IntroductionThe intestine must challenge the profuse daily flux ofcholesterol from dietary (300-600 mg), biliary (800-1200 mg)and sloughed intestinal cell (250-400 mg) sources (Davidsonand Magun, 1993). The absorption process takes place in aseries of orderly and interrelated steps. Cholesterol issolubilized in the lumen of the small intestine by bile acids,which are produced by the liver and secreted into the intestinallumen (Borgstrom, 1975; Simmonds et al., 1967; Thornton etal., 1968). Micellar dispersion of cholesterol by bile acidsassures its shuttle from the bulk water phase across theunstirred water layer to the region adjacent to the cell surface(Borgstrom, 1975; Simmonds et al., 1967; Thornton et al.,1968). Thereafter, cholesterol is taken up from micelles by theapical side of epithelial cells.

The abnormalities of intestinal cholesterol transport cancontribute to pathologic processes, including fat malabsorptionor atherosclerosis, and emphasize the importance of the smallgut in cholesterol homeostasis (Davidson and Magun, 1993;Levy and Roy, 1989; Levy, 1992; Thomson and Dietschy,1981). Notably, a positive relationship has been established

between cholesterol absorption magnitude, on the one hand,and plasma cholesterol levels and coronary disease, on theother hand (Turley et al., 1994; Kesaniemi and Miettinen,1987; McMurry et al., 1985). Similarly, increased dietary fatand cholesterol intake have been tightly linked to the elevatedincidence of other diseases, such as cancer, diabetes andobesity (Hannah and Howard, 1994; Vessby, 1995). Even ifthere is considerable interest in lowering cholesterol absorptionefficiency and, thereby, reducing morbidity and mortality,efforts are unfortunately hampered due to our incompleteunderstanding of the transport mechanisms. In fact, cholesteroluptake, the first critical limiting step in cholesterol absorption,and the mechanisms regulating net cholesterol translocationfrom the intestinal lumen into the enterocyte remain for themost part unclear.

Several lines of evidence allow investigators to call intoquestion the traditionally proposed passive diffusion model ofcholesterol incorporation into absorptive cells (Salen et al.,1970). There is now more and more support for an activetransport process mediated by protein(s) in the enterocytebrush border membrane, since (a) cholesterol uptake appears

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Studies employing human fetal intestine have yieldedremarkable information on the role of polarizedenterocytes in fat absorption. In this report, we investigatedthe intestinal expression, spatiotemporal distributions,ontogeny and function of the scavenger receptor, Class B,Type I (SR-BI) that plays a crucial role in cholesterolhomeostasis. SR-BI was detected as early as week 14 ofgestation in all gut segments and was almost entirelyconfined to the absorptive epithelial cells. By usingimmunofluorescence staining, the distribution of SR-BIrarely appeared as a gradient, increasing from thedeveloping crypt to the tip of the villus. Western blotshowed high levels of immunodetectable SR-BI in theduodenum, which progressively decreased toward thedistal colon. The high-resolution immunogold techniquerevealed labelling mainly over microvilli of the enterocyte.SR-BI was not associated with caveolin-1 and was notdetectable in caveolae. In order to define the role of SR-BI

in intestinal cholesterol absorption, Caco-2 cells weretransfected with a constitutive expression vector (pZeoSV)containing human SR-BI cDNA inserted in an antisenseorientation. As noted by immunoblotting and Protein A-gold techniques, stable transformants contained 40, 60 and80% the SR-BI level of control Caco-2 cells and exhibiteda proportional drop in free cholesterol uptake withoutaltering the capture of phospholipids or cholesteryl ester.Confirmation of these data was obtained in intestinal organculture where SR-BI antibodies lowered cholesterol uptake.These observations suggest that the human intestinepossesses a developmental and regional SR-BI pattern ofdistribution, and extends our knowledge in SR-BI-mediated cholesterol transport.

Key words: SR-BI, Enterocyte, Cholesterol transport, Caveolin-1,Malabsorption, Atherosclerosis

Summary

Ontogeny, immunolocalisation, distribution andfunction of SR-BI in the human intestineEmile Levy 1,*, Daniel Ménard 2, Isabelle Suc 1, Edgard Delvin 3, Valérie Marcil 1, Louise Brissette 4,Louise Thibault 1 and Moise Bendayan 5

Departments of 1Nutrition, 3Biochemistry, 5Pathology and Cell Biology, Hôpital Sainte-Justine and University of Montreal, Montreal QC H3T 1C5,Canada2Group on the Functional Development and Physiopathology of the Digestive Tract, Canadian Institute of Health Research and Department ofCellular Biology, Faculty of Medicine, Université de Sherbrooke, Sherbrooke QC J1H 5N4, Canada4Department of Biological Sciences, Université du Québec, Montréal QC H3C 3P8, Canada*Author for correspondence (e-mail: levye@justine.umontreal.ca)

