Scavenger receptor class B type I affects cholesterol homeostasis by magnifying cholesterol flux between cells and HDL
2001; Elsevier BV; Volume: 42; Issue: 12 Linguagem: Inglês
10.1016/s0022-2275(20)31525-x
ISSN1539-7262
AutoresMargarita de la Llera-Moya, Margery A. Connelly, Denise Drazul, Seth M. Klein, Elda Favari, Patricia G. Yancey, David L. Williams, George H. Rothblat,
Tópico(s)Drug Transport and Resistance Mechanisms
ResumoResults from several laboratories clearly indicate that expression of scavenger receptor class B type I (SR-BI) enhances the bidirectional flux of cholesterol between cells and lipoproteins. Because the activity of HMG-CoA reductase, the key enzyme in cholesterol biosynthesis, is regulated by cell cholesterol content, we designed experiments to investigate the effect of SR-BI expression on the activity of this enzyme and on net cellular cholesterol mass. In addition, we compared the function of SR-BI with its human homolog, CD36 and LIMPII analogous 1. Our experiments demonstrate that both receptors enhance the flux of unesterified or free cholesterol bidirectionally, down a concentration gradient. Receptor-mediated cholesterol flux can effectively modulate multiple aspects of cellular cholesterol metabolism, including the pool that regulates the activity of HMG-CoA reductase. We also found that constitutive expression of SR-BI alters the steady state level of cellular cholesterol and phospholipid when SR-BI-expressing cells are maintained in medium containing serum lipoproteins. All of these effects are proportional to the level of receptor on the cell surface. These data indicate that the level of SR-BI expression determines both the rate of free cholesterol flux and the steady state level of cellular cholesterol.—de la Llera-Moya, M., M. A. Connelly, D. Drazul, S. M. Klein, E. Favari, P. G. Yancey, D. L. Williams, and G. H. Rothblat. Scavenger receptor class B type I affects cholesterol homeostasis by magnifying cholesterol flux between cells and HDL. J. Lipid Res. 2001. 42: 1969–1978. Results from several laboratories clearly indicate that expression of scavenger receptor class B type I (SR-BI) enhances the bidirectional flux of cholesterol between cells and lipoproteins. Because the activity of HMG-CoA reductase, the key enzyme in cholesterol biosynthesis, is regulated by cell cholesterol content, we designed experiments to investigate the effect of SR-BI expression on the activity of this enzyme and on net cellular cholesterol mass. In addition, we compared the function of SR-BI with its human homolog, CD36 and LIMPII analogous 1. Our experiments demonstrate that both receptors enhance the flux of unesterified or free cholesterol bidirectionally, down a concentration gradient. Receptor-mediated cholesterol flux can effectively modulate multiple aspects of cellular cholesterol metabolism, including the pool that regulates the activity of HMG-CoA reductase. We also found that constitutive expression of SR-BI alters the steady state level of cellular cholesterol and phospholipid when SR-BI-expressing cells are maintained in medium containing serum lipoproteins. All of these effects are proportional to the level of receptor on the cell surface. These data indicate that the level of SR-BI expression determines both the rate of free cholesterol flux and the steady state level of cellular cholesterol. —de la Llera-Moya, M., M. A. Connelly, D. Drazul, S. M. Klein, E. Favari, P. G. Yancey, D. L. Williams, and G. H. Rothblat. Scavenger receptor class B type I affects cholesterol homeostasis by magnifying cholesterol flux between cells and HDL. J. Lipid Res. 2001. 42: 1969–1978. The maintenance of optimum cellular cholesterol content is basic to proper cell physiology. Cells reciprocally regulate cholesterol biosynthesis and the exchange of cholesterol with the extracellular lipoproteins (1Brown M.S. Dana S.E. Goldstein J.L. Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in human fibroblasts by lipoproteins.Proc. Natl. Acad. Sci. USA. 1973; 70: 2162-2166Google Scholar, 2Daerr W.H. Gianturco S.H. Patsch J.R. Smith L.C. Gotto Jr., A.M. Stimulation and suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase in normal fibroblasts by high density lipoprotein subclasses.Biochim. Biophys. Acta. 1980; 619: 287-301Google Scholar). All cells can exchange cholesterol with extracellular acceptors and a variety of mechanisms have been proposed to explain this movement or flux of cellular cholesterol (3Rothblat G.H. de la Llera-Moya M. Atger V. Kellner-Weibel G. Williams D.L. Phillips M.C. Cell cholesterol efflux: integration of old and new observations provides new insights.J. Lipid Res. 1999; 40: 781-796Google Scholar). Receptor-mediated cholesterol flux has been described in a number of different cell types (4Jian B. de la Llera-Moya M. Ji Y. Wang N. Phillips M.C. Swaney J.B. Tall A.R. Rothblat G.H. Scavenger receptor class B type I as a mediator of cellular cholesterol efflux to lipoproteins and phospholipid acceptors.J. Biol. Chem. 1998; 273: 5599-5606Google Scholar, 5Lawn R.M. Wade D.P. Garvin M.R. Wang X. Schwartz K. Porter J.G. Seilhamer J.J. Vaughan A.M. Oram J.F. The Tangier disease gene product ABC1 controls the cellular apolipoprotein-mediated lipid removal pathway.J. Clin. Invest. 1999; 104: R25-R31Google Scholar). More specifically, it has been repeatedly demonstrated that the expression of the scavenger receptor class B type I (SR-BI) can greatly enhance cholesterol movement between cells and phospholipid-containing acceptors such as lipoproteins (4Jian B. de la Llera-Moya M. Ji Y. Wang N. Phillips M.C. Swaney J.B. Tall A.R. Rothblat G.H. Scavenger receptor class B type I as a mediator of cellular cholesterol efflux to lipoproteins and phospholipid acceptors.J. Biol. Chem. 1998; 273: 5599-5606Google Scholar, 6Yancey P.G. de la Llera-Moya M. Swarnakar S. Monzo P. Klein S.M. Connelly M.A. Johnson W.J. Williams D.L. Rothblat G.H. HDL phospholipid composition is a major determinant of the bi-directional flux and net movement of cellular free cholesterol mediated by scavenger receptor-BI (SR-BI).J. Biol. Chem. 2000; 275: 36596-36604Google Scholar, 7Jian B. de la Llera-Moya M. Royer L. Rothblat G. Francone O. Swaney J.B. Modification of the cholesterol efflux properties of human serum by enrichment with phospholipid.J. Lipid Res. 1997; 38: 734-744Google Scholar). SR-BI interacts with both LDL and HDL to promote the bidirectional exchange of free or unesterified cholesterol (FC) between these lipoproteins and SR-BI-expressing cells (8de la Llera-Moya M. Rothblat G.H. Connelly M.A. Kellner-Weibel G. Sakr S.W. Phillips M.C. Williams D.L. Scavenger receptor BI (SR-BI) mediates free cholesterol flux independently of HDL tethering to the cell surface.J. Lipid Res. 1999; 40: 575-580Google Scholar, 9Ji Y. Jian B. Wang N. Sun Y. de la Llera Moya M. 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). In addition, SR-BI promotes the selective uptake of cholesteryl ester (CE) by interacting with HDL (10Williams D.L. Connelly M.A. Temel R.E. Swanakar S. Phillips M.C. de la Llera-Moya M. Rothblat G.H. Scavenger receptor BI and cholesterol trafficking.Curr. Opin. Lipidol. 1999; 10: 329-339Google Scholar, 11Rigotti A. Trigatti B. Babitt J. Penman M. Xu S. Krieger M. Scavenger receptor BI—a cell surface receptor for high density lipoprotein.Curr. Opin. Lipidol. 1997; 8: 181-188Google Scholar). Although the mechanism whereby SR-BI promotes movement of cholesterol between cells and lipoproteins is not well understood, it is apparent that cholesterol flux mediated by SR-BI can effectively modulate cellular cholesterol content. Because the activity of HMG-CoA reductase (HMGR), a key enzyme in cholesterol biosynthesis, is tightly regulated by cell cholesterol content we designed experiments to investigate the effect of SR-BI expression on both cell cholesterol and the activity of this enzyme. Human serum was obtained by the Lipoprotein Core Laboratory under an approved protocol and with approved consent from healthy, normolipemic volunteers. High density lipoprotein class 3 (HDL3) and lipoprotein-deficient serum (LPDS) were isolated from human serum by sequential centrifugation in the Lipoprotein Core Laboratory (8de la Llera-Moya M. Rothblat G.H. Connelly M.A. Kellner-Weibel G. Sakr S.W. Phillips M.C. Williams D.L. Scavenger receptor BI (SR-BI) mediates free cholesterol flux independently of HDL tethering to the cell surface.J. Lipid Res. 1999; 40: 575-580Google Scholar). HDL3 was modified as previously described by incubation