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INTRODUCTION |
The reverse cholesterol transport pathway removes excess
cholesterol from extrahepatic tissues including vessel wall, thus preventing development of atherosclerosis. The first and most likely
rate-limiting step of reverse cholesterol transport is cholesterol
efflux, the transfer of cholesterol from cells to acceptors in plasma.
Two key pathways of cholesterol efflux are currently known. The first
involves lipidation of lipid-free or lipid-poor apolipoprotein A-I
(apoA-I),1 and is most likely
mediated by the ABCA1 transporter (for review see Ref. 1). The second
involves transfer of cholesterol from plasma membrane caveolae to
lipidated apoA-I or mature high density lipoprotein (2) and may
be mediated by scavenger receptor B1. Induction of ABCA1 (3), scavenger
receptor B1 (4), and caveolin (5) results in a stimulation of
cholesterol efflux. Cholesterol efflux may also be stimulated by
inducing, or introducing into cells, elements of a pathway capable of
cholesterol catabolism. Bjorkhem and co-workers (6, 7) demonstrated
that limited oxidation of cholesterol in human macrophages may
represent such a mechanism; however, its contribution to overall
cholesterol efflux remains unclear. In this work we demonstrate
for the first time that expression of transfected sterol 27-hydroxylase
(CYP27A1) enhances efflux of non-oxidized cholesterol from cultured cells.
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MATERIALS AND METHODS |
Cells--
CHOP-C4 cells (8) were grown in RPMI 1640 medium
containing 10% fetal bovine serum, 2 mM
L-glutamine, penicillin/streptomycin (50 units/ml), and 0.2 mg/ml G418. The day before transfection, cells were plated in 12-well
plates at a density of 0.6 × 105 cells per well.
Transfection was performed with DEAE-dextran as described previously
(9), using 200 ng of plasmid DNA (CYP27A1 in pcDNA1 or pcDNA1
alone) per well. The efficiency of transfection checked with
-galactosidase reporter gene was 80% or better. CYP27A1 was
isolated from a human kidney cDNA library during studies of
progesterone metabolism using a screening protocol described previously
(9) for the isolation of 11
HSD2. The CYP27A1 clone was sequenced in
its entirety and found to contain the full-length coding region with a
three-nucleotide 5' untranslated region and full-length polyadenylated
3' sequence. There were no missense mutations.
Cholesterol Acceptors--
Blood from healthy normolipidemic
(plasma total cholesterol values ranging from 3.4 to 5.0 mmol/liter)
volunteers was collected in saline containing streptokinase (Sigma;
final concentration 150 units/ml), and plasma was isolated by repeated
centrifugation for 15 min at 1500 × g at 4 °C.
Plasma samples were not pooled but rather used individually, which, in
part, explains variations in cholesterol efflux levels. Apolipoprotein
A-I was isolated as described previously (10).
Cholesterol Efflux--
Transfected, non-transfected, and
mock-transfected CHOP cells were grown to 80% confluence prior to
experiments. The cultures were 100% confluent by the time of
incubation with cholesterol acceptors. Two methods were used to label
cellular cholesterol. Metabolic labeling was conducted by incubating
cells in serum-free medium with [1-14C]acetate (Amersham
Biosciences), specific radioactivity 2.07 GBq/mmol, final
radioactivity 0.8 MBq/ml) for 24 h at 37 °C in a
CO2 incubator. Alternatively, cells were incubated in
serum-containing medium with [1
,2
(n)-3H]cholesterol
(Amersham Biosciences; specific radioactivity 1.81 TBq/mmol, final
radioactivity 0.2 MBq/ml) or [4-14C] cholesterol
(Amersham Biosciences; specific radioactivity 1.96 GBq/mmol, final
radioactivity 50 KBq/ml) for 48 h in a CO2 incubator. After labeling, cells were washed six times with PBS and further incubated for 18 h in serum-free medium. In some experiments cells were incubated for 18 h at 37 °C in serum-free medium
containing 27-hydroxycholesterol added in methanol solution (the final
concentration of methanol was below 0.1%; the same amount of methanol
was added to the control incubation). Cells were washed and further
incubated for 2 h or for the indicated periods of time at 37 °C
in serum-free medium containing either lipid-free apoA-I (final
concentration 30 µg/ml) or the indicated concentrations of human
serum. The medium was then collected, centrifuged for 15 min at 4 °C
at 30,000 × g to remove cellular debris, and the
supernatant was counted or used for further analysis. Cells were
harvested using a cell scraper, dispensed in 0.5 ml of distilled water,
and aliquots were counted or used for further analysis. Cholesterol
efflux was expressed as a percentage of labeled cholesterol transferred from cells to the medium.
