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J. Biol. Chem., Vol. 281, Issue 16, 11193-11204, April 21, 2006
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23
From the
Center for Physiology and Pathophysiology, Department of Medical Chemistry and ¶Department of Physiology, Medical University of Vienna, Währingerstrasse 10, A-1090 Vienna, Austria, ||Center for Anatomy and Cell Biology, Department of Cell Biology and Ultrastructure Research, Medical University of Vienna, Schwarzspanierstrasse 17, A-1090 Vienna, Austria, Institute of Biophysics, Johannes-Kepler-University Linz, Altenbergerstrasse 69, A-4040 Linz, Austria, and the **Departments of Internal Medicine and Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky 40536-0200
Received for publication, September 19, 2005 , and in revised form, February 16, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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0.8% of the HDL added to the media is taken up by the ldlA7-SRBI cells within 1 h, and almost all HDL is finally resecreted. 125I-Labeled low density lipoprotein showed a very similar association, uptake, and resecretion pattern in ldlA7-SRBI cells that do not express any low density lipoprotein receptor. Moreover, we demonstrate that the process of HDL cell association, uptake, and resecretion occurs in three physiologically relevant cell systems, the liver cell line HepG2, the adrenal cell line Y1BS1, and phorbol myristate acetate-differentiated THP-1 cells as a model for macrophages. Finally, we present evidence that HDL retroendocytosis represents one of the pathways for cholesterol efflux. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The scavenger receptor class B, type I (SR-BI), a cell surface glycoprotein that binds HDL, LDL, very low density lipoprotein, modified LDL, and anionic phospholipids (3-6), can mediate the last step in reverse cholesterol transport, namely the delivery of cholesteryl esters from HDL to liver without HDL degradation, termed selective cholesteryl ester uptake (7-9). SR-BI is highly expressed in liver, adrenals, and ovaries with the highest mass of SR-BI protein localized in the liver (7). In addition to cholesterol uptake, SR-BI participates in the internalization of hepatitis C virus particles (10, 11) and lipopolysaccharide (12, 13). Serum amyloid A, a ligand of SR-BI, blocks the selective cholesteryl ester uptake (14). Furthermore, several small chemical inhibitors termed BLTs (block lipid transports) have been described to enhance the apparent affinity of HDL binding to SR-BI but to decrease selective cholesteryl ester uptake (15, 16). Besides its role in cholesterol delivery, SR-BI mediates cholesterol efflux for example from macrophages to HDL particles (17-22). Thus, SR-BI acts as a bidirectional cholesterol transporter (21, 23-25). Several other proteins such as ABCA1, which is defective in Tangier disease, a severe HDL deficiency syndrome characterized by accumulation of cholesterol in tissue macrophages, have also been shown to mediate cholesterol efflux (26-33). SR-BI and ABCA1 were reported to have differential and competing roles in HDL cholesterol efflux in macrophages (34). In nonpolarized cells, SR-BI promotes the reuptake of cholesterol actively secreted by ABCA1, creating a kind of futile cycle of cholesterol transport (34). Thus, the precise role and quantitative importance of SR-BI in cholesterol efflux in vivo still remains enigmatic.
The retroendocytosis of HDL was first postulated to occur in cultured rat aortic smooth muscle cells (35), in a process involving receptor-mediated internalization of HDL into endosomal compartments and its subsequent resecretion. 20 years ago, Schmitz et al. (36) described the uptake and subsequent resecretion of HDL particles by macrophages using electron microscopy. Several years later, DeLamatre et al. (37) provided evidence for a retroendocytic pathway using iodinated HDL particles in a rat liver cell line. DeLamatre et al. (37) showed that HDL particles can transit cells, but the receptor(s) responsible for the uptake and the physiologic consequences remained unknown. Recently, Silver et al. (38) described that selective cholesteryl ester uptake mediated by SR-BI is linked to this holo-HDL uptake process. Silver et al. (38) demonstrated that cholesterol transfer from HDL particles to polarized hepatocytes via SR-BI was accompanied by a transport of HDL particles to the endosomal recycling compartment. This is in contrast to findings of Nieland et al. (39) suggesting that endocytosis is not required for selective lipid uptake mediated by SR-BI. Recently, we followed HDL holoparticle uptake in a CHO cell line overexpressing SR-BI via ultrasensitive fluorescence microscopy (40).
The present study was designed to: 1) analyze HDL holoparticle uptake, its relation to selective uptake, and the role of SR-BI for this uptake process; 2) describe the intracellular trafficking of HDL and LDL during retroendocytosis in more detail; and 3) study the resecretion of HDL and its role for cholesterol efflux.
