Apolipoprotein A-I-stimulated Apolipoprotein E Secretion from Human Macrophages Is Independent of Cholesterol Efflux
2004; Elsevier BV; Volume: 279; Issue: 25 Linguagem: Inglês
10.1074/jbc.m401177200
ISSN1083-351X
AutoresMaaike Kockx, Kerry‐Anne Rye, Katharina Gaus, Carmel M. Quinn, Janelle Wright, T. Sloane, Dmitri Sviridov, Ying Fu, David Sullivan, John R. Burnett, Stephan Rust, Gerd Assmann, G.M. Anantharamaiah, Mayakonda N. Palgunachari, Sissel Lund Katz, Michael C. Phillips, Roger T. Dean, Wendy Jessup, Leonard Kritharides,
Tópico(s)Drug Transport and Resistance Mechanisms
ResumoApolipoprotein A-I (apoA-I)-mediated cholesterol efflux involves the binding of apoA-I to the plasma membrane via its C terminus and requires cellular ATP-binding cassette transporter (ABCA1) activity. ApoA-I also stimulates secretion of apolipoprotein E (apoE) from macrophage foam cells, although the mechanism of this process is not understood. In this study, we demonstrate that apoA-I stimulates secretion of apoE independently of both ABCA1-mediated cholesterol efflux and of lipid binding by its C terminus. Pulse-chase experiments using 35S-labeled cellular apoE demonstrate that macrophage apoE exists in both relatively mobile (Em) and stable (Es) pools, that apoA-I diverts apoE from degradation to secretion, and that only a small proportion of apoA-I-mobilized apoE is derived from the cell surface. The structural requirements for induction of apoE secretion and cholesterol efflux are clearly dissociated, as C-terminal deletions in recombinant apoA-I reduce cholesterol efflux but increase apoE secretion, and deletion of central helices 5 and 6 decreases apoE secretion without perturbing cholesterol efflux. Moreover, a range of 11- and 22-mer α-helical peptides representing amphipathic α-helical segments of apoA-I stimulate apoE secretion whereas only the C-terminal α-helix (domains 220–241) stimulates cholesterol efflux. Other α-helix-containing apolipoproteins (apoA-II, apoA-IV, apoE2, apoE3, apoE4) also stimulate apoE secretion, implying a positive feedback autocrine loop for apoE secretion, although apoE4 is less effective. Finally, apoA-I stimulates apoE secretion normally from macrophages of two unrelated subjects with genetically confirmed Tangier Disease (mutations C733R and c.5220–5222delTCT; and mutations A1046D and c.4629–4630insA), despite severely inhibited cholesterol efflux. We conclude that apoA-I stimulates secretion of apoE independently of cholesterol efflux, and that this represents a novel, ABCA-1-independent, positive feedback pathway for stimulation of potentially anti-atherogenic apoE secretion by α-helix-containing molecules including apoA-I and apoE. Apolipoprotein A-I (apoA-I)-mediated cholesterol efflux involves the binding of apoA-I to the plasma membrane via its C terminus and requires cellular ATP-binding cassette transporter (ABCA1) activity. ApoA-I also stimulates secretion of apolipoprotein E (apoE) from macrophage foam cells, although the mechanism of this process is not understood. In this study, we demonstrate that apoA-I stimulates secretion of apoE independently of both ABCA1-mediated cholesterol efflux and of lipid binding by its C terminus. Pulse-chase experiments using 35S-labeled cellular apoE demonstrate that macrophage apoE exists in both relatively mobile (Em) and stable (Es) pools, that apoA-I diverts apoE from degradation to secretion, and that only a small proportion of apoA-I-mobilized apoE is derived from the cell surface. The structural requirements for induction of apoE secretion and cholesterol efflux are clearly dissociated, as C-terminal deletions in recombinant apoA-I reduce cholesterol efflux but increase apoE secretion, and deletion of central helices 5 and 6 decreases apoE secretion without perturbing cholesterol efflux. Moreover, a range of 11- and 22-mer α-helical peptides representing amphipathic α-helical segments of apoA-I stimulate apoE secretion whereas only the C-terminal