Lipid Analysis by TLC--
Lipids were extracted with 3 volumes
of ethyl acetate and separated using TLC (chloroform/ethyl acetate 4:1)
(v/v) (11). TLC plates containing labeled lipids were exposed to a
phosphorimaging plate and analyzed on the Bioimager BAS-1000 (Fuji),
and the radioactivity in each spot was quantified. Spots of cholesterol
and 27-hydroxycholesterol identified by standards (Research Plus) were
also scraped and counted.
Sterol Analysis by Gas Chromatography Mass Spectrometry
(GCMS)--
Sterol analysis by GCMS was conducted as described
previously (12). Briefly, suspensions (1-ml) were hydrolyzed by
incubation with 2 ml of 1 M methanolic KOH at 45 °C for
1 h in sealed tubes, which were flushed with nitrogen. Samples
were cooled and diluted with 2 ml of water, and lipids were extracted
with ether:hexane (50:50) (v/v). Lipid extracts were dried under
nitrogen and then treated with
bis(trimethylsilyl)trifluoroacetamide (50 µl) and pyridine (50 µl) at 60 °C for 20 min to form trimethylsilyl
derivatives. For analysis of cell cholesterol, an internal standard of
cholestane (2.5 µg) was added to each sample prior to GCMS analysis.
Samples were analyzed by full scan electron impact GCMS using an
Agilent 5973 GCMS fitted with a 30-m × 0.25-mm inner diameter
HP-MS5 column with helium carrier gas at a flow rate of 1 ml/min. The
initial column temperature was 165 °C, increased at 20 degrees/min
to 280 °C, and then held. 27-Hydroxycholesterol was identified by comparison to an authentic standard, which showed a molecular ion at
546 m/z for the bis TMS ether. Other
characteristic ions included 456 (M
90), 441 (M
90,
15),
and 417 (M
90,
39).
Reverse Transcriptase PCR and Northern Blotting--
Total RNA
was extracted from CHOP cells following a modification of the
guanidinium thiocyanate method (13). The RNA concentration was
determined by measuring the absorption at 260 nm. Reverse transcription
was carried out in 20 µl containing 20 mM Tris-HCl, pH
8.4, 50 mM KCl, 5 mM MgCl2, 10 mM dithiothreitol, 40 units of RNase inhibitor, and 50 units of Superscript II reverse transcriptase (Invitrogen). Briefly, 2 µg of total RNA were incubated for 5 min at 65 °C with 0.5 µg of
oligo(dT)12-18 and 0.5 mM dNTPs and placed on
ice for 1 min. The remaining components of the reaction were added and
incubated for 1 h at 42 °C. The reaction was terminated at
70 °C for 15 min, and RNA was digested with 2 units of RNase H for
20 min at 37 °C.
PCR was performed in a total volume of 50 µl containing 10 mM Tris-HCl, pH 8.4, 50 mM KCl, 1.5 mM (GAPDH) or 5 mM (CYP27A1) MgCl2,
200 µM dNTPS, 100 ng of the appropriate forward and
reverse primer (CYP27A1 forward, GCAGCGCTCTATACGGAT; CYP27A1
reverse, GCAACACTAGGCCGTCGGTGCACTGTCTCTGCAGGAA; GAPDH forward,
ACGGCAAATTCAACGGCACAGTCA; GAPDH reverse,
CATTGGGGGTAGGAACACGGAAGG), 2 µl of reverse-transcribed cDNA, and 1 unit of Taq polymerase (Invitrogen). The
reaction was amplified with a DNA thermal cycler (PerkinElmer Life
Sciences) for 35 cycles. The amplification profile involved
denaturation at 94 °C for 15 s, primer annealing at 55 °C
for 30 s, and elongation at 72 °C for 1 min. 10 µl of each
PCR reaction was mixed with 2 µl of 6-fold concentrated loading
buffer and loaded on a 1% agarose gel containing ethidium bromide.