We present evidence that holo-HDL particle uptake facilitated by SR-BI is followed by resecretion in cell lines derived from tissues of central importance in cholesterol metabolism. Moreover, our data indicate that HDL uptake and resecretion may be involved in the efflux of cellular cholesterol.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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300 cpm/ng. Resecreted HDL was precipitated from tissue culture media using 12% trichloroacetic acid. To specifically precipitate lipoproteins, a precipitation method using sodium phosphotungstate and MgCl2, according to Burstein et al. (41), with a final concentration of 1.8% phosphotungstate and 180 mM MgCl2 was used.
The lipid part of the HDL particles was labeled with the fluorescent phospholipid DiI (Molecular Probes). The labeling procedure included incubation of HDL, diluted in lipoprotein-deficient serum, with DiI overnight at 37 °C followed by ultracentrifugation (42). To obtain double-labeled Alexa647-DiI HDL particles, the DiI-HDL particles were conjugated with Alexa647 as described above. HDL particles were covalently labeled with peroxidase using a peroxidase labeling kit (Roche Applied Science) according to the manufacturer's protocol. Gold-HDL conjugates were prepared by incubation of HDL with gold colloid (20 nm; Sigma) for 10 min at room temperature as described by Handley et al. (43). HDL was labeled with [3H]cholesteryl-oleate (Amersham Biosciences) by the Celite exchange method as described previously (44).
To check the integrity of HDL particles after tissue culture experiments, media obtained from association, displacement, and chase experiments were size-fractionated on 4-20% nondenaturing Tris-HCl polyacrylamide gels (Criterion; Bio-Rad) using a constant current of 30 mA for 4 h (37). The gels were analyzed by autoradiography using the Bio-Rad Personal Molecular Imager FX. Size analysis was performed using the high molecular weight calibration kit (Amersham Biosciences).
Cell Lines and Tissue Culture—Chinese hamster ovary cells (CHOKI) and CHO cells lacking LDL receptor activity (ldlA7 cells) and expressing high levels of recombinant SR-BI (ldlA7-SRBI) were used (22). For all experiments cells were plated in 6-well plates at a cell density of 300,000 cells/well in a 1:1 mixture of Dulbecco's minimal essential medium and Ham's F-12 medium (medium A) with 100 units/ml penicillin and 100 mg/ml streptomycin sulfate, supplemented with 5% (v/v) fetal calf serum (FCS) (Invitrogen). On day 2, cells were switched to medium A containing 5% (v/v) human lipoprotein-deficient serum. On day 3, cells were washed with PBS and refed with medium A supplemented with 2 mg/ml fatty acid-free bovine serum albumin (faf-BSA; Sigma). For inhibitor treatment cells were incubated with 150 µM BLT-4 (Chembridge Corp., San Diego), 500 µM glyburide (Biomol, Hamburg, Germany), or 50 µM chloroquine (Sigma) for 15 min before adding 125I-HDL. ldlA7-SRBI-EGFP cells were grown as described by Eckhardt et al. (45). HepG2 cells were grown in Dulbecco's minimal essential medium with 10% FCS and 2 mM glutamine. Y1BS1 cells, kindly provided by Dr. B. P. Schimmer (University of Toronto, Canada), were grown in medium A supplemented with 2% FCS and 15% horse serum (46). Cells were seeded in 6-well plates at a density of 1 million cells/well and further treated as described for the CHO cell lines. The human THP-1 monocyte leukemia cell line was cultured in RPMI 1640 medium containing 10% FCS and 2 mM glutamine. For induction of cell differentiation, 2 x 106 cells per well were seeded in 6-well plates and treated with 160 nM phorbol myristate acetate (PMA, stock 500 µg/ml in Me2SO; Calbiochem) for 4 days (47). After differentiation, nonattached cells were removed by aspiration, and adherent cells were washed with PBS and used in subsequent experiments.
Association Experiments—On the day of the experiment, cells were incubated with 125I-HDL or 125I-LDL at the indicated concentrations in duplicate for 1 h at 37 °C. To estimate the unspecific binding of lipoproteins to the cell surface, cells were incubated with iodinated lipoproteins in the presence of a 40-fold excess of unlabeled lipoprotein. Finally, media were recovered, and the cells were washed twice with 50 mM Tris-HCl, pH 7.4, containing 0.9% NaCl and 2 mg/ml BSA (buffer A) and twice with buffer A without BSA. Cells were lysed using 0.1 M NaOH, and radioactivity associated with the lysate and the media was analyzed using a Cobra II gamma counter (PerkinElmer Life Sciences). Cell protein was estimated using the Bradford reagent (Bio-Rad). Specific cell association was calculated in ng of lipoprotein/mg of cell protein by subtracting unspecific binding.
