.g., SAA4 in human) is identified only in human and mouse, acute-phase SAA (e.g., SAA1 and SAA2 in human) is found in all of the vertebrates investigated and is highly conserved across evolutionarily distinct species (1Uhlar C.M. Whitehead A.S. Serum amyloid A, the major vertebrate acute-phase reactant.Eur. J. Biochem. 1999; 265: 501-523Crossref PubMed Scopus (877) Google Scholar). Acute-phase SAA is a major acute-phase reactant, showing up to 1,000-fold increase in human plasma during inflammation (2Kushner I. The phenomenon of the acute phase response.Ann. N. Y. Acad. Sci. 1982; 389: 39-48Crossref PubMed Scopus (1158) Google Scholar). Increase of this protein is also found with chronic inflammatory diseases. Acute-phase SAA is experimentally induced in mice by intraperitoneal injection of lipopolysaccharide (LPS), a potent endotoxin in septic shock (3Cabana V.G. Reardon C.A. Wei B. Lukens J.R. Getz G.S. SAA-only HDL formed during the acute phase response in apoA-I+/+ and apoA-I−/− mice.J. Lipid Res. 1999; 40: 1090-1103Abstract Full Text Full Text PDF PubMed Google Scholar). SAA has a multisegment structure of amphiphilic helix that is very similar to helical apolipoprotein (4Turnell W. Sarra R. Glover I.D. Baum J.O. Caspi D. Baltz M.L. Pepys M.B. Secondary structure prediction of human SAA1. Presumptive identification of calcium and lipid binding sites.Mol. Biol. Med. 1986; 3: 387-407PubMed Google Scholar) and is found associated with HDL, especially HDL3, in the circulation (5Coetzee G.A. Strachan A.F. van der Westhuyzen D.R. Hoppe H.C. Jeenah M.S. de Beer F.C. Serum amyloid A-containing human high density lipoprotein 3. Density, size, and apolipoprotein composition.J. Biol. Chem. 1986; 261: 9644-9651Abstract Full Text PDF PubMed Google Scholar).SAA may play an antipathogenic role in inflammatory processes as it binds to outer membrane protein A of Gram-negative bacteria (6Hari-Dass R. Shah C. Meyer D.J. Raynes J.G. Serum amyloid A protein binds to outer membrane protein A of Gram-negative bacteria.J. Biol. Chem. 2005; 280: 18562-18567Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). However, it could also be proinflammatory. LPS binds the scavenger receptor class B type I (SR-BI) and is internalized to stimulate cells to produce cytokines such as tumor necrosis factor-α (TNF-α), interleukin(IL)-1α, IL-1β, IL-6, and IL-8, IL-1ra, and soluble tumor necrosis factor receptor-II (TNFR-II) (7Patel H. Fellowes R. Coade S. Woo P. Human serum amyloid A has cytokine-like properties.Scand. J. Immunol. 1998; 48: 410-418Crossref PubMed Scopus (143) Google Scholar, 8Vishnyakova T.G. Bocharov A.V. Baranova I.N. Chen Z. Remaley A.T. Csako G. Eggerman T.L. Patterson A.P. Binding and internalization of lipopolysaccharide by Cla-1, a human orthologue of rodent scavenger receptor B1.J. Biol. Chem. 2003; 278: 22771-22780Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 9Bocharov A.V. Baranova I.N. Vishnyakova T.G. Remaley A.T. Csako G. Thomas F. Patterson A.P. Eggerman T.L. Targeting of scavenger receptor class B type I by synthetic amphipathic alpha-helical-containing peptides blocks lipopolysaccharide (LPS) uptake and LPS-induced pro-inflammatory cytokine responses in THP-1 monocyte cells.J. Biol. Chem. 2004; 279: 36072-36082Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), and SAA itself may also contribute to the stimulation of cells via SR-BI to produce cytokines such as IL-1β, IL-8, IL-1ra, and soluble TNFR-II but not TNFα or IL-6, although to a lesser extent than LPS (7Patel H. Fellowes R. Coade S. Woo P. Human serum amyloid A has cytokine-like properties.Scand. J. Immunol. 1998; 48: 410-418Crossref PubMed Scopus (143) Google Scholar, 10Baranova I.N. Vishnyakova T.G. Bocharov A.V. Kurlander R. Chen Z. Kimelman M.L. Remaley A.T. Csako G. Thomas F. Eggerman T.L. et al.Serum amyloid A binding to CLA-1 (CD36 and LIMPII analogous-1) mediates serum amyloid A protein-induced activation of ERK1/2 and p38 mitogen-activated protein kinases.J. Biol. Chem. 2005; 280: 8031-8040Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). In addition, SAA is a precursor of amyloid A protein, the major component of amyloid fibrils in secondary amyloidosis (11Levin M. Franklin E.C. Frangione B. Pras M. The amino acid sequence of a major nonimmunoglobulin component of some amyloid fibrils.J. Clin. Invest. 