Reduction in apolipoprotein-mediated removal of cellular lipids by immortalization of human fibroblasts and its reversion by cAMP: lack of effect with Tangier disease cells
1999; Elsevier BV; Volume: 40; Issue: 10 Linguagem: Inglês
10.1016/s0022-2275(20)34893-8
ISSN1539-7262
AutoresJohn F. Oram, Armando J. Mendez, James Lymp, Terrance J. Kavanagh, Christine L. Halbert,
Tópico(s)Caveolin-1 and cellular processes
ResumoHigh density lipoprotein (HDL) phospholipids and apolipoproteins remove cellular lipids by two distinct mechanisms, but their relative contribution to reverse cholesterol transport is unknown. Whereas phospholipid-mediated cholesterol efflux from cultured cells reflects the activity of the HDL receptor SR-BI, apolipoprotein-mediated lipid removal is regulated in response to changes in cellular cholesterol content (positive) and cell proliferation rates (negative). Here we show that immortalization of human skin fibroblast lines with the papillomavirus E6/E7 oncogenes increased their proliferation rates and selectively reduced the activity of the apolipoprotein-mediated lipid removal pathway. This reduction was accompanied by a decrease in cellular cAMP levels and was reversed by treatment with a cAMP analog. The stimulatory effect of cAMP was independent of changes in cellular phenotype or activities of cholesteryl ester cycle enzymes. The severely impaired apolipoprotein-mediated lipid removal pathway in Tangier disease fibroblasts, which persisted after immortalization, was not improved by treatment with a cAMP analog, implying that the cellular defect in Tangier disease is upstream from this cAMP-dependent signaling pathway. These results indicate that papillomavirus-induced immortalization of fibroblasts selectively reduces the activity of the apolipoprotein-mediated lipid removal pathway by a cAMP-dependent process, perhaps to prevent loss of cellular lipids needed for continual membrane synthesis.—Oram, J. F., A. J. Mendez, J. Lymp, T. J. Kavanagh, and C. L. Halbert. Reduction in apolipoprotein-mediated removal of cellular lipids by immortalization of human fibroblasts and its reversion by cAMP: lack of effect with Tangier disease cells. J. Lipid Res. 1999. 40: 1769–1781. High density lipoprotein (HDL) phospholipids and apolipoproteins remove cellular lipids by two distinct mechanisms, but their relative contribution to reverse cholesterol transport is unknown. Whereas phospholipid-mediated cholesterol efflux from cultured cells reflects the activity of the HDL receptor SR-BI, apolipoprotein-mediated lipid removal is regulated in response to changes in cellular cholesterol content (positive) and cell proliferation rates (negative). Here we show that immortalization of human skin fibroblast lines with the papillomavirus E6/E7 oncogenes increased their proliferation rates and selectively reduced the activity of the apolipoprotein-mediated lipid removal pathway. This reduction was accompanied by a decrease in cellular cAMP levels and was reversed by treatment with a cAMP analog. The stimulatory effect of cAMP was independent of changes in cellular phenotype or activities of cholesteryl ester cycle enzymes. The severely impaired apolipoprotein-mediated lipid removal pathway in Tangier disease fibroblasts, which persisted after immortalization, was not improved by treatment with a cAMP analog, implying that the cellular defect in Tangier disease is upstream from this cAMP-dependent signaling pathway. These results indicate that papillomavirus-induced immortalization of fibroblasts selectively reduces the activity of the apolipoprotein-mediated lipid removal pathway by a cAMP-dependent process, perhaps to prevent loss of cellular lipids needed for continual membrane synthesis.—Oram, J. F., A. J. Mendez, J. Lymp, T. J. Kavanagh, and C. L. Halbert. Reduction in apolipoprotein-mediated removal of cellular lipids by immortalization of human fibroblasts and its reversion by cAMP: lack of effect with Tangier disease cells. J. Lipid Res. 1999. 