Research Article
3095
Localization and regulation of SR-BI in membrane
rafts of HepG2 cells
David Rhainds, Philippe Bourgeois, Geneviève Bourret, Karine Huard, Louise Falstrault and
Louise Brissette*
Département des Sciences Biologiques, Université du Québec à Montréal, 1200 Saint-Alexandre, Montréal, Québec, H3B 3H5, Canada
*Author for correspondence (e-mail: [email protected])
Accepted 24 February 2004
Journal of Cell Science 117, 3095-3105 Published by The Company of Biologists 2004
doi:10.1242/jcs.01182
Summary
The scavenger receptor class B, type I (SR-BI) mediates
cholesteryl esters (CE) selective uptake from low density
lipoprotein (LDL) and high-density lipoprotein (HDL)
particles. In a number of tissues expressing caveolin, SRBI is localized in caveolae. We show using detergent-free
sucrose gradients that SR-BI is found in membrane rafts
devoid of caveolin-1 in the human hepatoma HepG2 cell.
Perturbation of the structure of HepG2 cell membrane
rafts with cholesterol oxidase or sphingomyelinase
decreased LDL-CE association due to selective uptake by
60%, while HDL3-CE selective uptake was increased 2.3fold by cholesterol oxidase but was not affected by
sphingomyelinase. Sequestration of membrane cholesterol
with filipin III decreased LDL-CE selective uptake by 25%,
while it had no effect on HDL3-CE selective uptake.
Extraction of cell membrane cholesterol with βcyclodextrin increased LDL- and HDL3-CE selective
uptake by 1.6-fold and 3-fold, respectively. We found that
Key words: SR-BI, Cholesterol, Rafts, Caveolae, HepG2 cell,
Lipoprotein
Introduction
The liver is the major organ in the clearance of lipoprotein
cholesteryl esters (CE) by both endocytosis of whole
lipoprotein particles and lipid selective uptake. As such, it
participates in the final step of reverse cholesterol transport, a
process by which excess peripheral cholesterol is first cleared
by receptor-mediated pathways and then excreted as free
cholesterol and bile acids. The scavenger receptor class B, type
I (SR-BI) is expressed in the liver and steroidogenic organs
(Landschulz et al., 1996) and in a number of tissues with
important roles in lipid transport, such as the intestine (Cai et
al., 2001). SR-BI is a highly glycosylated 82-85 kDa protein
(Acton et al., 1994) that mediates selective uptake of CE and
other lipids from high density lipoprotein (HDL) (Acton et al.,
1996) and low density lipoprotein (LDL) (Swarnakar et al.,
1999). The mechanism of CE selective uptake by SR-BI is not
precisely defined. It appears to involve an initial transfer of CE
into membranes. In the next step, CE becomes inaccessible to
extracellular acceptors and enters intracellular compartments
(Rinninger et al., 1993). SR-BI has an intrinsic capacity to
transfer CE into membranes (Liu and Krieger, 2002).
Caveolin-1 is a cholesterol and fatty acid binding protein
(Murata et al., 1995; Trigatti et al., 1999). Caveolin-1 is
involved in lipid trafficking, membrane trafficking and signal
transduction (reviewed by Liu et al., 2002). This 21 kDa
protein oligomerizes with itself and caveolin-2 to form a
striated coat around flask-shaped membrane invaginations (50100 nm in diameter) called caveolae (Lisanti et al., 1993).
Owing to their insolubility in non-ionic detergents like Triton
X-100 and buoyancy in gradient media, caveolae may be
defined as caveolin-containing membrane rafts. Membrane
rafts are rich in (glyco)sphingolipids and cholesterol and are
likely to be present in all cell types, in contrast to caveolae
(Brown and London, 1998). In tissues where caveolin-1 is
expressed at the cell membrane and forms caveolae, such as
adrenals, SR-BI is localized in caveolae and copurifies with
caveolin-1 (Babitt et al., 1997). Another study has linked CE
selective uptake by SR-BI to its localization in caveolae (Graf
et al., 1999). Remarkably, hepatocytes exhibit very few
caveolae (Fielding and Fielding, 2000; Calvo et al., 2001) and
express small amounts of caveolin-1 compared to other tissues
(Li et al., 2001). There are conflicting reports about caveolin
expression in human hepatoma HepG2 cells: one study shows
a complete absence of caveolins (Fujimoto et al., 2000), while
a recent one shows that caveolin-1 is readily detectable in these
cells (Pohl et al., 2002).
We have shown that SR-BI is the major receptor for CE
selective uptake from LDL and HDL3 in HepG2 cells (Rhainds
et al., 1999; Rhainds et al., 2003) and that LDL-CE selective
uptake brings regulatory cholesterol to HepG2 cells (Charest
CE-selective uptake from both HDL and LDL occurs by a
pathway involving retro-endocytosis in HepG2 cells. An
analysis of the effect of SR-BI level on the expression of
critical lipid sensor and lipid binding proteins was
conducted with stable transformants of HepG2 cell
overexpressing SR-BI. We found that liver-type fatty acid
binding protein expression level is higher in SR-BIoverexpressing cells and that caveolin-1 and sterol response
element binding protein-2 levels are reduced. Thus, in this
hepatic cell model, SR-BI is associated with membrane
rafts devoid of caveolin and its expression affects
intracellular lipid binding and lipid sensor proteins. SR-BIdependent LDL- and HDL-CE selective uptake are affected
differently by the integrity of membrane rafts, but both
occur by a retroendocytic pathway in HepG2 cells.
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Journal of Cell Science 117 (15)
et al., 1999). The first aim of our study was to determine
whether SR-BI is located in lipid rafts of HepG2 cells along
with caveolin-1 and how CE selective uptake is modulated by
lipid raft structure and lipid composition. Our other aims were
to define the mechanism of CE selective uptake in HepG2 cells
and to identify intracellular proteins that may be linked with
SR-BI activity in HepG2 cells. Overall, our results show that
SR-BI and caveolin-1 are not colocalized in HepG2 cells. SRBI is found in lipid rafts, while caveolin-1 behaves as a
cytosolic protein. Although both LDL- and HDL-CE selective
uptake occur by a retroendocytic pathway in HepG2 cells,
LDL-CE selective uptake is sensitive to perturbation of
membrane rafts structure, while HDL3-CE selective uptake is
increased by raft disruption. Overexpression of SR-BI in
HepG2 cells leads to higher levels of liver-type fatty acid
binding protein (L-FABP) and to a reduction in the expression
of caveolin-1 and of both mature and precursor forms of sterol
response element binding protein-2 (SREBP-2). Thus, SR-BI
activity regulates the expression of various genes implicated in
cell lipid homeostasis.