1972; 51: 2773-2776Crossref PubMed Scopus (268) Google Scholar, 12Levin M. Pras M. Franklin E.C. Immunologic studies of the major nonimmunoglobulin protein of amyloid. I. Identification and partial characterization of a related serum component.J. Exp. Med. 1973; 138: 373-380Crossref PubMed Scopus (144) Google Scholar).A more complicated argument is that SAA may modulate the effect of LPS through HDL metabolism. The effect of LPS is blocked by physiological ligands of SR-BI, HDL, apolipoprotein A-I (apoA-I), and amphiphilic α-helical peptides (9Bocharov A.V. Baranova I.N. Vishnyakova T.G. Remaley A.T. Csako G. Thomas F. Patterson A.P. Eggerman T.L. Targeting of scavenger receptor class B type I by synthetic amphipathic alpha-helical-containing peptides blocks lipopolysaccharide (LPS) uptake and LPS-induced pro-inflammatory cytokine responses in THP-1 monocyte cells.J. Biol. Chem. 2004; 279: 36072-36082Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). As the HDL containing SAA competes with normal HDL for SR-BI binding (10Baranova I.N. Vishnyakova T.G. Bocharov A.V. Kurlander R. Chen Z. Kimelman M.L. Remaley A.T. Csako G. Thomas F. Eggerman T.L. et al.Serum amyloid A binding to CLA-1 (CD36 and LIMPII analogous-1) mediates serum amyloid A protein-induced activation of ERK1/2 and p38 mitogen-activated protein kinases.J. Biol. Chem. 2005; 280: 8031-8040Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 13Cai L. de Beer M.C. de Beer F.C. van der Westhuyzen D.R. Serum amyloid A is a ligand for scavenger receptor class B type I and inhibits high density lipoprotein binding and selective lipid uptake.J. Biol. Chem. 2005; 280: 2954-2961Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), an increase of such HDL may interfere with the effect of LPS. Infusion of HDL protected mice from the LPS-induced endotoxin shock status (14Levine D.M. Parker T.S. Donnelly T.M. Walsh A. Rubin A.L. In vivo protection against endotoxin by plasma high density lipoprotein.Proc. Natl. Acad. Sci. USA. 1993; 90: 12040-12044Crossref PubMed Scopus (424) Google Scholar), which seems to be consistent with the view that HDL traps LPS from direct interaction with inflammatory cells in the interstitial space (15Ulevitch R.J. Johnston A.R. Weinstein D.B. New function for high density lipoproteins. Their participation in intravascular reactions of bacterial lipopolysaccharides.J. Clin. Invest. 1979; 64: 1516-1524Crossref PubMed Scopus (217) Google Scholar, 16Cavaillon J.M. Fitting C. Haeffner-Cavaillon N. Kirsch S.J. Warren H.S. Cytokine response by monocytes and macrophages to free and lipoprotein-bound lipopolysaccharide.Infect. Immun. 1990; 58: 2375-2382Crossref PubMed Google Scholar). HDL particles containing SAA are different from regular apoA-I-containing HDLs in their physicochemical properties (3Cabana V.G. Reardon C.A. Wei B. Lukens J.R. Getz G.S. SAA-only HDL formed during the acute phase response in apoA-I+/+ and apoA-I−/− mice.J. Lipid Res. 1999; 40: 1090-1103Abstract Full Text Full Text PDF PubMed Google Scholar, 5Coetzee G.A. Strachan A.F. van der Westhuyzen D.R. Hoppe H.C. Jeenah M.S. de Beer F.C. Serum amyloid A-containing human high density lipoprotein 3. Density, size, and apolipoprotein composition.J. Biol. Chem. 1986; 261: 9644-9651Abstract Full Text PDF PubMed Google Scholar), and they may result in different biological functions (1Uhlar C.M. Whitehead A.S. Serum amyloid A, the major vertebrate acute-phase reactant.Eur. J. Biochem. 