40: 1769–1781. It is widely believed that HDL protects against cardiovascular disease by removing excess cholesterol from cells, but the proposed mechanisms involved have been controversial. It now appears that HDL components remove cellular lipids by at least two distinct mechanisms (1Oram J.F. Yokoyama S. Apolipoprotein-mediated removal of cellular cholesterol and phospholipids.J. Lipid Res. 1996; 37: 2473-2491Google Scholar). First, HDL phospholipids pick up cholesterol that desorbs from the plasma membrane (2Rothblat G.H. Phillips M.C. Cholesterol efflux from arterial wall cells.Curr. Opin. Lipidol. 1991; 2: 288-294Google Scholar). This process is facilitated by binding of HDL to scavenger receptor BI (SR-BI) (3Ji Y. Jian B. Wang N. Sun Y. Moya M.D.L.L. Phillips M.C. Rothblat G.H. Swaney J.B. Tall A.R. Scavenger receptor B1 promotes high density lipoprotein-mediated cellular cholesterol efflux.J. Biol. Chem. 1997; 272: 20982-20985Google Scholar) and may involve caveolae (4Fielding P.E. Fielding C.J. Plasma membrane caveolae mediate the efflux of cellular free cholesterol.Biochemistry. 1995; 34: 14288-14292Google Scholar). Second, HDL apolipoproteins remove both cholesterol and phospholipids from cells (5Hara H. Yokoyama S. Interaction of free apolipoproteins with macrophages.J. Biol. Chem. 1991; 266: 3080-3086Google Scholar, 6Forte T.M. Bielicki J.K. Goth-Goldstein R. Selmek J. McCall M.R. Recruitment of cell phospholipids and cholesterol by apolipoproteins A-II and A-I: formation of nascent apolipoprotein-specific HDL that differ in size, phospholipid composition, and reactivity with LCAT.J. Lipid Res. 1995; 36: 148-157Google Scholar, 7Bielicki J.K. Johnson W.J. Weinberg R.B. Glick J.M. Rothblat G.H. Efflux of lipid from fibroblasts to apolipoproteins: Dependence on elevated levels of cellular unesterified cholesterol.J. Lipid Res. 1992; 33: 1699-1709Google Scholar, 8Mendez A.J. Anantharamaiah G.M. Segrest J.P. Oram J.F. Synthetic amphipathic helical peptides that mimic apolipoprotein A-I in clearing cellular cholesterol.J. Clin. Invest. 1994; 94: 1698-1705Google Scholar, 9Yancey P.G. Bielicki J.K. Johnson W.J. Lund-Katz S. Palgunachari M.N. Anantharamaiah G.M. Segrest J.P. Phillips M.C. Rothblat G.H. Efflux of cellular cholesterol and phospholipid to lipid-free apolipoproteins and class A amphipathic peptides.Biochemistry. 1995; 34: 7955-7965Google Scholar) by an active excretory pathway (10Mendez A.J. Monensin and Brefeldin A inhibit high density lipoprotein-mediated cholesterol efflux from cholesterol-enriched cells. Implications for intracellular cholesterol transport.J. Biol. Chem. 1995; 270: 5891-5900Google Scholar, 11Mendez A.J. Uint L. Apolipoprotein-mediated cellular cholesterol and phospholipid efflux depend on a functional Golgi apparatus.J. Lipid Res. 1996; 37: 2510-2524Google Scholar, 12Mendez A.J. Cholesterol efflux mediated by apolipoproteins is an active cellular process distinct from efflux mediated by passive diffusion.J. Lipid Res. 1997; 38: 1807-1821Google Scholar). This mechanism may be mediated by lipid-poor apolipoproteins that transfer from HDL particles to cell-surface binding sites (1Oram J.F. Yokoyama S. Apolipoprotein-mediated removal of cellular cholesterol and phospholipids.J. Lipid Res. 1996; 37: 2473-2491Google Scholar, 8Mendez A.J. Anantharamaiah G.M. Segrest J.P. Oram J.F. Synthetic amphipathic helical peptides that mimic apolipoprotein A-I in clearing cellular cholesterol.J. Clin. Invest. 1994; 94: 1698-1705Google Scholar, 13Oram J.F. Mendez A.J. Slotte J.P. Johnson T.F. High density lipoprotein apolipoproteins mediate removal of sterol from intracellular pools but not from plasma membranes of cholesterol-loaded fibroblasts.Arterioscler. Thromb. 1991; 11: 403-414Google Scholar, 14Mendez A.J. Oram J.F. Limited proteolysis of high density lipoprotein abolishes its interaction with cell-surface binding sites that promote cholesterol efflux.Biochim. Biophys. Acta. 1997; 1346: 285-299Google Scholar). Although the molecular properties of the apolipoprotein-mediated pathway are unknown, it does not appear to involve SR-BI (3Ji Y. Jian B. Wang N. Sun Y. Moya M.D.L.L. Phillips M.C. Rothblat G.H. Swaney J.B. Tall A.R. Scavenger receptor B1 promotes high density lipoprotein-mediated cellular cholesterol efflux.J. Biol. Chem. 1997; 272: 20982-20985Google Scholar) or caveolae (15Mendez A.J. Lin G. Oram J.F. Apolipoprotein-mediated lipid efflux does not derive from membrane caveolae domains.Circulation. 