Displacement Experiments—To study HDL particle uptake, cells were prepared and incubated as described for association studies. After incubation with iodinated lipoproteins, cells were washed twice with PBS containing 2 mg/ml faf-BSA and twice with PBS without faf-BSA. Cells were then incubated for another 2 h at 0°C in medium A with 2 mg/ml faf-BSA, 10 mM Hepes, and a 100-fold excess of unlabeled lipoprotein to replace all surface-bound 125I-labeled lipoproteins. Finally, the media were collected, and the cells were washed with buffer A and harvested. Specific lipoprotein uptake was calculated in ng of lipoprotein/mg of cell protein by subtraction of unspecific binding.
Chase Experiments—To study HDL resecretion, cells were washed after the displacement procedure twice with PBS containing 2 mg/ml faf-BSA and twice with PBS without faf-BSA. Cells were incubated for the indicated time in medium A containing 2 mg/ml faf-BSA and a 20-fold excess of unlabeled lipoprotein at 37 °C. All media were recovered, and cells were washed with buffer A and lysed. The media and cell lysate were analyzed for their content of radiolabel.
Degradation Experiments—The media of association, displacement, or chase experiments were recovered, and proteins in the media were precipitated with trichloroacetic acid, and the supernatant was analyzed for degraded lipoproteins (48).
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Cholesterol Transfer Studies—On day 3, cells were incubated with the indicated concentrations of HDL labeled with [3H]cholesteryl-oleate for 1 or 5 h with or without the addition of inhibitors (3). The cells were then harvested, and lipids were extracted and analyzed by TLC (48) to determine cellular cholesteryl ester uptake and subsequent hydrolysis of the cholesteryl esters to free cholesterol.
Flow Cytometry—Cells were incubated either with 10 µg/ml Alexa647- or Alexa647-/DiI-HDL for 1 h, and an association, displacement, or chase experiment was performed. After the experiment media were discarded, and cells were washed with buffer A and harvested by trypsinization for 1 min. 1% paraformaldehyde was added to stop the reaction and preserve the cells, and cells were further incubated for 15 min at 4 °C and then centrifuged at 400 x g for 3 min at 4 °C. The supernatant was removed; cells were resuspended in 200 µl of PBS and kept at 4 °C. For flow cytometry measurement, 100 µl of cell suspension was diluted in 500 µl of PBS buffer and analyzed using a FACSCalibur (BD Biosciences). Two laser lines (emission 488 and 635 nm, respectively) were used to detect DiI (at 585 ± 21 nm in FL-2) and Alexa647 (at >670 nm in FL-4).
Electron Microscopy—After incubation for periods of 15 and 30 min and 1-3 h in media A with faf-BSA containing HDL colloidal gold conjugates (43) or peroxidase-labeled HDL, cells were prepared for electron microscopic examination. Fixation was performed in 2.5% glutaraldehyde, pH 7.4. After an overnight rinse in PBS, cells were postfixed in 1% OsO4, dehydrated in a graded series of ethanol, and embedded in Epon. In the case of peroxidase-HDL studies, prior to postfixation, peroxidase activities were visualized by means of the diaminobenzidine reaction (51). Ultrathin sections were analyzed in a transmission electron microscope. For controls, HDL was omitted from the incubation media; cells were incubated in media either containing the same amount of peroxidase or colloidal gold alone.
Ultrasensitive Fluorescence Microscopy—A modified epifluorescence microscope (Axiovert 200 TV; Zeiss, Oberkochen, Germany) was used for imaging fluorescence-labeled lipoproteins as described previously (40). Cy5 was excited with 647 nm light from a krypton laser (Innova 300; Coherent, Santa Clara, CA). The 528 nm line of an argon laser (2020 series; Spectra Physics, Mountain View, CA) was used for Cy3 excitation. Emitted fluorescence was collected using appropriate filter combinations and an oil 100x immersion objective (Plan-Apochromat, NA = 1.4; Zeiss). For two-color measurements, both images were obtained simultaneously by a liquid nitrogen-cooled slow-scan CCD camera (Micro Max 1300-PB; Roper Scientific, Trenton, NJ). Images were analyzed using algorithms implemented in MATLAB (The Math-Works, Natick, MA). The position of isolated fluorescence peaks corresponding to individual particles or endocytosed objects was automatically determined on each image by fitting with a Gaussian intensity profile. From subsequent images single particle trajectories were reconstructed, yielding the mean square displacements (MSD) as a function of the time lag. Diffusion was analyzed according to MSD = 4 D time lag, with D the lateral diffusion constant, directed transport according to 
time lag, with v the average velocity.