Accepted 5 September 2003Journal of Cell Science 117, 327-337 Published by The Company of Biologists 2004doi:10.1242/jcs.00856

Research Article

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to be a saturable process that is sensitive to protease treatment(Bhattacharyya and Connor, 1974); (b) potent saponins, suchas pamaqueside, are effective in inhibiting cholesterol transportat concentrations below doses required to complex cholesterolin a 1:1 molar ratio, possibly by interfering directly with aputative cholesterol transporter molecule in the intestine(Salisbury et al., 1995; Morehouse et al., 1999); (c) the poorintestinal absorption of closely related plant sterol moleculessuggests discrimination in absorption (Salen et al., 1970); (d)patients with sitosterolemia, a rare inherited disorder, lose theability to discriminate between plant sterols and cholesterol,resulting in the accumulation of sistosterol in plasma andtissues (Bhattacharyya and Connor, 1974), and (e) theheterogeneity of cholesterol absorption efficiency in animalspecies and humans remains an enigma (Bhattacharyya andEggen, 1980; Kushwaha et al., 1993; St Clair et al., 1981;Turley et al., 1997; Shwarz et al., 1998), which suggests agenetic component to the transport process.

Despite the above observations, the exact identity of theputative cholesterol transporter remains elusive. A few recentstudies have proposed the scavenger receptor Class B, Type I

(SR-BI) as a candidate protein for the uptake ofdietary cholesterol (Cai et al., 2001; Hauser et al.,1998; Altmann et al., 2002). SR-BI is a cellsurface receptor that binds high-densitylipoproteins (HDL) particles and mediates theselective uptake of HDL-cholesteryl ester in manytissues (Acton et al., 1996). Immunocytochemicalanalysis of SR-BI indicates that it is expressedmost abundantly in the liver and steroidogeniccells of the adrenal gland and ovary. Althoughemerging information indicated the location ofSR- BI in intestinal cells, its physiologicsignificance is unclear and its implication incholesterol absorption remains controversial. Forexample, SR-BI was not identified in the intestinalmucosa (Acton et al., 1996) and its absence inknockout mice did not affect intestinal cholesteroltransport (Mardones et al., 2001). Althoughemerging information indicates the location ofSR-BI in intestinal epithelial cells, its physiologicsignificance remains unclear. Additionally, despiteinformation concerning the location in both theyolk sac and placenta, no thorough studies areavailable on the developing humangastrointestinal tract. Yet our exhaustive work hasshown that very early in gestation, the smallintestine exhibits the capacity to absorb lipids,

elaborates most of the major lipoprotein classes and efficientlyexports these lipoproteins from the intestinal cells (Levy et al.,1992; Levy et al., 1996; Levy et al., 2001a).

In the present study, we planned to investigate the ontogenyof SR-BI expression developing human intestine and detectwhether regional differences exist among the duodenum,jejunum and ileum. We also examined SR-BI content in thecolon that has shown great ability to elaborate lipids andlipoproteins during the gestational period (Levy et al., 1996).We further investigated whether SR-BI concentrates in caveolæand colocalizes with caveolin-1 as it has been observed inmurine adrenocortical cells (Babitt et al., 1997), in whichcaveolin-1 regulates SR-BI-mediated selective HDL-cholesteryl ester uptake. Finally, we tested the possibility thatSR-BI mediates dietary cholesterol absorption and other lipidclasses. To tackle these issues, we used human small gut tissuesat different periods of gestation, immunofluorescence,electronic microscopic techniques coupled to protein A-gold,the successful maintenance of human fetal intestine tissues inserum-free organ culture, as well as Caco-2 cell line that wasgenetically manipulated to inactivate SR-BI gene expression.

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Fig. 1. Expression and distribution of SR-BI proteinalong the duodenal crypt-villus axis. Representativeindirect immunofluorescence micrographs ofcryosections are shown for 14 weeks (A,B) and 20weeks (C) of gestation, as well as for the adult period(D). Immunofluorescence was also visualized in themicrovascular endothelial cells of the lamina propria(E) and muscle cells (F). In all cases, no fluorescentstaining was observed when the primary SR-BIantibody was replaced with the appropriate nonimmuneserial (data not shown).

