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INTRODUCTION |
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-
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.
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EXPERIMENTAL PROCEDURES |
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.
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RESULTS |
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."
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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.
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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.
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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.
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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.
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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.
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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.
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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. , p < 0.001 by Student's t
test compared with the same medium without apoA-I.
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DISCUSSION |
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-
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.