α-helix (domains 220–241) stimulates cholesterol efflux. Other α-helix-containing apolipoproteins (apoA-II, apoA-IV, apoE2, apoE3, apoE4) also stimulate apoE secretion, implying a positive feedback autocrine loop for apoE secretion, although apoE4 is less effective. Finally, apoA-I stimulates apoE secretion normally from macrophages of two unrelated subjects with genetically confirmed Tangier Disease (mutations C733R and c.5220–5222delTCT; and mutations A1046D and c.4629–4630insA), despite severely inhibited cholesterol efflux. We conclude that apoA-I stimulates secretion of apoE independently of cholesterol efflux, and that this represents a novel, ABCA-1-independent, positive feedback pathway for stimulation of potentially anti-atherogenic apoE secretion by α-helix-containing molecules including apoA-I and apoE. The anti-atherogenic effects of high density lipoprotein (HDL) 1The abbreviations used are: HDL, high density lipoprotein; PLV, phospholipid vesicles; apoA, apolipoprotein; ABCA1, ATP-binding cassette transporter; SPM, sphingomyelin; PC, phosphatidylcholine; TD, Tangier disease; LDL, low density lipoprotein; apo, apolipoprotein; ABCA1, ATP-binding cassette transporter A1; RCT, reverse cholesterol transport; AcLDL, acetylated LDL; HMDM, human primary monocyte-derived macrophages; POPC, 1-palmitoyl-2-oleoyl phosphatidylcholine; SPM, egg yolk sphingomyelin; BCA, bicinchoninic acid; PBS, phosphate-buffered saline; AU, arbitrary units; RT-PCR, reverse transcriptase-PCR. (1Asztalos B.F. Roheim P.S. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1419-1423Crossref PubMed Scopus (68) Google Scholar) are at least in part attributed to the ability of HDL to stimulate cholesterol and phospholipid efflux from lipid-loaded macrophages, providing the initial step of reverse cholesterol transport (RCT). Apolipoprotein A-I (apoA-I), the major protein component of HDL, is understood to play an important role in this process (1Asztalos B.F. Roheim P.S. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1419-1423Crossref PubMed Scopus (68) Google Scholar). Lipid removal by apoA-I involves a cAMP-inducible active transport pathway (2Smith J.D. Miyata M. Ginsberg M. Grigaux C. Shmookler E. Plump A.S. J. Biol. Chem. 1996; 271: 30647-30655Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar) and is known to be mediated by the ATP-binding cassette transporter-1 (ABCA1/ABC-1) (3Lawn R.M. Wade D.P. Garvin M.R. Wang X. Schwartz K. Porter J.G. Seilhamer J.J. Vaughan A.M. Oram J.F. J. Clin. Invest. 1999; 104: R25-R31Crossref PubMed Scopus (654) Google Scholar). These transporters are membrane proteins that utilize ATP hydrolysis to transport substrates across membranes (4Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3375) Google Scholar). Mutations in ABCA1/ABC-1 cause Tangier disease (TD), a severe HDL deficiency syndrome characterized by very low plasma levels of HDL and apoA-I, accumulation of cholesterol in tissue macrophages, and a predisposition to atherosclerosis (5Bodzioch 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 (1345) Google Scholar, 6Brooks-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 (1505) Google Scholar, 7Rust S. Rosier M. Funke H. Real J. Amoura Z. Piette J.C. Deleuze J.F. Brewer H.B. Duverger N. Denefle P. 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Genet. 1999; 22: 336-345Crossref PubMed Scopus (1505) Google Scholar). Properties additional to induction of cholesterol efflux may contribute to the anti-atherogenic effect of apoA-1. Human atherosclerotic lesions contain apoE protein and mRNA, especially in association with macrophage foam cells (9O'Brien K.D. Deeb S.S. Ferguson M. McDonald T.O. Allen M.D. Alpers C.E. Chait A. Am. J. Pathol. 1994; 144: 538-548PubMed Google Scholar). Secretion of apoE by macrophages may protect against atherosclerosis, as indicated by the effects of transplantation of apoE-expressing bone marrow into apoE-null mice (10Linton M.F. Atkinson J.B. Fazio S. Science. 