Electrophoresis was carried out with a constant voltage for 1 h.
The sequences of the fragments amplified by PCR were confirmed by DNA sequencing.
For Northern blotting, RNA was separated on a 1% agarose gel,
transferred to a nylon membrane, and probed with
32P-labeled mouse ABCA1 cDNA (14) (gift of Dr. G. Chimini) and 32P-labeled GAPDH cDNA as an internal
standard. The membrane was exposed to a phosphorimaging plate and
analyzed on the Bioimager BAS-1000 (Fuji).
Western Blotting--
Cells were lysed in radioimmune
precipitation assay buffer, and proteins were separated on a 7.5%
(ABCA1) or 10% (CYP27A1) SDS polyacrylamide gel followed by
immunoblotting, using either rabbit anti-ABCA1 serum (Novus, Littelton,
CO) or QE10 monoclonal anti-Myc antibody. Bands were visualized
by chemiluminescence development and quantitated by densitometry.
Statistical Analysis--
All experiments were reproduced
three-four times, and representative experiments are shown. Unless
otherwise indicated, experiments were performed in quadruplicate.
Means ± S.D. are presented. The Student's t test or
one-way analysis of variance were used to determine statistically
significant differences between groups.
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RESULTS |
Expression of CYP27A1 in CHOP Cells--
CHOP cells were
transiently transfected with CYP27A1 as described under "Materials
and Methods." Expression of CYP27A1 was analyzed by reverse
transcriptase PCR, and abundance of CYP27A1 protein was analyzed by
Western blot. When CYP27A1 mRNA content was analyzed, a strong
signal was detected in transfected cells whereas no signal was detected
in mock-transfected cells (Fig. 1A). GAPDH was used as
internal standard, and a fragment of 561 bp of identical intensity was
found in both transfected and mock-transfected cells (not shown).
Expression of CYP27A1 tagged with a Myc epitope was also analyzed with
Western blot using an anti-Myc antibody. The presence of a band
migrating at 58 kDa was detected in transfected but not in
mock-transfected cells, consistent with the size of CYP27A1 (Fig.
1B). The antibody cross-reacted with an unknown protein
migrating at 35 kDa, but this band was of the same intensity in
mock-transfected and CYP27A1-transfected cells (Fig.
1B).

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Fig. 1.
Expression of CYP27A1 mRNA
(A) and protein (B) in
mock-transfected and CYP27A1-transfected CHOP cells. A,
expression of CYP27A1 mRNA in mock-transfected (left)
and CYP27A1-transfected (right) was analyzed by reverse
transcriptase PCR as described under "Materials and Methods." The
expected size of the amplicon is 1094 bp. B, Western blot of
mock-transfected cells (left) and cells transfected with
CYP27A1-Myc (right) using anti Myc antibody. Upper
band is CYP27A1 migrating with the expected molecular size of 58 kDa. Lower band is an unknown protein.
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CYP27A1 Activity in CHOP Cells--
The presence of
27-hydroxycholesterol was analyzed in transfected and mock-transfected
cells using gas chromatography mass spectrometry. About 0.8% of total
sterols in transfected cells were found in the 27-hydroxycholesterol
peak; no 27-hydroxycholesterol was found in the mock-transfected cells
(Table I). In HepG2 cells, used as
a positive control as they contain CYP27A1 as part of bile acid
synthesis pathway, 0.16% of sterols was found in the 27-hydroxycholesterol peak.
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Table I
Quantification of 27-hydroxycholesterol in transfected and
mock-transfected cells
Lipids were extracted from cells and analyzed using GCMS as described
under "Materials and Methods." ND, not detected.