1997; 96: I-485Google Scholar). It is unclear to what extent these lipid transport pathways contribute to overall mobilization of cholesterol from tissues in vivo. Studies with mice showed that plasma HDL cholesterol levels correlate inversely with SR-BI expression (16Kozarsky K.F. Donahee M.H. Rigotti A. Iqbal S.N. Edelman E.R. Krieger M. Overexpression of the HDL receptor SR-BI alters plasma HDL and bile cholesterol levels.Nature. 1997; 387: 414-417Google Scholar, 17Rigotti A. Trigatti B.L. Penman M. Rayburn H. Herz J. Krieger M. A targeted mutation in the murine gene encoding the high density lipoprotein (HDL) receptor scavenger receptor class B type I reveals its key role in HDL metabolism.Proc. Natl. Acad. Sci. USA. 1997; 94: 12610-12615Google Scholar), indicating that SR-BI-mediated selective uptake of HDL cholesteryl esters by tissues overshadows any possible role of this receptor in promoting transport of cholesterol from cells. Support for the physiological relevance of the apolipoprotein-mediated lipid removal pathway comes from studies of a rare HDL deficiency syndrome called Tangier disease (TD). Fibroblasts from TD homozygotes have a molecular defect that almost completely abolishes the ability of apolipoproteins to remove cellular cholesterol and phospholipids (18Walter M. Gerdes U. Seedorf U. Assmann G. The high density lipoprotein apolipoprotein A-I-induced mobilization of cellular cholesterol is impaired in fibroblasts from Tangier disease subjects.Biochem. Biophys. Res. Commun. 1994; 205: 850-856Google Scholar, 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: 78-87Google Scholar, 20Rogler G. Trumbach B. Klima B. Lackner K.J. Schmitz G. HDL-mediated efflux of intracellular cholesterol is impaired in fibroblasts from Tangier disease patients.Arterioscler. Thromb. Vasc. Biol. 1995; 15: 683-690Google Scholar, 21Remaley A.T. Schumacher U.K. Stonik J.A. Farsi B.D. Nazih H. Brewer 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: 1813-1821Google Scholar). These patients are characterized by a rapid turnover of apoA-I and deposition of cholesteryl esters in tissue macrophages (22Assmann G.A. von Eckardstein A. Brewer H.B. Familial HDL deficiency: Tangier disease.in: Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic Basis of Inherited Disease. McGraw-Hill Book Co., New York1995: 2053-2072Google Scholar), implying that an inability of newly synthesized apoA-I to acquire cellular lipids leads to accumulation of sterols in tissues and degradation of apoA-I. Thus the apolipoprotein-mediated lipid removal pathway clearly plays an important role in mobilizing tissue cholesterol and generating HDL particles. These recent findings illustrate the importance of developing cell culture systems to characterize the different HDL-mediated lipid removal pathways and to identify the participating cellular proteins. Many laboratories have relied on the use of immortalized cell lines to study HDL-mediated cholesterol efflux. Previous studies have shown, however, that the activity of the lipid removal pathway mediated by lipid-free apolipoproteins is sensitive to the proliferative state of cells, with quiescent cells exhibiting higher activities than proliferating cells (12Mendez A.J. Cholesterol efflux mediated by apolipoproteins is an active cellular process distinct from efflux mediated by passive diffusion.J. Lipid Res. 1997; 38: 1807-1821Google Scholar, 13Oram J.F. Mendez A.J. Slotte J.P. Johnson T.F. High density lipoprotein apolipoproteins mediate removal of sterol from intracellular pools but not from plasma membranes of cholesterol-loaded fibroblasts.Arterioscler. Thromb. 