Materials and Methods
Materials
The human hepatoma cell line HepG2 was obtained from the
American Type Culture Collection (Rockville, MD). The mouse
adrenocortical cell line Y1-BS1 was generously provided by Dr
Bernard Schimmer (University of Toronto, Ontario, Canada). Fetal
Clone I was from Hyclone Laboratories (Logan, UT, USA). Glutamin,
trypsin, penicillin-streptomycin and Zeocin were purchased from
Invitrogen Life Technologies (Burlington, Ontario). 1,2-[3H]cholesteryl oleate (50 mCi/mmol) was bought from Amersham
Pharmacia Biotech (Laval, Quebec, Canada) and 125Iodine (as sodium
iodide, 100 mCi/mmol) was bought from ICN Canada (Montreal,
Quebec). Saponin, filipin III, β-cyclodextrin (β-CD), cholesterol
oxidase (COase), neutral sphingomyelinase (SMase), Nethylmaleimide (NEM) and anti-clathrin heavy chain monoclonal
antibody (clone TD.1) were purchased from Sigma-Aldrich
(Mississauga, Ontario). Restriction and modification enzymes were
all from Amersham Pharmacia Biotech (Laval, Quebec). Anti-SR-BI
(495-509) and anti-ATP-binding cassette A1 (ABCA1) rabbit
polyclonal antibodies were from Novus Biologicals (Littleton, CO,
USA), anti-L-FABP monoclonal antibody (clone L2E3) was from
Research Diagnostics (Flanders, NJ, USA), anti-caveolin-1 polyclonal
and monoclonal (clone 2297) and anti-caveolin-2 monoclonal (clone
65) antibodies were from BD Transduction Laboratories
(Mississauga, Ontario). Anti-cytosolic neutral CE hydrolase (NCEH)
rabbit polyclonal antibody was a kind gift from Dr W. M. Grogan
(Virginia Commonwealth University, VA, Canada) and anti-bile saltdependent lipase/carboxyl esterase (BSDL/CEL) rabbit polyclonal
antibody was generously provided by Dr D. Y. Hui (University of
Cincinnati College of Medicine, OH, USA). Enhanced
chemiluminescence substrate and complete protease inhibitor cocktail
tablets were from Roche Diagnostics (Laval, Quebec). Goat antirabbit and anti-mouse IgG coupled to horseradish peroxidase were
from Chemicon (Temecula, CA, USA).
HepG2 cell culture
HepG2 cells were grown in 75 cm2 flasks containing 15 ml of Eagle’s
Minimal Essential Medium (MEM) supplemented with 10% fetal
bovine serum (FBS), 100 Units/ml penicillin, 100 µg/ml streptomycin
and 4 mM glutamine. Five days prior to the association or protein
degradation assays, 3.0×105 cells were seeded in 3.8 cm2 culture
dishes (12-well plates). When protein recycling (retroendocytosis)
had to be investigated, 7.3×105 cells were seeded in 9.4 cm2 dishes
(6-well plates). In all cases, the cells were used when they were 8090% confluent.
Preparation of HepG2 cell clones overexpressing human
SR-BI
The full (2.5 kb) CLA-1 (CD36- and LIMPII-analogous-1) cDNA
(hereafter referred to as human SR-BI) was recovered by partial
EcoRI digestion of the pCEXV-3 vector (a gift from Dr Miguel Angel
Vega, Hospital de la Princesa, Madrid, Spain) (Calvo and Vega, 1993).
The cDNA was subcloned in the eucaryotic expression vector pZeoSV
(Invitrogen) and verified for sense orientation. HepG2 cells at 80%
confluency were stably transfected with the vector expressing SR-BI
full cDNA or with the empty pZeoSV vector by the classic calciumphosphate method (Sambrook et al., 1989). Cells were selected for
using 800 µg/ml Zeocin for 3-4 weeks. Cell foci (clones) were then
isolated and propagated. The maintenance medium contained 500
µg/ml Zeocin. Twelve clones were analysed of which four were high
SR-BI expressors, five were moderate expressors and three clones
showed no increase in SR-BI expression.
Immunoblotting of HepG2 cell proteins
Total cell proteins from either normal HepG2 cells, vector-transfected
cells or SR-BI-overexpressing cells were extracted with 1% Triton X100 (Yoshimura et al., 1987). Proteins were separated on 10%
reducing SDS-PAGE and blotted on nitrocellulose. The blots were
incubated with either anti-SR-BI antibody (1:5000), anti-caveolin-1
(1:2000), anti-caveolin-2 (1:500), anti-clathrin heavy chain (1:1000),
anti-L-FABP (1:250), anti-NCEH (1:2500), anti-BSDL/CEL (1:1000)
or anti-ABCA1 (1:250) followed by enhanced chemiluminescence
detection captured on Kodak Biomax ML film. Protein expression was
measured by densitometric scanning and analyzed with ImageQuant
5.2 software (Molecular Dynamics, Sunnyvale, CA, USA).
Isolation of lipid rafts by discontinuous sucrose gradient
HepG2 cells were fractionated by sucrose gradients as described
elsewhere (Song et al., 1996). Briefly, three 75 cm2 flasks were
washed twice with 15 ml of phosphate-buffered saline (PBS). Cells
were scraped and suspended in 2 ml of 500 mM Na2CO3, pH 11. Cells
on ice were disrupted by 20 strokes of a 5 ml tight-fitting glass Dounce
homogenizer, three 10-second bursts with a Polytron homogenizer
(Brinkman) and three 20-second sonication bursts at 50% maximum
power (Branson Sonifier 250). The homogenate was rapidly mixed
with 2 ml of 85% sucrose in morpholinoethanesulfonic acid (MES)buffered saline (MBS), pH 6.5 in a 12 ml ultracentrifuge tube. The
top was layered with 4 ml of 35% sucrose in MBS plus 250 mM
Na2CO3 and 4 ml of 5% sucrose in MBS-Na2CO3. Gradients were
spun at 190,000 g for 18 hours at 4°C in an SW41 rotor (Beckman).
Twelve 1 ml fractions were collected from the top. Rafts floated at the
5-35% sucrose interface (fractions 4-6) and were visible as a cloudy
white band. Proteins of fractions 1-12 were precipitated with 21%
TCA and suspended in 300 µl 0.2 N NaOH plus 1% SDS for
immunoblotting. Fraction 13 (pellet) was suspended in 300 µl MBS,
pH 6.5. 50 µl aliquots of each fraction were loaded on gels. Lipid rafts
(fractions 4-6) typically contained 15% of the total protein in the
gradient. All fractions were analysed by immunoblotting.
Preparation and labelling of lipoproteins
Human normolipidemic plasma (Royal Victoria Hospital, Montreal,
Quebec) was supplemented with 0.01% ethylenediamine tetraacetate
(EDTA), 0.02% sodium azide, 10 µM phenylmethylsulfonylfluoride
(PMSF), 10 µM Trolox before the isolation of lipoproteins, which was
SR-BI function in membrane rafts of HepG2 cells
achieved by ultracentrifugation as described in Hatch and Lees (Hatch
and Lees, 1968). Human LDL (density=1.025-1.063 g/ml) and HDL3
(density 1.125-1.21 g/ml) were prepared as described by Brissette et
al. (Brissette et al., 1996). LDL and HDL3 contained no detectable
amount of apoE as assessed by immunoblotting. LDL and HDL3 were
iodinated by a modification (Langer et al., 1972) of the iodine
monochloride method (McFarlane, 1948). 1 mCi of sodium 125iodide
was used to iodinate 2.5 mg of LDL or HDL3 in the presence of 30
nmoles (10 nmoles for HDL3) of iodine monochloride in 0.5 M
glycine-NaOH, pH 10. Free iodine was removed by gel filtration on
Sephadex G-25 followed by dialysis in Tris-buffered saline (TBS).