Statistics—The results are expressed as means ± S.D. Mean values were compared using analysis of variance followed by Newman-Keuls test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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HDL Resecretion in ldlA7-SRBI Cells—To characterize the details of this pathway, we posed the question whether HDL particles internalized by the cells can transit the cell back to the media. Again cells were incubated with 10 µg of 125I-HDL/ml for 1 h at 37°C,andthe 125I-HDL associated to the cell surface was displaced by a 100-fold excess of unlabeled HDL at 0 °C. The cells were then warmed to 37 °C and chased with a 20-fold excess of unlabeled HDL for the indicated times (Fig. 1C). A time-dependent resecretion of HDL particles to the culture media was seen (Fig. 1C, open circles), which reached a plateau at about 3 h. Conversely, the amount of HDL particles in the cells decreased in a commensurate way (Fig. 1C, closed circles). 85% of the radioactivity recovered in the media was trichloroacetic acid-precipitable, indicating that the majority of the radioactivity was within intact lipoprotein particles. To calculate the amount of radioactivity derived from detached cells during longer incubation points, media were centrifuged at 500 x g. About 7% of the radioactivity in the media originated from detached cells. In further experiments a chase period of 30 min was chosen; at this time point resecretion of HDL did not reach its plateau. For the chase, a 20-fold excess of HDL was sufficient, as similar results were obtained using a 20-, 40-, or 100-fold excess of HDL (data not shown). During the 30-min chase period, 43% of the HDL particles internalized by the ldlA7-SRBI cells were resecreted (Fig. 2A). HDL resecretion was still occurring without the addition of an excess of unlabeled HDL but to a lesser extent (20% of the amount resecreted during the chase period with an addition of a 20-fold excess of HDL).
To exclude release of radioactivity not related to intracellular trafficking, we blocked intracellular movement during the chase by using a temperature shift to 0 °C. Very little radioactivity (only 10% of the amount resecreted during chase at 37 °C) was found in the media of ldlA7-SRBI cells incubated during the 30-min chase at 0 °C with a 20-fold excess of unlabeled HDL (data not shown).
To confirm the results obtained with radiolabeled HDL, we applied HDL fluorescently labeled with Alexa647 to measure the amount of HDL protein remaining within ldlA7-SRBI cells after association, uptake, and chase (Fig. 2B). During displacement and chase, cellular Alexa-HDL decreased to a similar extent as did 125I-HDL in ldlA7-SRBI cells.
To demonstrate that HDL holoparticle uptake observed in ldlA7-SRBI cells is mediated via SR-BI, we used the two parental cell lines, ldlA7 and CHOKI, both of which express SR-BI at a much lower level (22). Using a 30-min chase period all parts of HDL retroendocytosis could be seen in both cell lines (Fig. 2A), although to a much lower extent than in ldlA7-SRBI cells.
To prove the integrity of the 125I-HDL particles resecreted, we used nondenaturing gel electrophoresis (Fig. 2C). Media containing 125I-HDL after association, displacement, and chase experiments were size-fractionated on a 4-20% gradient gel for 4 h. The electrophoretic mobility of the 125I-labeled proteins corresponded to HDL indicating that the radioactivity released after the chase is contained in intact HDL particles. HDL particle size decreased after association probably because of selective cholesteryl ester uptake, whereas the particles analyzed after displacement showed an electrophoretic mobility similar to control HDL. Interestingly, HDL particle size after the chase became more heterogeneous indicating a change in HDL composition.
Taken together these results clearly demonstrate that HDL is internalized via an SR-BI-dependent mechanism and that this internalized HDL can be resecreted by the cell. Thus, SR-BI is a receptor facilitating uptake of HDL particles.
HDL Uptake and Resecretion Occurs in Physiologically Relevant Cell Lines—To test if HDL retroendocytosis exists in physiologically relevant cell lines, we first used HepG2 cells as a model for human hepatocytes. HepG2 cells showed all parts of the uptake and exocytosis process but to a lesser extent than the ldlA7-SRBI cells (Fig. 3, left, compare with Fig. 2A, left). HDL cell association was about 86 ng/mg cell protein after incubation with 10 µg of HDL/ml for 1 h in HepG2 cells compared with 190 ng/mg cell protein in ldlA7-SRBI. About 20% of cell-associated HDL was displaced within 2 h in HepG2 cells. Therefore, 80% of the associated radioactivity was HDL taken up by the cells, which is more than seen in the SR-BI overexpressing cell line. After chase about 77% of the HDL taken up by HepG2 cells was resecreted within 30 min.
Next, we used murine Y1BS1 cells derived from an adrenocortical tumor (46) as a model for the steroid hormone-producing cells of the adrenal cortex; Y1BS1 cells express more SR-BI protein than HepG2 cells and showed a higher association but lower resecretion of holo-HDL particles (Fig. 3, middle). In Y1BS1 cells 42% of the HDL associated was displaced, leaving 58% of HDL particles within the cells. About 47% of the HDL taken up was resecreted to the media.
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30% of the HDL was re-secreted. Thus, all three cell lines showed all parts of the HDL retroendocytosis pathway with HepG2 cells having the highest rate of HDL uptake and resecretion.
