Membrane Lipid Domains Distinct from Cholesterol/Sphingomyelin-Rich Rafts Are Involved in the ABCA1-mediated Lipid Secretory Pathway*

Armando J. MendezDagger §, Guorong Lin, David P. Wade||, Richard M. Lawn||, and John F. Oram

From the Dagger  University of Miami School of Medicine, Diabetes Research Institute, Miami, Florida 33101, the  University of Washington, Department of Medicine, Seattle, Washington 98195, and || CV Therapeutics Inc., Palo Alto, California 94304

Received for publication, August 23, 2000, and in revised form, October 13, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Efflux of excess cellular cholesterol mediated by lipid-poor apolipoproteins occurs by an active mechanism distinct from passive diffusion and is controlled by the ATP-binding cassette transporter ABCA1. Here we examined whether ABCA1-mediated lipid efflux involves the selective removal of lipids associated with membrane rafts, plasma membrane domains enriched in cholesterol and sphingomyelin. ABCA1 was not associated with cholesterol and sphingolipid-rich membrane raft domains based on detergent solubility and lack of colocalization with marker proteins associated with raft domains. Lipid efflux to apoA-I was accounted for by decreases in cellular lipids not associated with cholesterol/sphingomyelin-rich membranes. Treating cells with filipin, to disrupt raft structure, or with sphingomyelinase, to digest plasma membrane sphingomyelin, did not impair apoA-I-mediated cholesterol or phosphatidylcholine efflux. In contrast, efflux of cholesterol to high density lipoproteins (HDL) or plasma was partially accounted for by depletion of cholesterol from membrane rafts. Additionally, HDL-mediated cholesterol efflux was partially inhibited by filipin and sphingomyelinase treatment. Apo-A-I-mediated cholesterol efflux was absent from fibroblasts with nonfunctional ABCA1 (Tangier disease cells), despite near normal amounts of cholesterol associated with raft domains and normal abilities of plasma and HDL to deplete cholesterol from these domains. Thus, the involvement of membrane rafts in cholesterol efflux applies to lipidated HDL particles but not to lipid-free apoA-I. We conclude that cholesterol and sphingomyelin-rich membrane rafts do not provide lipid for efflux promoted by apolipoproteins through the ABCA1-mediated lipid secretory pathway and that ABCA1 is not associated with these domains.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cellular cholesterol efflux occurs by at least two distinct mechanisms (reviewed in Refs. 1-3). Aqueous diffusion involves desorption of membrane cholesterol into the aqueous compartment surrounding cells followed by absorption to an appropriate acceptor, such as high density lipoproteins (HDL).1 Efflux by aqueous diffusion is independent of cell growth state, cholesterol content, and metabolic energy and does not involve active cellular transport pathways (4-8). This process is facilitated by the interaction of HDL particles with scavenger receptor B1 (SR-B1) (9, 10). A second pathway is mediated by lipid-free apolipoproteins and involves specific cellular events distinct from aqueous diffusion. Apolipoprotein-mediated cholesterol efflux is only apparent in growth-arrested or cholesterol-enriched cells, and it requires metabolic energy (5, 6, 11, 12) and a functional Golgi apparatus transport system (5-8). Apolipoprotein-dependent cholesterol efflux is absent in fibroblasts from patients with Tangier disease (TD), whereas efflux by aqueous diffusion mechanisms occurs normally in cells from affected individuals (8, 13, 14). Mutations in the gene encoding the ATP-binding cassette transporter ABCA1 are the underlying cause of TD (15-20), implicating this protein as a rate-controlling step in the apolipoprotein-mediated lipid efflux pathway. This conclusion was further supported by studies showing that the activity of the apolipoprotein-mediated lipid efflux pathway paralleled the level of expression of ABCA1 in cells (15, 18, 21). Although the mechanisms involved in cholesterol efflux are becoming better understood, there are limited data describing the cellular pools of lipids removed from cells by extracellular acceptors.

When cultured cells are incubated with whole plasma, cholesterol released from cells during early times (less than 30 min) preferentially associate with a minor subfraction of lipoproteins called pre-beta 1 HDL (22-24). These particles, composed mostly of apoA-I, may promote cholesterol efflux by a mechanism similar to that mediated by lipid-free apolipoproteins. Cell membrane caveolae domains may play a role in providing cholesterol to plasma acceptors during the initial efflux period (24, 25). Caveolin mRNA levels were increased in response to free-cholesterol enrichment of fibroblasts, and this was proportional to increased free cholesterol efflux promoted by HDL present in whole plasma (26, 27).

These observations raise the possibility that caveolae participate in delivery of intracellular lipids to the plasma membrane for removal by ABCA1-dependent efflux mechanisms. Caveolae are plasma membrane structures enriched in cholesterol and sphingomyelin and contain a variety of distinct proteins (reviewed in Refs. 28-30). Caveolae are one component of cellular membranes broadly termed membrane rafts, discreet microdomains present on the cell surface. Raft domains exist in cells that do not express caveolin, and use of detergent-free systems have allowed the separation of caveolae from other raft microdomains (31, 32).

Simons and Ikonen (33) proposed the "raft hypothesis," in which sphingolipid-cholesterol microdomains are involved in numerous cellular functions, including membrane trafficking and signaling. According to this hypothesis, the lateral assembly of sphingolipids and cholesterol creates rafts floating in a glycerophospholipid-rich environment. These domains were first identified as membrane complexes insoluble in Triton X-100 (TX-100) at 4 °C (34, 35), and detergent insolubility has been used as a tool to identify lipid rafts and associated proteins (36). These TX-100-insoluble cellular membranes are enriched in glycosphingolipid and sphingomyelin and contain a significant fraction of the membrane cholesterol. In contrast, most of the cell glycerophospholipids are soluble in TX-100 (37, 38). The integrity of rafts depends on the presence of both cholesterol and sphingomyelin (39, 40).