The specific radioactivity ranged from 100,000 to 250,000 cpm/µg
protein. LDL and HDL3 were labelled with [3H]cholesteryl oleate
(CE) as described by Roberts et al. (Roberts et al., 1985). Thereafter,
the labelled lipoproteins were re-isolated by ultracentrifugation. The
specific activity of labelled lipoproteins ranged from 6800 to 11,900
cpm/µg protein.
Lipoprotein cell association and degradation assays
HepG2 cells were washed twice with 1 ml of PBS and were incubated
for 3 hours at 37°C with 20 µg protein/ml of [125I]lipoprotein or
[3H]CE-lipoprotein (LDL or HDL3) in a total volume of 250 µl
containing 125 µl of MEM (2×) plus 4% bovine serum albumin
(MEM-BSA), pH 7.4 (total association). After incubation, the cells
were washed twice with 1 ml of PBS plus 0.2% BSA (PBS-BSA)
followed by one wash with 1 ml of PBS. Cells incubated with 125Ilipoprotein were then homogenized in 1.5 ml of 0.2 N NaOH.
Radioactivity in the homogenates were measured with a Cobra II
counter (Canberra-Packard) and cell protein content was estimated.
The specific association was calculated by subtracting the non-specific
association of 125I-lipoprotein, as determined by the addition of 2 mg
protein/ml of unlabelled ligand, from the total association. [3H]CElipoprotein association (20 µg/ml) was determined by in situ
delipidation of cell monolayers with hexane/isopropanol 3:2 (v/v).
Associated [3H]CE mass was measured by liquid scintillation
counting (Wallack Beta Counter). [3H]CE-lipoprotein association was
also estimated as µg lipoprotein protein/mg cell protein (apparent
uptake). To achieve this, the specific activity of [3H]CE-lipoprotein
was calculated in cpm/µg lipoprotein protein. [3H]CE association due
to selective uptake was calculated as the total [3H]CE association
minus (125I-protein association + 125I-protein degradation). To
measure 125I-lipoprotein degradation, trichloroacetic acid (TCA) was
used at a final concentration of 12% and degradation was estimated
from the incubation medium as the non-iodine TCA-soluble fraction.
In some experiments, incubation was conducted in the presence of 2
mg/ml of maleylated-BSA (M-BSA) prepared as described by
Rhainds et al. (Rhainds et al., 1999). In other experiments, cells in 12well plates were preincubated with either COase (1 U/ml) (Smart et
al., 1994) or SMase (0.5 U/ml) (Scheek et al., 1997) or both enzymes
for 1 hour at 37°C; with either filipin III (5 µg/ml, 7.6 µM) (Schnitzer
et al., 1994) or β-CD (10 mM) (Kilsdonk et al., 1995) for 1 hour at
37°C, or with NEM (1 mM) (Reaven et al., 1996) for 30 minutes at
37°C. Appropriate controls were done with vehicle-treated cells. After
pre-treatment with enzymes or drugs, cells were washed twice with
PBS and processed for lipoprotein association assays.
Lipoprotein recycling (retroendocytosis) assays
Lipoprotein recycling (retroendocytosis) assays were conducted
separately with both 125I-lipoprotein and [3H]CE-lipoprotein, by a
pulse-chase protocol adapted from Kambouris et al. (Kambouris et al.,
1990) and Greenspan and St Clair (Greenspan and St Clair, 1984).
Firstly, radiolabelled LDL (20 µg protein/ml), HDL3 (40 µg
protein/ml) or M-BSA were incubated as described for the cell
association assay, for 2 hours at 37°C in a 500 µl total volume (pulse
phase). HDL3 working concentration was higher in order to obtain
3097
reliable lipoprotein degradation and recycling values. Secondly, the
dishes were put on ice and the medium was discarded. After
incubation, cells were washed once with cold PBS-BSA and once with
PBS. The incubation was continued for 1 hour at 4°C with 5 mg/ml
heparin (850-900 U/ml) in PBS in order to detach a maximal amount
of bound lipoproteins. The released radioactive protein mass was
similar for all the experiments. The use of heparin in this protocol has
been validated in previously published results in which heparin
abolished IDL binding to rat liver membranes (Adam and Brissette,
1994). Efficiency of the heparin treatment to detach LDL and HDL3
bound to HepG2 cells was assessed after binding experiments at 4°C.
Heparin detaches 54±7% of bound 125I-LDL (20 µg protein/ml) and
54±5% and 125I-HDL (40 µg protein/ml) (n=3) and thus reduces the
number of bound lipoprotein particles before the recycling/chase
phase. After the heparin wash, the cells were fed again with fresh
incubation medium (MEM-BSA) and incubated for 2 hours at 37°C
(chase phase). Then the cells were processed for measurement of
remaining cell association (NaOH solubilization) and the medium for
lipoprotein degradation (non-iodine TCA-soluble fraction) and
lipoprotein recycling (TCA-precipitable fraction). The TCAprecipitable fraction was suspended in 0.2 N NaOH for radioactivity
measurement. Wells containing [3H]CE-lipoprotein were treated
identically, except that free iodine extraction on the TCA-soluble
fraction was omitted. To confirm that the pulse-chase protocol
measures re-secretion, recycling at 0°C (2 hours) was compared to
recycling at 37°C (2 hours) after a 2 hour-pulse without heparin wash
as described by Silver et al. (Silver et al., 2000). At the end of the
chase at 0°C, 0.052±0.009 (cell associated) and 0.007±0.005
(detached from the cells) µg protein/mg cell protein for 125I-LDL and
0.0056±0.006 (cell associated) and 0.006±0.002 (detached) µg
protein/mg cell protein for 125I-HDL3 were obtained. After the chase
at 37°C, cell association was 0.031±0.002 and 0.044±0.006 µg
protein/mg cell protein, while recycling accounted for 0.015±0.004
and 0.012±0.003 µg protein/mg cell protein for 125I-LDL and 125IHDL3, respectively. There was no degradation at 0°C, but
0.008±0.002 and 0.0006±0.0002 µg protein/mg cell protein were
measured at 37°C for 125I-LDL and 125I-HDL3 respectively. Thus, at
37°C, cell association falls by 40% (LDL) and (20%) HDL3, while
there is a twofold increase in recycling of LDL and HDL3.
