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: brissette.louise{at}uqam.ca)

Accepted 24 February 2004


    Summary
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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, SR-BI 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 2.3-fold 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 ß-cyclodextrin increased LDL- and HDL3-CE selective uptake by 1.6-fold and 3-fold, respectively. We found that 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-BI-overexpressing 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-BI-dependent 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.

Key words: SR-BI, Cholesterol, Rafts, Caveolae, HepG2 cell, Lipoprotein


    Introduction
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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., 1996Go) and in a number of tissues with important roles in lipid transport, such as the intestine (Cai et al., 2001Go). SR-BI is a highly glycosylated 82-85 kDa protein (Acton et al., 1994Go) that mediates selective uptake of CE and other lipids from high density lipoprotein (HDL) (Acton et al., 1996Go) and low density lipoprotein (LDL) (Swarnakar et al., 1999Go). 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., 1993Go). SR-BI has an intrinsic capacity to transfer CE into membranes (Liu and Krieger, 2002Go).

Caveolin-1 is a cholesterol and fatty acid binding protein (Murata et al., 1995Go; Trigatti et al., 1999Go). Caveolin-1 is involved in lipid trafficking, membrane trafficking and signal transduction (reviewed by Liu et al., 2002Go). This 21 kDa protein oligomerizes with itself and caveolin-2 to form a striated coat around flask-shaped membrane invaginations (50-100 nm in diameter) called caveolae (Lisanti et al., 1993Go). 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, 1998Go). 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., 1997Go). Another study has linked CE selective uptake by SR-BI to its localization in caveolae (Graf et al., 1999Go). Remarkably, hepatocytes exhibit very few caveolae (Fielding and Fielding, 2000Go; Calvo et al., 2001Go) and express small amounts of caveolin-1 compared to other tissues (Li et al., 2001Go). There are conflicting reports about caveolin expression in human hepatoma HepG2 cells: one study shows a complete absence of caveolins (Fujimoto et al., 2000Go), while a recent one shows that caveolin-1 is readily detectable in these cells (Pohl et al., 2002Go).

We have shown that SR-BI is the major receptor for CE selective uptake from LDL and HDL3 in HepG2 cells (Rhainds et al., 1999Go; Rhainds et al., 2003Go) and that LDL-CE selective uptake brings regulatory cholesterol to HepG2 cells (Charest et al., 1999Go). 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. SR-BI 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
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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, ß-cyclodextrin (ß-CD), cholesterol oxidase (COase), neutral sphingomyelinase (SMase), N-ethylmaleimide (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 salt-dependent 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 anti-rabbit 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, 3.0x105 cells were seeded in 3.8 cm2 culture dishes (12-well plates). When protein recycling (retroendocytosis) had to be investigated, 7.3x105 cells were seeded in 9.4 cm2 dishes (6-well plates). In all cases, the cells were used when they were 80-90% 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, 1993Go). 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 calcium-phosphate method (Sambrook et al., 1989Go). 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 X-100 (Yoshimura et al., 1987Go). 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., 1996Go). 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 achieved by ultracentrifugation as described in Hatch and Lees (Hatch and Lees, 1968Go). 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., 1996Go). LDL and HDL3 contained no detectable amount of apoE as assessed by immunoblotting. LDL and HDL3 were iodinated by a modification (Langer et al., 1972Go) of the iodine monochloride method (McFarlane, 1948Go). 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., 1985Go). 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 (2x) 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 125I-lipoprotein 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]CE-lipoprotein 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., 1999Go). In other experiments, cells in 12-well plates were preincubated with either COase (1 U/ml) (Smart et al., 1994Go) or SMase (0.5 U/ml) (Scheek et al., 1997Go) or both enzymes for 1 hour at 37°C; with either filipin III (5 µg/ml, 7.6 µM) (Schnitzer et al., 1994Go) or ß-CD (10 mM) (Kilsdonk et al., 1995Go) for 1 hour at 37°C, or with NEM (1 mM) (Reaven et al., 1996Go) 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., 1990Go) and Greenspan and St Clair (Greenspan and St Clair, 1984Go). 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 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, 1994Go). 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 TCA-precipitable 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., 2000Go). 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 125I-HDL3, 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., 1957Go). 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., 1951Go) with BSA as standard. Paired Student's t-test or ANOVA-1 (with Tukey's post-test) were used to obtain statistical comparison of means. Differences were considered significant at P<0.05.


    Results
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 Materials and Methods
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 References
 
It is known that SR-BI is expressed in HepG2 cells (Rhainds et al., 2003Go), however caveolin-1 expression in this cell type is 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 SR-BI 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, caveolin-1 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., 1997Go). 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.



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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.

 


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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.

 

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) co-localized 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., 1995Go), which is thought to be implicated in extralysosomal CE hydrolysis (Delamatre et al., 1993Go) 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., 1997Go). 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 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, 2001Go). SMase treatment produces ceramide from sphingomyelin, which is mainly found in membrane rafts (Yu et al., 1973Go). 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, 1997Go). 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 non-specific 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.