with phospholipid micelles at the transition temperature (6Yancey P.G. de la Llera-Moya M. Swarnakar S. Monzo P. Klein S.M. Connelly M.A. Johnson W.J. Williams D.L. Rothblat G.H. HDL phospholipid composition is a major determinant of the bi-directional flux and net movement of cellular free cholesterol mediated by scavenger receptor-BI (SR-BI).J. Biol. Chem. 2000; 275: 36596-36604Google Scholar); both native and modified HDL were characterized by measuring cholesterol and phospholipid content relative to total protein. Phospholipids were obtained from Avanti Polar Lipids (Alabaster, AL). Chemicals used in the HMGR assay were purchased from Sigma (St. Louis, MO) except for hydroxy-3-methylglutaryl-coenzyme A, dl-3-[glutaryl-3-12C] and mevalonic acid, ammonium salt, (R)-[5-3H], which were purchased from NEN Life Sciences Products (Boston, MA). [1,2-3H]-cholesterol and 125I-labeled iodine (125I-iodine) were also purchased from NEN Life Sciences Products. Whatman LK6 silica gel 60A thin-layer chromatography plates were from Whatman (Clifton, NJ). All organic solvents were purchased from Fisher Scientific (Pittsburgh, PA). Sterols used as gas–liquid chromatography (GLC) standards such as cholestenone, cholesterol, and cholesterol methyl ether were from Sigma. Cholesterol oxidase from Nocardia erythropolis were purchased from Boehringer Mannheim (Indianapolis, IN). DMEM, Eagle's minimal essential medium (EMEM), PBS, and geneticin (G418) were obtained from Cellgro-Mediatech (Fisher Scientific). FBS, calf serum (CS), trypsin-EDTA, penicillin-streptomycin, and gentamicin were purchased from Sigma. Penicillin-streptomycin-glutamine antibiotic solution was purchased from GIBCO (Grand Island, NY). Tissue culture flasks and plates were obtained from Corning (Corning, NY). Other materials and reagents were obtained as noted below. COS-7 cells were grown and transiently transfected as previously described (8de la Llera-Moya M. Rothblat G.H. Connelly M.A. Kellner-Weibel G. Sakr S.W. Phillips M.C. Williams D.L. Scavenger receptor BI (SR-BI) mediates free cholesterol flux independently of HDL tethering to the cell surface.J. Lipid Res. 1999; 40: 575-580Google Scholar). Briefly, 1.5 × 106 cells were seeded in 100-mm plates in growth medium (DMEM, 10% CS, 1 mM sodium pyruvate, antibiotics). Cells were transfected with a mixture of 10 μg of the desired plasmid diluted in 470 μl of serum-free medium and 30 μl of FuGENE 6 (Boehringer Mannheim, Indianapolis, IN) in a sterile polystyrene tube (Falcon 2058; Becton Dickinson Labware, Lincoln Park, NJ) after 18 h of incubation (37°C, humidified 95% air–5% CO2). Two different plasmids were used to express SR-BI. Plasmid pSG5(SR-BI) has been described (8de la Llera-Moya M. Rothblat G.H. Connelly M.A. Kellner-Weibel G. Sakr S.W. Phillips M.C. Williams D.L. Scavenger receptor BI (SR-BI) mediates free cholesterol flux independently of HDL tethering to the cell surface.J. Lipid Res. 1999; 40: 575-580Google Scholar). Plasmid pcDNA3(SR-BI) was used to express SR-BI in experiments comparing SR-BI with CD36 and LIMPII analogous 1 (CLA-1). This plasmid was constructed from pmSR-BI 77, a gift from M. Krieger (Massachusetts Institute of Technology, Cambridge, MA). The SR-BI-coding region was digested with HindIII and XbaI and inserted into a pcDNA3 vector modified to contain a cytomegalovirus translational enhancer upstream of the multiple cloning site to boost protein expression. The plasmid used to express CLA-1, pcDNA3(Cla-I), was a gift from O. Quehenberger (University of California, San Diego). All plasmids were prepared with endotoxin-free Qiagen (Valencia, CA) Maxi-prep kits and were sequenced throughout the coding region to confirm correct fragment insertion and that no point mutations had occurred. DNA sequencing was performed by the automated sequencing facility at the State University of New York (SUNY, Stony Brook, NY). Reactions were performed with a dye termination cycle sequencing kit and were analyzed on an Applied Biosystems model 373 DNA sequencer (PE Applied Biosystems, Foster City, CA). Transfections with empty vector were done to yield control cells. Experiments were typically carried out 48–72 h after transfection. SR-BI expression was confirmed by Western blot assays of whole cell lysates