Cholesterol and cholesterol ester content (hydrolysis) of
incubated and recycled lipoproteins
For this purpose, recycling assays were conducted in parallel with
[3H]CE-LDL and [3H]CE-HDL. Non-specific values were determined
by the addition of 2 mg protein/ml of unlabelled lipoprotein. At the end
of the 2-hour pulse phase, medium from triplicate wells were collected,
pooled and a 250 µl aliquot was extracted by the method of Folch et al.
(Folch et al., 1957). At the end of the chase phase, media from triplicate
wells were pooled and extracted similarly after addition of 20 µg of free
cholesterol and cholesterol oleate as carriers. Extracts were evaporated
under nitrogen and separated by thin layer chromatography with
petroleum ether/diethyl ether/acetic acid (90/10/1, v/v/v) as the mobile
phase. Lipids were revealed by iodination, scraped from the plates and
their radioactivity was measured.
Other methods
Protein content was determined by the method of Lowry et al. (Lowry
et al., 1951) with BSA as standard. Paired Student’s t-test or ANOVA1 (with Tukey’s post-test) were used to obtain statistical comparison
of means. Differences were considered significant at P<0.05.
Results
It is known that SR-BI is expressed in HepG2 cells (Rhainds
et al., 2003), however caveolin-1 expression in this cell type is
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Journal of Cell Science 117 (15)
Fig. 1. Immunoblotting of HepG2 cell proteins fractionated by
discontinuous sucrose gradient. HepG2 cell proteins were
fractionated with detergent-free sodium carbonate
discontinuous 5-40% sucrose gradients as described in the
Materials and Methods. The 12 gradient fractions were
concentrated with TCA and 50 µl sample of each fraction was
loaded on 10% reducing SDS-polyacrylamide gel. After
transfer on nitrocellulose, proteins were immunoblotted and
detected by enhanced chemiluminescence. (A) SR-BI of
control cells. (B) SR-BI of cells treated with 0.5% saponin (30
minutes at 4°C). C-F are from control cells: (C) caveolin-1;
(D) clathrin heavy chain; (E) ABCA1; (F) L-FABP;
(G) cytosolic NCEH; H, BSDL/CEL. The images are
representative of three independent experiments.
uncertain. Our first aim was to determine if SR-BI resides in
cholesterol and sphingomyelin-rich membrane rafts of HepG2
cells and if caveolin-1 is expressed and copurified with SR-BI
in these cells. Fig. 1A shows that SR-BI is mainly found in
low-density, buoyant fractions (fractions 4-6) of HepG2 cells
fractionated by sucrose gradients in the presence of carbonate,
thus in fractions commonly referred to as low-density
membrane rafts or caveolin-rich membranes. Some SR-BI
dimers were also observed. Densitometric scanning of SR-BI
blots shows that at least 80% (n=3) of SR-BI protein was found
in fractions 4-6. Since lipid raft protein accounted for ~15% of
total proteins on the gradient, there is a net enrichment of SRBI of at least 20-fold in fractions 4-6. Pretreatment of HepG2
cells with saponin (0.5%), a detergent that complexes with
membrane cholesterol, disrupted lipid rafts so that SR-BI was
recovered in the denser fractions of the sucrose gradient (Fig.
1B). No SR-BI protein could be found in cytosolic fractions
(100,000 g supernatant) nor in soluble fractions after carbonate
extraction, while saponin treatment of membranes did not shift
SR-BI toward cytosolic extracts (data not shown). Saponin
probably acts on SR-BI localization in gradients by
sequestering membrane cholesterol and increasing the density
of membranes where SR-BI is localized. Thus, SR-BI behaves
completely as an integral membrane protein in HepG2 cells.
Interestingly, SR-BI immunoreactivity at ~55 kDa was
exclusively recovered in denser fractions (8-12),
indicating that only the mature glycosylated SR-BI (~82
kDa) is likely to enter lipid rafts of HepG2 cells. The next
step was to determine if the HepG2 cells express
detectable caveolin-1 in membrane rafts. Caveolin-1
immunoblotting with both a polyclonal and a monoclonal
antibody revealed no enrichment of caveolin in fractions
4-6 corresponding to membrane rafts. Instead, caveolin1 was found in dense gradient fractions corresponding to
cytosolic proteins both as monomers and, probably,
dimers (Fig. 1C). As a positive control, mouse
adrenocortical Y1 cells (clone BS1) were fractionated
since SR-BI and caveolin co-purify in these cells (Babitt
et al., 1997). Accordingly, Fig. 2 shows that both SR-BI
and caveolin-1 are detected in fraction 5. As for HepG2
cells, SR-BI precursor (~55 kDa) of Y1 cells is
exclusively recovered in dense membranes of the pellet
(ER microsomes) (Fig. 2A). We conclude that in HepG2
cells, SR-BI is localized to non-caveolae membrane rafts
as a mature, functional protein, while caveolin-1 behaves
like a cytosolic protein.
The localization of other important proteins was analysed.
L-FABP, a typical cytosolic protein, was detected in fractions
9-11 (Fig. 1F) and clathrin heavy chain in fractions 9-12 (Fig.
1D). We found that ATP-binding cassette A1 (ABCA1) colocalized with clathrin heavy chain in fractions 9-12 (Fig. 1E).
Thus, in HepG2 cells, ABCA1 is not in the rafts with SR-BI.
None of these proteins was found in the gradient pellet (P)
where we could expect to see microsomal proteins and
cytoskeletal insoluble proteins. Hepatic cytosolic NCEH
(Ghosh et al., 1995), which is thought to be implicated in
extralysosomal CE hydrolysis (Delamatre et al., 1993) and
hydrolysis of CE from cytosolic lipid droplets, was found in
the gradient pellet and in lighter fractions compared to lipid
rafts (Fig. 1G), indicating that it may follow the lipid droplets
found at the top of the gradient and may also be a peripheral
microsomal protein. The pancreatic bile salt-dependent lipase
(BSDL) is also known as carboxyl ester lipase (CEL) or
secreted NCEH and was found intracellularly in retrosomes
(Hornick et al., 1997). Although mainly a cytosolic protein, a
small amount of BSDL/CEL was in fact associated with low
density membrane rafts (fraction 5 of the gradient, Fig. 1H)
and is partially copurified with SR-BI.