1999; 265: 501-523Crossref PubMed Scopus (877) Google Scholar). Displacement of apoA-I from HDL by SAA causes an enhancement of HDL clearance and thereby a reduction of HDL (5Coetzee G.A. Strachan A.F. van der Westhuyzen D.R. Hoppe H.C. Jeenah M.S. de Beer F.C. Serum amyloid A-containing human high density lipoprotein 3. Density, size, and apolipoprotein composition.J. Biol. Chem. 1986; 261: 9644-9651Abstract Full Text PDF PubMed Google Scholar, 17Salazar A. Mana J. Fiol C. Hurtado I. Argimon J.M. Pujol R. Pinto X. Influence of serum amyloid A on the decrease of high density lipoprotein-cholesterol in active sarcoidosis.Atherosclerosis. 2000; 152: 497-502Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). A recent report, however, indicated that SAA, whether free or lipid-bound, could compete with HDL binding to SR-BI and interfere with cellular cholesteryl ester uptake from HDL (10Baranova I.N. Vishnyakova T.G. Bocharov A.V. Kurlander R. Chen Z. Kimelman M.L. Remaley A.T. Csako G. Thomas F. Eggerman T.L. et al.Serum amyloid A binding to CLA-1 (CD36 and LIMPII analogous-1) mediates serum amyloid A protein-induced activation of ERK1/2 and p38 mitogen-activated protein kinases.J. Biol. Chem. 2005; 280: 8031-8040Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 13Cai L. de Beer M.C. de Beer F.C. van der Westhuyzen D.R. Serum amyloid A is a ligand for scavenger receptor class B type I and inhibits high density lipoprotein binding and selective lipid uptake.J. Biol. Chem. 2005; 280: 2954-2961Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Thus, reports of the roles of SAA in the pathogenesis of inflammation and HDL metabolism are controversial and unclear.HDL plays a central role in cholesterol transport from extrahepatic peripheral cells to the liver as a major pathway for cholesterol catabolism. This pathway is also thought important to act against atherogenesis, as intracellularly accumulated cholesterol in the vascular wall can be removed by this mechanism. One of the rate-limiting steps of this pathway is the release of cellular cholesterol to HDL, and two independent mechanisms have been identified for this step (18Yokoyama S. Release of cellular cholesterol: molecular mechanism for cholesterol homeostasis in cells and in the body.Biochim. Biophys. Acta. 2000; 1529: 231-244Crossref PubMed Scopus (117) Google Scholar): nonspecific cholesterol exchange between cell membrane and extracellular lipoproteins, and assembly of new HDL particles with cellular lipid and lipid-free helical apolipoproteins. The latter seems a major source of plasma HDL, as the cells from patients with familial HDL deficiency (Tangier disease) lack this reaction (19Francis G.A. Knopp R.H. Oram J.F. Defective removal of cellular cholesterol and phospholipids by apolipoprotein A-I in Tangier disease.J. Clin. Invest. 1995; 96: 78-87Crossref PubMed Scopus (370) Google Scholar, 20Remaley A.T. Schumacher U.K. Stonik J.A. Farsi B.D. Nazih H.B. Brewer Jr., H.B. Decreased reverse cholesterol transport from Tangier disease fibroblasts: acceptor specificity and effect of brefeldin on lipid efflux.Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1813-1821Crossref PubMed Scopus (191) Google Scholar). ABCA1 was identified as essential for HDL biogenesis (21Bodzioch M. Orso E. Klucken J. Langmann T. Bottcher A. Diederich W. Drobnik W. Barlage S. Buchler C. Porsch-Ozcurumez M. et al.The gene encoding ATP-binding cassette transporter 1 is mutated in Tangier disease.Nat. 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Chem. 2004; 279: 604-611Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar).Recently, SAA was reported to induce cellular lipid release both in ABCA1-dependent and -independent manners (28Stonik J.A. Remaley A.T. Demosky S.J. Neufeld E.B. Bocharov A. Brewer H.B. Serum amyloid A promotes ABCA1-dependent and ABCA1-independent lipid efflux from cells.Biochem. Biophys. Res. Commun. 