1995; 267: 1034-1037Crossref PubMed Scopus (410) Google Scholar, 11Bellosta S. Mahley R.W. Sanan D.A. Murata J. Newland D.L. Taylor J.M. Pitas R.E. J. Clin. Investig. 1995; 96: 2170-2179Crossref PubMed Scopus (251) Google Scholar). This may depend upon enhanced local clearance of cellular lipids and/or inhibition of local inflammatory responses, in addition to lowering plasma concentrations of atherogenic plasma lipoproteins. Recent studies demonstrated that apoA-I stimulates both apoE secretion and cholesterol efflux from human and mouse foam cell macrophages (12Bielicki J.K. McCall M.R. Forte T.M. J. Lipid Res. 1999; 40: 85-92Abstract Full Text Full Text PDF PubMed Google Scholar, 13Rees D. Sloane T. Jessup W. Dean R.T. Kritharides L. J. Biol. Chem. 1999; 274: 27925-27933Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Stimulation of apoE secretion by apoA-I and by HDL is most likely post-transcriptionally regulated and is observed for both endogenous apoE (macrophages) and recycled apoE (hepatocytes) (12Bielicki J.K. McCall M.R. Forte T.M. J. Lipid Res. 1999; 40: 85-92Abstract Full Text Full Text PDF PubMed Google Scholar, 13Rees D. Sloane T. Jessup W. Dean R.T. Kritharides L. J. Biol. 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We demonstrate that apoA-I promotes secretion of a mobile pool of apoE, which is otherwise destined for intracellular degradation, that this process does not require interactions between the C terminus of apoA-I and the plasma membrane and occurs normally in macrophages of patients with Tangier disease, implying independence from ABCA1 activity. Reagents—All solvents were high performance liquid chromatography (HPLC) grade (Mallinckrodt). Polyclonal goat anti-human antibodies to human apoE were obtained from Chemicon International Inc. For immunoblotting of nitrocellulose membranes after non-denaturing gel electrophoresis, polyclonal goat antibodies to human apoA-I and polyclonal sheep antibodies to human apoA-II (Roche Applied Science) were used. Secondary rabbit polyclonal anti-goat or anti-sheep horseradish peroxidase-linked antibodies, nitrocellulose membranes (0.45 μm), and enhanced chemiluminescence (ECL) reagents and Hyperfilm were obtained from Amersham Biosciences. Human apoE standard was from Biodesign. [1α,2β(n)-3H]cholesterol and [methyl-3H]choline chloride were supplied by Amersham Biosciences (specific activity: 44 Ci/mmol and 79 Ci/mmol, respectively). LDL, acetylated LDL (AcLDL), [3H]cholesterol-labeled AcLDL were prepared as described (17Kritharides L. Jessup W. Mander E.L. Dean R.T. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 276-289Crossref PubMed Scopus (89) Google Scholar, 18Kritharides L. Christian A. Stoudt G. Morel D. Rothblat G.H. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 1589-1599Crossref PubMed Scopus (94) Google Scholar). 1-Palmitoyl-2-oleoyl phosphatidylcholine (POPC), egg yolk sphingomyelin (SPM), and sodium cholate were purchased from Sigma. Bicinchoninic acid (BCA) reagent for protein determination was also supplied by Sigma-Aldrich. For pulse-chase studies, methionine-free medium was supplied by Invitrogen and [35S]TRAN-label (1175 Ci/mmol) from ICN. Apolipoproteins—Human apoA-I and apoA-II were isolated from HDL by ultracentrifugation and anion exchange chromatography (17Kritharides L. Jessup W. Mander E.L. Dean R.T. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 276-289Crossref PubMed Scopus (89) Google Scholar, 19Rye K.A. Barter P.J. J. Biol. Chem. 1994; 269: 10298-10303Abstract Full Text PDF PubMed Google Scholar). Human apoA-IV was isolated from the density >1.25 g/ml fraction of plasma by adsorption onto synthetic triglyceride-rich microemulsions. The microemulsions were delipidated and the apoA-IV purified by anion exchange chromatography on a Q-Sepharose fast flow column (20Steinmetz A. Clavey V. Vu-Dac N. Kaffarnik H. Fruchart J.C. J. Chromatogr. 