In the current study, we examined whether ABCA1 was present in membrane rafts and if plasma membrane raft domains were involved in or required for ABCA1-mediated cholesterol and phospholipid efflux.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- All cell culture incubations were performed at 37 °C in a humidified 5% CO2 incubator. Human skin fibroblasts from patients having TD and from normal subjects were grown and maintained as described previously (7, 13, 41). Cellular cholesterol and choline-containing phospholipids were labeled as described previously (5). Labeled cells were cholesterol-enriched by incubation with DMEM containing 2 mg/ml fatty acid-free bovine serum albumin (BSA) and 30 µg/ml of cholesterol for 24 h, followed by 48 h in serum-free DMEM containing 1 mg/ml BSA (DMEM/BSA) to allow equilibration of cholesterol pools. For immortalized fibroblast lines NL1 and TD1 (18), ABCA1 expression was induced by cholesterol loading for 48 h as above followed by 24-h incubations with DMEM/BSA containing 1 mM 8-Br-cAMP as described (18, 41).

Plasma, Lipoproteins, and Apolipoproteins-- Blood was collected into vacutainer tubes containing streptokinase (160 units/ml final concentration) and chilled on ice, and plasma was separated by centrifugation (2000 × g for 20 min). Plasma was used for experiments within 60 min of isolation.

LDL and HDL were prepared from pooled EDTA plasma by ultracentrifugation in the density intervals 1.019-1.063 and 1.12-1.21 g/ml, respectively. HDL was depleted of apoE- and apoB-containing particles by heparin-agarose chromatography (7). LDL was acetylated by the method of Goldstein et al. (42). ApoA-I was purified from HDL as described previously (43). Protein was measured by the method of Lowry et al. (44) using BSA as the standard.

Cholesterol and Phospholipid Efflux-- Efflux of [3H]cholesterol from cells was measured by the appearance of label in medium after incubation with acceptors as described in detail previously (5-7). Medium cholesterol (efflux) and cellular free and esterified cholesterol were expressed as the percentage of total [3H]cholesterol. Cell proteins were dissolved in 0.1 M NaOH and quantified by the method of Lowry et al. (44).

Choline-labeled phospholipids were determined in cell medium after extraction with CHCl3:CH3OH and in cell layers after extraction with isopropanol (5). Extracts were separated by thin layer chromatography (TLC) developed with CHCl3:CH3OH:H20 (65:35:4; v/v), and radioactivity corresponding to sphingomyelin and phosphatidylcholine was measured. Phospholipid efflux was calculated as the percentage of total individual [3H]phospholipid.

Cholesterol and Phospholipid Mass-- Cellular free and esterified cholesterol mass were measured enzymatically (45) after separation by TLC (5). To measure phosphatidylcholine and sphingomyelin mass, phospholipids were separated by TLC as above, and phospholipid phosphorous was quantified by the method of Bartlett (46).

Cellular Cholesterol Esterification-- Esterification of cellular cholesterol by acyl-CoA:cholesterol O-acyltransferase was measured after the incubation with experimental medium by the incorporation of [14C]oleate (56 mCi/ml, Amersham Pharmacia Biotech, Arlington Heights, IL) into cholesteryl esters during an additional 1-h incubation at 37 °C in DMEM containing 9 µM [14C]oleate and 3 µM BSA (47). Cholesterol esterification was expressed as nanomoles of [14C]oleate incorporated into [14C]cholesteryl esters per milligram of cell protein.

Triton X-100 Cell Solubilization-- Cells were separated into TX-100-soluble and -insoluble fractions by published methods (37). Confluent cell monolayers were washed in phosphate-buffered saline and then scraped into MES-buffered saline (25 mM MES, pH 6.5, 0.15 M NaCl) containing 1% Triton X-100 and 1 mM phenylmethylsulfonyl fluoride (for lipid analysis) and 1 µM leupeptin, 1 µM pepstatin, and 1 µM aprotinin (for protein analysis). The suspension was homogenized by 10 strokes with a Dounce homogenizer, kept on ice for 20 min, and centrifuged at 14,000 × g for 20 min at 4 °C. The supernatant (containing the TX-100-soluble fraction) was removed. For lipid analysis, supernatants and pellets were extracted with CHCl3:CH3OH (3:2; v/v), and lipids were subjected to TLC as above to separate free and esterified cholesterol or phospholipids as indicated in individual experiments. For protein analyses, the pellet was suspended in TX-100 buffer containing HEPES (pH 7.4) instead of MES and incubated at room temperature for 30 min to solubilize rafts. Aliquots of the supernatant and solubilized pellet were used for immunoblots and ABCA1 immunoprecipitation.

Isolation of Membrane Rafts-- Low density membrane rafts were separated from other cellular organelles by the sucrose density gradient method of Sargiacomo et al. (38). Briefly, cells grown in 150-mm dishes were cholesterol-loaded and solubilized in MES-buffered saline containing 1% TX-100 and 1 mM phenylmethylsulfonyl fluoride as above. The entire suspension was adjusted to 40% sucrose and placed in an ultracentrifuge tube. A 5-30% linear sucrose gradient was layered above the samples and centrifuged for 16 h at 39,000 rpm in a SW 41 rotor. After centrifugation, 1-ml fractions were collected from the top of the tube for [3H]cholesterol and caveolin-1 determinations.

