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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
1Division of Cancer Biology and Genetics, Cancer Research Institute and the Department of Pathology and Molecular Medicine, Queen’s University; and 2Centre d’Immunologie de Luminy, Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, Université de La Mediterranée, Marseille, France
Submitted 6 February 2006 ; accepted in final form 4 April 2006
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ABSTRACT |
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 TOP ABSTRACT EXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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5-fold more rapidly by wild-type cells. In addition, the rate of efflux from the wild-type but not the ABCA1–/– fibroblasts is increased a further twofold by inducers of ABCA1 expression. Thus under the experimental conditions employed, endogenous ABCA1 is a major contributor to 25-hydroxycholesterol efflux from wild-type fibroblasts. Evidence from in vitro studies indicates that oxysterols are potent inducers of genes involved in cellular cholesterol efflux and metabolism, including the ABCA1 gene, and repressors of genes involved in cholesterol synthesis or uptake. Our observations raise the possibility that efflux of oxysterols by ABCA1 could contribute to a homeostatic mechanism, which both attenuates oxysterol-induced expression of its cognate gene and alleviates repression of genes encoding proteins, such as HMG-CoA reductase and LDL receptor. active transport; cholesterol homeostasis
The mechanism by which ABCA1 mediates efflux of cholesterol and phospholipids remains poorly defined. ABC transporters typically use the energy of ATP binding and hydrolysis to drive the transport of substrate across cellular membranes (26). In the case of hydrophobic compounds, transport is thought to involve "flopping" of substrate from the inner to the outer leaflet of the membrane (55). In the presence of an acceptor molecule, this can result in a net flux of very hydrophobic compounds, such as phospholipids and cholesterol, across the lipid bilayer and into the luminal space. There is persuasive evidence that ABCA1 "flops" phospholipids, such as phosphatidylserine from the inner to the outer leaflet of the membrane (25), but it remains controversial whether cholesterol and possibly other sterols are direct substrates for ABCA1.
Most ABC proteins are energy-dependent active transporters, but there are exceptions. For example, the cystic fibrosis transmembrane transporter is an ATP-gated chloride channel (4), whereas the sulfonylurea receptors act as potassium channel regulators (67). To date, the evidence that ABCA1 can function as an ATP-dependent active transporter is indirect and based largely on studies of wild-type and mutant proteins in transfected cells. Mutations of ABCA1 that in other ABC proteins eliminate ATP hydrolysis and sometimes ATP binding markedly decrease the efflux of phospholipid and cholesterol to apoA-I or lipid-poor HDL (10). However, biochemical studies indicate that although ABCA1 can bind ATP with relatively high affinity, its ATPase activity, as evidenced by the ability to trap ADP in the presence of orthovanadate, is very low (63, 65). Consequently, it has been suggested that the protein may act as an ATP dependent apoA-I "receptor" rather than a direct active transporter (63). In addition, little is known of the substrate specificity of ABCA1, other than its ability to promote efflux of phospholipids and cholesterol (35). Much information has been gained about the substrate specificity and mechanism of transport of other ABC proteins using membrane vesicle preparations. On the basis of our previous studies using such systems to investigate the function of ABC transporters, such as the multidrug resistance proteins (MRPs) (22, 32, 73), we established an in vitro transport system for ABCA1 using membranes from insect Sf21 cells expressing wild-type and mutant forms of the protein. This system allowed us to identify certain biologically active oxysterols that in vitro, are potent regulators of cellular cholesterol homeostasis, as potential high affinity ABCA1 substrates.
Cellular cholesterol is maintained within narrow limits by regulatory mechanisms that control synthesis and uptake, as well as cholesterol metabolism and efflux (9, 75). The response to decreased cellular cholesterol is mediated largely by sterol response-element binding proteins (SREBPs) in the endoplasmic reticulum (ER) that are mobilized and activated upon cholesterol depletion (13, 23, 58). Following trafficking to and activation in the Golgi, SREBPs stimulate transcription of genes involved in cholesterol synthesis and uptake, such as the HMG-CoA reductase and LDL receptor genes, respectively (27, 69, 77). SREBPs can also act as transcriptional repressors of genes involved in cholesterol efflux, such as the ABCA1 gene. SREBP activation is suppressed by binding of cholesterol to the SREBP escort protein, Scap, which results in retention of the SREBP/Scap complex in the ER by promoting association with an ER anchor protein (23). In vitro studies have shown that cholesterol derived oxysterols, such as 25-hydroxycholesterol (25-OHC), 24(S), 25-epoxycholesterol, and 27-OHC are potent regulators of this process (8, 20, 54). However, recent studies using 25-OHC, the most potent of the oxysterols, failed to detect direct binding to Scap suggesting that the mechanism of action of the oxysterols may involve an intermediary component, possibly an oxysterol binding protein (1). Oxysterols have also been implicated in the cellular response to increased levels of cholesterol by binding to and activating liver X receptors (LXRs) (12, 16, 20). LXRs function as heterodimers with the retinoid X receptors (RXRs) and induce transcription of genes involved in cholesterol catabolism (16, 49, 51). They also increase expression of several ABC transporter genes that play important roles in cholesterol elimination, including the ABCA1 gene (16, 17, 48, 79).