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Activity of CYP27A1 was first assessed by analyzing sterol synthesis in
transfected cells following metabolic labeling with [14C]acetate. The amount of newly synthesized
non-oxidized cholesterol in transfected cells was decreased by more
than 50% compared with mock-transfected cells (Fig.
2A). Because the amount of
oxidized cholesterol and cholesteryl esters was exceedingly low
compared with the amount of newly synthesized cholesterol (see below), it is unlikely that cholesterol metabolism is responsible for decreased
amounts of newly synthesized cholesterol. The inhibition of cholesterol
biosynthesis is a more likely explanation. This finding is consistent
with the report by Esterman et al. (15) who showed
previously that 27-hydroxycholesterol inhibits cholesterol biosynthesis
in Chinese hamster ovary cells. The amount of newly synthesized
27-hydroxycholesterol found in transfected cells was 2.5-fold higher
than in mock-transfected cells (Fig. 2B). However, the
overall amount of 27-hydroxycholesterol formed was low consisting of
only 1.5 and 0.6% for transfected and mock-transfected cells, respectively.

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Fig. 2.
Biosynthesis of cholesterol
(A) and 27-hydroxycholesterol (B) in
cells transfected with CYP27A1. CHOP cells were transfected with
CYP27A1 or mock-transfected with pcDNA1 and cultured for 48 h.
One day after transfection, cells were incubated for 24 h in
serum-free medium containing [1-14C]acetate. Sterols from
cells were extracted, separated with TLC as described under
"Materials and Methods," and counted. Means ± S.D. of
quadruplicate determinations are shown. *, p < 0.01 (versus mock-transfected cells).
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The activity of CYP27A1 was further assessed following the labeling of
cellular cholesterol pool with [3H]cholesterol. No
27-hydroxycholesterol was found in mock-transfected cells (Table
II) whereas 2.2% of
[3H]cholesterol was converted into 27-hydroxycholesterol
in transfected cells. The majority of 27-hydroxycholesterol formed in
transfected cells was released into medium following 2 h of
incubation with 5% human serum (Table. 2). An unidentified cholesterol
metabolite accounting for 3% of total radioactive lipids was found in
the medium of CHOP cells transfected with CYP27A1. A small amount of
cholesteryl esters was also found in the cells and in the medium of
both transfected and mock-transfected cells. The amount of cholesterol
and cholesteryl esters in the medium was higher, whereas the amount of
cholesterol and cholesteryl esters in the cells was lower in
transfected cells compared with mock-transfected cells (Table II).
Thus, expression of CYP27A1 in transfected cells was confirmed by
detecting CYP27A1 mRNA and protein and measuring enzyme activity
(assessed by the formation of 27-hydroxycholesterol) and its biological
effect (inhibition of cholesterol biosynthesis and stimulation of
cholesterol efflux).
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Table II
Conversion of [3H]cholesterol in transfected and
mock-transfected cells
CHOP cells transfected with CYP27A1 or mock-transfected were labeled
with [3H]cholesterol as described under "Materials and
Methods." Cells were then incubated with human plasma (final
concentration 5%), for 2 h at 37 °C in a CO2
incubator. Medium was collected, cells were harvested, and lipids from
cells and medium were extracted and separated by TLC as described under
"Materials and Methods." Values are percentages of total
radioactivity (cells plus medium) extracted to the organic phase. ND,
not detected.
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Effect of CYP27A1 on Cholesterol Efflux--
When cells
metabolically labeled with [14C]acetate were exposed to
5% human plasma for 2 h, significantly more cholesterol and 27-hydroxycholesterol was released from transfected cells compared with
mock-transfected cells (Fig. 3,
A and B). It should be noted that because the
amount of newly synthesized 27-hydroxycholesterol in the cells was
considerably lower than the amount of newly synthesized cholesterol,
the contribution of 27-hydroxycholesterol to overall sterol efflux
was less than 3%.

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Fig. 3.