Metabolic Labeling and Cell-surface Biotinylation-- Cellular proteins were labeled with 35S by including 10 µCi of [35S]methionine overnight in the equilibration medium. The different incubation conditions used had no effect on total cell [35S]protein-specific activity. For selective labeling of plasma membrane proteins, fibroblasts were incubated for 30 min at 0 °C with phosphate-buffered saline containing 1 mg/ml sulfo-N-hydroxysuccinimide-biotin to biotinylate cell-surface proteins (48). Cells were then solubilized in detergent, and ABCA1 was isolated by immunoprecipitation as described below.

Immunoprecipitation-- Rabbit antiserum for ABCA1 was raised against a synthetic peptide located at the COOH terminus of human ABCA1 (18). This antibody had immunoprecipitating activity but was ineffective on immunoblots (18). For immunoprecipitation, TX-100 extracts were incubated overnight at 4 °C with the ABCA1 antiserum (1:200 dilution) followed by an additional 1-h incubation with protein A-coated magnetic beads (Dynal, Lake Success, NY). The antibody-antigen complex was sedimented with a magnet, the beads were washed twice with TX-100 buffer, and proteins were eluted with 1% acetic acid. After neutralization with 1 M Tris (pH 7.4), proteins were solubilized in SDS buffer for electrophoresis.

SDS Polyacrylamide Gel Electrophoresis and Immunoblotting-- Proteins were solubilized in 50 mM Tris buffer containing 1% SDS, 0.1 M mercaptoethanol, and 0.5 mM EDTA, and resolved by polyacrylamide gel electrophoresis using 6 and 15% gels for ABCA1 and caveolin-1, respectively. For each cell extract, the volume of SDS buffer added per lane represented the fraction of protein partitioning between TX-100-soluble and -insoluble membranes. To ensure that the same amount of total cell protein was represented for each experimental condition and cell line, aliquots of the initial TX-100 extracts were assayed for protein content, and the volume of SDS buffer applied to each lane was adjusted accordingly. Immunoprecipitated [35S]methionine-labeled ABCA1 was visualized in dried gels by phosphorimaging. For immunoblots, proteins were transferred to nitrocellulose membranes and probed with a 1:5000 dilution of rabbit antiserum to human caveolin-1 (Transduction Laboratories, Lexington, KY), 0.25 µg/ml mouse IgG to human clathrin heavy chain (Transduction Laboratories), 1 µg/ml mouse IgG to human Transferrin Receptor (Zymed Laboratories Inc. Laboratories, San Francisco, CA), or 2 µg/ml rabbit IgG to human T-cadherin (raised against the 15 amino acids of the NH2 terminus of the mature protein). Antibody-positive bands were visualized by an enhanced chemiluminescence assay (ECL, Amersham Pharmacia Biotech, Piscataway, NJ). For detection of biotinylated ABCA1, proteins were transferred to nitrocellulose and visualized with a streptavidin-horseradish peroxidase ECL assay.

Other Methods-- Lactate dehydrogenase in cell culture medium or in cell layers solubilized with 1% TX-100 at room temperature was measured using a commercial kit (Roche Diagnostics, Indianapolis IN). Statistical comparisons were made by paired Student's t test with significance assumed for p values less than 0.05. All results were confirmed in at least two independent experiments.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Distribution of ABCA1 between TX-100-soluble and -insoluble Membranes-- ABCA1 plays a critical role in apolipoprotein-mediated cholesterol efflux (15-20), perhaps by transporting cellular lipids across the plasma membrane to apolipoproteins and/or shuttling lipids between other cellular compartments and the plasma membrane. We examined whether ABCA1 was associated with cholesterol and sphingomyelin-rich membrane raft domains. For these studies we used immortalized normal and TD fibroblast lines shown to exhibit massive increases in ABCA1 expression in response to cholesterol loading and 8-Br-cAMP treatment (18). The fractional distribution of cholesterol and phospholipids in membrane rafts in these cells is identical to that in the parental primary cell lines (41). Cells were maintained in growth medium containing serum (controls) or were induced by cholesterol and 8-Br-cAMP treatment, and the distribution of ABCA1 and domain-specific membrane proteins between TX-100-soluble and -insoluble fractions was examined. The TX-100-insoluble fraction in both control and treated cells contained ~60% of the total sphingomyelin and less than 10% of the phosphatidylcholine, consistent with the properties of membrane rafts (37-40).

We took two approaches to identify ABCA1. First, cellular proteins were labeled with [35S]methionine, and ABCA1 was immunoprecipitated from each fraction. By this method, nearly all of the detectable 35S-labeled ABCA1 was present in the TX-100-soluble fraction. ABCA1 levels markedly increased in induced cells compared with controls in both the normal and TD cells (Fig. 1), although the levels were significantly lower in TD fibroblasts, as reported previously (18). For the second approach, cell-surface proteins were biotin-labeled prior to detergent fractionation and ABCA1 was immunoprecipitated after detergent solubilization. Similar to 35S-labeling studies, levels of ABCA1 were increased by cholesterol and 8-Br-cAMP treatment, and all detectable biotin-labeled ABCA1 protein was present in the TX-100-soluble (i.e. nonraft) membrane fraction (Fig. 1). The distribution of other membrane proteins was also examined. In contrast to ABCA1, the vast majority of caveolin-1 was present in the detergent-insoluble or membrane raft fraction of both normal and TD cells, indicating a distinct localization compared with membranes containing ABCA1 (Fig. 1). The relative immunoreactivity of caveolin-1 was not markedly different between the immortalized normal and TD cells incubated without or with cholesterol and 8-Br-cAMP.