329SR-BI and intestinal cholesterol transport

Materials and Methods Human gut specimensSmall intestine and large bowel tissues were obtained from fetusesranging from 14 to 20 weeks following legal or therapeutic abortionwith informed patient consent. No tissues were collected from casesassociated with known fetal abnormalities or fetal death. Jejunaland colonic samples were obtained from patients who hadundergone surgical resection of different areas of the digestive tract.Normal tissue found adjacent to the resected pathological tissueswas used in all cases, as ascertained by routine hematoxylin/eosinstaining. Studies were approved by the Institutional ReviewCommittee for the use of human material from the ‘CentreHospitalier Universitaire de Sherbrooke/Faculté de Médecine’. Theproximal third of the fetal small intestine was defined as theduodenum, the second third as the jejunum and the residual thirdas the ileum. The colon was divided in two equivalent portions, theproximal and distal parts.

Indirect immunofluorescenceThe preparation and embedding of specimens for cryosectioningwere performed as described previously (Levy et al., 2001b) usingoptimum cutting temperature embedding compounds (Tissue Tek,

Miles Laboratories, Elkhat, IN). Cryosections (2-3 µm thick) cuton a Jung Frigocut 2800N Cryostat (Leica Canada, Saint-Laurent,Québec) were fixed on a glass slide with acetone-chloroform (1:1)for 5 minutes at 4°C, then blocked 30 minutes with fish gelatin0.1% in phosphate buffer containing 0.8% Bovine Serum Albumin(BSA) at room temperature. The staining procedure usingantibodies and fluorescence was performed at room temperature inhumid chambers. Sections were incubated for 60 minutes withpolyclonal antibodies (Novus Biologicals, Littleton, CO):scavenger receptor BI (SR-BI) 1:500 and caveolin-1:300 diluted inPBS. Fluorescein isothiocyanate-conjugated sheep anti-rabbit IgG(Chemicon International, Temecula, CA) was used as secondaryantibody at the working dilution of 1:75 and added for 45 minutes.After extensive washing with PBS, sections were then contrastedwith 0.01% Evans blue in PBS, mounted in glycerol-PBS (9:1)containing 1% paraphenylenediamine and viewed with a ReichertPolyvar Microscope equipped for epifluorescence (Leica Canada,Saint-Laurent, Québec). Finally, the primary antibodies wereomitted or replaced by non-immune rabbit serum at 1:700 dilutionand all the control experiments confirmed the specificity of theresults.

Fig. 2. Expression and distribution of SR-BI protein along thejejunal, ileal and colonic crypt-villus axis for 14 weeks of gestation.Representative indirect immunofluorescence micrographs ofcryosections of human fetal jejunum (A), ileum (B), proximal colon(C) and distal colon (D). In all cases, no fluorescent staining wasobserved when the primary SR-BI antibody was replaced with theappropriate nonimmune serial (data not shown). Bars, 50 µm.

Fig. 3. Expression and distribution of SR-BI protein along thejejunal, ileal and colonic crypt-villus axis for 20 weeks of gestation.Representative indirect immunofluorescence micrographs ofcryosections of human fetal jejunum (A), ileum (B), proximal colon(C) and distal colon (D). In all cases, no fluorescent staining wasobserved when the primary SR-BI antibody was replaced with theappropriate nonimmune serial (data not shown). Bars, 50 µm.

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Tissue preparation for electron microscopyIntestinal specimens were fixed by immersion in 1% glutaraldehyde,0.1 M phosphate-buffered saline (pH 7.4) for 2 hours at 4°C andembedded in Lowicryl K4M at –20°C according to our previouslydescribed procedures (Bendayan, 1984). Tissue blocks were examinedby light microscopy to select well-oriented villous tips. Thin sections(60-80 nm) of the different tissue blocks were mounted on nickel gridswith a carbon-coated Parlodion film and processed forimmunocytochemistry.

Immunocytochemical labellingProtein A-gold immunocytochemical techniques were employed todetect the presence of SR-BI and caveolin-1 in intestinal tissue as wehave described previously (Levy et al., 2002). Briefly, the tissuesections were washed initially in distilled water, incubated for 5minutes on a drop of PBS containing 1% ovalbumin, and transferredsubsequently to a drop of the PBS-diluted antibody (see below). Afterincubation (90 min) at room temperature, the grids were rinsed withPBS to remove unbound antibodies. They were transferred to the PBS-ovalbumin (3 minutes) and incubated on a drop of protein A-gold (pH7.2) for 30 minutes at room temperature. The tissue sections were thenthoroughly washed with PBS, rinsed with distilled water and dried.Sections were stained with uranyl acetate and lead citrate beforeexamination with a Philips 410 electron microscope. Polyclonalantibodies were used at various dilutions (SR-BI 1/100 and caveolin-1 1/10, 1/50, 1/100, 1/1000) in combination with protein A-goldcomplexes, which were prepared using 10 or 5 nm gold particlesaccording to our established techniques (Levy et al., 2002). Controlexperiments were performed to assess the specificity of the results.Excess purified SR-BI (tenfold) was added to the antibody solution.Incubation with this solution was followed by the protein A-goldcomplex. Pre-immune rabbit serum (diluted 1:10) was used on tissuesections before incubation with protein A-gold complex. Incubationswere also performed with the protein A-gold complex alone, omittingthe antibody step to test for non-specific adsorption of the protein A-gold complex to tissue sections.