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Fig. 3. Effect of COase and/or SMase treatment on [3H]CE and 125I-protein 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.

 

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, 1982Go) and may disrupt cholesterol-rich domains (Schnitzer et al., 1994Go). Sequestering of membrane cholesterol with 5 µg/ml filipin III reduced LDL-CE association and selective uptake by 30% (Fig. 4A). A similar 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., 1999Go). Conversely, 125I-HDL 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 ß-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) 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 ß-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., 1999Go). 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.



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Fig. 4. Effect of filipin III or ß-CD treatment on [3H]CE and 125I-protein association of LDL and HDL3 in HepG2 cells. HepG2 cells were treated with 5 µg/ml filipin III or 10 mM ß-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]CE-HDL3 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.

 

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 reduce endosome acidification and associated vesicular transport. Fig. 5A shows that in HepG2 cells, NEM reduced 125I-LDL protein association by 58%, nearly abolished 125I-LDL 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.



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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.

 

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 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]CE-LDL (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 cholesterol rapidly equilibrates within the cells and may be available for efflux/retroendocytosis.


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Table 1. LDL and HDL3 particle recycling (retroendocytosis) in HepG2 cells

 

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 SR-BI expressing clone were chosen that exhibit normal growth 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, M-BSA, which is not targeted toward degradation in HepG2 cells (Rhainds et al., 1999Go). We found that gradually increasing SR-BI expression increased 125I-M-BSA protein association and recycling (Fig. 6C). Clone S1.1, a cell line with moderate SR-BI overexpression, shows a significant 73% increase in 125I-M-BSA 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 biosynthesis (Horton et al., 2002Go), were reduced in SR-BI-overexpressing 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.



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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-M-BSA 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.

 


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Fig. 7. Immunoblotting of lipid binding and/or sensor proteins in HepG2 cells overexpressing SR-BI. Total proteins were extracted by 1% Triton X-100 and were loaded on 10% reducing SDS-PAGE. 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.

 


    Discussion
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 SR-BI association with rafts, by its cholesterol-sequestering effect that reduces cholesterol-protein interactions (Schroeder et al., 1998Go). 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., 2002Go) 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, 1997Go). 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., 2003Go). 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 X-100 (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., 2001Go).

Our results show that CE selective uptake from LDL, which is a SR-BI-dependent pathway in HepG2 cells (Rhainds et al., 1999Go; Rhainds et al., 2003Go), 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 ß-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 ß-CD treatments did not modify SR-BI localization in gradients (data not shown). Therefore, differences in membrane rafts structure/composition that influence interaction between SR-BI 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 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., 2000Go) 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., 2003Go). 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., 2004Go) 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., 1987Go) that is supported by the intrinsic capacity of SR-BI to incorporate CE into membranes (Liu et al., 2002Go) and an endocytotic mechanism involving retroendocytosis of HDL particles depleted of CE in mouse hepatocytes (Silver et al., 2001Go), which is also present in HepG2 cells (Kambouris et al., 1990Go). 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., 2001Go) and that leads to degradation of oxidized LDL (Gillotte-Taylor et al., 2001Go) and advanced glycation end-product modified MSA (AGE-BSA) (Ohgami et al., 2001Go); (2) hydrolysis of LDL- and HDL-CE in HepG2 cells (Rhainds et al., 1999Go) and esterification of cholesterol by ACAT in CHO cells expressing SR-BI (Stangl et al., 1999Go) and HepG2 cells (Charest et al., 1999Go) is sensitive to lysosomal inhibitor chloroquine, and (3) membrane raft lipids can undergo endocytosis through a non-clathrin-dependent mechanism (Marks and Pagano, 2002Go). 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 clathrin-dependent pathway (Sharma et al., 2003Go). From early endosomes, lipids can be directed to the Golgi apparatus (Puri et al., 2001Go) or mainly recycled to the plasma membrane along with transferrin (Sharma et al., 2003Go). The endosomal recycling compartment is enriched with cholesterol (Hao et al., 2002Go), rafts lipids and proteins (Gagescu et al., 2000Go) and contains BSDL/CEL (Hornick et al., 1997Go), which can hydrolyze CE in the presence of bile salts (Hui and Howles, 2002Go). In mouse hepatocytes, HDL protein is found with SR-BI in transferrin-positive endosomes (Silver et al., 2001Go). 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 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 HEK-293T 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., 2001Go). 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., 2002Go), 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., 1999Go; Charest et al., 1999Go). 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 PPAR{alpha}/RXR{alpha} heterodimers (Poirier et al., 1997Go). L-FABP brings fatty acids to the nucleus where it binds to PPAR{alpha} (Wolfrum et al., 2001Go). Liganded PPAR{alpha} may in turn increase L-FABP expression via its PPRE. Our data suggest that this positive feedback loop was present in high SR-BI-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 ß-oxidation 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, 2002Go; Li et al., 1996Go), 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., 2001Go). 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 that may cooperate in response to increased SR-BI-dependent lipid flux.


    Acknowledgments
 
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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 Materials and Methods
 Results
 Discussion
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