2004; 321: 936-941Crossref PubMed Scopus (68) Google Scholar). Here, we have characterized the SAA-cell interaction in the context of HDL biogenesis using a series of cell lines. It was demonstrated that SAA is an analog of helical apolipoprotein and is fully capable of generating new HDL particles with cellular lipid in the presence of ABCA1 or ABCA7 in the cell membrane. HDLs generated with SAA appeared to have different physicochemical properties than those generated with apoA-I.MATERIALS AND METHODSMaterialsApoA-I was isolated from a human plasma HDL fraction (density, 1.09–1.21) and stored at −80°C until use as described previously (29Yokoyama S. Tajima S. Yamamoto A. The process of dissolving apolipoprotein A-I in an aqueous buffer.J. Biochem. (Tokyo). 1982; 91: 1267-1272Crossref PubMed Scopus (73) Google Scholar). A stock solution (1 mg/ml) was prepared and stored at 4°C as described previously (30Abe-Dohmae S. Suzuki S. Wada Y. Aburatani H. Vance D.E. Yokoyama S. Characterization of apolipoprotein-mediated HDL generation induced by cAMP in a murine macrophage cell line.Biochemistry. 2000; 39: 11092-11099Crossref PubMed Scopus (100) Google Scholar). SAA used in this study was recombinant human SAA corresponding to human SAA1α except for three amino acids (catalog number 300-13; PeproTech EC, London, UK), and a stock solution (1 mg/ml) was prepared according to the manufacturer's instructions and stored at 4°C. ApoA-I protein was undetectable in the SAA stock solution by Coomassie Brilliant Blue staining or Western blotting (data not shown). The sources of the other reagents were as follows: phorbol 12-myristate 13-acetate (PMA) and 9-cis retinoic acid were from Wako (Osaka, Japan); liver X receptor agonist TO901317 was from Sigma; dibutyryl cyclic AMP (dBcAMP) was from Seikagaku Corp. (Tokyo, Japan); and protein kinase C inhibitor Goä6976 was from Biomol Research Laboratories.Cell cultureTHP-1, HEK293, and L929 cells were obtained from the Health Science Research Resources Bank. HEK293 clones of 293/2c and 293/6c were established in our laboratory; they stably express human ABCA1-green fluorescent protein and ABCA7-green fluorescent protein, respectively (27Abe-Dohmae S. Ikeda Y. Matsuo M. Hayashi M. Okuhira K. Ueda K. Yokoyama S. Human ABCA7 supports apolipoprotein-mediated release of cellular cholesterol and phospholipid to generate high density lipoprotein.J. Biol. Chem. 2004; 279: 604-611Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). CHO-K1 cells, which produce cholesterol-containing HDL were from the American Type Culture Collection. The cells were maintained in medium supplemented with 10% (v/v) fetal calf serum under a humidified atmosphere of 5% CO2 and 95% air at 37°C. RPMI 1640 medium was used for THP-1 cells, and a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium (DF medium) was used for all other cells. For differentiation of THP-1 cells, cells were subcultured in six-well trays (Techno Plastic Products AG, Trasadingen, Switzerland, catalog number 92406) at a density of 3.0 × 106 cells/well in the presence of 320 nM PMA for 72 h (31Arakawa R. Abe-Dohmae S. Asai M. Ito J. Yokoyama S. Involvement of caveolin-1 in cholesterol-enrichment of HDL during its assembly by apolipoprotein and THP-1 cells.J. Lipid Res. 2000; 41: 1952-1962Abstract Full Text Full Text PDF PubMed Google Scholar).Cellular lipid release assayCells were subcultured in six-well trays at the indicated density with 10% fetal calf serum-containing medium. After 48 h of incubation, the cells were washed once with Dulbecco's phosphate-buffered saline (D-PBS), except that buffer H [Hank's balanced salt solution containing 20 mM HEPES-KOH (pH 7.5) and 14 mM glucose] was used for HEK293, 293/2c, and 293/6c (27Abe-Dohmae S. Ikeda Y. Matsuo M. Hayashi M. Okuhira K. Ueda K. Yokoyama S. Human ABCA7 supports apolipoprotein-mediated release of cellular cholesterol and phospholipid to generate high density lipoprotein.J. Biol. Chem. 2004; 279: 604-611Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), and incubated in 1 ml/well DF medium containing 0.02% BSA (0.02% BSA/DF medium) and the compounds indicated. The PMA-treated THP-1 cells were further washed with D-PBS and incubated with apoA-I or SAA. The PMA-untreated THP-1 cells were directly subcultured in six-well trays at a density of 3.0 × 106 cells/well and cultured in 0.02% BSA/DF medium containing apoA-I or SAA. Lipid content in the medium and cells was determined after 24 h of incubation. Procedures for lipid extraction and enzymatic assays for cholesterol, cholesteryl ester, and choline-phospholipids were described previously (30Abe-Dohmae S. Suzuki S. Wada Y. Aburatani H. Vance D.E. Yokoyama S. Characterization of apolipoprotein-mediated HDL generation induced by cAMP in a murine macrophage cell line.Biochemistry. 