To validate the results of vesicle transport studies, we used stably transfected human embryonic kidney (HEK) cells to demonstrate that expression of wild-type ABCA1 increases rapid efflux of 25-OHC in the presence of BSA or apoA-I as an acceptor. Under the same experimental conditions, we observed no increase in ABCA1-dependent efflux of cholesterol or phospholipid in the presence of BSA, although such an increase was readily demonstrable in the presence of apoA-I. The increased expression of ABCA1 also prevented the downregulation of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and LDL receptor mRNA levels that occurs when control HEK cells are treated with submicromolar concentrations of 25-OHC. To assess whether physiological levels of ABCA1 contribute significantly to oxysterol efflux, we compared the rates of efflux of [3H]25-OHC by wild-type and ABCA1–/– fibroblasts in the presence and absence of the RXR and LXR agonists, 9-cis retinoic acid and 22R-hydroxycholesterol. Overall, the results strongly suggest that the affinity of ABCA1 for 25-OHC, and possibly certain other oxysterols, is in a biologically relevant range and that physiological levels of the protein are sufficient to influence oxysterol efflux. These in vitro data raise the intriguing possibility that ABCA1 levels may be subject to a negative autoregulatory loop, in which the protein attenuates its own synthesis by effluxing molecules that activate expression of its cognate gene.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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]dATP (3,000 Ci/mmol) were obtained from Perkin-Elmer (Woodbridge, ONT, Canada) and [methyl-14C]choline chloride (56 mCi/mmol) was obtained from GE Healthcare (Montreal, QC, Canada). Generation of expression vectors. The murine wild-type ABCA1-GFP (in expression vector pBI from Clontech) and three ABCA1-GFP mutants (MK, KM, and MM), harboring a methionine (M) substitution for lysine (K) in the Walker A motif of the first, second, or both nucleotide domains were constructed as described previously (25). To generate recombinant baculovirus, the wild-type pBI-ABCA1-GFP and the three ABCA1-GFP mutants were digested with Not1. The digested vectors were purified and ligated individually into the Not1 fragment of the donor plasmid pFASTBAC1. In addition, the wild-type ABCA1-GFP and the three mutants’ constructs were moved into the mammalian expression vector pcDNA3.1(–) via a Not1 digestion of the pFASTBCA1 and Not1 digestion of the vector.
Production of recombinant baculovirus. The conditions used to generate recombinant bacmids and for viral infection of Sf21 cells were as described previously (22).
Membrane vesicle preparation and immunoblotting of ABCA1 proteins.
Membrane vesicles were prepared from Sf21 cells infected with virus encoding wild-type ABCA1 or Walker A mutant proteins, as well as from control cells transfected with virus encoding 
-glucuronidase (-gus). Cells were disrupted by nitrogen cavitation to generate membrane vesicles, as described elsewhere (33). Briefly, cells were homogenized in buffer containing (in mM) 10 Tris·HCl, 250 sucrose, 3 KCl, 0.25 MgCl2, pH 7.5, and protease inhibitor cocktail. Cell pellets were frozen at –70°C for at least 1 h, thawed, and then disrupted by N2 cavitation. Membrane vesicles were purified by sucrose gradient centrifugation, as detailed previously (22, 32). For vesicle transport assay of [3H]25-OHC, vesicles were prepared in homogenization buffer containing 10 mg/ml BSA or 50 µg/ml purified human apoA-I. After determination of protein levels by Bradford assay (Bio-Rad), comparable amounts of vesicle protein were subjected to SDS-PAGE (7.5% gel) and transferred to HyBond-XL membrane (Amersham/Pharmacia) by electroblot analysis (22). The wild-type and mutant ABCA1 proteins were detected using rabbit polyclonal antibody to ABCA1 (Novus Biologicals, Littleton, CO) and detected by horseradish peroxidase-conjugated secondary antibodies utilizing an enhanced chemiluminescence technique and subsequently exposed to film. Relative expression of ABCA1 and its mutants was determined by densitometry.
Membrane vesicle uptake of 25-OHC.
Uptake of 25-OHC into membrane vesicles was measured at 37°C in either a 25- or 120-µl reaction volume for single time point or kinetic measurements, respectively. Assays of wild-type ABCA1 typically contained 0.2 µg/µl of total vesicular protein. Assays of mutant ABCA1 contained from 0.2–0.5 µg/µl total vesicle protein, depending on the relative level of expression of the mutant compared with wild-type ABCA1, as determined by immunoblot analysis. Standard assays contained [3H]25-OHC (2.5 µM; 100 nCi/25 µl), ATP or AMP (4 mM), and MgCl2 (10 mM) in transport buffer (50 mM Tris·HCl, 250 mM sucrose, pH 7.4). At the indicated times, 20-µl samples were removed, diluted with 1 ml of ice-cold transport buffer, and filtered under vacuum through glass fiber filters (type A/E; Gelman Sciences, Montreal, PQ, Canada). Filters were presoaked in transport buffer containing 10 µg/ml ethidium bromide for 2 h, to reduce nonspecific binding of free oxysterols and were washed twice under vacuum with 4 ml of transport buffer. Filter-retained radioactivity was determined by scintillation counting. ATP-dependent 25-OHC uptake was defined as the difference between filter bound [3H]25-OHC in the presence of ATP minus retained radioactivity from corresponding assays carried out in the presence of AMP. As a control for ABCA1-independent partitioning of 25-OHC between vesicles and the aqueous medium, assays were also carried out in the presence of AMP and ATP using vesicles prepared from Sf21 cells infected with a control vector encoding -gus.