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Fig. 1.   Localization of ABCA1 to TX-100-soluble membrane domains. For control cells, normal and TD fibroblasts were incubated for 48 h with 10% fetal bovine serum followed by 24-h incubations with DMEM containing 1 mg/ml BSA. To induce ABCA1 expression, fibroblasts were incubated for 48 h with DMEM containing 2 mg/ml BSA and 30 µg/ml cholesterol followed by 24-h incubations with DMEM containing 1 mg/ml BSA and 1 mM 8-Br-cAMP. Total cell proteins were labeled by including [35S]methionine in the medium during the final 24-h incubations (35S-ABCA1). Cell surface proteins were labeled by with sulfo-N-hydroxysuccinimide-biotin (bt-ABCA1). After appropriate pretreatment, the TX-100-soluble (+) and -insoluble (-) proteins were isolated. Immunoprecipitated [35S]ABCA1 and immunodetectable caveolin-1 (CAV1) and clathrin (CLATH) were identified as described under "Experimental Procedures."

To further verify that TX-100 solubility provided separation of membrane rafts from other membranes, the distribution of additional marker proteins were also examined by immunoblotting. The GPI-anchored protein t-cadherin, shown to localize to noncaveolae membrane raft domains (49, 50), had the same membrane distribution as caveolin-1 (not shown). In contrast, clathrin (Fig. 1) and the transferrin receptor (not shown), associated with membranes distinct from membrane rafts, (50, 51) were present in the TX-100-soluble (nonraft) membrane fraction. These data indicate that the TX-100 solubility of cell membranes under the described condition separated membrane raft-associated proteins from nonraft-associated proteins.

Cholesterol Efflux from TX-soluble and -insoluble Membranes-- To assess which cellular pool(s) provide(s) cholesterol for efflux, we incubated [3H]cholesterol-labeled and cholesterol-enriched fibroblasts with purified apoA-I, isolated HDL, or plasma. After incubation, cells were homogenized with TX-100 buffer, and the detergent-soluble and -insoluble fractions were separated. Changes in cholesterol label and mass in each fraction were compared relative to cells incubated without cholesterol acceptors (Fig. 2). In the control cells 32 ± 4% and 38 ± 2% of the [3H]cholesterol and cholesterol mass were recovered in the TX-100-insoluble fraction, respectively. The detergent-insoluble fraction contained about 20% of total cellular protein. The TX-100-insoluble fraction was significantly enriched in cholesterol compared with the TX-100-soluble fraction when normalized for protein, 253 ± 16 and 61 ± 2 µg of cholesterol/mg of protein, respectively. Efflux of cholesterol mediated by apoA-I was accounted for by decreased free cholesterol in the TX-100-soluble (i.e. nonraft) cell fraction without significant change in the TX-100-insoluble (raft) fraction assessed by changes in radiolabel or mass. In contrast, cells incubated with plasma or HDL showed a significant decrease of [3H]cholesterol in both the TX-100-soluble and -insoluble fractions. Cholesterol efflux (label or mass) was greatest for plasma followed by HDL then apoA-I. Approximately 30% of the decrease in cell cholesterol mediated by HDL or plasma was due to depletion of cholesterol from the TX-insoluble fraction. However, when plasma was used as the acceptor, the change in cholesterol mass was underestimated as compared with efflux of [3H]cholesterol. This apparent discrepancy could be accounted for by a 15 and 11% decrease in the specific activity of labeled cholesterol in the TX-100-insoluble and -soluble fractions, respectively, after incubation with plasma, and most likely accounted for by the exchange of cholesterol by diffusional pathways. In contrast, apoA-I and HDL had no significant effect on the specific activity of cellular [3H]cholesterol. These data show that efflux mediated by apoA-I was limited to removal of cholesterol from membranes distinct from cholesterol and sphingomyelin-rich rafts. In contrast, HDL and acceptors present in whole plasma could also mediate the depletion of cholesterol associated with membrane rafts.



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Fig. 2.   Cellular distribution of cholesterol between Triton X-100-soluble and -insoluble membranes after incubation with apoA-I, HDL, or plasma. [3H]Cholesterol-labeled and cholesterol-enriched human skin fibroblasts were incubated with control medium (DMEM) alone or containing 5 µg/ml apoA-I, 50 µg/ml HDL, or 5% whole human plasma for 6 h. After incubation, medium was collected for determination of [3H]cholesterol efflux, and cells were subjected to Triton X-100 (TX) solubilization as described under "Experimental Procedures." Unesterified [3H]cholesterol and cholesterol mass in the TX-Soluble and TX-Insoluble fractions were determined after lipid extraction and TLC separation. Results are the mean ± S.D. of four dishes expressed as micrograms of cholesterol/dish for mass determinations or cpm [3H]cholesterol/dish for radiolabel. Cholesterol mass efflux was calculated as the decrease in cell cholesterol relative to cells incubated with DMEM for each acceptor. *, p < 0.025 compared with cells incubated with DMEM.

In a similar study, we examined apoA-I-mediated efflux of phospholipids from cholesterol-enriched cells (Fig. 3). A decrease in the TX-100-soluble fraction accounted for apoA-I-mediated efflux of radioactive phosphatidylcholine and sphingomyelin (although the sphingomyelin decrease did not reach significance), and there was no change in the phospholipid content of the TX-100-insoluble fraction. These results are consistent with the notion that cellular lipids removed by apoA-I arise from membrane fractions with detergent solubility properties distinct from membrane rafts.