1991; 11: 403-414Google Scholar). This implies that the apolipoprotein-dependent component of HDL-mediated cholesterol efflux would be relatively inactive in rapidly proliferating cells. Because immortalized cell lines proliferate rapidly even in the absence of serum growth factors, they may be poor models for studying apolipoprotein-mediated lipid removal. In the current study, we examined the effects of immortalization of human skin fibroblasts on the relative activities of the different lipid efflux pathways. Results show that immortalization of fibroblasts with papillomavirus E6/E7 oncogenes selectively reduced lipid efflux promoted by lipid-free apoA-I, and that treatment of immortalized fibroblasts with a cAMP analog largely reversed this reduction in lipid efflux. The severely impaired apolipoprotein-mediated lipid efflux in TD fibroblasts, which persisted after immortalization, was not improved by treatment with a cAMP analog. These findings indicate that papillomavirus-induced immortalization of cells selectively inactivates the apolipoprotein-mediated lipid removal pathway by a cAMP-dependent process downstream from the cellular defect in TD. LDL and HDL3 (hereafter referred to as HDL) were prepared by sequential ultracentrifugation in the density intervals 1.019–1.063 and 1.125–1.21g/ml, respectively, and HDL was depleted of apoE and apoB as previously described (18Walter M. Gerdes U. Seedorf U. Assmann G. The high density lipoprotein apolipoprotein A-I-induced mobilization of cellular cholesterol is impaired in fibroblasts from Tangier disease subjects.Biochem. Biophys. Res. Commun. 1994; 205: 850-856Google Scholar, 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: 78-87Google Scholar). ApoA-I was purified from isolated HDL as described previously (8Mendez A.J. Anantharamaiah G.M. Segrest J.P. Oram J.F. Synthetic amphipathic helical peptides that mimic apolipoprotein A-I in clearing cellular cholesterol.J. Clin. Invest. 1994; 94: 1698-1705Google Scholar) and was iodinated by the iodine monochloride method (23Bilheimer D.W. Eisenberg S. Levy R.I. The metabolism of very low density lipoprotein proteins.Biochim. Biophys. Acta. 1972; 260: 212-221Google Scholar). Trypsinized HDL was prepared as previously described (13Oram J.F. Mendez A.J. Slotte J.P. Johnson T.F. High density lipoprotein apolipoproteins mediate removal of sterol from intracellular pools but not from plasma membranes of cholesterol-loaded fibroblasts.Arterioscler. Thromb. 1991; 11: 403-414Google Scholar, 14Mendez A.J. Oram J.F. Limited proteolysis of high density lipoprotein abolishes its interaction with cell-surface binding sites that promote cholesterol efflux.Biochim. Biophys. Acta. 1997; 1346: 285-299Google Scholar) by treating HDL with trypsin for 30 min at 37°C at an HDL:trypsin protein ratio of 40:1. This procedure digests approximately 25% of the total HDL protein content of HDL particles without disturbing its lipid composition (14Mendez A.J. Oram J.F. Limited proteolysis of high density lipoprotein abolishes its interaction with cell-surface binding sites that promote cholesterol efflux.Biochim. Biophys. Acta. 1997; 1346: 285-299Google Scholar). For experiments comparing HDL and trypsinized HDL, molar concentrations were based on particle phospholipid composition measured by the method of Bartlett (24Bartlett G.R. Phosphorus assay in column chromatography.J. Biol. Chem. 1959; 234: 466-468Google Scholar). Molarities of HDL particles and apoA-I were calculated based on protein molecular weights of 100,000 for HDL and 28,000 for apoA-I. LDL was acetylated by the method of Goldstein et al. (25Goldstein J.L. Ho Y.K. Basu S.K. Brown M.S. Binding site on macrophages that mediate uptake and degradation of acetylated low density lipoprotein, producing massive cholesterol deposition.Proc. Natl. Acad. Sci. USA. 1979; 76: 333-337Google Scholar). Human fibroblasts were obtained from skin explants from two normal subjects (NL4, NL7) and two patients with homozygous Tangier disease (IM, previously TG1; JW, previously TG2) as described (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: 78-87Google Scholar). BHK cells and PD388 and RAW247 macrophages were obtained from ATCC, and Fu5AH cells were a gift from Dr. G. Rothblat (Allegheny University of Health Sciences, Philadelphia). Cultured cells were grown and maintained in either DMEM (fibroblasts, Fu5AH cells, and BHK cells) or Ham's F-12 (PD388 and RAW247 macrophages) supplemented with 5–10% fetal bovine serum (growth medium). The same medium supplemented with 1 mg/ml bovine serum albumin (BSA) instead of serum (serum-free medium) was used for all cholesterol loading, equilibration, and efflux incubations. Primary human skin fibroblasts were immortalized as described previously (26Halbert C.L. Demers G.W. Galloway D.A. The E6 and E7 genes of human papillomavirus type 6 have weak immortalizing activity in human epithelial cells.J. Virol. 