Efflux of newly synthesized cholesterol
(A) and 27-hydroxycholesterol (B)
from cells transfected with CYP27A1. CHOP cells were transfected
with CYP27A1 or mock-transfected and cultured for 48 h. One day
after transfection, cells were incubated for 24 h in serum-free
medium containing [1-14C]acetate. Cells were then washed
and incubated for 2 h with 5% human plasma. Sterols from both
cells and medium were extracted and separated by TLC as described under
"Materials and Methods," and radioactivity was counted. Background
values (i.e. the efflux to the medium alone) were
subtracted. Means ± S.D. of quadruplicate determinations are
shown. *, p < 0.01 (versus mock-transfected
cells).
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To further study the effect of transfection with CYP27A1 on cholesterol
efflux, cellular cholesterol was labeled with
[3H]cholesterol, and cells were incubated in the presence
or absence of whole human plasma or lipid-free human apoA-I. The level
of cholesterol efflux from cells labeled with
[3H]cholesterol was considerably lower than after
metabolic labeling. We speculate that this may be because of
preferential distribution of newly synthesized cholesterol to pools
more readily accessible to the efflux in rapidly dividing CHOP cells
(e.g. plasma membrane). Differences in cholesterol efflux
from different cellular pools have been observed previously (16, 17).
Transfection of cells with CYP27A1 resulted in a 3-fold increase in
cholesterol efflux to whole plasma and a doubling of the efflux to
apoA-I compared with both non-transfected and mock-transfected cells
(Fig. 4). This difference was not because
of differences in the amount of labeled cholesterol loaded into cells,
which was 437 ± 87 versus 342 ± 150 dpm/µg
cell protein for transfected and non-transfected cells, respectively.
Cholesterol efflux to lipid-free apoA-I was considerably lower than to
plasma. This is consistent with our previous findings (17) and reflects
the requirement for apoA-I to take up cellular phospholipid before it
can accept cholesterol (2), as well as lack of cholesterol
esterification activity in pure apoA-I. Cholesterol efflux to the
medium without acceptors was not affected by transfection.

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Fig. 4.
Cholesterol efflux to human plasma and
lipid-free apolipoprotein A-I. CHOP cells transfected with
CYP27A1, mock-transfected, and non-transfected were labeled with
[3H]cholesterol as described under "Materials and
Methods." Cells were then incubated with human plasma (final
concentration 5%), human lipid-free apoA-I (final concentration 30 µg/ml), or serum-free medium alone for 2 h at 37 °C in a
CO2 incubator. Medium was collected, cells were washed, and
the amount of radioactivity in the cells and medium was determined by
liquid scintillation spectrometry. Cholesterol efflux is expressed as
the percentage of labeled cholesterol moved from cells to medium
(i.e. radioactivity in the medium/radioactivity in the
medium + radioactivity in the cells). Means ± S.D. of
quadruplicate determinations are shown. *, p < 0.001 versus mock-transfected and non-transfected cells.
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The time course of cholesterol efflux to human plasma is shown in Fig.
5A. The kinetics of
cholesterol efflux consists of fast (<20 min) and slow (>20 min)
phases as reported by Gaus et al. (18). The difference in
cholesterol efflux between transfected and mock-transfected cells was
greater during the fast phase (2.5-3-fold stimulation within the first
20 min) compared with the slow phase (40-80% stimulation at 1-2 h).
However, the effect of transfection on the absolute amount of
cholesterol released was greater in the slow phase. The effect of
plasma concentration on cholesterol efflux is shown in Fig.
5B. Relatively more cholesterol was released from
transfected cells at low concentrations of plasma, and the difference
between transfected and non-transfected cells gradually disappeared as
the concentration of plasma increased.

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Fig. 5.
Time course (A) and dose
dependence (B) of cholesterol efflux to human
plasma. CHOP cells transfected with CYP27A1 or mock-transfected
were labeled with [3H]cholesterol as described under
"Materials and Methods." Cells were then incubated with human
plasma added at a final concentration of 5% (A) or at the
indicated concentrations (B) or serum-free medium alone for
the indicated periods of time (A) or for 2 h
(B) at 37 °C in a CO2 incubator. Medium was
then collected, cells were washed, and the amount of radioactivity in
the cells and medium was determined in a -counter. Cholesterol
efflux is expressed as the percentage of labeled cholesterol moved from
cells to medium (i.e. radioactivity in the
medium/radioactivity in the medium + radioactivity in the cells).