Cell lines and tissue culture. J774 murine macrophages obtained from the American Type Tissue Collection were maintained as described previously (64). HEK-293 cells were stably transfected with pcDNA3.1 vector containing the wild-type and mutant ABCA1 cDNAs as described previously (28). They were then maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and Geneticin (400 µg/ml). Primary embryonic mouse fibroblasts were obtained from wild-type and ABCA1-deficient mice (25) and cultured in DMEM containing 10% FBS, supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate, and 1% nonessential amino acids.
Confocal microscopy. Approximately 5 x 105 stably transfected HEK-293 cells were seeded in each well of a six-well tissue culture plate on coverslips coated with 0.1% PolyLys. When cells had grown to confluence, they were washed twice with phosphate-buffered saline (PBS) and monitored by confocal microscopy using a Leica TCS MP2 confocal microscope.
Efflux of 25-OHC from J774 cells, ABCA1 transfectants, and mouse fibroblasts. J774 cells were maintained in DMEM supplemented with 10% FBS. HEK stable ABCA1 transfectants were cultured in the same medium plus Geneticin (200 µg/ml). Mouse fibroblasts from wild-type and ABCA1-deficient animals were cultured in media described above. In some experiments, ABCA1 expression in mouse fibroblasts was stimulated for 16 h by incubation with media containing 10 µM 9-cis-retinoic acid, a RXR ligand and 10 µM 22R-hydroxycholesterol, a natural LXR agonist. Both ligands were removed from the cells by extensive washing with PBS/BSA before 25-OHC efflux studies. With the exception of mouse fibroblasts, cells were plated in a 6-well plate and allowed to reach 80% confluency. Mouse fibroblasts were plated at a density of one million cells per well. Cells were then preloaded for 24 h with [3H]25-OHC (0.5 µCi/ml) in the presence of unlabeled 25-OHC (4 µg/ml) and in the absence or presence of Sandoz 58-035 (1 µg/ml) to block esterification of 25-OHC (53). Cells were washed twice with PBS containing 0.1% BSA and once with PBS alone. They were then exposed to serum-free DMEM, or the same medium containing various acceptors with and without 2-deoxy-D-glucose (10 mM) or glybenclamide (1.0 mM), for periods up to 6 h to determine the efflux of 25-OHC from cells. Aliquots of medium were removed at various times as indicated, centrifuged to remove cellular debris, and radioactivity was quantified by liquid scintillation. Cell layers were washed twice with PBS/BSA and once with PBS. The cell-associated radioactivity was then determined after extraction with 1 ml hexane/isopropanol (3:2 vol:vol). Efflux of radioactive label to the medium was calculated as the percentage of the total count in each well.
Effect of BSA and apoA-I on the efflux of [3H]cholesterol and [14C]phospholipids by HEK ABCA1 transfectants. Wild-type and mutant (MM) HEK stable ABCA1 transfectants were labeled as described above except using [3H]cholesterol and [methyl-14C]choline and conditions such that the labeling medium contained 0.5 µCi/ml [3H]cholesterol and 1.0 µCi/ml [methyl-14C]choline. After 16 h, the labeling medium was removed and the cells were washed extensively. They were then cultured in DMEM in the absence or presence of either 10 mg/ml BSA or 20 µg/ml human apoA-I for 0–24 h. At various time points, radioactivity of the chase medium was determined after lipid extraction in 1 vol of methanol and 2 vol of chloroform. The solvents were dried down in scintillation vials before radioactivity determination. All samples were counted with the use of a Beckman LS6500 scintillation counter using quench and dual-label spill corrections. Cell radioactivity was determined by extraction in 1 ml of hexane-isopropanol (3:2) and evaporation of the solvent in a scintillation vial. The fractional efflux of [3H]cholesterol and [14C]phospholipids to the medium was calculated as the percentage of total (medium + cell) 3H and 14C radioactivity, respectively.
Preparation of RNA and Northern blot analyses.
Total RNA was extracted using TRIzol reagent (Invitrogen) and polyA+ RNA fraction was subsequently isolated using the polyATract system (Promega). PolyA+ RNA (5 µg) was subjected to electrophoresis through 1.2% formaldehyde agarose gels and transferred onto nylon membrane, as described previously (74). RNA blots were probed with cDNA fragments that had been radiolabeled by random priming with [32P-]dATP. The cDNA fragments included an EcoRI + BglII coding region fragment of LDLR cDNA from pLDLR-2 (ATCC) and an ApaI + BglII fragment of HMG-CoA reductase cDNA from pHRed-102 (ATCC). Northern blots were also probed with a radiolabeled Xba + Pst1 fragment of human GAPDH cDNA from pHcGAP (ATCC) to correct for loading variations.