1992; 66: 2125-2134Google Scholar, 27Perez-Reyes N. Halbert C.L. Smith P.P. Benditt E.P. McDougall J.K. Immortalization of primary human smooth muscle cells.Proc. Natl. Acad. Sci. USA. 1992; 89: 1224-1228Google Scholar) by infection with amphotrophic retroviruses containing vectors with inserts of human papillomavirus 16 oncogenes E6 and E7 and a neomycin selectable marker. Pooled cell populations were selected in the presence of G418 for two passages, after which G418 was excluded from the medium. In some cases, cells were infected with vector alone as controls (mock-infected). Fibroblasts were used between the fifth and thirteenth passage (primary) or sixth and fourteenth passage (immortalized). Cell number per dish was quantified using a Coulter Counter (Hialeah, FA) after cells were removed from the dishes by trypsinization. Cell protein was quantified by the method of Lowry et al. (28Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 1951; 193: 265-275Google Scholar) after digestion of cells with 0.1 N NaOH. For most experiments, cells were seeded into 16-mm wells and grown to confluence. Cells were loaded with cholesterol by incubation for 24 h to 48 h in serum-free medium supplemented with either nonlipoprotein cholesterol (30 μg/ml), LDL (100 μg/ml), or acetylated LDL (50 μg/ml), followed by an 18–20 h incubation with the same medium lacking cholesterol to allow equilibration of cholesterol pools. In some experiments, 8-bromo-cAMP (8-Br-cAMP) was added to the medium during the equilibration incubations. Unless indicated otherwise, cellular cholesterol pools were radiolabeled by addition of 0.2–0.5 μCi/ml [1,2-3H]cholesterol (40–60 Ci/mmol, Amersham Corp., Arlington Heights, IL) to the growth medium 3 days prior to cholesterol loading. To radiolabel cellular phospholipids in cholesterol-loaded cells, 10 μCi/ml [3H]choline chloride (75–85 Ci/mmol; Amersham Corp.) was added to the equilibration medium. Cells were washed with phosphate-buffered saline (PBS) containing BSA twice or four times prior to measurements of cholesterol and phospholipid efflux, respectively. Cells were incubated at 37°C with serum-free medium and the indicated additions. After the indicated times, the efflux media were collected and centrifuged to remove cell debris, and cell layers were rinsed twice with ice-cold PBS/BSA and twice with PBS. Media and cells were stored frozen at -20°C until extraction of lipid and protein. Efflux media was either counted directly (for cells labeled with [3H]cholesterol) or extracted by the method of Folch et al. (29Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipids from animal tissues.J. Biol. Chem. 1957; 226: 497-509Google Scholar) for [3H]phospholipid or cholesterol mass measurements. Cell layers were extracted with hexane–isopropanol 3:2 (vol/vol) as described (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: 78-87Google Scholar). Sterol species were separated by thin-layer chromatography on silica gel G plates developed in hexane–diethyl ether–methanol–acetic acid 120:30:10:1.5 (vol/vol/vol/vol). Choline-containing phospholipids were separated by thin-layer chromatography on silica gel plates developed in chloroform–methanol–water 65:35:4 (vol/vol/vol). Lipid spots were identified by staining with I2 vapor and by co-migration with standards. Appropriate spots were taken for determination of sterol and phospholipid radioactivity or sterol mass (8Mendez A.J. Anantharamaiah G.M. Segrest J.P. Oram J.F. Synthetic amphipathic helical peptides that mimic apolipoprotein A-I in clearing cellular cholesterol.J. Clin. Invest. 1994; 94: 1698-1705Google Scholar, 9Yancey P.G. Bielicki J.K. Johnson W.J. Lund-Katz S. Palgunachari M.N. Anantharamaiah G.M. Segrest J.P. Phillips M.C. Rothblat G.H. Efflux of cellular cholesterol and phospholipid to lipid-free apolipoproteins and class A amphipathic peptides.Biochemistry. 