LDL Association, Uptake, and Resecretion Exists in ldlA7-SRBI Cells— It is well known that several ligands bind to SR-BI. Previously, we reported that LDL-derived cholesterol is taken up by ldlA7-SRBI cells and transported to the endoplasmic reticulum as effectively as HDL-derived cholesterol (22). To check whether SR-BI also mediates LDL retroendocytosis, we followed the uptake of LDL labeled covalently with Cy5 by using ultrasensitive microscopy (Fig. 4). There was a strong staining of Cy5 on certain areas of the cell surface (Fig. 4A). Individual LDL particles were observed inside ldlA7-SRBI cells that overexpress SR-BI but completely lack LDL receptor-mediated endocytosis (see Fig. 4, blue line, and supplemental video). Accordingly, there was almost no degradation of LDL in this cell line; only 10 ng of LDL/mg of cell protein/h at a concentration of 10 µg of LDL/ml was degraded. The movement of endocytosed LDL molecules was characterized by long periods of directed movement with v = 0.38 ± 0.3 µm/s indicating active transport. The calculated diffusion constant for trapped particles at the cell surface was D = 0.0118 ± 0.022 µm2/s.
To provide biochemical evidence for LDL retroendocytosis, an association, uptake, and chase study was performed using 10 µg of 125I-LDL/ml (Fig. 4B). Association of LDL in ldlA7-SRBI cells was higher than HDL association (compare Figs. 4B and 2A). Similar to HDL, about two-thirds of the LDL specifically associated with the cell were actually internalized. This indicates that SR-BI facilitates internalization of LDL like that of HDL. Furthermore, 40% of the LDL internalized was readily recovered in the media after 30 min of chase indicating retroendocytosis of the LDL particle. This uptake and resecretion of LDL was also observed in the ldlA7 cells, expressing SR-BI at lower levels but lacking any functional LDL receptors (Fig. 4B). Thus, not only HDL but also LDL uptake is mediated by SR-BI.
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In addition, both detection methods clearly showed intracellular compartments reactive for HDL. The number of stained organelles increased with increasing incubation periods. At all periods of treatment, labeled endocytic vesicles and organelles were apparent in regions located close to the cell surfaces and were concentrated in perinuclear areas neighboring the Golgi apparatus (Fig. 7). Many of the compartments reactive for HDL could be classified as multivesiculated or multilamellated organelles.
HDL Holoparticle Uptake Proceeds while Selective Cholesteryl Ester Uptake Is Blocked—To dissect the holo-HDL particle uptake and resecretion pathway described above from the selective cholesteryl ester uptake pathway, we employed several substances referred to as BLTs (15) known to modulate binding of HDL to SR-BI and selective cholesteryl ester uptake.
First, we used glyburide and BLT-4, both described to enhance the affinity of HDL for SR-BI but to decrease selective cholesteryl ester uptake (15, 16). As expected both glyburide and BLT-4 increased HDL cell association significantly in ldlA7-SRBI cells (2.6- and 2.1-fold, respectively) (Fig. 8), although selective cholesteryl ester uptake was decreased by 90 or 50%, respectively (data not shown). Interestingly, after displacement of cell surface-bound HDL, more HDL particles were found in the cell treated with one or the other substance; finally, about 40% of the HDL taken up by the cells was excreted during the 30-min chase period. This is in the same range as observed in untreated ldlA7-SRBI cells. Furthermore, HepG2 cells pretreated with glyburide, like ldlA7-SRBI cells, showed a higher degree of HDL cell association (2.5-fold), but uptake and resecretion of HDL particles were within the same range as in untreated HepG2 cells (data not shown). Thus, both glyburide- and BLT-4-treated cells show all parts of the HDL endocytosis and resecretion pathway, suggesting that selective cholesteryl ester uptake is not required for HDL retroendocytosis. Furthermore, we used chloroquine, a substance that raises intracellular pH leading to an accumulation of HDL in the cell (40). In our study the influence of chloroquine on selective cholesteryl ester uptake differed from that of the BLTs, as chloroquine did not block selective cholesteryl ester uptake and its subsequent hydrolysis to cholesterol (Fig. 9, A and B) (53). However, chloroquine inhibits the transport of the cholesterol moiety to the endoplasmic reticulum and its subsequent reesterification (22). After chloroquine treatment ldlA7-SRBI cells showed higher HDL association and uptake but not resecretion compared with control cells (Fig. 8). Taken together these experiments show that retroendocytosis of HDL occurs in the presence of selective cholesteryl ester uptake but is not altered when selective cholesteryl ester uptake is substantially reduced. Thus, these two pathways cannot be tightly linked in ldlA7-SRBI cells. Furthermore, chloroquine did not significantly inhibit efflux of cholesterol to HDL as shown using trace labeling with [3H]cholesterol or with endogenously synthesized cholesterol derived from [14C]acetate (Fig. 9, C and D, respectively).