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RESULTS |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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50-fold more rapidly than cholesterol (39). In addition, these studies showed that although the presence of BSA resulted in a major increase in oxysterol efflux, the rate of cholesterol efflux was essentially unaffected. Whether this disparity in the rates of oxysterol and cholesterol efflux in the presence of BSA is solely attributable to the greater rate of passive exchange of the more hydrophilic oxysterol, or whether an energy-dependent mechanism may be involved, has not been established. To investigate the possible contribution of an energy dependent mechanism to the rapid efflux of 25-OHC, we examined the effect of ATP depletion. Cells were cultured in medium containing [3H]25-OHC (10 µM, BSA 50 mCi/mmol) for 24 h, in the presence of 58–035 to inhibit esterification, as described previously (53). After transfer to fresh medium lacking oxysterols but containing 50 µg/ml apoA-I, 100 µg/ml HDL, or 5 mg/ml BSA as an acceptor, 25%, 35%, and 20%, respectively, of the total [3H]25-OHC was effluxed over a 10-min period. Virtually no efflux of [3H]25-OHC occurred when the labeled cells were transferred to DMEM alone, as previously reported (39) (see Fig. 1A). When cells were cultured in the presence of 2-deoxy-D-glucose (10 mM) to deplete ATP, [3H]25-OHC efflux in the presence of BSA, decreased 2- to 3-fold, suggesting that there was an energy-dependent component to the rapid efflux phase (Fig. 1A). J774 cells have been reported to express relatively high levels of ABCA1 (6). To obtain a preliminary assessment of the possible involvement of a transporter such as ABCA1, we examined the effect of the inhibitor glybenclamide on [3H]25-OHC efflux. As observed following treatment with 2-deoxy-D-glucose, glybenclamide (1 mM) decreased the rate of efflux of [3H]25-OHC to BSA or HDL by 2 to 3 fold (Fig. 1A). Because BSA has not been reported to function as an acceptor for ABCA1 mediated efflux of either phospholipids or cholesterol, we investigated whether the preparation used may be contaminated with apoA-I by immunoblot analysis with a polyclonal anti-apoA-I antiserum. As shown in Fig. 1B, we were readily able to detect apoA-I when mixed at a ratio of 1:500 with BSA, whereas no apoA-I could be detected in the BSA preparation used. On the basis of these and other results described below, we conclude that the ability of BSA to function as an effective acceptor for 25-OHC is not attributable to low levels of contaminating apoA-I.
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50% that of wild-type ABCA1 (Fig. 2A). Membrane vesicles were prepared from infected Sf21 cells expressing wild-type ABCA1 and the three mutant proteins, as well as from control cells that had been infected with a vector encoding -gus, using nitrogen cavitation, and sucrose density gradient fractionation, as described (22). On the basis of the results of efflux experiments carried out with J774 cells, we also produced vesicles in the presence of either 50 µg/ml apoA-I or 10 mg/ml BSA to encapsulate the protein as an intravesicular acceptor for the oxysterol. The procedure used generates a mixture of inside-out and right-side-out vesicles. However, only the inside-out vesicles are capable of ATP dependent substrate uptake, because the NBDs of any ABC transporter present in right-side-out vesicles are within the lumen of the vesicle and thus are inaccessible to nucleotide. In addition to the inclusion of an intravesicular acceptor, the procedure was modified by pretreatment of the filters used for collection of vesicles after transport assays with ethidium bromide (10 µg/ml) to decrease the otherwise prohibitively high level of nonspecific binding of free oxysterol (Fig. 2B). A similar treatment has been used previously to decrease nonspecific filter binding of hydrophobic substrates of drug transporters (47).
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-gus vector, as well as vesicles containing wild-type and mutant ABCA1, with and without intravesicular BSA, incubated in the presence of AMP rather than ATP. Approximately 0.1 nmol 25-OHC mg–1 total vesicle protein min–1 was recovered in association with vesicles incubated in the presence of AMP, regardless of whether they contained wild-type or mutant ABCA1 or -gus (Fig. 2B). Thus the presence of ABCA1 did not detectably influence the energy-independent vesicular partitioning of [3H]25-OHC. In the case of control vesicles, this amount did not change significantly with the inclusion of intravesicular BSA, or with the substitution of ATP for AMP (Fig. 2B), indicating that there was no detectable ATP or BSA dependent uptake by these vesicles. In contrast, when AMP was replaced by ATP, uptake by vesicles containing wild-type ABCA1 increased 2-fold and 10-fold in the absence and presence, respectively, of intravesicular BSA. Thus ATP dependent uptake by vesicles containing wild-type ABCA1 was 0.1 nmol mg–1 total vesicle protein min–1 in the absence of BSA and 0.9 nmol mg–1 total vesicle protein min–1 in its presence. The results indicate that [3H]25-OHC uptake by vesicles containing ABCA1 is strongly dependent on ATP and the presence of intravesicular BSA. Comparison of ATP-dependent [3H]25-OHC uptake by wild-type ABCA1 vesicles containing BSA with vesicles preloaded with apoA-I (50 µg/ml) yielded similar levels of uptake (Fig. 2C). To determine whether ATP-dependent uptake required ATP hydrolysis, we also carried out assays in the presence of a comparable concentration of the poorly hydrolyzable ATP analog ATP-
-S. After correction for the level of [3H]25-OHC association with the vesicles in the presence of AMP, uptake in the presence of ATP--S was <10% of that observed with ATP (data not shown). In addition to wild-type ABCA1, we examined the ability of the three Walker A mutant proteins to support uptake of [3H]25-OHC. The single mutations (KM and MK) each decreased ATP-dependent uptake by 50–60%, whereas the double mutant (MM) was only 10% as active as the wild-type protein (Fig. 2C). Thus ATP dependent uptake is decreased by mutations in the Walker A motif of either NBD of ABCA1 and is virtually eliminated by mutation of both NBDs.