1989; 487: 154-160Crossref PubMed Scopus (13) Google Scholar, 21Weinberg R.B. Hopkins R.A. Jones J.B. Methods Enzymol. 1996; 263: 282-296Crossref PubMed Google Scholar). Human apoE2, apoE3, and apoE4 were expressed by BL21 Escherichia coli (provided by Dr. Karl Weisgraber, Gladstone Institute) and purified by gel filtration chromatography. ApoA-I Peptides and Recombinant Proteins—Pure peptides representing peptides of apoA-I were synthesized using an automated solid phase peptide synthesizer as described previously (22Anantharamaiah G.M. Methods Enzymol. 1986; 128: 627-647Crossref PubMed Scopus (81) Google Scholar, 23Palgunachari M.N. Mishra V.K. Lund-Katz S. Phillips M.C. Adeyeye S.O. Alluri S. Anantharamaiah G.M. Segrest J.P. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 328-338Crossref PubMed Scopus (203) Google Scholar, 24Mishra V.K. Palgunachari M.N. Datta G. Phillips M.C. Lund-Katz S. Adeyeye S.O. Segrest J.P. Anantharamaiah G.M. Biochemistry. 1998; 37: 10313-10324Crossref PubMed Scopus (78) Google Scholar). To promote the α-helical stability of the peptide molecules, the N and C termini were acetylated and amidated (except for peptide 1–33) respectively (25Venkatachalapathi Y.V. Phillips M.C. Epand R.M. Epand R.F. Tytler E.M. Segrest J.P. Anantharamaiah G.M. Proteins. 1993; 15: 349-359Crossref PubMed Scopus (114) Google Scholar). Truncated apoA-I propeptide mutants were expressed in an E. coli/PGEX expression system (26Pyle L.E. Barton P. Fujiwara Y. Mitchell A. Fidge N. J. Lipid Res. 1995; 36: 2355-2361Abstract Full Text PDF PubMed Google Scholar). Cholesterol efflux to these proteins has previously been published (27Sviridov D. Pyle L.E. Fidge N. J. Biol. Chem. 1996; 271: 33277-33283Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). ApoA-I deletion mutants were constructed as described previously (28Holvoet P. Zhao Z. Vanloo B. Vos R. Deridder E. Dhoest A. Taveirne J. Brouwers E. Demarsin E. Engelborghs Y. Biochemistry. 1995; 34: 13334-13342Crossref PubMed Scopus (87) Google Scholar, 29Saito H. Dhanasekaran P. Nguyen D. Holvoet P. Lund-Katz S. Phillips M.C. J. Biol. Chem. 2003; 278: 23227-23232Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Wild-type apoA-I and engineered variants of apoA-I were expressed in expression host E. coli strain Bl21-DE3 and isolated by gel filtration and anion exchange chromatography (29Saito H. Dhanasekaran P. Nguyen D. Holvoet P. Lund-Katz S. Phillips M.C. J. Biol. Chem. 2003; 278: 23227-23232Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Identical stimulation of apoE secretion from human macrophage foam cells was observed in control experiments using recombinant wild-type apoA-I and human apoA-I prepared from human plasma. In previous studies we established that 5–10 μg/ml apoA-I was saturating for cholesterol efflux and apoE secretion from human and murine macrophages (13Rees D. Sloane T. Jessup W. Dean R.T. Kritharides L. J. Biol. Chem. 1999; 274: 27925-27933Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). In the present studies, peptides and proteins were added to cells at a final concentration of 25 μg/ml culture medium after first establishing that this was in excess of saturating concentrations for induction of apoE secretion for all molecules. Preparation of Phospholipid ApoA-I and Phospholipid ApoA-II Discs—Discs were prepared as described previously (19Rye K.A. Barter P.J. J. Biol. Chem. 1994; 269: 10298-10303Abstract Full Text PDF PubMed Google Scholar) using the cholate dialysis method (30Matz C.E. Jonas A. J. Biol. Chem. 1982; 257: 4535-4540Abstract Full Text PDF PubMed Google Scholar). After preparation, all discs were dialyzed extensively against 0.01 m Tris-buffered saline containing 0.15 m NaCl, 0.005% (w/v) EDTA-Na2, 0,006% (w/v) NaN3, and stored under argon. Lipid-free apoA-I and apoA-II were not present in the preparations as judged by electrophoresis on non-denaturing 3–40% gradient gels. Phospholipid composition was varied by altering the proportions of phospholipids added at the time of preparation. All chemical analyses of reconstituted particles were carried out on a Roche/Hitachi 902 analyzer (Roche Diagnostics, Zurich, Switzerland). Phospholipid concentrations were determined enzymatically (31Trinder P. Ann. Clin. Biochem. 