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HDL Resecretion Is Linked to Cholesterol Efflux—As HDL retroendocytosis and selective cholesteryl ester uptake do not appear to be tightly linked, the role and function of HDL retroendocytosis in cellular cholesterol homeostasis remain unclear. Therefore, we asked if HDL retroendocytosis is able to facilitate cholesterol efflux. To answer this, we trace-labeled the intracellular cholesterol pool of differentiated THP-1 cells or ldlA7-SRBI cells with [3H]cholesterol and analyzed the media collected after displacement and chase for their cholesterol content.
The cholesterol pool of differentiated THP-1 macrophages or ldlA7-SRBI cells was labeled by the addition of 2 µCi of [3H]cholesterol per well overnight. The next day, cells were incubated with 10 µg of 125I-HDL/ml for 1 h followed by displacement of bound HDL with a 100-fold excess of unlabeled HDL and a chase with a 20-fold excess of unlabeled HDL for 30 min. Then the media were collected and analyzed for their 3H and 125I content (Fig. 11). After displacement, a considerable amount of [3H]cholesterol (5.4 ± 2 pmol/mg cell protein for ldlA7-SRBI cells and 1.2 ± 0,5 pmol/mg cell protein for THP-1 cells; Fig. 11) was found in the media. The [3H]cholesterol was contained within lipoprotein particles removed from the cell surface after the initial 1-h incubation as demonstrated by precipitation of both labels with trichloroacetic acid. These displaced particles represent HDL that was bound to the cell surface after 1 h of incubation at 37 °C. These HDL particles obtained their [3H]cholesterol through cell surface efflux as well as through retroendocytosis.
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To assess if the [3H]cholesterol removed from the cells is indeed contained in HDL particles, we applied several methods to demonstrate the integrity of the particle. First, the electrophoretic mobility of the displaced and resecreted HDL particles was in the same range as control HDL (not shown; results were similar to those obtained with 125I-HDL only as shown in Fig. 2). Second, we applied the phosphotungstate/MgCl2 precipitation method to precipitate all lipoproteins (41). 80% of both radiolabels was precipitated, indicating a loading of HDL particles with [3H]cholesterol derived from cells.
To check if cholesterol efflux in the chase experiment is not dependent solely on the presence of an excess of unlabeled HDL in the media, we followed the HDL resecretion without the addition of a 20-fold excess of unlabeled HDL for 30 min. In this experiment [3H]cholesterol efflux was seen, although to a lesser extent (about 10% of the [3H]cholesterol amount seen in Fig. 11, upper middle panel, remained). 125I-HDL release was also decreased (to about 20% of the amount resecreted during the chase period with an addition of a 20-fold excess). The ratio of cholesterol to HDL protein was 18 in the HDL displaced from the cell surface and increased to 27 after resecretion. The disproportional decrease of HDL protein and cholesterol during resecretion indicates that about 50% of the cholesterol efflux seen in our experiment using ldlA7-SRBI cells derives from retroendocytosis.
Next, we tested if cholesterol efflux still occurs in the absence of endocytosis. Endocytosis was blocked by keeping the cells at 0 °C during incubation with 10 µg of 125I-HDL/ml for 1 h. Then cell surface-bound HDL was displaced; the cells were warmed to 37 °C, and a 20-fold excess of unlabeled HDL was added for 30 min (Fig. 11). Cholesterol efflux was 80% lower compared with the retroendocytosis experiment performed at 37 °C. Prolonged incubation of up to 2 h during the chase led to an increase, after a delay, in cholesterol efflux attributed to the restarting endocytosis (data not shown). These data indicate that cholesterol efflux depends on both endocytosis and exocytosis of HDL.
To assess if ABCA1, a protein known to be involved in cell surface cholesterol efflux, plays a role in the cholesterol efflux described here, Tangier fibroblast cell lines derived from two independent patients (kindly donated by Dr. Calandra, University Modena, Italy) (54, 55) were used. 125I-HDL retroendocytosis was in the same range in the two Tangier fibroblast cell lines as in control fibroblasts (data not shown). The ratio of cholesterol to HDL increased from 23 during displacement to about 45 during the chase, indicating that this cholesterol efflux pathway described here is not mediated by ABCA1.