Kinetic studies of ATP-dependent uptake of [3H]25-OHC.
To further characterize the ATP dependent uptake of [3H]25-OHC by vesicles containing ABCA1, we determined the time dependence and apparent Km of the process using vesicles containing wild-type protein. The time course of ATP-dependent [3H]25-OHC uptake by Sf21 membrane vesicles prepared from cells expressing wild-type ABCA1 is shown in Fig. 3A. ATP-dependent transport was linear for 60 s. During the linear phase, the rate of uptake at 37°C was 0.7 nmol·mg–1 total membrane protein·min–1. Rates of ATP-dependent uptake were also determined at various concentrations of [3H]25-OHC (0.1 to 10 µM) and yielded an apparent Km of 0.7 µM and Vmax of 1.1 nmol mg–1 min–1 for the wild-type protein (Fig. 3B). The apparent Km values of the single ABCA1 mutants (KM and MK) for 25-OHC are similar to the wild-type protein while the Vmax values were 0.45 nmol·mg–1·min–1. Thus the single mutations result in a 2.0–2.5 fold decrease in Vmax for 25-OHC but no change in apparent Km. Rates of transport by the MM mutant were too low to allow determination of reliable Km values. These results are consistent with the rate of vesicular uptake being dependent on the catalytic activity of ABCA1. The ATP dependence was characterized further by determining the apparent Km of the process with respect to ATP. The Km of wild-type ABCA1 for ATP was determined by measuring the initial rates of [3H]25-OHC uptake in the presence of concentrations of ATP ranging from 01–4.0 mM. Linear transformation of the data yielded an apparent Km for ATP of 280 µM (Fig. 3C, inset).
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80–90%. Vitamin D3 and vitamin E decreased uptake by 45% and 55%, respectively and weak inhibition was observed with 9- and 13-cis-retinoic acid. However, bile acids (chenodeoxycholic acid and cholic acid), steroid hormone derivatives (ethynyl estradiol and estradiol 17 glucuronide), natural product cytotoxic agents (vincristine and doxorubicin), and vitamin K had no effect.
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50% of total [3H]25-OHC in 1 h in the presence of BSA compared with <5% in its absence. During the same period, control transfected cells and cells expressing the single and double mutant forms of ABCA1 effluxed 15% of total [3H]25-OHC (Fig. 5). Thus efflux of 25-OHC from the HEK transfectants is strongly dependent on the presence of wild-type ABCA1 and either BSA or apoA-I as an acceptor.
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2-fold from transfectants expressing either wild-type or mutant ABCA1. In contrast, apoA-I (20 µg/ml) stimulated efflux of [3H] cholesterol and [14C] phospholipid 8–10 fold from transfectants expressing wild-type ABCA1, compared with 2- to 2.5-fold from cells expressing the mutant protein (Fig. 6, A and B). The kinetics of efflux of cholesterol and phosholipid from the wild-type ABCA1 transfectants in the presence of apoA-I were very similar and in both cases, considerably slower than the efflux of 25-OHC (compare Figs. 5 and 6). Thus although either BSA or apoA-I can function as acceptors for 25-OHC, BSA appears not to be an effective acceptor for the ABCA1 mediated efflux of either phospholipid or cholesterol.
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5-fold higher than observed with fibroblasts from ABCA1–/– mice and increased a further twofold after pretreatment with the RXR/LXR agonists (Fig. 8B). Pretreatment with the agonists did not affect the rate of oxysterol efflux from the ABCA1–/– fibroblasts. Similar results were obtained when BSA (10 mg/ml) was substituted for apoA-I, with the exception that the differential in the initial rate of efflux from wild-type type vs. ABCA1–/– fibroblasts under basal conditions was 2.5- rather than 5-fold, as observed with apoA-I (Fig. 8C).
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DISCUSSION |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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Regardless of the mechanism involved, it has been established that mutation of the conserved Walker A lysine residue of either NBD of ABCA1 abrogates the transfer of both phospholipid and cholesterol, as well as eliminating detectable interaction of the protein with apoA-I (10). Such mutations of the Walker A motif invariably decrease or eliminate the ATPase activity of ABC proteins, but they can have different effects on the ability of these proteins to bind nucleotide. The ATPase activity of wild-type ABCA1 appears to be relatively low and has proven difficult to detect. Attempts to measure ATPase activity of ABCA1 expressed in insect cells were unsuccessful (63). Furthermore, it was not possible to detect vanadate-dependent trapping of ADP, a sensitive indicator of ATPase activity, although Mg2+-dependent binding of azido-[32P]ATP by ABCA1 was readily detectable. A more recent study of ABCA1 expressed in HEK cells also detected binding of ATP by the protein and detected weak vanadate stimulated ADP trapping, but only in the presence of nonphysiological Mn2+ rather than Mg2+ (65).