1969; 6: 24-27Crossref Google Scholar, 32Takayama M. Itoh S. Nagasaki T. Tanimizu I. Clin. Chim. Acta. 1977; 79: 93-98Crossref PubMed Scopus (999) Google Scholar). The Stokes' diameter and surface charge of the particles was measured by non-denaturing PAGE and agarose gel electrophoresis (19Rye K.A. Barter P.J. J. Biol. Chem. 1994; 269: 10298-10303Abstract Full Text PDF PubMed Google Scholar, 33Liang H.Q. Rye K.A. Barter P.J. J. Lipid Res. 1994; 35: 1187-1199Abstract Full Text PDF PubMed Google Scholar). In some experiments, after cell efflux incubations, media containing apoA-I phospholipid or apoA-II phospholipid discs underwent non-denaturing polyacrylamide gel electrophoresis, followed by electrophoretic transfer nitrocellulose membranes and immunoblotting for apoE, apoA-I, or apoA-II to investigate possible association of apoE with phospholipid-containing particles. Isolation and Culture of Human Monocyte-derived Macrophages (HMDM)—Human monocytes (apoE3/E3 phenotype) were isolated from white cell concentrates from healthy donors using centrifugal elutriation as described (13Rees D. Sloane T. Jessup W. Dean R.T. Kritharides L. J. Biol. Chem. 1999; 274: 27925-27933Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Genotyping was completed by Taq polymerase chain reaction amplification of a 232-base pair fragment of the apoE gene followed by cutting with the restriction endonuclease Cfo1 by the Department of Biochemistry, Royal Prince Alfred Hospital. White cell buffy coat concentrates ( 95% purity by nonspecific esterase staining) were differentiated by plating at 1.5 × 106 cells per 22-mm diameter culture dish (Costar) in RPMI 1640 containing penicillin G and streptomycin (50 units/ml and 50 μg/ml, respectively), l-glutamine (2 mm), and 10% (v/v) heat-inactivated whole human serum for 6 days. Following differentiation, the cells were washed and enriched with unesterified cholesterol (FC) and cholesteryl ester (CE) by incubation in RPMI 1640 containing 10% LPDS (v/v) and AcLDL (50 μg protein/ml) for 4 days (13Rees D. Sloane T. Jessup W. Dean R.T. Kritharides L. J. Biol. Chem. 1999; 274: 27925-27933Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Mononuclear cells from individual donors (Tangier disease subjects and their controls) were isolated by minor modification of a previously described density separation method (34Boyum A. Methods Enzymol. 1984; 108: 88-102Crossref PubMed Scopus (179) Google Scholar, 35Becq F. Hamon Y. Bajetto A. Gola M. Verrier B. Chimini G. J. Biol. Chem. 1997; 272: 2695-2699Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar) using OptiPrep™ solution of density 1.068 g/ml and a solution of density 1.063 g/ml to remove platelets and lymphocytes, respectively. Total cells were plated at a density of 2 × 106 cells/22-mm diameter culture well (Falcon). Cells were differentiated to macrophages in RPMI 1640-containing antibiotics, glutamine, 50 ng/ml M-CSF (36Langmann 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), and heat-inactivated whole human serum (13Rees D. Sloane T. Jessup W. Dean R.T. Kritharides L. J. Biol. Chem. 1999; 274: 27925-27933Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 37Garner B. Baoutina A. Dean R.T. Jessup W. Atherosclerosis. 1997; 128: 47-58Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) for 6 days. At this time cells were washed prior to cholesterol enrichment (see above). Direct comparisons between HMDM isolated from normal donors by density centrifugation and centrifugal elutriation confirmed identical cholesterol efflux and apoE secretion by either method. Lipid Efflux and ApoE Secretion—To achieve cholesterol loading of macrophages, 2 μCi/ml [3H]cholesterol in ethanol was incorporated into AcLDL before incubating with cells for 4 days as described (18Kritharides L. Christian A. Stoudt G. Morel D. Rothblat G.H. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 1589-1599Crossref PubMed Scopus (94) Google Scholar). Triplicate cultures were harvested after loading to confirm efficient enrichment with FC and CE by HPLC. Cells were subsequently washed, equilibrated overnight in RPMI 1640 containing 0.1% (w/v) bovine serum albumin, to allow equilibration of [3H]cholesterol in FC and CE pools. In some experiments, cells were loaded with AcLDL without [3H]cholesterol, and phospholipids were labeled at the end of the 4-day cholesterol-loading period by incubating cells with RPMI 1640 containing 3 μCi/ml [3H]choline chloride in ethanol (final 2% (v/v)) and 0.1% bovine serum albumin (w/v) for 22 h, followed by equilibration for 1 h in RPMI 1640 containing 0.1% bovine serum albumin. Equilibrated, cholesterol-enriched cells were washed with warm PBS and incubated in 1.0 ml of efflux medium comprising RPMI 1640 with or without 25 μg/ml apoA-I or the appropriate additions. After 1–8 h, media were removed, mixed with Complete© protease inhibitor (Roche Applied Science) and 0.02 TIU of aprotinin (Sigma) and spun for 2 min at 16,000 × g to remove any detached cells. The cultures were washed twice with ice-cold PBS and then scraped in 0.8 ml of cold PBS containing Complete© protease inhibitor and aprotinin. Quantitation of Cholesterol and Cholesteryl Ester in Cells and Media—40-μl media aliquots and 5-μl cell aliquots were analyzed by scintillation counting to quantify efflux of [3H]cholesterol. The proportions of label in cell [3H]FC and [3H]CE were determined after separation by TLC in heptane/ethyl acetate (1:1, v/v). Standards of FC and CE were identified by charring at 100 °C in 10% CuSO4, 8.5%H3PO4. The masses of FC and CE in cells and media were determined by reverse-phase HPLC after extraction into methanol/hexane (17Kritharides L. Jessup W. Mander E.L. Dean R.T. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 276-289Crossref PubMed Scopus (89) Google Scholar, 38Kritharides L. Jessup W. Gifford J. Dean R.T. Anal. Biochem. 1993; 213: 79-89Crossref PubMed Scopus (178) Google Scholar). Radiochemical equilibrium was determined by calculating the specific activity of free and esterified cholesterol (dpm per nmol) (18Kritharides L. Christian A. Stoudt G. Morel D. Rothblat G.H. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 1589-1599Crossref PubMed Scopus (94) Google Scholar) and was reproducibly achieved after 4 days of cholesterol enrichment and overnight equilibration. Quantitation of Phospholipids in Cells and Media—Phospholipids were extracted from 150-μl aliquots of cell lysates and media samples using the method of Bligh and Dyer including two backwashes with methanol/water (10:9, v/v) as described previously (39Gelissen I.C. Rye K.A. Brown A.J. Dean R.T. Jessup W. J. Lipid Res. 1999; 40: 1636-1646Abstract Full Text Full Text PDF PubMed Google Scholar). After evaporation of the chloroform phase, lipids were dissolved in 200 μl of chloroform/methanol (2:1, v/v), and 50-μl aliquots were counted by scintillation counting to determine phospholipid efflux. Total phospholipid mass was determined using a modification of the Bartlett assay as described (40Sokoloff L. Rothblat G.H. Proc. Soc. Exp. Biol. Med. 1974; 146: 1166-1172Crossref PubMed Scopus (109) Google Scholar) and was used to determine specific activity (dpm per nmol phospholipid). Quantitation of ApoE in Cell Lysates and Efflux Media by Western Blot and by ELISA—Aliquots (55 μl) of cell culture medium were mixed with sample buffer (27.5 μl) containing 10 mm dithiothreitol, heated to 100 °C for 5 min, and separated by SDS-PAGE using a 4% stacking gel, and 12.5% polyacrylamide resolving gel under reducing conditions in Tris-glycine buffer as described (13Rees D. Sloane T. Jessup W. Dean R.T. Kritharides L. J. Biol. Chem. 1999; 274: 