2000; 39: 11092-11099Crossref PubMed Scopus (100) Google Scholar).Lecithin:cholesterol acyltransferase and lipase activities of the culture medium were determined using LCAT kit-S from Alfresa Pharma Corp. (catalog number 4987274862199; Tokyo, Japan) and an LIP Roche/Hitachi from Roche Diagnostics K.K. (catalog number 11821791 216; Tokyo, Japan), respectively.Western blottingA rabbit anti-ABCA1 antiserum (32Kojima K. Abe-Dohmae S. Arakawa R. Murakami I. Suzumori K. Yokoyama S. Progesterone inhibits apolipoprotein-mediated cellular lipid release: a putative mechanism for the decrease of HDL.Biochim. Biophys. Acta. 2001; 1532: 173-184Crossref PubMed Scopus (36) Google Scholar, 33Arakawa R. Yokoyama S. Helical apolipoproteins stabilize ATP-binding cassette transporter A1 by protecting it from thiol protease-mediated degradation.J. Biol. Chem. 2002; 277: 22426-22429Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar), a rat monoclonal anti-ABCA7 antibody, KM3095 (34Ikeda Y. Abe-Dohmae S. Munehira Y. Aoki R. Kawamoto S. Furuya A. Shitara K. Amachi T. Kioka N. Matsuo M. et al.Posttranscriptional regulation of human ABCA7 and its function for the apoA-I-dependent lipid release.Biochem. Biophys. Res. Commun. 2003; 311: 313-318Crossref PubMed Scopus (62) Google Scholar), and a rat monoclonal anti-SAA antibody (Cell Sciences) were used to detect the indicated proteins.Lipoprotein analysisABCA1- or ABCA7-transfected cells, 293/2c and 293/6c, were subcultured in 100 mm dishes at a density of 6.0 × 106 cells/dish, cultured as described above, and stimulated with 5 ml/dish 0.02% BSA/DF medium containing 10 μg/ml SAA for 24 h. The conditioned medium was analyzed by sucrose density gradient ultracentrifugation as described previously (27Abe-Dohmae S. Ikeda Y. Matsuo M. Hayashi M. Okuhira K. Ueda K. Yokoyama S. Human ABCA7 supports apolipoprotein-mediated release of cellular cholesterol and phospholipid to generate high density lipoprotein.J. Biol. Chem. 2004; 279: 604-611Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 35Hara H. Yokoyama S. Interaction of free apolipoproteins with macrophages: formation of high density lipoprotein-like lipoproteins and reduction of cellular cholesterol.J. Biol. Chem. 1991; 266: 3080-3086Abstract Full Text PDF PubMed Google Scholar). It was also analyzed by an HPLC system with two tandem gel permeation columns (Lipopropak XL, 7.5 mm × 300 mm; Tosoh) at a flow rate of 700 μl/min and two parallel online enzymatic lipid detection systems (350 μl/min each) (36Okazaki M. Usui S. Hosaki S. Analysis of plasma lipoproteins by gel permeation chromatography.in: Rifai N. Warnick G.R. Dominiczak M.H. Handbook of Lipoprotein Testing. AACC Press, Washington, DC2000: 647-669Google Scholar, 37Usui S. Hara Y. Hosaki S. Okazaki M. A new on-line dual enzymatic method for simultaneous quantification of cholesterol and triglycerides in lipoproteins by HPLC.J. Lipid Res. 2002; 43: 805-814Abstract Full Text Full Text PDF PubMed Google Scholar) to evaluate the size distribution of the lipoprotein particles (38Tsujita M. Wu C.A. Abe-Dohmae S. Usui S. Okazaki M. Yokoyama S. On the hepatic mechanism of HDL assembly by the ABCA1/apoA-I pathway.J. Lipid Res. 2005; 46: 154-162Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 39Hayashi M. Abe-Dohmae S. Okazaki M. Ueda K. Yokoyama S. Heterogeneity of high density lipoprotein generated by ABCA1 and ABCA7.J. Lipid Res. 2005; 46: 1703-1711Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) (Skylight Biotech, Inc., Akita, Japan). The system was calibrated by latex beads and high-molecular-weight standards for apparent spherical