Our next goal was to determine if manipulation of lipid rafts
composition affects CE selective uptake by SR-BI. We used a
SR-BI function in membrane rafts of HepG2 cells
3099
Fig. 2. Immunoblotting of Y1-BS1 cell proteins fractionated by
discontinuous sucrose gradient. Y1-BS1 cell proteins were
fractionated and immunoblotted as described in Fig. 1. (A) SR-BI;
(B) caveolin-1 in Y1-BS1 cells. The images are one of two
independent experiments.
panel of treatments known to interfere with lipid rafts structure
and function. In the first set of experiments, cells were pretreated with COase or SMase or both enzymes. COase
treatment produces membrane cholestenone, which is thought
to break the hydrogen bonding pattern of cholesterol hydroxyl
group with (glyco)sphingolipids amido group (Masserini and
Ravasi, 2001). SMase treatment produces ceramide from
sphingomyelin, which is mainly found in membrane rafts (Yu
et al., 1973). Ceramide has two important effects on
membranes. It self-aggregates and forms microdomains and
acts as a fusogen between membranes. Cholesterol is excluded
from these ceramide patches and returns to intracellular
membranes (Lange and Steck, 1997). Both substrates in the
outer leaflet of the plasma membrane are found abundantly in
lipid rafts. Fig. 3A shows that both enzymes reduced LDL-CE
selective uptake by 60%. Although 125I-LDL protein
association was increased by more than 2.1-fold, protein
degradation by the LDL receptor pathway was reduced. Thus,
disruption of cholesterol and sphingomyelin-rich lipid rafts by
enzymatic treatment impairs LDL-CE association by both
selective uptake and endocytosis pathways. COase had a
different effect on HDL3-CE selective uptake: the 1.7-fold
increase in 125I-HDL3 protein association was accompanied by
a 2.2-fold increase in CE selective uptake. SMase treatment
had no significant effect on CE and protein associations, but
increased the effect of COase alone (Fig. 3B) since there was
a 3.4-fold increase in CE selective uptake. We do not believe
that modulation of CE selective uptake was due to traces of
enzymes that may have been present during the association
assay since omission of the PBS washes, removal of the
treatment media and replacement by fresh media did not affect
total association values. Rather they tended to increase nonspecific association values (data not shown), which remained
below 20% during the assays. Thus, disruption of lipid rafts
favours CE selective uptake from HDL, while it impairs CE
selective uptake from LDL.
We next turned to cholesterol binding agents to manipulate
cholesterol content of HepG2 cell membranes. First, we used
filipin III, a polyene antibiotic that clusters free membrane
sterols (Friend, 1982) and may disrupt cholesterol-rich
domains (Schnitzer et al., 1994). Sequestering of membrane
cholesterol with 5 µg/ml filipin III reduced LDL-CE
association and selective uptake by 30% (Fig. 4A). A similar
Fig. 3. Effect of COase and/or SMase treatment on [3H]CE and 125Iprotein association of LDL and HDL3 in HepG2 cells. HepG2 cells
were treated with 1 U/ml COase or 0.5 U/ml SMase or both enzymes
for 1 hour at 37°C. The cells were then processed for [3H]CE
association (black bars), CE selective uptake (CE association –
protein association and degradation) (white bars), 125I-protein
association (hatched bars) and degradation (cross-hatched bars), with
20 µg/ml radiolabelled LDL (A) or HDL for 3 hours at 37°C (B).
Results are shown as mean percentage±s.e.m. Control values in µg
protein/mg cell protein were set as 100% and were: 0.507±0.066,
0.444±0.066, 0.123±0.015, 0.029±0.001 for [3H]CE-LDL
association (n=7), LDL-CE selective uptake, 125I-LDL protein
association (n=7) and degradation (n=2) respectively; and
0.332±0.058, 0.271±0.058, 0.067±0.008 for [3H]CE-HDL3
association (n=7), HDL3-CE selective uptake and 125I-HDL3 protein
association (n=7) respectively (mean±s.e.m.). Statistically different
values from the control values (without enzymes) are indicated as:
aP<0.05; bP<0.01 and cP<0.001.
reduction in 125I-LDL protein association was coupled to a
substantial reduction in LDL protein degradation by 75%, as
reported in other studies (Subtil et al., 1999). Conversely, 125IHDL protein association increased by 1.4-fold, while HDL-CE
association, selective uptake and protein degradation were
unaffected (Fig. 4B). The effects observed with filipin III are
likely to be maximal since concentrations higher than 10 µg/ml
of filipin III had toxic effects on cells. Pre-treatment with 10
mM β-CD, which extracts membrane cholesterol, increased
LDL-CE selective uptake by 1.6-fold, however this did not
correlate with an higher selective uptake efficiency (CE
association/protein association), since both the protein and CE
associations showed equivalent rises (Fig. 4). Therefore, it is
likely that cholesterol depletion increases either the number of
LDL bound to SR-BI, or the number of SR-BI-dependent
binding sites on HepG2 cells. Even though the same treatment
also increases HDL3-CE selective uptake by 3-fold, its effect
is different on these particles since 125I-HDL3-protein (particle)
3100
Journal of Cell Science 117 (15)
Fig. 4. Effect of filipin III or β-CD treatment on [3H]CE and 125Iprotein association of LDL and HDL3 in HepG2 cells. HepG2 cells
were treated with 5 µg/ml filipin III or 10 mM β-cyclodextrin for 1
hour at 37°C. The cells were then processed as described in Fig. 3.
[3H]CE association (black bars), CE selective uptake (CE association
– protein association and degradation) (white bars), 125I-protein
association (hatched bars) and degradation (cross-hatched bars).
Results are shown as mean percentage±s.e.m. Control values in µg
protein/mg cell protein were set as 100% and were: 0.467±0.029,
0.311±0.025, 0.159±0.009, 0.041±0.004 for [3H]CE-LDL
association (n=6), LDL-CE selective uptake, 125I-LDL protein
association (n=6) and degradation (n=6) respectively; and
0.233±0.021, 0.159±0.023, 0.074±0.008, 0.002±0.004 for [3H]CEHDL3 association (n=7), HDL3-CE selective uptake, 125I-HDL3
protein association (n=7) and degradation (n=7) respectively
(mean±s.e.m.). Statistically different values from the control values
(without treatment) are indicated as: aP<0.05; bP<0.01 and cP<0.001.
association (Fig. 4B) remained the same and an increase in
selective uptake efficiency is observed (from 3 to 5.5). We
addressed the implication of SR-BI in this rise of selective
uptake efficiency towards HDL. Cells pre-treated with β-CD
were assayed in the presence of 2 mg/ml of M-BSA, a known
competitor of SR-BI-mediated CE selective uptake (Rhainds et
al., 1999). A 72% reduction in HDL-CE selective uptake (n=2)
was observed, indicating that the increase in selective uptake
is mainly due to SR-BI activity. We conclude that LDL-CE
selective uptake efficiency is impaired when membrane
cholesterol is sequestered but is not influenced by cholesterol
depletion, while HDL3-CE selective uptake is not affected by
cholesterol sequestering but is favoured by cholesterol
depletion.