Taken together our data indicate that during resecretion HDL is loaded with intracellular cholesterol, suggesting that HDL retroendocytosis may be one general mechanism contributing to cholesterol efflux from cells to HDL particles.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Experiments presented here show that holo-HDL particle uptake can be mediated by SR-BI (Fig. 1). This observation is in agreement with previous data showing that in hepatocyte couplets SR-BI and HDL are taken up together and that HDL is transported to the canalicular side (38). Linked endocytosis of HDL and SR-BI, similar to the data presented in Fig. 6, was reported in CHO cells by Eckhardt et al. (45). In our experiments about 0.8% of the HDL added to the media was taken up by ldlA7-SRBI cells after 1 h of incubation. Subsequently, HDL particles were resecreted into the media as demonstrated by a decrease of 125I-HDL in the cell lysate and a concomitant increase in the media (Fig. 1C). As there is hardly any degradation of HDL in the ldlA7-SRBI cells, almost all HDL particles taken up via SR-BI are resecreted after 7 h. The calculated initial rate for resecretion in SR-BI overexpressing cells is 1.28 ng of HDL/mg of cell protein and per minute. It is important to mention that the holo-HDL particle uptake and resecretion process described here is different from apoE recycling reported to occur in hepatocytes and macrophages where a part of the apoE associated with HDL escapes degradation (56, 57). HDL used in our study did not contain any apoE as demonstrated by silver staining. Moreover, we demonstrated previously that HDL2 as well as acetylated and methylated HDL, which do not bind to the apolipoprotein B/E receptor family, showed a similar uptake and resecretion pattern (40).
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HDL holoparticle uptake and resecretion is not limited to the ldlA7-SRBI cell line; on the contrary, it can be seen in HepG2 cells, a macrophage cell line, and an adrenal cell line (Fig. 3). The relevance of this holoparticle uptake in tissues with different roles in cholesterol homeostasis needs to be investigated.
Furthermore, our data shed light on the fate and the intracellular trafficking of holo-HDL particles during the uptake process. First, we demonstrated that SR-BI directly mediates HDL holoparticle uptake; fluorescently labeled HDL and SR-BI visualized as enhanced green fluorescent fusion protein were endocytosed together showing a similar uptake path and velocity. Second, by using electron microscopy we demonstrated that HDL binds to the cell surface at plane and microvillar areas; then HDL is taken up by endosomal vesicles and transported to the perinuclear area, where it is often found in multivesiculated or multilamellated vesicles. This is in line with the data of Schmitz et al. (36) who showed that HDL is taken up by macrophages and transported into nonlysosomal endosome compartments and that HDL-containing vesicles are located in close contact with lipid droplets. Similarly, DeLamatre et al. (37) reported that HDL is taken up into endosomes negative for acid phosphatase in hepatocytes. These earlier data are also in agreement with our observation that HDL uptake and resecretion were still functional when lysosomal cholesterol transport was blocked by chloroquine (Figs. 8 and 9).
It is controversial whether a linkage of HDL particle uptake and selective lipid uptake exists. The data in this study leads to the conclusion that retroendocytosis is not tightly linked to selective uptake; inhibitors that block selective cholesteryl ester uptake did not inhibit HDL endocytosis and resecretion. This is in line with Nieland et al. (39) who showed that HDL particle uptake and selective lipid uptake, followed using DiI, are not linked. Another study reported that HDL particles were taken up by HepG2 cells but that their cholesterol moiety arrived earlier at the apical membrane and the biliary canaliculi than the HDL protein (61); even when HDL uptake was blocked by ATP depletion, the cholesterol uptake was not impaired.
Although evidence accumulates that selective lipid uptake and holoparticle uptake are separate pathways, a role for retroendocytosis in cholesterol homeostasis has not been described so far. In this study we provide evidence from chase experiments that HDL retroendocytosis is one pathway enabling the cell to export excess intracellular cholesterol. This is strongly confirmed by data showing a significant drop of cholesterol efflux by 80% when retroendocytosis was blocked in ldlA7-SRBI cells using incubation at 0 °C. Furthermore, we present evidence that the retroendocytosis-mediated cholesterol efflux described here clearly differs from cell surface cholesterol efflux as follows. 1) Cholesterol efflux still occurred in the absence of unlabeled HDL required as an acceptor for cell surface cholesterol efflux during the chase. This excludes that warming of the cells after displacement only triggers cell surface efflux when excess HDL is present. Moreover, the temperature dependence of various modes of cell surface cholesterol efflux (62, 63) was taken into account by performing all cholesterol efflux experiments at 37 °C. 2) Cell surface efflux mediated by SR-BI or ABCA1 was ruled out by using glyburide. In addition to inhibiting selective uptake, glyburide was reported to block cell surface efflux of cholesterol to HDL via SR-BI and ABCA1 (16, 64). In our experiments, however, a considerable amount of cholesterol efflux during the chase still occurred in the presence of glyburide (Fig. 11). In the displacement media we did measure a lower amount of [3H]cholesterol after glyburide treatment, which suggests that HDL in the media obtained cholesterol in part by cell surface efflux during the preceding 1-h association period at 37 °C. Thus, the cholesterol efflux seen in the chase period is not because of cell surface efflux either mediated by SR-B1 or ABCA1. 3) Finally cholesterol efflux mediated by retroendocytosis was still present in fibroblast cells derived from patients with Tangier disease that are defective in ABCA1. This again confirms that the cholesterol efflux seen in our study is independent of ABCA1.