27925-27933Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Electrophoretic Western blot transfer onto 0.45-μm nitrocellulose membranes was performed in Trisglycine buffer for 1 h at 25–30 V, blocked before incubating with primary antibody to apoE (goat anti-human polyclonal, 1:5000 dilution), and washing and incubating with 1:5000 dilution of secondary antibody (rabbit anti-goat IgG) conjugated with horseradish peroxidase (13Rees D. Sloane T. Jessup W. Dean R.T. Kritharides L. J. Biol. Chem. 1999; 274: 27925-27933Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Membrane chemiluminescence signal was quantified using Kodak Digital Science 1D and expressed as arbitrary units (AU) per mg of cell protein. In most experiments, a serial dilution of authentic human apoE standard in sample buffer was separated and blotted in parallel to allow calculation of secreted apoE mass as microgram per culture or μg per mg of cell protein. The linear response range of chemiluminescence signal to mass of authentic human apoE standard was defined for each Western blot in each experiment. In addition, apoE secretion was measured in some experiments using ELISA. NUNC 96 well immunoplates were coated with 1.5 μg/ml mouse monoclonal anti-human apoE antibody (Biodesign) in 0.2 m Na2CO3/NaHCO3 buffer. Standards were prepared from purified human apoE (Biodesign), whereas antibodies used in Western analysis were used as tagging antibodies (goat anti-human polyclonal, 1:3750 and rabbit antigoat horseradish peroxidase, 1:2500). Metabolic Labeling of Cell Proteins with [35S]Methionine/Cysteine— To study the fate of newly synthesized apoE, cholesterol-enriched HMDM were labeled in methionine-free medium (Invitrogen) containing 250 μCi/ml [35S]TRAN-label (1175 Ci/mmol, ICN) for 3 h, then washed and chased in RPMI 1640 media containing 2 mm methionine (Sigma) and cysteine (Sigma). 35S-labeled apoE in cell lysates and culture medium were determined by immunoprecipitation using 1:10,000 dilution of the polyclonal goat antibody to human apoE and protein A-Sepharose (Amersham Biosciences) after separation by SDS-PAGE, quantified by phosphorimaging (Photostimulated Luminescence, Fujix BAS 1000) and expressed as AU of apoE/mg of cell protein (13Rees D. Sloane T. Jessup W. Dean R.T. Kritharides L. J. Biol. Chem. 1999; 274: 27925-27933Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Where indicated, in experiments detecting small amounts of secreted apoE requiring increased sensitivity, immunoprecipitated 35S-labeled apoE was directly quantified by radiometric detection, after confirming a single band on SDS-PAGE and phosphorimaging. [35S]Methionine/cysteine labeling was also used to discriminate between cell derived (secreted) and exogenous apoE in experiments investigating the secretion of apoE by recombinant apoE2, E3, and E4. Biotinylation and Isolation of Biotinylated ApoE—Cholesterol-enriched HMDM were incubated with 200 μCi/ml [35S]TRAN-label in methionine-free Dulbecco's modified Eagle's medium (DMEM) containing 10% DMEM for 16 h. Surface proteins were then biotinylated by placing the cells on ice for 5 min, followed by incubation with 1 mg/ml sulfo-NHS-SS-Biotin (Pierce) in PBS at 4 °C for 45 min. Cells were washed twice with ice-cold PBS and subsequently incubated with or without 25 μg/ml apoA-I for 30 min at 37 °C. ApoE was immunoprecipitated as described above, and biotinylated apoE separated from total immunoprecipitated apoE as described (41Zhao Y. Mazzone T. J. Biol. Chem. 2000; 275: 4759-4765Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). In short, total apoE was released from protein A-Sepharose beads by boiling in 100 μl of HEPES-buffered saline containing 1% SDS at 90 °C for 3 min. Biotinylated apoE was separated from unbiotinylated apoE using ImmunoPure Immobilized Streptavidin (Pierce) and both were quantified using scintillation counting and SDS-PAGE/phosphorimaging. Biotinylated apoE was
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