In vitro transport assays using inside-out membrane vesicles have proven to be extremely useful for characterizing the substrate specificity and kinetic properties of several ABC transporters. To date, no such assay has been developed for ABCA1. One of the difficulties with respect to ABCA1 is that such assays are not readily applicable to studying transport of compounds that are major structural constituents of the membrane, or which cannot be maintained in solution without the use of agents that may compromise the integrity of the vesicle. Consequently, we sought potential substrates of ABCA1 with higher aqueous solubility than cholesterol or phospholipids. Oxysterols represent interesting candidates, both biologically because of their in vitro potency as regulators of cholesterol homeostasis and experimentally because of their relatively high hydrophilicity, particularly in the case of 25-OHC, when compared with cholesterol. The partition coefficient of 25-OHC between aqueous and liposomal phases, determined with liposomes containing various cholesterol: phospholipid ratios, is between 0.32 and 0.39 compared with zero for cholesterol and 25-OHC can exist as a monomer in aqueous solution at physiologically relevant concentrations (61). The relatively polar 25-OHC, as well as certain other oxysterols, has been shown to exchange rapidly between artificial liposomes and between liposomes and acceptors such as HDL3, but interestingly, lipid free apoA-I and apoE did not stimulate oxysterol release (36, 66). On the basis of these and other studies, it has been widely assumed that the relatively rapid efflux of oxysterols such as 25-OHC and 27-OHC from various cell types to exogenous acceptors is the result of a passive exchange process and oxidation of cholesterol to products, such as 27-hydroxycholesterol has been proposed as a mechanism by which some cell types enhance cholesterol elimination (18, 60). However, as far as we have been able to ascertain, there have been no published investigations of whether an energy-dependent transport mechanism may contribute to this process.
The kinetics of efflux of 25-OHC from the murine macrophage-like cell line, J774, have been well characterized (39) and consequently, we used them as a model for preliminary studies. As reported previously, we found that efflux of the oxysterol was markedly enhanced by the presence of an acceptor, such as HDL or BSA, compared with efflux to protein-free medium (39). We also found that lipid-free apoA-I was able to function as an efficient acceptor. However, prior depletion of ATP by treatment of the cells with 2-deoxy-D-glucose decreased the rate of rapid efflux of 25-OHC efflux 2.5- to 3.0-fold and a similar decrease was observed in the presence of glybenclamide. These observations supported the possible involvement of an ABC transporter. Because ABCA1 is expressed at relatively high-levels in macrophages and macrophage-like cell lines such as J774 (6), we established an in vitro transport assay using membrane vesicles prepared from Sf21 insect cells expressing wild-type or mutant ABCA1, similar to that we have described previously for the MRPs (21, 22, 45, 73). The extent of passive association of [3H] 25-OHC with the membrane vesicles was determined by carrying out assays in the presence of AMP. We found that passive association was not significantly affected by the presence of ABCA1 in the vesicles, or by the inclusion of BSA or apoA-I as an intravesicular acceptor. Furthermore, no ATP-dependent uptake mediated by endogenous proteins could be detected using control vesicles prepared from cells expressing -gus, either in the presence or absence of an intravesicular acceptor. In contrast, membrane vesicles containing wild-type ABCA1 that were preloaded with apoA-I or BSA revealed a 10-fold, ATP-dependent increase in uptake of [3H]25-OHC compared with the background level of association observed in the presence of AMP, which was typically in the order of 2–3,000 dpm. Substitution of ATP with a comparable concentration of the poorly hydrolyzable analog, ATP--S, decreased uptake to <10% of that observed in the presence of ATP after correction for [3H]25-OHC association in the presence of AMP, indicating that uptake was dependent on ATP hydrolysis rather than the result of ATP binding. In addition, the level of uptake by vesicles containing mutant forms of the protein was reduced 50–60% by the single Walker A Lys to Met mutations and by 90% by the double mutation. More detailed kinetic analyses revealed a Vmax for ATP-dependent [3H]25-OHC uptake by vesicles containing wild-type ABCA1 of 1.0 nmol·mg–1 membrane protein·min–1 that was reduced 2-fold by the single NBD mutations. As observed with other ABC proteins, the Walker A mutations did not affect the apparent affinity for substrate, and Km values of between 0.6 and 1.0 µM were obtained for 25-OHC with both the wild-type and the two single Walker A mutant proteins. Thus, overall, the results indicate that the ABCA1 mediated uptake of 25-OHC is dependent on the ability of both NBDs of the protein to hydrolyze ATP. Analysis of the ATP dependence of 25-OHC transport by wild-type ABCA1 revealed a Km for ATP of 280 µM. This value is in the same range as that reported for ABC transporters which display relatively high affinity for ATP, such as the ABCC proteins (33), and is consistent with the ability to demonstrate relatively strong nucleotide binding by ABCA1 using the photoactivateable ATP analog 8-azido-ATP (63, 65).