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Fig. 3.   Effects of apoA-I on phospholipid efflux and the cellular distribution of phospholipids between Triton X-100-soluble and -insoluble membranes. [3H]Choline-labeled and cholesterol-enriched fibroblasts were incubated with medium alone (DMEM) or with medium containing 5 µg/ml apoA-I for 6 h. After incubation, medium was collected and cells were subjected to Triton X-100 (TX) solubilization to obtain the soluble and insoluble fractions. Phosphatidyl[3H]choline (PC) and [3H]sphingomyelin (SM) were measured after lipid extraction and TLC separation. Results are expressed as the percentage of total [3H]lipid and are the mean and S.D. of three dishes. *, p < 0.02 by Student's t test compared with cells incubated with DMEM.

Efflux Studies in TD Fibroblasts-- Fibroblasts from patients with TD lack the apolipoprotein-mediated cholesterol efflux pathway (13) due to mutations in the gene for ABCA1 (15-20), indicating that this transporter has a direct role in modulating apolipoprotein-mediated lipid efflux. We examined whether defective ABCA1 function in TD produced alterations in the properties of membrane rafts or affected efflux by other pathways. The distribution of [3H]cholesterol between TX-100-soluble and -insoluble fractions and pools accessible to cholesterol oxidase was compared in TD and normal fibroblasts after incubation with acceptors (Fig. 4). Cholesterol accessible to oxidation by exogenous cholesterol oxidase is believed to be localized to caveolae (24, 52). Fibroblasts from TD patients had similar proportions of [3H]cholesterol present in TX-100-insoluble fraction as did normal cells (34.2 ± 4.8 versus 37.1 ± 2.5%, respectively, for all cell lines). However, less cholesterol was present in a cellular pool accessible to cholesterol oxidase in the TD cells (20.3 ± 4.7%, n = 2 lines) compared with the normal cells (32.6 ± 3.6%, n = 6 lines), suggesting that TD cells have subnormal levels of caveolae cholesterol.



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Fig. 4.   Fraction of cholesterol insoluble in Triton X-100 and accessible to cholesterol oxidase in cholesterol-loaded normal and TD fibroblasts. Normal and TD fibroblasts were radiolabeled with [3H]cholesterol and cholesterol-loaded as described under "Experimental Procedures." After the incubation to equilibrate cholesterol pools, cells were treated with Triton X-100 buffer or cholesterol oxidase, and the fraction of free [3H]cholesterol insoluble in detergent or converted to [3H]cholestenone, respectively, was measured as described under "Experimental Procedures." Each value is the mean ± S.D. of four to eight dishes.

We used sucrose gradients to further examine the association of cholesterol with membrane rafts in normal and TD fibroblasts. Cells labeled and enriched with cholesterol were homogenized in buffer containing TX-100, and the cell suspensions were subjected to density gradient centrifugation to separate the membrane raft fraction from other cellular membranes (Fig. 5). In both normal and TD cells, we observed a peak of cholesterol in the low density region of the gradient. The peak of cholesterol was coincident with caveolin (one component of caveolae, a specialized raft domain) detected by immunoblotting (not shown). Cholesterol distribution in the low density raft fraction in TD fibroblasts was identical to that in normal cells.



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Fig. 5.   Density gradient separation of the Triton X-100-soluble membranes from normal and TD fibroblasts. Cholesterol-loaded normal and TD fibroblast were solubilized with Triton X-100 buffer and subjected to sucrose-gradient ultracentrifugation as described under "Experimental Procedures." Gradient fractions were collected from the top of the tube and analyzed for [3H]cholesterol. Results represent the percentage of total [3H]cholesterol appearing in each fraction. The fraction corresponding to the bottom of the tube was excluded. Values are representative of three different experiments with similar results.

Fielding et al. (24) reported that the pool of cholesterol removed from cells by whole plasma during the initial period of efflux was derived from caveolae. Caveolae cholesterol was defined as the pool of plasma membrane cholesterol accessible to cholesterol oxidase at 37 °C (24, 52). To further assess the role of membrane rafts in promoting cholesterol efflux from TD cells, we measured the contribution of cholesterol oxidase-accessible cholesterol to cholesterol efflux in fibroblasts from normal and TD fibroblasts (Table I). Efflux was examined in cells without or with cholesterol enrichment, the latter condition inducing ABCA1 expression (18) and apolipoprotein-mediated cholesterol efflux (5). After incubation, we measured cholesterol oxidase accessibility of the remaining cellular labeled cholesterol to assess whether this pool of cholesterol was preferentially depleted from cells.


                              
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Table I
Efflux and cholesterol (Chol) oxidase sensitivity of [3H]cholesterol in Tangler disease and normal fibroblasts
Cells were labeled with [3H]cholesterol as described under "Experimental Procedures" then received medium with DMEM containing 2 mg/ml BSA with either 0 or 30 µg/ml cholesterol (Chol pre-treat) for 24 h. Cells receiving excess cholesterol were incubated an additional 48 h to equilibrate cholesterol pools. Cultures were incubated for 6 h with DMEM containing 1 mg/ml BSA (DMEM) or the same medium containing 5 µg/ml apoA-I or 5% plasma (total cholesterol = 189 mg/dl, HDL-cholesterol = 65 mg/dl). After incubation, medium was removed; cells were rinsed twice with PBS then incubated for 30 min in PBS containing 0.5 units/ml cholesterol oxidase. Cell layers were extracted, and lipids were subjected to TLC to separate cholesterol (Oxidase-resistant [3H]Chol), cholestenone (Oxidase-sensitive [3H]Chol), and cholesteryl esters ([3H]Chol esters) for quantitation of radioactivity. Results are expressed as the percentage of total [3H] and are the means ± S.D. of three dishes.