1995; 34: 7955-7965Google Scholar). Unless indicated otherwise, efflux of radiolabeled lipids represents the fraction of total radiolabeled lipid (cells plus medium) appearing in the medium. Cholesterol oxidase sensitivity of plasma membrane [3H]cholesterol was determined by treating cells at 37°C with DMEM containing 1 U/ml cholesterol oxidase (30Smart E.J. Ying Y-S. Conrad P.A. Anderson R.G.W. Caveolin moves from caveolae to the Golgi apparatus in response to cholesterol oxidation.J. Cell Biol. 1994; 127: 1185-1197Google Scholar) and measuring fractional conversion of free [3H]cholesterol to [3H]cholestenone as described (12Mendez A.J. Cholesterol efflux mediated by apolipoproteins is an active cellular process distinct from efflux mediated by passive diffusion.J. Lipid Res. 1997; 38: 1807-1821Google Scholar). Accessibility of cholesterol to cyclodextrin was assessed by measuring the fraction of cellular free [3H]cholesterol released into DMEM containing 25 mm hydroxypropyl-β-cyclodextrin (Research Plus, Bayonne, NJ) during 30-min incubations at 37°C. The fraction of cellular [3H]sphingomyelin accessible to sphingomyelinase was determined by treating [3H]choline-labeled cells with DMEM plus or minus 0.1 U/ml sphingomyelinase for 30 min at 37°C and measuring cellular [3H]sphingomyelin content. The percent sphingomyelin-sensitive phospholipid was calculated as 100× the difference in [3H]sphingomyelin content between enzyme-treated and untreated cells divided by values for untreated cells. To determine the radiolabeled lipid composition of caveolaerich membranes, washed fibroblasts were dislodged from the wells and pelleted in a microfuge, cells were solubilized in 1% Triton X-100 (in MES buffer, pH 6.5) at 0°C using a 25-g needle to disperse cells, detergent insoluble membranes were sedimented at 4°C by microfugation at 20,000 g for 10 min, and lipids in the pellets were Folch-extracted (29Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipids from animal tissues.J. Biol. Chem. 1957; 226: 497-509Google Scholar) for separation by thin-layer chromatography and scintillation counting. Detergent-soluble radiolabeled lipids were measured in aliquots representing 10% of the total volume of the microfuge supertants to minimize the interference of Triton X-100 with the lipid isolation procedures. The detergent-insoluble pellet contained over 60% of the total cellular sphingomyelin and immunoblot-detectable caveolin-1 and less than 10% of the total phosphatidylcholine and membrane protein. Cholesterol-loaded fibroblasts were incubated at 37°C for 6 h with serum-free medium containing 125I-labeled apoA-I and unlabeled apoA-I. Cells were washed twice with ice-cold PBS/BSA and twice with PBS. Cell layers were dissolved in 0.1 N NaOH and aliquots were taken for quantitation of radioactivity and protein (8Mendez A.J. Anantharamaiah G.M. Segrest J.P. Oram J.F. Synthetic amphipathic helical peptides that mimic apolipoprotein A-I in clearing cellular cholesterol.J. Clin. Invest. 1994; 94: 1698-1705Google Scholar). Cells were seeded into covered glass chambers and grown to approximately 50% confluence. Cells were washed and treated with PBS containing 4 μg/ml acetoxymethyl ester of calcein (calcein-AM, molecular probes, Eugene, OR) for 10 min at 37°C. After washing with PBS and adding growth medium, the cover-slip chambers were mounted on the stage of an ACAS Ultima confocal scanning microscope. Fields of 540 × 540 microns were scanned at a resolution of 2 microns/pixel using a 100× oil immersion objective with 488 nm excitation from an argon–ion laser (300 mwatts). Calcein fluorescence emission was detected with a 530/30 nm band pass filter using a pinhole setting of 100 microns. Area and perimeter values were determined for individual cells on a DASY 9,000 Workstation (Meridian Instruments, Okemos, MI) using the Single Cell Analysis software package provided by the manufacturer. Shape factor was calculated as perimeter2/4πarea. The shape factor is one for a circle and increases with shape complexity. For each cell line, mean area and shape factor were calculated as the grand mean over all cells. Statistical analysis was done using S-PLUS, Version 3.1 Release 1 for Sun SPARC, Sun OS 4.x (Statistical Sciences, Inc., Seattle, WA), and the Statistical Analysis System, Proprietary