The inhibition of endocytosis affects HDL-lipid uptake mediated by the human scavenger receptor class B type I

Abstract

The scavenger receptor SR-BI plays an important role in the hepatic clearance of HDL cholesterol and other lipids, driving reverse cholesterol transport and contributing to protection against atherosclerosis in mouse models. We characterized the role of endocytosis in lipid uptake from HDL, mediated by the human SR-BI, using a variety of approaches to inhibit endocytosis, including hypertonic shock, potassium or energy depletion and disassembly of the actin cytoskeleton. Our studies revealed that unlike mouse SR-BI, human SR-BI-mediated HDL-lipid uptake was reduced by inhibition of endocytosis. This was not dependent on the cytoplasmic C-terminus of SR-BI. Monitoring the uptake of both the protein and lipid components of HDL revealed that although overall lipid uptake was decreased, the degree of selective lipid uptake was increased. These data suggest that that endocytosis is a dynamic regulator of SR-BI's selective lipid uptake activity.

• Endocytosis
• HDL
• lipoprotein
• selective uptake
• SR-BI

Introduction

HDL plays an important role in protection against atherosclerosis in part by mediating reverse cholesterol transport from macrophage foam cells in atherosclerotic plaque, to the liver [1]. Studies in gene-targeted and transgenic mice revealed that the multi-ligand receptor SR-BI (scavenger receptor, class B type I) plays an important role in HDL metabolism, control of plasma HDL-cholesterol levels and reverse cholesterol transport ([1] and references therein). Elimination of SR-BI expression increases and hepatic overexpression reduces atherosclerosis in mouse models ([1] and references therein). Mice deficient in both SR-BI and apoE develop severe occlusive coronary artery atherosclerosis and myocardial infarction and exhibit cardiac functional and conductance abnormalities prior to early death by 8 weeks of age [2]. Elimination of SR-BI in bone marrow derived cells, including macrophages, also results in increased atherosclerosis but without altering plasma lipoprotein cholesterol levels [3] (also [1] and references therein). Thus, both hepatic and macrophage SR-BI protect against atherosclerosis, and reverse cholesterol transport may be only one of multiple pathways involved.

SR-BI is heavily glycosylated, fatty acylated and is localized to lipid rafts and/or caveolae in many but not all cell types [4–9]. It undergoes internalization and recycling back to the cell surface. In polarized cells, cholesterol depletion induces SR-BI redistribution from the basal to apical membrane surface in a protein kinase A dependent manner [9]. In differentiating 3T3-L1 adipocytes and HepG2 human hepatoma cells, recruitment of SR-BI to the cell surface from internal sites is induced by insulin and/or serum and is dependent on the phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B) signaling pathway [10], [11]. In a variety of cell types, HDL is internalized and undergoes resecretion or retro-endocytosis back to the cell surface, in an SR-BI-dependent manner [12–14]. HDL-dependent cholesterol efflux mediated by murine SR-BI appears to involve HDL internalization and retroendocytosis [15]. On the other hand, murine SR-BI-mediated lipid uptake from either HDL or LDL is not affected by inhibition of endocytosis, and can occur in a purified reconstituted system, containing only SR-BI in proteoliposomes devoid of other cellular facotors [16–18]. This suggests that endocytosis is not required for murine SR-BI mediated lipid uptake from either HDL or LDL. The role of endocytosis in HDL lipid uptake mediated by the human (h) SR-BI, (also called CLA-1, or CD36 and LIMP-2 related antigen 1) is not known. Analysis of the role of endocytosis in human SR-BI-mediated lipid uptake from lipoproteins will provide insight into whether the lipid uptake pathway is conserved between mouse and human SR-BI and may help to identify functionally important regions of the molecule.

In this study, we examine the role of endocytosis in human SR-BI-mediated HDL-lipid uptake in transfected Chinese hamster ovary derived cells. We demonstrate that human SR-BI-mediated HDL-lipid uptake is sensitive to inhibition of endocytosis by hypertonic shock or depletion of potassium or energy. This suggests that endocytosis is normally required for HDL-lipid uptake mediated by human SR-BI. Despite the decrease in overall HDL lipid uptake when endocytosis was inhibited, there was an increase in the degree of selective lipid uptake, measured as the ratio of HDL lipid/HDL protein taken up. We also demonstrate that the endocytosis-dependent HDL lipid uptake was not affected by mutation or deletion of the cytoplasmic C-terminus of human SR-BI. These findings suggest that endocytosis is a dynamic regulator of SR-BI's selective lipid uptake activity.