Double-labeling techniqueTo simultaneously reveal the existence of SR-BI and caveolin-1 withinthe cellular compartments, the double-labeling technique was applied.The tissue sections were labeled concomitantly for SR-BI andcaveolin-1. The two-phase labeling technique (Bendayan, 1982;Bendayan, 1995) was applied to avoid any cross-reaction betweenreagents. The small protein A-gold complex (5 nm) was used for thefirst labeling protocol, and the larger (10 nm) protein A-gold complexwas used for the second. This protocol allows for the simultaneousvisualization of two antigens (SR-BI and caveolin-1) in the sametissue section. Anti-scavenger BI and caveolin-1 polyclonal antibodieswere from Novus Biologicals. They were purified on a sepharosecolumn and non-immune rabbit IgG was utilized as a negative control.

Cell cultureCaco-2 cells (American Type Culture Collection, Rockville, MD)were grown at 37°C with 5% CO2 in MEM (Gibco-BRL, GrandIsland, NY) containing 1% penicillin/streptomycin and 1% MEMnonessential amino acids (GIBCO BRL) and supplemented with 10%decomplemented fetal bovine serum (FBS; Flow, McLean, VA).Caco-2 cells (passages 30-40) were maintained in T-75 cm2 flasks(Corning Glass Works, Corning, NY). Cultures were split (1:6) whenthey reached 70-90% confluence, using 0.05% trypsin-0.5 mM EDTA(GIBCO BRL). For individual experiments, cells were plated at adensity of 1×106 cells/well on 24.5 mm polycarbonate transwell filterinserts with 0.4 µm pores (Costar, Cambridge, MA) in MEM (asdescribed above) supplemented with 5% FBS. The inserts were placed

into six-well culture plates and cultured for 20 days, a period at whichthe Caco-2 cells are highly differentiated and appropriate for lipidmetabolism (Marcil et al., 2002; Courtois et al., 2000).

Preparation of stable transformants expressing various levelsof Apo SR-BITo obtain Caco-2 cells deficient in SR-BI expression, two vectors werecreated, one pZeoSV containing the full human SR-BI cDNA andanother containing a cDNA, inserted in the antisense orientation. Thetwo different constructs and the expression vector without insert wereseparately transfected to Caco-2 cells using SuperfectTM TransfectionReagent (Quiagen) after a 72 h incubation period, Zeocine-resistant celllines were selected and isolated from the pools with cloning cylindersand then propagated to study cells in appearance and in growth rate.

SR-BI neutralization by antibodySR-BI antibody was obtained by Novus. A titration experiment wasperformed to determine the optimal antibody concentration to inhibitSR-BI activity using Caco-2 cells expressing SR-BI. It shows that a1:1000 dilution of immune serum was sufficient to maximally reducecholesterol transport. This concentration equaled ∼ 10-20 µg/mlprotein A-purified IgG.

Western blotsTo assess the presence of SR-BI and caveolin-1 and evaluate theirmass, intestinal tissue was homogenized and adequately prepared forwestern blotting as described previously (Levy et al., 2001a). Proteinswere denatured in sample buffer containing SDS and β-

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Fig. 4. Expression and distribution of SR-BI in adult human guttissues. The staining is present in all epithelial cells of the crypt-villus axis in the jejunum (A) and ileum (B) as well as in the crypt-surface epithelium of the proximal (C) and distal (D) colon. Bars,30µm.

331SR-BI and intestinal cholesterol transport

mercaptoethanol, separated on a 4-20% gradient SDS/PAGE, andelectroblotted onto nitrocellulose membranes. Nonspecific bindingsites of the membranes were blocked using defeated milk proteinsfollowed by the addition of primary antibodies directed against SR-BI and caveolin-1. The relative amount of primary antibody wasdetected with species-specific horseradish peroxidase-conjugatedsecondary antibody. Blots were developed and the mass of SR-BI andcaveolin-1 was quantitated using an HP Scanjet scanner equipped witha transparency adapter and software.