diameters of the subjects (36Okazaki M. Usui S. Hosaki S. Analysis of plasma lipoproteins by gel permeation chromatography.in: Rifai N. Warnick G.R. Dominiczak M.H. Handbook of Lipoprotein Testing. AACC Press, Washington, DC2000: 647-669Google Scholar, 37Usui S. Hara Y. Hosaki S. Okazaki M. A new on-line dual enzymatic method for simultaneous quantification of cholesterol and triglycerides in lipoproteins by HPLC.J. Lipid Res. 2002; 43: 805-814Abstract Full Text Full Text PDF PubMed Google Scholar).For electron microscopy, the HDL fraction was isolated by ultracentrifugation from the conditioned medium at a density of 1.21 g/ml adjusted with NaBr or sucrose at 90,000 rpm in a Hitachi CS100 ultracentrifuge with an S100AT6 rotor (4.8 × 105 g) for 16 h (NaBr) or 48 h (sucrose) at 4°C. The top fraction (300 μl) of each tube (3 ml) was collected as HDL. The HDL fractions from eight tubes of the sucrose solution were pooled and further concentrated by centrifugation for another 48 h at 1.21 g/ml with sucrose. HDL samples were dialyzed against 10 mM ammonium bicarbonate, pH 7.9, at 4°C overnight. They were negatively stained with 0.5% uranium acetate and examined with a Hitachi 7100 electron microscope as described (40Ito J. Nagayasu Y. Kato K. Sato R. Yokoyama S. Apolipoprotein A-I induces translocation of cholesterol, phospholipid and caveolin-1 to cytosol in rat astrocytes.J. Biol. Chem. 2002; 277: 7929-7935Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 41Okuhira K. Tsujita M. Yamauchi Y. Abe-Dohmae S. Kato K. Handa T. Yokoyama S. Potential involvement of dissociated apoA-I in the ABCA1-dependent cellular lipid release by HDL.J. Lipid Res. 2004; 45: 645-652Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Long diameters (major axes) of the HDL particles were measured graphically using NIH Image 1.63 software.To evaluate the electrophoretic mobility of lipoproteins, the conditioned medium was analyzed using an agarose gel electrophoresis system (Paragon System; Beckman) after concentration by an ultrafiltration filter (Amicon Ultra-15; Millipore).Statistical analysisData were analyzed by one-way ANOVA followed by Scheffeé's test. P < 0.05 was accepted as statistically significant.RESULTSCellular lipid was released by SAA and apoA-I from THP-1 cells (Fig. 1). From the undifferentiated cells, choline-phospholipid was a major component, suggesting that the HDL generated was cholesterol-poor. When the cells were differentiated with PMA, the lipid release was increased but more prominently in cholesterol, indicating the generation of cholesterol-rich HDL. The lipid-release profile was also examined for CHO-K1, L929, and HEK293 cells (Fig. 2). These cell lines represent cells that release both phospholipid and cholesterol, phospholipid only, and no lipid, respectively, by apoA-I, as we reported in previous works (27Abe-Dohmae S. Ikeda Y. Matsuo M. Hayashi M. Okuhira K. Ueda K. Yokoyama S. Human ABCA7 supports apolipoprotein-mediated release of cellular cholesterol and phospholipid to generate high density lipoprotein.J. Biol. Chem. 2004; 279: 604-611Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 42Yamauchi Y. Abe-Dohmae S. Yokoyama S. Differential regulation of apolipoprotein A-I/ATP binding cassette transporter A1-mediated cholesterol and phospholipid release.Biochim. Biophys. Acta. 2002; 1585: 1-10Crossref PubMed Scopus (35) Google Scholar). The results with SAA were the same as those with apoA-I.Fig. 2Release of cholesterol (Ch; A, C, E) and choline-phospholipids (PL; B, D, F) from CHO-K1 (A, B), L929 (C, D), and HEK293 (E, F) cells. CHO-K1, L929, and HEK293 cells were subcultured in six-well trays at densities of 5.0 × 105, 5.0 × 105, and 1.0 × 106 cells/well, respectively. After 48 h of incubation, cells were washed once and treated as described for Fig. 1. Data represent averages ± SD of duplicate measurements in one of two independent experiments yielding similar results.View Large Image Figure ViewerDownload Hi-res
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