In cells not enriched with cholesterol, apoA-I-mediated cholesterol efflux was not detected from control or TD cells as expected (not shown). When 5% human plasma was present in the culture medium, appreciable [3H]cholesterol appeared in the medium. The majority of the cholesterol efflux was accounted for by a decrease in the cholesterol oxidase-resistant cholesterol, but in both cell lines oxidase-sensitive cholesterol (i.e. caveolae-associated) decreased significantly. In cholesterol-enriched cells, efflux to plasma in both cell lines was again associated with a decrease in both cholesterol oxidase-resistant and -sensitive pools. Because efflux of radiolabeled cholesterol was monitored, the change in cell [3H]cholesterol could be due to exchange without changes in cell mass. Nevertheless, caveolae cholesterol content (based on cholesterol oxidase sensitivity) was affected in both cell lines to a similar extent when incubated with whole plasma. Similar results were obtained when isolated HDL was used as the acceptor (not shown). There was no apoA-I-mediated efflux activity in TD cells, as expected (15, 16), and no difference in cholesterol distribution after incubation with apoA-I. In contrast, apoA-I effectively promoted cholesterol efflux and decreased cholesterol present in the cholesterol oxidase-resistant (noncaveolae) pool without affecting the cholesterol oxidase-sensitive pool in the normal cells. Thus, efflux of cholesterol promoted by apoA-I was accounted for by a decrease in cholesterol not associated with caveolae. Furthermore, serum- and HDL-mediated efflux of caveolae cholesterol occurred similarly in normal and TD cells, suggesting that efflux of cholesterol from these sites occurs independently of a functional ABCA1 protein.

Filipin Treatment-- Filipin binds to cholesterol-rich domains of cellular membranes and disrupts raft structure and organization (53-57). We examined the effects of treating cells with filipin on [3H]cholesterol efflux from cholesterol-enriched fibroblasts by apoA-I or HDL (Fig. 6). Filipin had no effect on the recoveries of cell protein or [3H]cholesterol or release of lactate dehydrogenase (as a measure of cell permeability and viability) compared with controls (not shown). Filipin treatment had no significant effect on cholesterol efflux to control medium containing only albumin or the extent of efflux mediated by apoA-I. The lack of an effect of filipin suggests that apoA-I does not require structurally intact rafts to promote cholesterol efflux. Efflux of cellular [3H]cholesterol by HDL was decreased by ~25% (p < 0.05 at all doses of filipin) compared with control incubations. Decreased HDL-mediated efflux after filipin treatment could be due to depletion of cholesterol associated with membrane rafts. This possibility is supported by the observation that the decrease in efflux was similar to the proportion of efflux accounted for by cholesterol associated with membrane raft domains (i.e. TX-100-insoluble fraction, Fig. 2). These findings again show that, in contrast to lipid-free apoA-I, HDL can promote efflux of [3H]cholesterol associated with membrane rafts. These results, however, may be confounded by the fact that filipin will bind to HDL lipids and that such interactions may interfere with the ability of HDL to accommodate cholesterol.



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Fig. 6.   Effects of filipin on apoA-I- and HDL-mediated cholesterol efflux. Cholesterol-enriched fibroblasts were preincubated with DMEM containing the indicated concentrations of filipin for 30 min at 37 °C. Cells were then incubated with the same medium (DMEM) or with medium containing 5 µg/ml apoA-I for 6 h. After incubation, medium and cell [3H]cholesterol were measured as described under "Experimental Procedures." Results are the mean ± S.D. of three dishes. *, p < 0.05 by Student's t test compared with cells incubated with DMEM.

Sphingomyelinase Treatment-- A majority of membrane sphingomyelin is associated within raft domains in the plasma membrane (34). We reasoned that treating cells with sphingomyelinase would alter raft structure or composition and disrupt apolipoprotein-mediated lipid efflux from these domains, if they were involved in this process. The effect of sphingomyelinase treatment on apoA-I-mediated efflux of cellular [3H]-phospholipids was examined (Fig. 7). Sphingomyelinase treatment digested greater than 80% of cellular [3H]sphingomyelin without affecting levels of phosphatidyl[3H]choline (not shown). As expected, lipase treatment nearly abolished sphingomyelin efflux from cells. Loss of membrane sphingomyelin, presumed to be at least partially associated with membrane raft domains, had no appreciable effect on the ability of apoA-I to promote efflux of [3H]cholesterol and slightly increased phosphatidyl[3H]choline efflux. Cellular cholesterol esterification by acyl-CoA:cholesterol O-acyltransferase was measured in the same cells after the cholesterol efflux incubation. Acyl-CoA:cholesterol O-acyltransferase activity was significantly (p < 0.05) increased in sphingomyelinase-treated cells compared with controls, demonstrating the internalization of membrane cholesterol to acyl-CoA:cholesterol O-acyltransferase accessible pools by lipase action, as shown previously (58, 59). However, incubation with apoA-I decreased cholesterol esterification in both control and sphingomyelinase-treated cells, and the absolute decrease was similar for both conditions (a decrease of 1.2 ± 0.1 and 1.5 ± 0.3 nmol of cholesterol ester/mg of cell protein for control and treated cells, respectively). In the same study, cholesterol efflux by HDL decreased in sphingomyelinase-treated cells compared with controls, from 9.8 ± 0.6% to 7.9 ± 0.9% of total cell cholesterol. HDL decreased cholesterol esterification to a similar extent as apoA-I, and the extent of the decrease was not affected by sphingomyelin treatment (not shown) as occurred during incubations with apoA-I. These data suggest that digestion of sphingomyelin depleted the pool of cholesterol associated with membrane rafts available for efflux by HDL without affecting cholesterol available for efflux by apoA-I, similar to results obtained using filipin-treated cells. Because the lipase was present during the incubation with HDL and cells, we cannot rule out the possibility that the decreased efflux was due to a direct effect on HDL composition affecting the ability to promote cholesterol efflux.