[14C]-cholesterol absorptionTo study cholesterol uptake, 10 µCi [14C]-cholesterol, 10 µCi [14C]-cholesterol ester or 10 µCi phosphatidylcholine was added as a mixedbile salt micelle (6.6 mmol/L sodium taurocholate, 1 mmol/L oleicacid, 0.5 mmol/L monoolein, 0.1 mmol/L cholesterol and 0.6 mmol/Lphosphatidylcholine). Caco-2 cells were incubated at 37°C for 8-24hours.

Statistical analysisData from the experiments were analyzed by using a Student’s t-test.Reported values are expressed as mean ± s.e. Statistical significancewas accepted at P<0.05.

Results and DiscussionLocalization of SR-BI by immunofluorescent studiesOriginally, the expression of SR-BI was mostly described in

the liver and steroidogenic tissues as well as in macrophageswithin the vascular wall (Acton et al., 1996; Ji et al., 1997;Temel et al., 1997). A few studies showed the presence of SR-

Fig. 5. Immunocytochemical detection of SR-BI in fetal duodenalmucosa. Protein A-gold immunocytochemical technique was appliedto localize SR-BI in absorptive cells of duodenal tissue at 17 weeks(A) and 20 weeks (B) of gestation as well as at the adult period(C,D). The gold particles revealing SR-BI antigenic sites are mainlyassociated with the luminal plasma membrane lining the microvilli(mv). They are also present in endosomal invaginations and vesicles(e). The labelling within the cell, although of lower intensity, isobserved in the rough endoplasmic reticulum (RER), the Golgiapparatus (G) and the basolateral membrane (blm). Mitochondria (m)are devoid of labelling.

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BI in the intestine of rodents and Caco-2 cells (Cai et al., 2001;Hauser et al., 1998; Voshol et al., 2001). In the presentinvestigation, we focused on the maturational and allocationaspects of SR-BI in human fetal intestine.

To establish the expression of SR-BI in the different regionsof the gut and to determine their distribution along the crypt-villus axis, indirect immunofluorescence was carried out indeveloping as well as in adult human intestinal tissues. Figs 1-4 illustrate the presence and localization of SR-BI proteinalong the crypt-villus axis in the human fetal and adult smalland large intestine. Overall, this first series of studies supportsSR-BI expression in the developing gut. Immunofluorescentstaining was detected as early as the week 14 of gestation inboth the small and large intestine (Figs 1 and 2) and its patternof distribution was similar to the apparent profile observed at20 weeks of gestation (Fig. 3) and in the adult condition (Fig.4). Immune fluorescence staining was noted in the columnarepithelial cells of the small intestinal segments, i.e. duodenum,jejunum and ileum. Cytoplasmic immunofluorescence stainingwas visualized in most of the absorptive cells located in thecrypt-villus axis, but also in the microvascular endothelial cellsof the lamina propria (Fig. 1). However, only in fewexperiments, more immunofluorescence was noticeable in thecolumnar epithelial cells of the villi with a lower intensity incrypt cells. In the colon mucosa, all the epithelial cells liningthe crypt and the surface area of the mucosa expressed SR-BIprotein, which was mainly localized in the apical cytoplasm ofthe colonocytes. Taking into account the qualitative nature ofthis technique, it was not possible to speculate on quantitativeaspects relative to SR-BI signals in the different human gutsegments.

Detection of SR-BI by immunoelectron microscopyapproachIn an effort to better understand the cellular localization of SR-BI, Protein A-gold immunocytochemical techniques wereapplied on thin sections incubated with specific antibodies todisclose SR-BI in human fetal intestine. Electron microscopicimmunocytochemical studies revealed significant immunogoldlabelling in the luminal region of enterocytes, particularlyassociated with the apical plasma membrane lining themicrovilli (Fig. 5A,B,C). The labelling of SR-BI by goldparticles was also present in endosomal invaginations andvesicles. Within the cell, the labelling, although of lowerintensity, was present in the rough endoplasmic reticulum, theGolgi apparatus and the basolateral membrane (Fig. 5D).Under the control conditions tested, labelling was markedlyreduced or eliminated, demonstrating its specificity (Data notshown).