as described previously (12Temel R.E. Trigatti B. DeMattos R.B. Azhar S. Krieger M. Williams D.L. Scavenger receptor class B, type I (SR-BI) is the major route for the delivery of high density lipoprotein cholesterol to the steroidogenic pathway in cultured mouse adrenocortical cells.Proc. Natl. Acad. Sci. USA. 1997; 94: 13600-13605Google Scholar), using antibody directed against the C-terminal tail of SR-BI (a gift from S. Azhar, Veterans Affairs Palo Alto Health Care Systems, Palo Alto, CA). Stably transfected SR-BI-expressing clones were obtained from COS-7 cells transfected as described above with pcDNA3(SR-BI). Clones were maintained in growth medium supplemented with geneticin (400 μg/ml), which was used for selection. Stably transfected WI38-VA13 human fibroblastic cells were obtained after transfection with the same SR-BI-expressing plasmid, using geneticin selection (800 μg/ml). Cultures of WI38-VA13 human fibroblastic cells were maintained in DMEM supplemented with 10% FBS, antibiotics, and geneticin (800 μg/ml). SR-BI expression in stably transfected cell lines was confirmed by Western blots of whole cell lysates as described above. Cell surface SR-BI expression in COS-7 clones was measured using specific binding of HDL3 (see below). Frozen cell pellets were thawed and resuspended in 75 μl of TEDK buffer (50 mM Tris-HCl, 1 mM EDTA, 10 mM DTT, 10 M KCl, pH 7.5). The cells were lysed by sonication (10 min at room temperature). Lysates were incubated for 10 min at 37°C and then centrifuged for 1 min at 12,000 rpm to obtain postnuclear supernatants (PNS). PNS protein was measured by modified Lowry assay (13Markwell M.A.K. Haas S.M. Bieber L.L. Tolbert N.E. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples.Anal. Biochem. 1978; 87: 206-210Google Scholar). HMGR activity was assayed by a modification of the assay published by Harwood, Bridge, and Stacpoole (14Harwood H.J. Bridge D.M. Stacpoole P.W. In vivo regulation of human mononuclear leukocyte 3-hydroxy-3-methylglutaryl coenzyme A reductase.J. Clin. Invest. 1987; 79: 1125-1132Google Scholar). Typically, 0.1–0.2 mg of PNS protein was preincubated for 20 min at 37°C in a 75-μl reaction mixture containing 33 mM Tris (pH 7.5), 33.3 mM KCl, 3.3 mM DTT, and 68 mM EDTA (inhibits mevalonic acid kinase). After the preincubation, 30 mM glucose 6-phosphate, 3.4 mM NADP+, and 0.2 U of glucose-6-phosphate dehydrogenase were added to regenerate NADPH. To start the reaction, [3-14C]HMG-CoA (specific activity, ~15–30 cpm/pmol) was added, and the mixture was then incubated for an additional 90 min at 37°C. The reaction was stopped by adding 20 μl of 12 N HCl to inactivate the reductase, and convert the mevalonic acid to the lactone. The acidified mixture was placed on ice and [5-3H]mevalonic acid was added as an internal standard (0.6–1.2 Ci/mmol, 15,000–20,000 cpm per reaction). The mixture was incubated for 30 min at 37°C to assure complete lactonization. Unlabeled mevalonic acid lactone (10 μl, 100 mg/ml) was added as a carrier, and TLC was used to separate the product [silica gel plates run in toluene–acetone 1:1 (v/v)]. The lactone band was visualized with iodine and scraped. Radioactivity was detected by double isotope liquid scintillation counting. The reaction product obtained was corrected for recovery and enzyme activity was expressed as units (picomoles of mevalonic acid per minute per milligram cell protein). A reaction blank was always measured and subtracted. Cell cholesterol content was measured in aliquots of 2-propanol extracts from monolayers, using GLC as previously described (15Klansek J.J. Yancey P.G. St. Clair R.W. Fischer R.T. Johnson W.J. Glick J.M. Cholesterol quantitation by GLC: artifactual formation of short-chain esters.J. Lipid Res. 1995; 36: 2261-2266Google Scholar). Total cell phospholipids were measured by quantitating inorganic phosphorus in 2-propanol extracts as described previously (6Yancey P.G. de la Llera-Moya M. Swarnakar S. Monzo P. Klein S.M. Connelly M.A. Johnson W.J. Williams D.L. Rothblat G.H. HDL phospholipid composition is a major determinant of the bi-directional flux and net movement of cellular free cholesterol mediated by scavenger receptor-BI (SR-BI).J. Biol. Chem. 2000; 275: 36596-36604Google Scholar). Cell lipids were normalized to total cell protein measured by modified Lowry assay (13Markwell M.A.K. Haas S.M. Bieber L.L. Tolbert N.E. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples.Anal. Biochem. 1978; 87: 206-210Google Scholar). Cell cholesterol efflux to HDL3 was measured as previously described (8de la Llera-Moya M. Rothblat G.H. Connelly M.A. Kellner-Weibel G. Sakr S.W. Phillips M.C. Williams D.L. Scavenger receptor BI (SR-BI) mediates free cholesterol flux independently of HDL tethering to the cell surface.J. Lipid Res. 1999; 40: 575-580Google Scholar). Briefly, cells plated in multiwell plates were prelabeled by incubation for 24–48 h in growth medium containing serum labeled with [1,2-3H]-cholesterol. The radioactive cholesterol released to HDL3 at a given time was obtained by counting an aliquot of medium by liquid scintillation. Total cell radioactivity was measured in 2-propanol extracts obtained from washed monolayers. Radioactive cholesterol released to the medium was expressed as a fraction of the total radioactivity in the well. Selective uptake was measured with doubly labeled HDL to establish CE uptake independent of internalization as previously described (16Swarnakar S. Temel R.E. Connelly M.A. Azhar S. Williams D.L. Scavenger receptor class B, type II, mediates selective uptake of low density lipoprotein cholesteryl ester.J. Biol. Chem. 1999; 42: 29733-29739Google Scholar). The cholesterol oxidase-sensitive pool was measured as previously described, using a method that assays cholesterol found in caveolae (17Kellner-Weibel G. de la Llera-Moya M. Connelly M.A. Stoudt G. Christian A.E. Haynes M.P. Williams D.L. Rothblat G.H. Expression of scavenger receptor BI in COS-7 cells alters cholesterol content and distribution.Biochemistry. 2000; 39: 221-229Google Scholar). Briefly, cells were prelabeled with [1,2-3H]cholesterol and exposed to cholesterol oxidase, without previous fixation, for 1–4 h at 37°C. Extracts were prepared from washed monolayers, using 2-propanol, and the radioactive cholestenone and cholesterol were separated by TLC and quantitated by liquid scintillation counting. The radioactive cholestenone produced was expressed as a percentage of the total radioactive cell cholesterol. Cholestenone production after oxidase treatment was always compared with that found in replicate cell cultures harvested at the start of the experiment (T0 cells). Cholestenone formed in replicate cell cultures incubated at 37°C for up to 4 h without oxidase was no different than that found in T0 cells. In some experiments the fraction of cholesterol oxidized to cholestenone was measured by GLC. Cholestenone standards were used to establish its retention time. Both cholesterol and cholestenone were quantitated in the same chromatogram and the ratio of the area under the cholesterol peak to that under the cholestenone peak was used to estimate the fraction of cell cholesterol oxidized. In preliminary experiments estimates of cholestenone production, using TLC, of labeled cell extracts were compared with estimates obtained by GLC in identical cell extracts and the two estimates were found to be similar. Stably transfected COS-7 clones were characterized for SR-BI expression by measuring the specific binding of HDL3 labeled with 125I-iodine. Binding was measured by incubating cells and radioactive HDL3 (25 μg of HDL protein per ml) for 1.5 h at 4°C as described (18Williams D.L. de la Llera-Moya M. Thuahnai S.T. Lund-Katz S. Connelly M.A. Azhar S. Anantharamaiah G.M. Phillips M.C. Binding and cross-linking studies show that scavenger receptor BI (SR-BI) interacts with multiple sites in apolipoprotein A-I and identify the class A amphipathic α-helix in a recognition motif.J. Biol. Chem. 2000; 275: 18897-18904Google Scholar). Nonspecific binding was measured in duplicate incubations containing a 20× excess of nonradioactive ligand. Specific binding was calculated by subtraction of nonspecific binding from total binding. The mass of bound ligand was calculated from the specific activity of the radioactive HDL3. The iodine monochloride method was used to label HDL3 (19Goldstein J.L. Basu S.K. Brown M.S. Receptor-mediated endocytosis of low-density lipoprotein in cultured cells.Methods Enzymol. 