In order to distinguish further the mechanism of LDL- and
HDL-CE selective uptake, HepG2 cells were treated with 1
mM NEM and then processed for association assays. NEM is
a cysteine alkylating agent, which acts on V-type ATPases to
Fig. 5. Effect of NEM treatment on [3H]CE and 125I-protein
association of LDL and HDL3 in HepG2 cells. HepG2 cells were
treated with 1 mM NEM for 30 minutes at 37°C. The cells were then
processed as described in Fig. 3. [3H]CE association (black bars), CE
selective uptake (CE association – protein association and
degradation) (white bars), 125I-protein association (hatched bars) and
degradation (cross-hatched bars). Results are shown as mean
percentage±s.e.m. Control values in µg protein/mg cell protein were
set as 100% and were: 0.607±0.076, 0.470±0.083, 0.136±0.012,
0.0301±0.0003 for [3H]CE-LDL association (n=9), LDL-CE
selective uptake, 125I-LDL protein association (n=9) and degradation
(n=6) respectively; and 0.313±0.029, 0.243±0.085, 0.070±0.003,
0.0020±0.0003 for [3H]CE-HDL3 association (n=9), HDL3-CE
selective uptake, 125I-HDL3 protein association (n=9) and
degradation (n=6) respectively (mean±s.e.m.). Statistically different
values from the control values (without NEM) are indicated as:
aP<0.05; bP<0.01 and cP<0.001.
reduce endosome acidification and associated vesicular
transport. Fig. 5A shows that in HepG2 cells, NEM reduced
125I-LDL protein association by 58%, nearly abolished 125ILDL degradation but was not effective against LDL-CE
selective uptake. Fig. 5B shows that NEM reduced 125I-HDL3
degradation by 80%, increased 125I-HDL protein association
2.1-fold and HDL-CE selective uptake by 1.7-fold. Although
the reasons for these increases are unclear, overall these results
indicate that NEM impairs both LDL and HDL
endocytosis/degradation pathways and that CE selective uptake
pathway by HepG2 cells is not dependent on an acidic
endosomal pathway.
The mechanism of SR-BI-mediated CE selective uptake is
still being debated. We aimed to determine if recycling
(retroendocytosis) of both LDL and HDL3 occur in HepG2 cells
and if it was accompanied by a remodelling of lipoprotein
particles. The pulse-chase protocol allowed us, at the very end
of the experiment, to recover and sort radioactivity that was
mainly produced by intracellular metabolism of lipoproteins
during the chase incubation and minimally by bound
SR-BI function in membrane rafts of HepG2 cells
3101
Table 1. LDL and HDL3 particle recycling
(retroendocytosis) in HepG2 cells
125I-protein
(µg protein/
mg cell protein)
Lipoprotein
LDL(n=7)
Internalized*
Recycled‡
Degraded§
HDL3 (n=5) Internalized
Recycled
Degraded
[3H]CE
(µg protein/ CE/protein
mg cell protein)
ratio
0.089±0.012
0.022±0.002
0.025±0.002
0.483±0.051†
0.015±0.002†
0.0005±0.0001†
5.4
0.68
0.018±0.002
0.013±0.004
0.0015±0.0005
0.210±0.040†
0.008±0.001†
0.00014±0.0007†
11.7
0.62
*Internalized fraction is the radioactivity associated with cells after the
chase period.
†Statistically different from the 125I-protein value (at least P<0.05).
‡Recycled fraction is the TCA-precipitable radioactivity in the chase
medium.
§Degraded fraction is the TCA-soluble (non-iodine) radioactivity in the
chase medium.
125I-lipoprotein and [3H]CE-lipoprotein recycling were measured
separately according to a pulse-chase protocol as described in Materials and
Methods. Results are expressed as mean±s.e.m.
lipoproteins detached from the cell surface (see Materials and
Methods). We found that both 125I-LDL and 125I-HDL3 were
recycled in HepG2 cells. Indeed, 17% of LDL-protein was
recycled while 64% remained into the cell and 19% was
degraded. In contrast, 97% of [3H]CE-LDL remained in the
cells, while 3% was recycled (Table 1, upper part). The mass
of recycled LDL-protein, estimated by [3H]CE apparent uptake,
was significantly lower than 125I-protein recycling (P<0.05):
[3H]CE recycling/125I recycling ratio was 0.68, indicating a
selective retention of CE within the cells, since in the case of
recycling intact particles (a futile process), [3H]CE
recycling/125I recycling ratio would be near unity. 125I-HDL3
recycling accounted for 40% of the total radioactivity at the end
of the chase incubation, while 55% of the radioactivity
remained into the cells (Table 1, lower part). As for LDL, HDL3
protein mass estimated by [3H]CE-HDL3 recycling was lower
than 125I-HDL3 recycling (P<0.05): [3H]CE-recycling/125I
recycling ratio was 0.62. Since a degradable CE tracer was used
([3H]cholesteryl oleate) for both LDL- and HDL-CE, it seemed
possible that recycled lipoproteins contained free cholesterol
hydrolysed into the cells but still associated with the particle
and/or free cholesterol effluxed from cells during recycling.
Separation of free cholesterol and cholesteryl ester from the
recycling medium shows that 16±5% and 24±7% of [3H]CELDL (n=3) and [3H]CE-HDL3 (n=3) was free cholesterol.
Since part of CE in recycled lipoproteins is recovered as free
cholesterol, the CE/protein ratios of recycled lipoproteins
underestimates the CE depletion due to intracellular
accumulation of CE. CE/protein ratios of recycled lipoproteins,
corrected for free cholesterol content, are therefore 0.57 for
LDL and 0.47 for HDL3. Furthermore, we found that CE
hydrolysis occurred during the pulse phase, given that a small
but significant amount of radioactive free cholesterol was
recovered in incubated lipoproteins (1.6±0.4% for [3H]CE-LDL
and 1.6±0.6% for [3H]CE-HDL3, n=3). Taken together these
results indicate that delivery of CE by selective uptake in
HepG2 cells is a retroendocytotic process for both HDL3 and
LDL, that CE hydrolysis is an early phenomenon and that free
Fig. 6. Human SR-BI overexpression in HepG2 cells. HepG2 cells
were stably transfected with a eukaryotic expression vector
containing human SR-BI cDNA and clones were isolated. (A) SR-BI
levels of normal HepG2 cells and of two overexpressing clones (S1.1
and S1.7) determined by immunoblotting with anti-SR-BI polyclonal
antibody (1:5000) as described under Materials and Methods. Shown
here is a representative image from five different protein extractions.
(B) [3H]CE association (black bars) and 125I-protein association
(white bars) of LDL (n=9) and HDL3 (n=9) in HepG2 cells
overexpressing SR-BI measured as described in figure 3. (C) 125I-MBSA association (black bars) and recycling (retroendocytosis, white
bars) (n=4) in HepG2 cells overexpressing SR-BI measured by
incubating 125I-M-BSA with cells at 20 µg/ml for 2 hours at 37°C
according to the pulse-chase protocol described in Materials and
Methods. Statistically different values from the control HepG2 cell
values (or otherwise indicated) are indicated as: aP<0.05; bP<0.01
and cP<0.001.
cholesterol rapidly equilibrates within the cells and may be
available for efflux/retroendocytosis.