From these experiments we conclude that the cholesterol removal from ldlA7-SRBI cells seen in our study depends on HDL exocytosis and that the HDL particles resecreted are enriched in cholesterol tracers derived from the intracellular cholesterol pool. This is supported by the findings of Alam et al. (65) that show that apoE-free HDL3 is converted to a larger apoE-containing particle of HDL2 size and density during chase through cholesterol-laden macrophages, an observation that is consistent with an enrichment of HDL3 with cholesterol. Rinninger et al. (66) showed an increase in HDL uptake in cholesterol-loaded HepG2 cells and a decrease of their cholesterol content by 13% after 6 h of exposure to HDL. The internalization of HDL in these cells could not be mediated by apolipoprotein B/E receptor as it occurred in the presence of heparin that prevents apolipoprotein B/E receptor-mediated lipoprotein uptake. Considering the data from these studies, cholesterol efflux can occur through HDL uptake and resecretion as described here for ldlA7-SRBI cells.
We assume that this novel pathway could be a fine-tuning of cholesterol transfer reflecting the intracellular cholesterol status. It also could be an important pathway in cases where other cholesterol transfer mechanisms are blocked, e.g. in rare diseases of cholesterol metabolism.
To determine whether a link exists between selective cholesteryl ester uptake and cholesterol efflux mediated by retroendocytosis, we used glyburide, which increases HDL cell association but conversely decreases selective cholesteryl ester uptake. In this setting, enrichment of cholesterol in HDL particles resecreted was unaltered, indicating that there is no tight connection between the two pathways. Considering our data, one can further speculate that SR-BI seems not to be required for cholesterol efflux via retroendocytosis, although SR-BI seems to be necessary for mediating holo-HDL uptake. Interestingly, another study using an SR-BI-neutralizing antibody (34) reported that cholesterol efflux to HDL particles was not inhibited, although selective cholesteryl ester uptake as well as HDL cell association was blocked demonstrating that cholesterol efflux can be provided by many different pathways.
In summary, our studies demonstrate that retroendocytosis occurs with at least two different lipoproteins, HDL and LDL, and that the uptake in both cases can be mediated by SR-BI. Furthermore, our study implies that resecretion of the HDL particle is accompanied by an efflux of cellular cholesterol.
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1 Supported by National Institutes of Health Grant HL63763.
2 Supported by Austrian Science Foundation Grant P15053.
3 To whom correspondence should be addressed: Center for Physiology and Pathophysiology, Dept. of Medical Chemistry, Medical University of Vienna, Währingerstrasse 10, A-1090 Vienna, Austria. Tel.: 43-1-4277-60823; Fax: 43-1-4277-60881; E-mail: Herbert.Stangl{at}meduniwien.ac.at.
4 The abbreviations used are: HDL, high density lipoprotein; LDL, low density lipoprotein; PBS, phosphate buffered saline; MSD, mean square displacement; faf-BSA, fatty acid-free bovine serum albumin; FACS, fluorescence activated cell sorter; cpm, counts per min; PMA, phorbol myristate acetate; SR-BI, Scavenger receptor class B, type I; CHO, Chinese hamster ovary; FCS fetal calf serum; BLT, block lipid transport, DiI, 1,1'-dioctodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate.
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, 10 •, 50 
, and 100 µg/ml 
) at 37 °C for the indicated times. Subsequently, cells were washed with buffer A and lysed as described under "Experimental Procedures." Values are expressed as specific association calculated by subtracting the unspecific binding determined using a 40-fold excess of HDL from the total cell association given in ng of HDL/mg of cell protein. The inset shows a magnification of the two lowest doses. B, time course of holo-125I-HDL particle uptake. ldlA7-SRBI cells were incubated with 10 µg of HDL/ml at 37 °C for the indicated times. Cells were then washed and incubated with a 100-fold excess of unlabeled HDL at 0 °C for 2 h, lysed, and analyzed as described under "Experimental Procedures." Values are presented as specific 125I-HDL uptake in ng per mg of cell protein. C, time-dependent exocytosis of holo-HDL particles after chase with a 20-fold excess of unlabeled HDL at 37 °C. ldlA7-SRBI cells were first incubated for 1 h with 10 µgof 125I-HDL/ml, then with a 100-fold excess of HDL at 0 °C for 2 h, and finally warmed to 37 °C and chased with a 20-fold excess of HDL for different times. Media (
) and cells (•) were harvested and analyzed as described. Note the large increase in 125I-HDL with time in the media (