Software Release 6.09 (SAS Institute Inc., Carey, NC). Standard errors were weighted by the number of cells per image, and the associated degree of freedom was the number of image fields minus one. Each P value was obtained by ANOVA. Log transformations were taken on both area and shape factor to satisfy ANOVA assumptions and adjustment was made for image field. The P values are 2-sided and based on the standard ANOVA F-test. For the current study, 3 to 5 images containing at least five isolated cells per image were analyzed for each cell line. To compare cellular expression of SR-BI and caveolin-1, fibroblasts grown in 60-mm dishes were washed with PBS and dislodged from the dishes in ice-cold 50 mm Tris saline (pH 7.4) containing protease inhibitors and 0.5 mm EDTA (30Smart E.J. Ying Y-S. Conrad P.A. Anderson R.G.W. Caveolin moves from caveolae to the Golgi apparatus in response to cholesterol oxidation.J. Cell Biol. 1994; 127: 1185-1197Google Scholar). After centrifugation, cell pellets were solubilized in buffer containing 2% SDS, aliquots were assayed for protein content, and equal amounts of cellular protein (200 μg) were applied to adjacent lanes of a 15% polyacrylamide gel. After electrophoresis, proteins were transferred to nitrocellulose, and SR-BI and caveolin-1 were identified by immunoblot analysis as described previously (31Garver W.S. Deeg M.A. Bowen R.F. Culala M.M. Bierman E.L. Oram J.F. Phosphoproteins regulated by the interaction of high-density lipoprotein with human skin fibroblasts.Arterioscler. Thromb. Vasc. Biol. 1997; 17: 2698-2706Google Scholar) using 1:5,000 dilutions of rabbit antisera to human SR-BI (a gift from Dr. Monty Krieger, MIT, Boston) and a 1:5,000 dilution of rabbit antiserum to human caveolin-1 (Transduction Laboratories, Lexington, KY). Antibody-positive bands were visualized by enhanced chemiluminescence (ECL, Amersham). To assess cholesterol esterification rates, cells were washed once with PBS and incubated for 1 h at 37°C with DMEM containing 9 μm [14C]oleate (50–60 mCimmol, Amersham Corp.) bound to 3 μm BSA (8Mendez A.J. Anantharamaiah G.M. Segrest J.P. Oram J.F. Synthetic amphipathic helical peptides that mimic apolipoprotein A-I in clearing cellular cholesterol.J. Clin. Invest. 1994; 94: 1698-1705Google Scholar, 13Oram J.F. Mendez A.J. Slotte J.P. Johnson T.F. High density lipoprotein apolipoproteins mediate removal of sterol from intracellular pools but not from plasma membranes of cholesterol-loaded fibroblasts.Arterioscler. Thromb. 1991; 11: 403-414Google Scholar, 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: 78-87Google Scholar). Cell lipids were separated by thin-layer chromatography as described above to determine cholesteryl ester radioactivity. To measure cellular cAMP content, fibroblasts plated in 35-mm wells were treated as described in the text, cell layers were extracted with ice-cold 65% (v/v) ethanol, and cAMP was quantified using the Biotrak Enzyme Immunoassay (non-acetylated) system (Amersham Corp.) according to the manufacturer's directions. Student's t test was performed where indicated for determining significances between sets of data. Lipid-free apolipoproteins remove cellular cholesterol by an active process that is up-regulated by both loading cells with cholesterol and inhibiting cell proliferation (1Oram J.F. Yokoyama S. Apolipoprotein-mediated removal of cellular cholesterol and phospholipids.J. Lipid Res. 1996; 37: 2473-2491Google Scholar, 12Mendez A.J. Cholesterol efflux mediated by apolipoproteins is an active cellular process distinct from efflux mediated by passive diffusion.J. Lipid Res. 1997; 38: 1807-1821Google Scholar). We found that this lipid removal pathway was absent or had very low activity in many immortalized cell lines, even when previously incubated with cholesterol-rich media. When Fu5AH hepatoma cells were radiolabeled with [3H]cholesterol and then loaded with non-lipoprotein cholesterol, 6-h incubations with HDL led to a dose-dependent increase in [3H]cholesterol efflux (Fig. 1A). In contrast, purified apoA-I had virtually no ability to stimulate cholesterol efflux from these cells. We obtained the same
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