1983; 98: 241-260Google Scholar). The figure legends specify whether the data represent means of multiple assays or of multiple experiments and whether the error bars represent the standard error or the standard deviation. Significant differences were established by unpaired t-tests. Rate constants were estimated by nonlinear regression after the model described by Johnson et al. (20Johnson W.J. Bamberger M.J. Latta M.J. Rapp R.A. Phillips M.C. Rothblat G.H. The bidirectional flux of cholesterol between cells and lipoproteins.J. Biol. Chem. 1986; 261: 5766-5776Google Scholar). All statistical analyses were done using GraphPad (San Diego, CA) Prism. Initial experiments were designed to validate our HMGR assay by demonstrating the well-documented dependence of HMGR activity on medium cholesterol content (1Brown M.S. Dana S.E. Goldstein J.L. Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in human fibroblasts by lipoproteins.Proc. Natl. Acad. Sci. USA. 1973; 70: 2162-2166Google Scholar). Transiently transfected COS-7 cells were prepared as described in Materials and Methods and then incubated with medium containing either 5% CS, 2.5% CS, or LPDS (5 mg/ml). The control cells used in these experiments were transfected with empty vector and have no detectable endogenous SR-BI as measured by Western blots of whole cell lysates (data not shown). As expected, when COS-7 cells were incubated for 24 h with medium containing increasingly lower serum concentrations, a reciprocal increase in HMGR activity was obtained in both control and SR-BI-expressing cells (Fig. 1), signifying a drop in cell cholesterol content. The increase in enzyme activitywas most pronounced when COS-7 cells were exposed to LPDS, and HMGR activity measured in these cells was significantly greater than the activity measured in cells exposed to 5% CS in both control and SR-BI cells. The effect of incubation with HDL3 on cell cholesterol content and HMGR activity was measured in transiently transfected COS-7 cells that had been maintained for 48 h in growth medium supplemented with 10% CS (T0 cells). Figure 2 shows that overnight incubation with HDL3 (100 μg of protein per ml) had little effect on HMGR activity in control cells. On the other hand, the same treatment caused a significant increase in enzyme activity in cells expressing SR-BI (T0 cells, 0.34 ± 0.10 units; vs. HDL3-treated cells, 2.10 ± 0.5 units; P = 0.002). To test the effect of SR-BI expression on HMGR activity in cholesterol-depleted cells, either control or SR-BI COS-7 cells were exposed to LPDS (5 mg protein/ml) for 24 h to deplete cell cholesterol and increase HMGR activity (T0 cells). Replicate cultures were then incubated for an additional 18 h with HDL3. Enzyme activity was measured in both T0 and HDL3-treated cells; in some experiments cellular cholesterol content was measured in identically treated, replicate cell cultures. Because LPDS treatment was expected to cause a significant decrease in cell cholesterol, we first confirmed that, in LPDS-treated cells, SR-BI retained its ability to enhance cell cholesterol efflux to HDL3 (data not shown). Figure 3 summarizes results from four different experiments in which LPDS-treated control and SR-BI-expressing cells were incubated with HDL3 (100 μg of protein per ml) for an additional 18 h. Compared with the activity measured at T0 time, incubating control cells with HDL3 did not significantly change HMGR activity. This result is in agreement with experiments published by Brown, Dana, and Goldstein (1Brown M.S. Dana S.E. Goldstein J.L. Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in human fibroblasts by lipoproteins.Proc. Natl. Acad. Sci. USA. 1973; 70: 2162-2166Google Scholar) and Daerr et al. (21Daerr W.H. Gianturco S.H. Patsch J.R. Smith L.C. Gotto A.M. Effects of HDL subclasses on 3-hydroxy-3-methylglutaryl coenzyme A reductase in human fibroblasts.Nutr. Metab. 