The activity of ABC proteins is dependent on strong cooperative interactions between the two NBDs and, in contrast to the 40–50% retention of activity observed with the MK and KM ABCA1 mutants, mutation of the corresponding Walker Lys residue in either NBD of an ABC transporter, such as MDR1 (ABCB1), essentially inactivates the protein (59). However, this is not invariably the case. In ABCC proteins such as MRP1/ABCC1, a Walker A Lys to Met mutation in NBD1 reduces transport activity by 60–70%, although the comparable mutation in NBD2 essentially inactivates the protein (21, 45). In some transporters that have two identical NBDs, such as the histidine permease of Escherichia coli, one wild-type NBD, together with a mutant NBD that can bind but not hydrolyze ATP, can drive transport at 50% of the rate of a transporter with two normal NBDs, as we observed with the ABCA1 single mutants (42).
To investigate the specificity and saturability of the ABCA1 mediated uptake of [3H]25-OHC, we determined the ability of various sterols, steroid conjugates and other lipid soluble molecules, some of which are substrates of other ABC proteins, to inhibit the process. Among the compounds tested, the four naturally occurring oxysterols proved to be the most potent inhibitors. Approximately 90% inhibition was achieved with a nominal 50-fold excess of 25-OHC, 27-OHC, and 24S-OHC, whereas 22R-OHC reduced uptake by 80%. In contrast, the di- and tri-hydroxy bile acid products of oxysterol metabolism, chenodeoxycholic acid and cholic acid, which are substrates for the hepatocanalicular ABC transporter, Bsep (ABCB11) (72), had no significant effect on ABCA1 mediated [3H]25-OHC uptake. The only other compounds tested that displayed significant inhibition at a nominal 50-fold molar excess were vitamin D3 and vitamin E, which reduced transport by 45% and 55%, respectively. The inhibition by vitamin E is consistent with a previous study (43) using ABCA1 transfected BHK cells, which found that the protein promoted the efflux of vitamin E in the presence of an acceptor such as apoA-I. To date, there has been no report that ABCA1 may mediate the efflux of vitamin D3. The apparent specificity of the inhibition observed and the relatively low Km value for 25-OHC raises the possibility that oxysterols may be primary substrates of ABCA1, as opposed to their vesicular uptake being driven by the generation of phospholipid asymmetry in the membrane. Comparison of the characteristics of ABCA1 mediated efflux of 25-OHC by HEK transfectants with those of cholesterol and phospholipid strongly supports the possibility of primary, direct transport of the oxysterol. In these studies, BSA failed to enhance ABCA1 mediated efflux of either cholesterol or phospholipid. These observations, together with the immunoblot analysis experiments described, demonstrate that the ability of BSA to act as an acceptor for ABCA1 mediated efflux of 25-OHC cannot be attributable to contamination of the protein with apoA-I. The fact that BSA as well as apoA-I will act as an acceptor for 25-OHC also suggests that a specific/direct interaction with ABCA1 is not essential for the oxysterol efflux to occur. Although a direct interaction is apparently required for apoA-I to act as an acceptor for cholesterol and phospholipid, other proteins with amphipathic helixes can fulfill this function (46). This difference in behavior may be attributable to the greater hydrophilicity of 25-OHC, when compared with either cholesterol or phospholipid, which may allow oxysterol transported to the outer leaflet region of the membrane to more readily diffuse to an available acceptor.
Studies with stably transfected HEK cell lines expressing wild-type and mutant proteins confirmed the ability of wild-type ABCA1 to enhance efflux of 25-OHC. HEK-293 cells transfected with ABCA1 effluxed [3H]25-OHC to culture medium containing BSA, 2.5- to 3-fold faster than cells transfected with an empty vector or vectors expressing ABCA1 mutant proteins. However, the relative rate of efflux from transfectants expressing KM and MK mutant proteins was lower than observed with vesicle uptake assays and was only marginally higher than control transfectants. Why the single mutations have a more profound effect on activity in the HEK transfectants when compared with the vesicle uptake studies is presently not known. It is possible that membrane composition may be a factor. The cholesterol content of insect cell membranes is only 10% that of mammalian cell membranes and membrane composition is known to influence the transport characteristics of some ABC proteins including their ATPase activity (52, 68). The trafficking of the wild-type and mutant proteins may also differ. Although results of our confocal microscopy studies of HEK transfectants revealed no major differences in distribution between the wild-type and single mutant proteins, as reported previously, it is not known to what extent the mutations alter the dynamics of vesicular trafficking under different growth conditions. Wild-type ABCA1 shuttles between the plasma membrane and early and late endosomes and enhances the vesicular transport of cholesterol to the cell surface (40). The expression of wild-type ABCA1 also increases vesicular trafficking from the Golgi to the plasma membrane in fibroblasts cultured in the presence of apoA-I (80). Whether the NBD mutations affect these processes is currently under investigation.