Immunoblotting evaluation of SR-BI ontogeny anddistribution along the intestineExperiments were conducted to study the distribution of SR-BI along the intestine. Samples of the different regions of theintestine containing equal quantities of protein wereelectrophoresed on an SDS-polyacrylamide gel (Fig. 6). Animmunoblot of these samples showed immunoreactive bandscorresponding to SR-BI. Densitometric estimation of the SR-BI visualized on the immunoblot showed that the jejunum,

ileum, proximal colon and distal colon contained 76.2%,57.1%, 16.7% and 9.5% of SR-BI amounts measured in theduodenum (Fig. 6). We also explored the maturation aspect ofSR-BI in each region of the human small and large intestineusing equal protein amount within each intestinal segmentonly. An increase in SR-BI was noted only in the duodenaltissue as a function of fetal age (Fig. 7). The duodenumexhibited a significant progressive rise in SR-BI proteincontent: 35.5% at the end of week 15 and 59.3% at the end ofthe week 17 compared with the value (100%) noted at the endof week 20. No marked ontogenic differences in SR-BI proteinexpression were recorded in the other intestinal segmentsexcept for the distal colon where a decline was apparent at theend of week 17. On the whole, SR-BI content appearedpredominant in the duodenum, whereas it seemed mostimpoverished in the colon, thus indicating an increasing distal-to-proximal gradient. This is in accordance with our previousfindings, which documented the colon’s reduced capacity ofthe colon to synthesize lipids and assemble lipoproteins ascompared with the small intestine during development (Levyet al., 1996; Loirdighi et al., 1997). Additionally, our dataclearly demonstrated that only the duodenum is endowed witha developmental SR-BI expression profile, whereas SR-BIprotein expression was stable throughout the other gestationalages considered in our studies.

Co-expression of scavenger receptor-BI and caveolin-1In this report we have addressed three additional questions: Dointestinal fetal tissues express caveolin-1 and contain caveolae?Does SR-BI concentrate in caveolæ and colocalize withcaveolin-1? Is this co-localization associated with enhancedcholesterol uptake? Babitt et al., demonstrated that SR-BIcolocalized with caveolin-1, a constituent of caveolae inChinese hamster ovary cells or in murine adrenocortical Y1-BS1 cells (Babitt et al., 1997). Other studies on macrophage

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Fig. 6. Pattern of SR-BI distribution in different regions of humanfetal intestine. Immunoblotting analysis was performed at week 20 ofthe gestational period. The intestinal regions are: duodenum (D),jejunum (J), ileum (I), proximal colon (PC) and distal colon (DC).Values are means ± s.e. of 4-6 experiments/group.

333SR-BI and intestinal cholesterol transport

cell lines documented an association between SR-BI andcaveolin-1 (Matveev et al., 1999). The localization of SR-BI incaveolin-1 enriched membrane domains on the cell surfacemay have implications for the mechanism of SR-BI-mediatedselective lipid uptake by cells. Numerous investigators have

suggested that caveolae may serve as sites of cholesterol effluxfrom cells (Fielding and Fielding, 1995) where caveolin-1appears to be a cholesterol binding protein (Murata et al., 1995;Li et al., 1996) involved in intracellular cholesterol transport(Smart et al., 1994; Smart et al., 1996). The concentration ofSR-BI in caveolae may bring the receptor into proximity withcaveolin-1 or other resident proteins that may be critical fordirect lipid transfer into the cell. Furthermore, the ability ofcaveolae to internalize molecules may be essential for efficientlipid transport. Here we have used immunochemical andelectrophoretic methods to study the subcellular localization ofcaveolin-1. The assessment of intestinal tissues byimmunofluorescent techniques at various ages of gestation andin the postnatal period could not reveal caveolin-1 in theenterocyte (Fig. 8). Staining either in the fetal or adult smallintestine and colon did not reveal any signal of caveolin-1 atthe epithelial level. However, intense staining was detected inthe sub-epithelial layer, i.e. the endothelial cells of the bloodvessels and the smooth-muscle cells of the muscula layer.Because of the potential association or physical interaction ofSR-BI and caveolin-1, we reasoned that theimmunoprecipitation of one of the two proteins would drag theother. For this purpose, intestinal epithelial cells (detachedfrom the duodenum) or Caco-2 cells were homogenized in anondenaturing buffer and immunoprecipitation was alsocarried out with an anti-SR-BI antibody under nondenaturingconditions. The immunoprecipitates were run onto SDS-PAGEand transferred to a nitrocellulose membrane (Fig. 9).Immunoblotting the membrane with anti-SR-BI (a-SR-BI) andanti-caveolin-1 (a-cav-1) antibodies confirmed the presence ofSR-BI, but could not detect any traces of caveolin-1. Thus, theimmunoprecipitation procedure refutes an interaction betweenSR-BI and caveolin-1. Furthermore, combined high-resolution immunoelectron microscopy with specificpolyclonal antibodies could not disclose the presence ofcaveolin-1 (data not shown). Repeated manipulations anddouble-labelling techniques revealed morphologicallyidentifiable plasma membrane invaginations and endosomesin the apical side of the enterocyte, which contained SR-BIwithout caveolin-1 (data not shown). Altogether, our findingsindicate that SR-BI localizes in microvilli, plasma membraneinvaginations and endosomes, which do not exhibit caveolin-1. If our observations garner little supporting evidence for thecooperation between caveolin-1 and SR-BI, they at leastsuggest that endosomes may function as signalling platformscapable of delivering endocytosed molecules to specificorganelles. However, further work is needed to establish theprecise molecular mechanisms that couple SR-BI-mediatedligand uptake and intracellular destination in the enterocyte.Interestingly, a recent investigation has shown that SR-BI–stimulated cholesterol efflux or selective uptake is notaffected by caveolin-1 expression in Fisher rat thyroid cellsor human embryonic kidney cells (Wang et al., 2003). Instead,SR-BI-mediated cholesterol uptake may occur selectively in