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Fig. 7.   Effects of cellular sphingomyelin degradation on apoA-I-mediated phospholipid efflux. Cholesterol-enriched cells labeled with [3H]choline or [3H]cholesterol were preincubated with or without 0.4 units/ml sphingomyelinase (SMase) for 30 min at 37 °C. Cells were then incubated for 6 h with DMEM alone (Control) or DMEM plus SMase or with the same media containing 5 µg/ml apoA-I for 6 h. SMase was present during the entire incubation with treated cells. After incubation, medium and cellular [3H]sphingomyelin and phosphatidyl[3H]choline or [3H]cholesterol were quantitated as described under "Experimental Procedures." Lipid efflux was calculated as cpm of 3H-lipid in the medium/mg of cell protein. After removal of efflux medium, cellular acyl-CoA:cholesterol O-acyltransferase activity (ACAT) was measured during a 1-h incubation with [14C]oleate as described under "Experimental Procedures." Cholesterol esterification is expressed as nanomoles of [14C]oleate incorporated into cholesteryl esters per milligram of cell protein per hour. Results are the mean ± S.D. from three dishes. Sphingomyelinase treatment had no effect on the recovery of cellular phosphatidyl[3H]choline, [3H]cholesterol, or protein. *, p < 0.01 by Student's t test compared with cells incubated with DMEM control. Dagger , p < 0.001 by Student's t test compared with the same medium without apoA-I.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

HDL components can remove cholesterol from cells by at least two distinct mechanisms. First, HDL phospholipids promote efflux of plasma membrane cholesterol by a passive diffusion process that is facilitated by HDL binding to SR-BI (9, 10) and suggested to involve caveolae (60). Second, lipid-poor HDL apolipoproteins remove excess cellular cholesterol by an active, Golgi-dependent pathway (5-7) that is severely impaired in TD (13) and depends on a functional ABCA1 protein (15, 18, 20, 61). Although passive diffusion mechanisms may be significant in some cells, the massive accumulation of cholesterol in macrophages of TD patients demonstrates the physiological importance of the ABCA1 pathway in clearing cholesterol from macrophages. Previous studies showed that the ability of lipid-free apoA-I to promote cholesterol efflux from cells is independent of the level of SR-BI expression (9) and that expression of SR-B1 can inhibit the extent of ABCA1-mediated lipid efflux (62), suggesting that this receptor does not play a role in the ABCA1-mediated lipid secretory pathway. Here we show that the ABCA1 efflux pathway also does not involve cholesterol and sphingomyelin-rich raft membrane domains.

Several lines of evidence support the lack of involvement of membrane rafts in the apolipoprotein-mediated lipid removal pathway. First, ABCA1 was associated with a membrane fraction distinct from cholesterol and sphingomyelin-rich rafts. Second, efflux of lipids from cultured fibroblasts by apoA-I was accounted for by decreases in cellular lipids not associated with raft domains as defined by both detergent insolubility and accessibility to cholesterol oxidase. Third, apoA-I-mediated cholesterol efflux was completely absent from cultured TD fibroblasts despite normal amounts of raft lipids. Fourth, treating cells with filipin, which disrupts raft structure in other cell types (53-57), had no effect on apoA-I-mediated cholesterol efflux. In contrast, filipin treatment partially inhibited HDL-mediated cholesterol efflux, possibly by interfering with diffusional efflux of cholesterol associated with membrane raft domains. Fifth, sphingomyelinase treatment, which depleted over 80% of the cellular sphingomyelin, had no inhibitory effect on apoA-I-mediated cholesterol and phosphatidylcholine efflux. Because sphingomyelin is required to maintain the integrity of rafts (39, 40), these results provide additional evidence that disrupting these structures does not impair the ABCA1 lipid secretory pathway. Interestingly, these data also showed that apoA-I-mediated lipid efflux can occur without the concomitant efflux of cellular sphingomyelin. These data imply that removal of lipids from membrane raft domains is not involved in apolipoprotein-mediated cholesterol efflux.

Mutations in ABCA1 account for the severe impairment of this pathway in TD and other familial HDL deficiencies (15, 18, 20, 61). We also show that compartments distinct from membrane raft domains contain all of the cellular and plasma membrane ABCA1, the rate-controlling protein in the apolipoprotein-mediated lipid removal pathway. As shown previously, treatment of immortalized normal fibroblasts with cholesterol and a cAMP analog, which enhances apoA-I-mediated lipid efflux, markedly induced ABCA1 expression and incorporation into the plasma membrane (18, 41). ABCA1 was present only in membranes soluble in TX-100, whether expressed at low or high levels. This membrane fraction contained nearly all of the phosphatidylcholine and nonraft protein markers (clathrin and the transferrin receptor), and it excluded most of the sphingomyelin and nearly all of the marker for caveolae (caveolin) and for noncaveolae rafts (T-cadherin) (49). Thus, these ABCA1-associated membranes do not contain significant amounts of caveolae or other types of sphingomyelin-rich rafts. ABCA1 expressed by one TD cell line, which contains a single amino acid substitution (15, 18), also localized to these nonraft membranes. Although this mutation nearly abolishes apoA-I-mediated lipid efflux (13, 41) and impairs ABCA1 expression (18), the processing and membrane distribution of this protein does not appear to be grossly defective in these cells.

It is noteworthy that caveolin-1 expression was not markedly affected by cholesterol and cAMP or the mutation in ABCA1, providing further evidence for unrelated functions of these two proteins. Based on accessibility to cholesterol oxidase, however, TD cells appeared to have below normal levels of caveolae cholesterol, suggesting that ABCA1 may have some effect on caveolae composition.