Development of the in vitro transport system described and the identification of oxysterols as potential substrates should facilitate studies of the mechanism of transport by ABCA1, which to date remains relatively poorly characterized. Our results also indicate that in vitro and in intact cells, the apparent affinity of ABCA1 for 25-OHC and possibly other oxysterols is in a physiologically relevant range and that under certain conditions ABCA1 mediated efflux could serve to attenuate their effects. In addition to stimulating expression of genes involved in cholesterol metabolism and efflux, submicromolar concentration of some oxysterols, including 25-OHC have been shown in cultured cells to repress genes encoding proteins involved in cholesterol synthesis and uptake, such as HMG-CoA reductase and LDL receptor by acting as ligands for the LXRs (12, 16, 20). Because the Km value for 25-OHC obtained from vesicle uptake studies was in the concentration range over which the oxysterol is biologically active, we determined whether 25-OHC efflux by ABCA1 could potentially influence expression of genes involved in cholesterol homeostasis. To do so, we examined the ability of ABCA1 to prevent downregulation of HMG-CoA reductase and LDL receptor mRNA levels in response to exposure of transfected HEK cells to submicromolar concentrations of 25-OHC. The levels of both mRNAs in control HEK transfectants and cells transfected with mutant ABCA1 decreased progressively to 50% of those in untreated cells, over a concentration range of 25-OHC from 0.1 to 1.0 µM. Consistent with the high apparent affinity for 25-OHC determined by vesicle uptake studies and the ability of ABCA1 to maintain intracellular concentrations below those required to repress the HMG-CoA reductase and LDL receptor genes, this decrease was not observed in cells transfected with the wild-type protein. Because the HEK transfectants used in this study express abnormally high levels of ABCA1, we also examined the influence of endogenous ABCA1 on efflux of 25-OHC by comparing fibroblasts from wild-type and ABCA1–/– mice. The results of these experiments indicated that the basal level of ABCA1 in the wild-type fibroblasts was responsible for 80% of the rapid oxysterol efflux. As additional confirmation that the [3H]25-OHC efflux was mediated by ABCA1, treatment of the wild-type fibroblasts with a combination of RXR/LXR agonists to enhance expression of the protein increased the rate of efflux twofold, without affecting efflux by fibroblasts from ABCA1 null mice. Thus, in these two cell models, the data indicate that ABCA1 is a major contributor to the rate of efflux of 25-OHC. The results also raise the possibility that oxysterol efflux by ABCA1 could attenuate the effect of 25-OHC, and possibly other oxysterols, on expression of LXR responsive genes. However, although studies with cultured cells have provided compelling data that certain oxysterols are high affinity ligands for LXRs and potent suppressors of SREBP activation, ablation, or manipulation of expression of murine genes encoding key enzymes involved in oxysterol synthesis, such as 27-hydroxylase and 7-ketohydroxylase has not provided definitive proof of their in vivo role in total cholesterol homeostasis (30, 38, 50). In ABCA1–/– mice, two studies have found that hepatic HMG-CoA reductase activity and/or mRNA levels are substantially decreased (15, 37). In one case, in which sterol levels were also determined, this decrease was observed with no change in total liver cholesterol (15). However, a third report (24), which also observed no changes in liver cholesterol levels, also detected no changes in hepatic HMG-CoA reductase mRNA. To date, there have been no reports directly examining alterations in oxysterol metabolism in ABCA1–/– mice.
Finally, independent of a role in cholesterol homeostasis, the possibility that oxysterol efflux by ABCA1 serves a protective function should also be considered. The substrate specificity of many ABC transporters, particularly those implicated in multidrug resistance, is thought to reflect their evolution as a protective mechanism against potentially toxic endo- and xenobiotics (31). However, such a function is clearly not limited to the multidrug transporters. ABCG5 and ABCG8 provide examples of sterol transporters that fulfill a major protective role in preventing absorption of plant sterols in the intestine, while contributing to cholesterol homeostasis by mediating biliary cholesterol efflux (7, 76, 78). Some oxysterols, including 25-OHC, are toxic to normal and tumor cells at micromolar concentrations and induce apoptosis, possibly by triggering an increase in intracellular Ca2+ concentration (44). Interestingly, Chinese hamster ovary cells selected for resistance to 25-OHC are also resistant to apoptosis induced by oxidized LDL, suggesting that they are resistant to multiple oxysterols (57). Whether ABCA1 may contribute to such resistance is currently under investigation.
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), BSA (5 mg/ml) + glybenclamide (1.0 mM, 
), HDL (100 µg/ml), or apoA-I (50 µg/ml, 
). Efflux to the medium was expressed as a percentage of total counts in each well. Results are means ± SE of 3 independent experiments. B: immunoblot analysis of BSA with anti-apolipoprotein A (apoA)-I antibody was carried out to determine whether the albumin preparation used in the studies described contained detectable levels of apoA-I. BSA (50 µg) alone and mixed with various amounts of purified apoA-I (0.1, 0.5, and 1.0 µg) was subjected to SDS-PAGE and immunoblotted with a species cross-reactive polyclonal rabbit anti-apoA-I antibody (Santa Cruz Biotechnology) that also displayed some cross-reactivity with serum albumin. ApoA-I could not be detected in purified BSA but was readily detectable in a 500:1 BSA:apoA-I mixture.
) were incubated at 37°C in transport buffer containing [3H]25-OHC (2.5 µM, 100 nCi) and in the presence of 4 mM AMP or ATP for the times indicated. ATP-dependent transport of 25-OHC was determined as described in 
), were used as a control. Efflux of radioactivity to the medium was calculated as the percentage of the total count in each well. Results are means ± SE of 3 independent experiments.
) or presence of either 10 mg/ml BSA (