Fig. 7. SR-BI protein expressionin human small intestine andcolon from 15-20 weeks ofgestation. Each intestinalsegment was analyzedseparately with similar proteinamounts at the various periods

of gestation. However, protein amounts were different amongintestinal regions in order to increase the electrophoretic resolution.

Fig. 8. Expression and distribution of caveolin-1 along the duodenaland colonic crypt-villus axis. Representative indirectimmunofluorescence micrographs of cryosections are shown for the20 weeks of gestation (A,C) as well as for the adult period (B,D).

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rats, without a specific requirement for caveolin-1 orinvaginated caveolae (Briand et al., 2003).

Role of SR-BI in cholesterol absorptionAs stated above, the molecular mechanisms of cholesterolabsorption in the intestine are poorly understood. With the goal

of defining whether SR-BI plays a role in this process, twostrategies were devised. We first used the organ culture system,allowing the morphological and physiological preservation ofthe cultured fetal tissues to study lipid transport and lipoproteinassembly (Levy et al., 1992; Levy et al., 2001a; Loirdighi etal., 1997). The incubation of intestinal explants with SR-BIantibody partially inhibited (34%) the uptake of micellar free[14C]-cholesterol (results not shown). The same amount ofantibody had no effect on the uptake of labelled cholesterylesters or phospholipids. Of note were also the preferentialuptake of micellar cholesterol (compared with cholesterolbound to albumin), the efficient cholesterol absorption withintact micelles (without missing components) and the saturatedcholesterol transport process as a function of cholesterolconcentration (data not shown). Secondly, we utilized theCaco-2 cell line, an excellent in vitro model for theinvestigation of intestinal lipoprotein metabolism (Levy et al.,1995). In order to determine the importance of SR-BI inintestinal cholesterol transport, Caco-2 cells were transfectedwith a constitutive expression vector (pZeoSV) containing thehuman SR-BI cDNA inserted in an antisense orientation for

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Fig. 11. Effect of SR-BI antisense treatment on cell proliferation anddifferentiation. As examined by [3H]-thymidine incorporation (A),sucrase activity (B) and transepithelial resistance (C), antisensetreatment did not affect cell growth differentiation and cellintegrity.Values are means ± s.e. of three experiments.

Fig. 10. Disruption of SR-BI expression using SR-BI antisense (AS)oligodeoxynucleotides in Caco-2 cells. Following antisensetreatment, cells were incubated with fresh MEM medium allowed togrow and tested for SR-BI expression at the differentiated state.Three clones (AS1, AS2, AS3) presenting decreased SR-BI proteinlevels (compared with cells transfected with vector without insert)were selected. Values are means ± s.e. of three experiments.

Fig. 9. Association of SR-BI with caveolin-1 in intestinal epithelialcells. Homogenates of epithelial cells (detached from the duodenum)and Caco-2 cells were incubated with anti-SR-BI (a-SR-BI)antibody. The immunoprecipitates were run on SDS-PAGE andtransferred to a nitrocellulose membrane. The membrane was blottedwith anti-SR-BI (a-SR-BI) or anti-caveolin-1 (a-cav-1) antibodies.Immunoblotting with a-SR-BI revealed the SR-BI protein, whereasimmunoblotting with a-cav-1 did not display any signal. The lack ofco-precipitation and the absence of caveolin-1 signal refuted thepresence of caveolin-1 and potential physical interaction betweenSR-BI and caveolin-1. STD, standard.