Our other main goal was to identify lipid sensor proteins and
lipid binding proteins that are regulated by cholesterol and fatty
acids derived from SR-BI-dependent CE selective uptake. For
this, HepG2 cells were stably transfected with an expression
vector containing the full human SR-BI cDNA. Cells
containing the empty vector were tested in parallel for SR-BI
expression and gave very similar results to HepG2 cells (data
not shown). To conduct our study, a moderate and a high SRBI expressing clone were chosen that exhibit normal growth
3102
Journal of Cell Science 117 (15)
biosynthesis (Horton et al., 2002), were reduced in SR-BIoverexpressing cells (Fig. 7D). Thus, SR-BI-dependent CE
influx or SR-BI expression itself increases the level of L-FABP
and reduces that of caveolin-1 and SREBP-2.
Fig. 7. Immunoblotting of lipid
binding and/or sensor proteins in
HepG2 cells overexpressing SRBI. Total proteins were extracted
by 1% Triton X-100 and were
loaded on 10% reducing SDSPAGE. 150 µg of protein were
loaded for the caveolin-1
immunoblot (A), while 50 µg were
loaded for L-FABP (B), cytosolic
NCEH (C) and SREBP-2 (D)
immunoblots. Shown here are
representative images of at least
four independent protein
extractions.
rate. These were clones S1.1 and S1.7 showing 2.1±0.3 fold
(P<0.01) and 4.5±1.1 fold (P<0.001) increases in SR-BI
expression compared to HepG2 cells, respectively (Fig. 6A).
We found that CE selective uptake from both LDL and HDL3
parallels SR-BI expression in HepG2 cells (Fig. 6A,B). Clones
S1.1 and S1.7 had 50% and 2.2-fold increases in CE selective
uptake from both HDL and LDL, respectively. As anticipated
SR-BI overexpression did not change the selective uptake
efficiency but the mass of CE entering the cells. We also
assessed the relationship between SR-BI expression in our
clones and retroendocytosis of the SR-BI protein ligand, MBSA, which is not targeted toward degradation in HepG2 cells
(Rhainds et al., 1999). We found that gradually increasing SRBI expression increased 125I-M-BSA protein association and
recycling (Fig. 6C). Clone S1.1, a cell line with moderate SRBI overexpression, shows a significant 73% increase in 125I-MBSA recycling, while S1.7, a cell line with marked increase in
SR-BI expression, shows a 2.6-fold increase in protein
recycling, in good agreement with increases in LDL and HDL
protein association data. Thus, SR-BI overexpressing cell lines
had significant increases in SR-BI-dependent ligand
processing. We proceeded to measure the expression of lipid
sensor and lipid binding proteins in these cells and the parental
HepG2 cells. As shown in Fig. 7A, high SR-BI expressing cells
(S1.7) had an unexpected ~70% reduction in caveolin-1
expression, while no reduction was observed in moderate
expressing cells (S1.1). SR-BI expression has also an important
effect on L-FABP expression in HepG2 cells (Fig. 7B). High
SR-BI-expressing cells had a very strong increase in L-FABP
content, while moderate SR-BI-expressing cells had a modest
but reproducible increase in L-FABP. Cytosolic NCEH
expression was not modified by an increase in SR-BI
expression (Fig. 7C), suggesting that either its activity was
modulated by CE influx or that NCEH is not involved directly
in the hydrolysis of CE from the selective uptake pathway.
Both the precursor and mature forms of SREBP-2, the latter
being the major transcription factor controlling cholesterol
Discussion
Our results show that HepG2 cell SR-BI is present in lipid rafts
devoid of caveolin. We believe that this association is not
artifactual for three reasons. Firstly, the sucrose gradient
protocol in the presence of carbonate is not based on detergent
insolubility of raft membranes, but rather on their intrinsic
buoyancy. Secondly, saponin (0.5%) efficiently disrupted SRBI association with rafts, by its cholesterol-sequestering effect
that reduces cholesterol-protein interactions (Schroeder et al.,
1998). Thirdly, COase treatment, while modulating CE
selective uptake, did not affect SR-BI localization on sucrose
gradients (data not shown). Our finding that caveolin-1 in
HepG2 cell is a cytosolic protein is in line with the results of
Pohl et al. (Pohl et al., 2002) showing that caveolin-1
immunofluorescence is not visible at the cell membrane. These
results, as well as our own, suggest that caveolin-1 does not
form membrane caveolae in HepG2 cells. Interestingly, our
findings fit the Fielding’s concept that cells enriched with
lipoprotein receptors actively engaged in cholesterol uptake
may be caveolae-deficient (Fielding and Fielding, 1997).
Moreover our results show that membrane rafts may be the
preferred platform to control flux between cells and donor
lipoproteins at least when SR-BI activity is concerned.
We found that SR-BI is recovered in Triton X-100 soluble
fractions of HepG2 cells (Rhainds et al., 2003). It is worth
noting that a concentration of Triton X-100 as low as 0.25%
was sufficient to solubilize >80% of SR-BI in HepG2 cells and
that SR-BI in the Triton X-100 insoluble fraction was
detectable only in HepG2 cells extracted with 0.1% Triton X100 (data not shown). This suggests that the SR-BI membrane
domain is not a cholesterol-binding domain and that SR-BI
loosely interacts with cholesterol in membrane rafts. However,
this does not exclude that SR-BI extracellular domain binds
cholesterol as part of its lipid transfer activity (Wang et al.,
2001).
Our results show that CE selective uptake from LDL, which
is a SR-BI-dependent pathway in HepG2 cells (Rhainds et al.,
1999; Rhainds et al., 2003), was sensitive to enzymatic
disruption of rafts structure by either COase, SMase or to
sequestration of membrane cholesterol by filipin III treatment.
Surprisingly, CE selective uptake from HDL3 increased with
COase, SMase and β-CD. These results could be interpreted
by the exit of SR-BI from rafts that would impair LDL-CE
selective uptake but favour HDL-CE selective uptake. This
possibility has to be rejected since we found that the disordered
raft structure created by COase and β-CD treatments did not
modify SR-BI localization in gradients (data not shown).
Therefore,
differences
in
membrane
rafts
structure/composition that influence interaction between SRBI and its ligands HDL and LDL are likely to explain the
different effects on CE selective uptake. Based on our data,
rafts rich in sphingomyelin and unoxidized cholesterol are
likely to favour LDL association and CE selective uptake,
while rafts poor in cholesterol or containing oxidized
cholesterol or rich in fusogenic ceramide molecules favour
SR-BI function in membrane rafts of HepG2 cells
HDL-CE selective uptake. Thus, when SR-BI is in a normal
raft it shows an optimal activity towards LDL but not to HDL.
It can be suggested that modification of the raft
structure/composition changes the conformation of SR-BI in a
way that will differently affect its action towards LDL and
HDL. Indeed it has to be remembered that Gu et al. (Gu et al.,
2000) obtained data suggesting that the LDL binding site on
SR-BI differs from that of HDL. This notion of SR-BI having
multiple binding sites is also supported by the study of
Thuahnai et al. (Thuahnai et al., 2003). Alternatively,
modification of the rafts structure/composition could induce
dimerization of SR-BI molecules leading to an improved and
an impaired CE selective uptake from HDL and LDL,
respectively. Indeed Reaven et al. (Reaven et al., 2004) have
recently shown in various cells types that SR-BI dimerization
is linked to an upregulation of the HDL-CE selective uptake
pathway.