Background values (i.e. the efflux to the medium alone) were
subtracted. Means ± S.D. of triplicate determinations are
shown.
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Effect of 27-Hydroxycholesterol on Cholesterol Efflux--
To
ascertain whether the effect of transfection with CYP27A1 can be
reproduced with exogenous 27-hydroxycholesterol, non-transfected cells
were preincubated for 18 h at 37 °C with 10
9 or
10
7 M 27-hydroxycholesterol added into the
incubation medium. Assuming that this incubation results in nearly
equilibration between extracellular and intracellular
27-hydroxycholesterol, the concentrations of 27-hydroxycholesterol used
are within the range of its expected concentration in cells transfected
with CYP27A1 (Table I). Preincubation of cells with
27-hydroxycholesterol led to stimulation of cholesterol efflux by 24 and 60%, respectively (Fig. 6).

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Fig. 6.
The effect of exogenous 27-hydroxycholesterol
on cholesterol efflux. Non-transfected CHOP cells were labeled
with [14C]cholesterol as described under "Materials and
Methods." After label was removed, cells were incubated for 18 h
in serum-free medium containing the indicated concentrations of
27-hydroxycholesterol added in methanol solution (the final
concentration of methanol was below 0.1%; the same amount of methanol
was added to the control incubation). Cells were then washed and
incubated with human plasma added at a final concentration of 5% or
serum-free medium for 2 h at 37 °C in a CO2
incubator. Medium was then collected, cells were washed, and the amount
of radioactivity in the cells and medium was determined in a
-counter. Cholesterol efflux is expressed as the percentage of
labeled cholesterol moved from cells to medium (i.e.
radioactivity in the medium/radioactivity in the medium + radioactivity
in the cells). Background values (i.e. the efflux to the
medium alone) were subtracted. Means ± S.D. of quadruplicate
determinations are shown. *, p < 0.05; **,
p < 0.001 (versus untreated cells).
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Effect of CYP27A1 on Expression of
ABCA1--
27-Hydroxycholesterol and possibly another product of
CYP27A1, cholestenoic acid, may be ligands of the liver X-activated receptor, regulating a number of genes involved in lipid metabolism including ABCA1 (19). Therefore expression of ABCA1 was evaluated by
Northern blot in transfected and mock-transfected cells to determine
the effect of CYP27A1. ABCA1 mRNA was identified as a single band
migrating at the expected size (9.5 kb) by Northern blot analysis when
using mouse ABCA1 cDNA as a probe (Fig.
7A). The expression of ABCA1
in transfected cells was slightly less than in mock-transfected cells;
the ratio of ABCA1 mRNA to GAPDH mRNA was 0.84 and 1.36 for
transfected and mock-transfected cells, respectively (Fig.
7A). The abundance of ABCA1 protein in transfected and
mock-transfected cells was analyzed using Western blot (Fig. 7B). The size of CHOP cell ABCA1 was found to be over 200 kDa, which is similar to the calculated molecular mass of 248 kDa for the human protein (20). When the same amount of cellular
protein was loaded, no significant difference in ABCA1 abundance was
found between transfected and mock-transfected cells.

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Fig. 7.
Expression of ABCA1 mRNA
(A) and protein (B) in cells
transfected with CYP27A1. A, expression of ABCA1
mRNA in mock-transfected (left lane) and
CYP27A1-transfected (right lane) cells was analyzed by
Northern blot as described under "Materials and Methods."
Upper panel, ABCA1; lower panel, GAPDH.
B, Western blot of mock-transfected cells (left
lane) and cells transfected with CYP27A1 (right lane)
using anti-ABCA1 antibody.