335SR-BI and intestinal cholesterol transport

SR-BI gene inactivation. Control Caco-2 cellswere transfected with the vector pZeoSVwithout insert. Cellular clones were obtainedfrom each pool of transformants, and theirlevel of SR-BI was determined byimmunoblotting. As observed in Fig. 10,stable transformants generated by thisconstitutive antisense RNA technology wereobtained and they expressed lower levels ofSR-BI than control Caco-2 cells.Densitometric analysis of these clones, takingthe control Caco-2 cells (infected with vectorwithout insert) SR-BI level as 100%, revealedthat the corresponding experimental cellsexpressed 40, 60 and 80% the control level.This genetic manipulation did notsignificantly modify cell growth anddifferentiation, as observed in Fig. 11, by theincorporation of [3H]-thymidine, sucraseactivity and monolayer resistance. Weconfirmed the reduction of endogenous SR-BIprotein expression in Caco-2 intestinal cellswith EM immunogold microscopy. Indeed,the use of antisense led to an evident decreasein protein A-gold labelling over apical plasmamembrane as well as over the basolateralmembrane in transfected (Fig. 12B) comparedwith control (Fig. 12A) Caco-2 cells. Thequantitative evaluation of gold particlesrevealed less labeling density in transfectedCaco-2 cells (Table 1). Our next aim was todefine whether the changes of SR-BI levels inCaco-2 cells would affect their ability tocapture labelled cholesterol. As illustrated inFig. 13A, the disruption of SR-BI in Caco-2cells resulted in decreased [14C]-freecholesterol uptake. Under identicalconditions, no changes were noted inphospholipid and cholesteryl ester uptake(Fig. 13A,B). Our findings are consistent within vitro studies that showed that SR-BI canfacilitate the cellular uptake of nonlipoproteinunesterified cholesterol in a hamster ovary

Fig. 12. Immunocytochemical labelling of SR-BIin Caco-2 cells following antisense treatment. Toconfirm the disruption of SR-BI expression usingSR-BI antisense oligodeoxynucleotides, weapplied the immunogold protein A technique onCaco-2 cells transfected with the vector pZeoSVwithout insert (A) or with the human SR-BIcDNA inserted in an antisense orientation (B).Upper panels show the apical region of the cell,and lower panels illustrate the basolateral regionof the cell. Reduced labelling is noticed inmicrovilli (mv) and the basolateral membrane aswell as within endocytotic vesicles (arrows).Magnification: A, upper panel ×38,000; lowerpanel ×36,000; B, upper panel ×40,000; lowerpanel ×56,000.

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cell line (Bruneau et al., 2003). However, our observations aredivergent from the results reported by Mardones et al.(Mardones et al., 2001). These investigators addressed the roleof SR-BI in SR-BI knockout mice and concluded that SR-BIis not essential for intestinal cholesterol absorption.Nevertheless, they suggested that in vivo SR-BI-independentmechanisms must have been able to efficiently compensate forthe loss of SR-BI expression in the mutant mice subjected toa cholesterol-enriched diet. Furthermore, whereas there was atight coupling between faulty free cholesterol transport andSR-BI disruption, the SR-BI receptor level was not linked to avariation in the uptake of other lipid classes. As SR-BIantisense expression specifically reduced endogenous SR-BIprotein expression in Caco-2 cells, and this decrease correlatedwith a significant decline in protein A-gold labelling andcholesterol uptake, we propose that SR-BI functionallymediates alimentary cholesterol uptake in the intestine.

Over the last decade, our knowledge of the biosyntheticevents essential for the formation and secretion of lipoproteinshas significantly increased in the human gut in general and inthe developing gut in particular. It has been established that the

fetal small intestine exhibits very early the capacity to absorblipids, elaborate most of the major lipoprotein classes andefficiently export these lipid vehicles from the intestinal cells.The present work allows us to demonstrate an additionalprotein component in the apical membrane of the human fetaland adult SR-BI intestine.

SR-BI was detected in all segments of the small and largeintestine. However, Western blot analyses revealed that SR-BIis preponderantly expressed in the duodenum wherecholesterol absorption is optimal. Immunofluorescenceexperiments showed that SR-BI is found not only in the villus,but also in the crypt cells. Based on gene expression, it seemsthat SR-BI mRNA is mostly expressed in differentiated Caco-2 cells. The observed relation between SR-BI expression andcholesterol uptake supports the hypothesis that SR-BItransports alimentary free cholesterol, and not phospholipidsand cholesteryl esters. It seems, therefore, that carboxyl esterlipase, a lipolytic enzyme synthesized in the acinar cells of thepancreas (Bruneau et al., 2003), is needed for hydrolysis ofcholesteryl ester, thereby facilitating free cholesterol transferto SR-BI. This is particularly important given the absence ofcaveolin-1 in our studies, which normally binds to cholesteroland is involved in intracellular cholesterol trafficking.

This work was supported by grants from Canadian Institutes ofHealth Research and Canadian Heart Association. The authors thankMrs Schohraya Spahis for her expert secretarial assistance.

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