1980; 24: 26-33Google Scholar), who have shown that incubation of LPDS-treated skin fibroblasts with a broad range of HDL concentrations did not change HMGR activity. In contrast, when LPDS-treated COS-7 cells expressing SR-BI were incubated with HDL3, a statistically significant decrease in HMGR activity was consistently obtained. Because changes in HMGR activity typically signal a change in cell cholesterol content, both cell cholesterol content and HMGR activity were measured in LPDS-treated COS-7 cells incubated with increasing concentrations of HDL3 for 18 h. As shown in Fig. 4, SR-BI expression promoted net influx of cholesterol from HDL3, resulting in significant increases in cell cholesterol content and a reciprocal decrease in HMGR activity. In these experiments identical treatmentsusing control cells resulted in minimal changes in cell cholesterol content and no change in HMGR activity (data not shown, P > 0.05).Fig. 4.Effect of incubation with increasing concentrations of HDL3 on HMGR activity and cell cholesterol content in LPDS-treated SR-BI COS-7 cells. Control and SR-BI-expressing COS-7 cells were transfected as described in Fig. 1. All the cells were then exposed for 24 h to LPDS (5 mg/ml), which was considered the T0 treatment. Some cultures were then incubated for an additional 18 h with increasing concentrations of HDL3. HMGR activity and cell cholesterol content were measured both at T0 and after HDL3 treatment as described in Materials and Methods. The graph shows the average of triplicate assays of HMGR activity and cell cholesterol levels obtained with SR-BI-expressing cells in two experiments. Significant change (P < 0.05) in HMGR (*) and cell cholesterol (++) obtained as compared with values measured in T0 cells.View Large Image Figure ViewerDownload (PPT) The results described above indicate that, although SR-BI enhances FC flux in or out of cells, the direction of this movement depends on the FC concentration gradient between the cell and HDL in the medium. To better characterize the effect of the cellular cholesterol status on the gradient for FC flux, experiments were done in which control and SR-BI-expressing cells having different initial cell cholesterol mass were incubated in parallel with the same HDL3 preparation. In these experiments cell cholesterol content and HMGR activity were measured in transiently transfected COS-7 cells that had been either maintained in growth medium supplemented with 10% CS or exposed to LPDS for 24 h before incubation with HDL3 for 18 h. The data in Fig. 5 show that, compared with T0 cells, when cholesterol-depleted, SR-BI-expressing cells were incubated with HDL3 (100 μg of protein per ml) there was net cholesterol influx (Fig. 5B, hatched column), and a reciprocal decrease in HMGR activity (Fig. 5A, hatched column). On the other hand, incubation of cholesterol-rich, SR-BI-expressing cells with HDL3 resulted in net efflux of cell cholesterol (Fig. 5B, solid column), and a reciprocal increase in HMGR activity (Fig. 5A, solid column). Overnight incubation with 100 μg of HDL3 protein per ml did not change either cholesterol mass or HMGR activity in LPDS-treated control cells and caused minimal changes in these parameters in control cells thathad been maintained in 10% CS (data not shown, P > 0.05). These results confirm our previous observations (Figs. 2, Fig. 3., Fig. 4.). Because significant changes in cell cholesterol content could be measured only in cells expressing SR-BI, we can conclude that under these conditions SR-BI magnifies the effect of HDL3 on cell cholesterol homeostasis. We can also conclude that, although SR-BI enhances both the bidirectional flux of FC and CE uptake, the gradient that determines net cholesterol movement depends not only on the concentration of extracellular lipoproteins that can interact with this receptor but also on factors that affect cellular cholesterol content. Our results demonstrate that SR-BI expression can profoundly affect cellular cholesterol homeostasis. Specifically, SR-BI expression markedly enhanced the exchange of cellular cholesterol with lipoproteins and changed the distribution of cellular cholesterol as evidenced by the increased sensitivity of intact cells to cholesterol oxidase (17Kellner-Weibel G. de la Llera-Moya M. Connelly M.A. Stoudt G. Christian A.E. Haynes M.P. Williams D.L. Rothblat G.H. Expression of scavenger receptor BI in COS-7 cells alters cholesterol content and distribution.Biochemistry. 2000; 39: 221-229Google Scholar). To test whether CLA-1, the human homolog of rodent SR-BI (22Calvo D. Vega M.A. Identification, primary structure, and dis
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