The mechanism for CE selective uptake in hepatocytes
remains obscure. There have been some reports arguing both
for a non-endocytotic, cell surface mechanism (Pittman et al.,
1987) that is supported by the intrinsic capacity of SR-BI to
incorporate CE into membranes (Liu et al., 2002) and an
endocytotic mechanism involving retroendocytosis of HDL
particles depleted of CE in mouse hepatocytes (Silver et al.,
2001), which is also present in HepG2 cells (Kambouris et al.,
1990). Our results favour the retroendocytosis model, which
does not exclude a direct role for SR-BI in transferring CE to
membranes. We found that both LDL and HDL3 can deliver
CE to HepG2 cells before being re-secreted as CE-depleted
particles. The existence of an endocytotic mechanism for CE
selective uptake is supported by a number of facts: (1) SR-BI
itself is an endocytotic receptor that traffics into hepatocytes
(Silver et al., 2001) and that leads to degradation of oxidized
LDL (Gillotte-Taylor et al., 2001) and advanced glycation
end-product modified MSA (AGE-BSA) (Ohgami et al.,
2001); (2) hydrolysis of LDL- and HDL-CE in HepG2 cells
(Rhainds et al., 1999) and esterification of cholesterol by
ACAT in CHO cells expressing SR-BI (Stangl et al., 1999)
and HepG2 cells (Charest et al., 1999) is sensitive to
lysosomal inhibitor chloroquine, and (3) membrane raft lipids
can undergo endocytosis through a non-clathrin-dependent
mechanism (Marks and Pagano, 2002). Recent studies on the
dynamic nature of membrane raft glycosphingolipids show
that they can be endocytosed independently of clathrin-coated
endosomes and directed to early endosomes, where the
clathrin-independent pathway merges with the clathrindependent pathway (Sharma et al., 2003). From early
endosomes, lipids can be directed to the Golgi apparatus (Puri
et al., 2001) or mainly recycled to the plasma membrane along
with transferrin (Sharma et al., 2003). The endosomal
recycling compartment is enriched with cholesterol (Hao et
al., 2002), rafts lipids and proteins (Gagescu et al., 2000) and
contains BSDL/CEL (Hornick et al., 1997), which can
hydrolyze CE in the presence of bile salts (Hui and Howles,
2002). In mouse hepatocytes, HDL protein is found with SRBI in transferrin-positive endosomes (Silver et al., 2001).
Thus, we propose that retroendocytosis as the mechanism for
CE selective uptake arises from the presence of SR-BI in
membrane rafts.
We found that stable overexpression of SR-BI in HepG2
cells leads to increases in LDL- and HDL3-CE selective uptake
3103
and 125I-M-BSA retroendocytosis that are well correlated with
SR-BI expression levels. While others have shown that SR-BI
increases caveolin-1 stability in plasma membranes of HEK293T cells (Frank et al., 2002), our high SR-BI expressor cells
(clone S1.7) had reduced caveolin-1 expression. This may be
explained by the subcellular localization of caveolin-1, which
is known to depend on the cell type, caveolin being expressed
in mitochondria, cytosol, secretory granules or the plasma
membrane (Li et al., 2001). Indeed, the relationship between
SR-BI and caveolin-1 expression in general and especially in
hepatocytes is not well understood. If caveolin-1 is in
mitochondria of HepG2 cells as reported by others (Pohl et al.,
2002), its down-regulation may prevent mitochondrial damage
because of an excess of free cholesterol or fatty acid
transported or stabilized by caveolin-1 in mitochondria.
It was shown that SR-BI brings regulatory cholesterol which
down-regulates cholesterol biosynthesis by HMG-CoA
reductase (Stangl et al., 1999; Charest et al., 1999). This may
occur via a reduction in SREBP-2 transcriptional activity.
Accordingly, we show that SR-BI expression decreases the
level of SREBP-2 in HepG2 cells. Our study is also the first to
report that SR-BI may also modulate intracellular fatty acid
transport proteins as an increase in L-FABP expression in both
moderate and high SR-BI-overexpressing cells was observed
in this study. L-FABP promoter is induced by fatty acids via
its peroxisome proliferator-activated receptor response element
(PPRE) bound by PPARα/RXRα heterodimers (Poirier et al.,
1997). L-FABP brings fatty acids to the nucleus where it binds
to PPARα (Wolfrum et al., 2001). Liganded PPARα may in
turn increase L-FABP expression via its PPRE. Our data
suggest that this positive feedback loop was present in high SRBI-overexpressing cells since the increase in L-FABP protein
was 10-fold higher than in moderate SR-BI-overexpressing
cells. In high SR-BI-overexpressing cells, fatty acid levels
influx due to CE selective uptake and hydrolysis may reach a
threshold level where L-FABP and other genes involved in
fatty acid metabolism (mitochondrial transporters and βoxidation enzymes) are induced. Other candidate genes like
sterol carrier protein-2 (SCP-2) and hepatic bile salt-dependent
lipase (BSDL, also known as carboxyl ester lipase, CEL) may
also work in close association with SR-BI as intracellular sinks
for CE. BSDL may help in CE hydrolysis (Hui and Howles,
2002; Li et al., 1996), while SCP-2 undoubtedly plays a role
in sterol transport to a number of intracellular sites in
hepatocytes and especially in biliary cholesterol efflux (Fuchs
et al., 2001). Additionally, ABC transporters are likely to
participate in cholesterol flux and cholesterol secretion into bile
at the apical pole of hepatocytes. We are now exploring the
relationship between SR-BI expression, selective uptake and
these proteins.
In conclusion, our study shows that SR-BI is present in
membrane rafts of HepG2 cells, where it drives CE selective
uptake independently of caveolin-1 protein, which is a
cytosolic protein in HepG2 cells. Modification of membrane
raft lipid contents affects CE selective uptake from LDL and
HDL particles differently in HepG2 cells. SR-BI
overexpression duly increased CE selective uptake, which was
proportional to SR-BI expression. In these cells, L-FABP
protein expression was increased, while caveolin-1 and
SREBP-2 expression were decreased, revealing that SR-BI
expression influences lipid binding and lipid sensor proteins
3104
Journal of Cell Science 117 (15)
that may cooperate in response to increased SR-BI-dependent
lipid flux.
We thank Dr William M. Grogan and his staff (B. Langston) for
performing the NCEH blots. D.R. would like to thank Ms Lucie
Simoneau for helpful scientific discussion. The work from our
laboratory was supported by the Canadian Institutes for Health
Research (CIHR) grant MOP-53095 to L.B. L.B. was the recipient of
a senior scientist scholarship from Fonds de la Recherche en Santé du
Québec (FRSQ). D.R. was the recipient of scholarships from the
NSERC of Canada, the Fonds pour la Formation des Chercheurs et
l’Aide à la Recherche (FCAR) and FRSQ-FCAR-Health.
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