The precise function of ABCA1 is still unknown, but the structural similarities with other ABC transporters suggest a role in facilitating transmembrane movement of lipids. Its appearance on the cell surface supports the possibility that it may transport cholesterol and phospholipid directly to cell-surface-bound apolipoproteins, and this possibility is strengthened by recent reports showing that ABCA1 is an apoA-I-binding protein (63, 64). Whether ABCA1 also participates in intracellular lipid transport pathways leading to efflux independent of apolipoprotein binding cannot be excluded. Whatever the mechanism, our data indicate that ABCA1-mediated lipid secretion does not require membrane rafts.

Evidence that caveolae, a specialized type of membrane raft domain, play a role in cellular cholesterol transport and efflux comes from several studies showing that caveolin modulates sterol trafficking in cells and that HDL can selectively remove cholesterol from caveolar membranes. Caveolin mediates transport of newly synthesized cholesterol from the endoplasmic reticulum to caveolae (65) by a Golgi-independent process (66). This cholesterol then redistributes to other plasma membrane sites without accumulating in caveolae, implicating a direct role for caveolin in the transport of cholesterol from sites of synthesis to caveolae and other membrane domains (65). Fielding and colleagues (24, 25) showed that LDL-derived free cholesterol and newly synthesized sterol was selectively enriched in caveolae. Caveolae-associated cholesterol was subsequently depleted by incubations with plasma or HDL, suggesting that caveolae represent an important site for sterol efflux from both of these sources. They also showed that the rate of HDL-mediated cholesterol efflux was positively correlated with the levels of cholesterol present in caveolae and with caveolin mRNA. Reduction of caveolin mRNA levels inhibited efflux of free cholesterol, and the free cholesterol content of cells regulated caveolin mRNA levels (26, 27). The observation that SR-BI is localized to caveolae (60) is consistent with the concept that cholesterol efflux mediated by HDL interactions with this receptor is derived from caveolae. Together these data support the hypothesis that caveolin serves as a regulator of cellular free cholesterol and that caveolae are involved in cholesterol efflux by lipidated HDL.

The current study confirms the involvement of membrane rafts in cholesterol efflux to lipidated HDL particles. Incubation of cholesterol-loaded fibroblasts with isolated HDL or plasma promoted efflux of cholesterol and phospholipids from membrane rafts, although lipid efflux also occurred from other membrane domains. ApoA-I, however, had no measurable effect on raft cholesterol content under conditions in which the same pool of cholesterol could be depleted by plasma or isolated HDL. The inability of apoA-I to cause an apparent reduction in raft cholesterol would occur if the labeled sterol initially present in caveolae were removed by apoA-I then rapidly replenished by cholesterol from other cellular pools. This seems unlikely, however, as such a mechanism would also be expected to operate when plasma or lipoproteins were present in the medium to promote efflux, conditions where we found a clear decrease in raft cholesterol. In addition, HDL and plasma removed cholesterol from membrane rafts both in normal fibroblasts not loaded with cholesterol and in TD fibroblasts, cells that lack the active apolipoprotein-mediated lipid removal pathway (6, 11, 13). These observations may reflect the ability of HDL and other acceptors present in plasma to promote efflux by diffusional mechanisms dependent on the exchange of cholesterol between the plasma membrane and the lipoprotein surface, a process in which lipid-free apoA-I does not participate.

Asztalos et al. (68) showed that the interaction of lipid-free apoA-I with fibroblasts leads to the formation of HDL particles with pre-beta mobility that have an unusual phospholipid composition (67). This composition differed from that of both raft and nonraft membranes, suggesting that apoA-I may remove lipid from other specialized domains of the plasma membrane or from intracellular compartments. These experiments, however, were performed with cells maintained on serum where the activity of the apolipoprotein-mediated lipid removal pathway would be expected to be low. Thus, it remains to be determined if the same particles are formed with cholesterol-loaded cells.

In summary, our studies indicate that membrane raft lipids are not directly available for lipid efflux by apolipoprotein-dependent mechanisms. In contrast, cholesterol efflux from cells to HDL particles or plasma was partially accounted for by depletion of cholesterol from membrane rafts. These findings suggest that cellular cholesterol efflux mediated by lipid-poor apolipoproteins and involving ABCA1 occurs by mechanisms distinct from those mediated by lipoprotein particles containing lipids as their major constituent. Further studies are needed to elucidate the nature of the different cholesterol efflux pathways, their contributions to cellular cholesterol homeostasis, and their roles in depletion of excess cellular cholesterol that accumulates during atherogenesis.


    FOOTNOTES

* This work was supported by National Institutes of Health Grant HL53451 and an American Heart Association Grant-in-Aid 9950534N (to A. J. M.), by National Institutes of Health Grants HL18645, HL55362, and DK02456 (to J. F. O. and G. L.), and by CV Therapeutics, Inc. (to D. P. W. and R. M. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: University of Miami School of Medicine, Diabetes Research Inst. (R-134), 1450 NW 10th Ave., Miami, FL 33138. Tel.: 305-243-5342; Fax: 305-243-5351; E-mail: amendez2@med.miami.edu.

Published, JBC Papers in Press, November 9, 2000, DOI 10.1074/jbc.M007717200


    ABBREVIATIONS

The abbreviations used are: HDL, high density lipoprotein; SR-B1, scavenger receptor B1; TD, Tangier disease; TX-100, Triton X-100; DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; 8-Br-cAMP, 8-bromo-cAMP; LDL, low density lipoprotein; MES, 4-morpholineethanesulfonic acid.


    REFERENCES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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