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DISCUSSION |
Oxidation of cholesterol in liver is the major pathway of
cholesterol catabolism (21) resulting in formation of water-soluble bile acids and their subsequent excretion. It was assumed that liver is
the only tissue capable of this process. Bjorkhem et al. (6)
first suggested that this mechanism may not be unique for liver but may
also be present in macrophages and endothelial cells, contributing
therefore to the protection against cholesterol accumulation in the
vessel wall (7, 22, 23). According to this hypothesis the more
hydrophilic 27-hydroxycholesterol and cholestenoic acid may be released
from cells more readily (22). Cholestenoic acid may also be released to
albumin instead of high density lipoprotein (24). This hypothesis was
supported by findings that a genetic CYP27A1 deficiency in humans
(cerebrotendinous xanthomatosis) is associated with lipid deposition in
connective tissue and an increased risk of cardiovascular disease (6, 25), classic symptoms of deficiency of reverse cholesterol transport. In CYP27A1 knock-out mice, however, most abnormalities were localized in the liver with little effect of the gene deletion on cholesterol homeostasis in extrahepatic tissues (26). Furthermore, overexpression of CYP27A1 in transgenic mice did not result in major changes in
lipoprotein metabolism (27). These differences might be a result of
profound dissimilarities in lipoprotein metabolism in mice and humans
and may also reflect the need to distinguish between roles of CYP27A1
in liver and non-hepatic cells. We addressed this issue by
overexpression of CYP27A1 in an in vitro system and measured
cholesterol efflux.
The major finding of this work is that transfection of cells with
sterol 27-hydroxylase stimulates the efflux of non-oxidized cholesterol. The stimulation of cholesterol efflux could be explained by two mechanisms. First, the appearance of hydroxycholesterol in the
plasma membrane may facilitate the release of cholesterol and/or
27-hydroxycholesterol to an acceptor in plasma. Second, 27-hydroxycholesterol or its derivative cholestenoic acid, may be a
ligand of the liver X-activated receptor, which regulates a number of
genes involved in cholesterol homeostasis, in particular ABCA1 (19).
Several findings in our study point to the likelihood of the first
mechanism. First, there was no significant increase of ABCA1 expression
and abundance in transfected cells. Second, the effect of transfection
was more pronounced with whole plasma than with lipid-free apoA-I. An
opposite result would be expected if the enhanced cholesterol efflux
would result from stimulation of the expression of ABCA1. Third,
transfection mainly stimulated the rapid phase of the efflux,
i.e. release of cholesterol already present in the plasma
membrane (18), making involvement of pathways required for mobilization
of intracellular cholesterol less likely. Fourth, treatment of cells
with exogenous 27-hydroxycholesterol also stimulated cholesterol
efflux, although to a lesser degree.
It should be emphasized that only a small amount of
27-hydroxycholesterol (less than 3%) was formed after transfection
with CYP27, and increased efflux was found to be mainly, if not
entirely, because of stimulation of the efflux of non-oxidized
cholesterol. We speculate that small amounts of 27-hydroxycholesterol
may change the properties of the plasma membrane and could trigger an
increased partition of cholesterol into pools readily accessible to the efflux process e.g. caveolae. Consistent with our data
Westman et al. (23) demonstrated that increased formation of
27-hydroxycholesterol in cholesterol-laden macrophages was associated
with increased efflux of both cholesterol and oxysterols. Another
oxyderivative of cholesterol, 7-ketocholesterol, was not released to
the extracellular acceptor and inhibited efflux of cholesterol; the
effect was most likely mediated by changes in lipid composition of the
plasma membrane (28). However, the effect of 27-hydroxycholesterol or
other cholesterol derivatives on genes other than ABCA1, regulated by
the liver X-activated receptor and involved in lipid metabolism (for
review see Ref. 29), cannot be excluded.
In this paper we demonstrate for the first time that introduction of
CYP27A1 into cultured cells results in a dramatic increase in
cholesterol efflux. This finding has possible implications for the
treatment of atherosclerosis where enhancing reverse cholesterol transport by inserting genes into cells of the vessel wall could be of
benefit. Providing that cholesterol released from cells proceeds
further along the reverse cholesterol transport pathway, this finding
offers a potentially effective way to enhance protection against
accumulation of cholesterol, development of atherosclerosis, and heart diseases.