Materials and methods

Materials

Plasmids peGFP-C1 and pDsRed-N1 were from BD Clontech (Palo Alto, CA, USA). The Quickchange site-directed mutagenesis kit was from Stratagene (Palo Alto, CA, USA). Plasmid, pmRFP, containing monomeric red fluorescent protein (mRFP) [19] was from Dr R. Tsien (University of California, San Diego, USA). The eGFP-clathrin light chain expression plasmid [20] was from Dr J. H. Keen (Thomas Jefferson University, Philadelphia, USA). Anti-SR-BI⁴⁹⁵ antibody, and the ldlA7 and ldlA[mSR-BI] cell lines were from Dr M. Krieger (Massachusetts Institute of Technology, Cambridge, USA). Rabbit anti-SR-BI-C-terminus polyclonal antibody NB 101–400 was from Novus Biologicals (Littleton, CO, USA). The mouse anti-GFP monoclonal antibody was from Stressgen Bioreagents Inc. (Cedarlane Laboratories Inc, Hornby ON, Canada). Polyclonal rat saposin A antibody was from CR Morales (McGill University, Montreal Canada) and cathepsin A antibody was from Y Suzuki (Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan). Alexa 596-transferrin, 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI), TA-cloning vector and Lipofectin transfection reagent were from Invitrogen Canada Inc (Burlington, ON, Canada). All other reagents were from sources previously described [23].

Methods

Generation of expression plasmids

For peGFP-hSR-BI, (human SR-BI tagged on its N-terminus with enhanced green fluorescent protein (eGFP)), PCR was used to introduce Sal1 and Kpn1 restriction sites on either end of the human SR-BI cDNA, which was inserted into the TA cloning vector, released with Sal1 and Kpn1 and ligated to peGFP-C1 similarly digested. Sequence alterations arising from PCR [21] were corrected by site-directed mutagenesis. Plasmid pmRFP-hSR-BI (hSR-BI tagged on its N-terminus with mRFP) was generated from peGFP-hSR-BI by replacing the coding sequence of eGFP with that for mRFP. Plasmid phSR-BI (encoding untagged hSR-BI) was generated by inserting hSR-BI cDNA with its stop codon into pDsRed-N1. The mutant plasmids peGFP-hSR-BI Y⁴⁷¹D, peGFP-hSR-BI Y⁴⁷¹F and peGFP-hSR-BIΔC (containing a stop codon after the codon encoding I⁴⁶⁴) were generated from peGFP-hSR-BI by site-directed mutagenesis. All plasmids were sequenced (MOBIX core facility, McMaster University).

Cell lines and culture conditions

LdlA7 Chinese hamster ovary (CHO) mutant cells, lacking functional LDL receptor, and ldlA7 cells stably overexpressing mouse [22] or human SR-BI (tagged or untagged) were cultured as described previously [4], [23]. LdlA7 cells were Lipofectin-transfected with appropriate plasmids and exposed to G418 (0.5 mg/ml) selection. EGFP-hSR-BI- or mRFP-hSR-BI–transfected cells were subjected to fluorescence activated cell sorting (FACS) for expressing cells. Cells transfected with untagged human SR-BI, as well as ldlA[mSR-BI] cells were incubated with DiI-HDL (5 µg/ml) for 2 h at 37°C and subjected to FACS to enrich for cells expressing functional SR-BI. In each case, cells with the highest levels of fluorescence were collected, pooled and expanded.

HDL preparation and labeling

HDL was prepared from human plasma by KBr density gradient ultracentrifugation and was labeled with DiI according to standard protocols [22], [24]. DiI/alexa 488-double labeled HDL was prepared as described previously [23]. Newborn calf lipoprotein deficient serum (NCLPDS) was prepared by KBr density gradient centrifugation of newborn calf serum [25]. Maleyl-BSA was prepared as described [26].

Cell surface biotinylation

Cells, cultured in media containing 3% NCLPDS, were released from dishes by mild trypsinization, washed and allowed to recover for 20 min at 37°C in Ham's F12 medium without additions. Cells were subjected to energy depletion (see below) and cell surface proteins were biotinylated by treatment of cells in suspension with sulfo-NHS-biotin as described previously [23]. Preparation of cell lysates, capture of biotinylated proteins and analysis by SDS-PAGE and immunoblotting were as described previously [4], [23].

Treatment of cells and analysis of lipoprotein lipid and protein uptake

Potassium depletion [27], exposure to hypertonic (0.45 M) sucrose [28], and treatment with 10 µM of either cytochalasin D or colchicine were done as described previously [23]. Cells were treated with Ro31-8220 or wortmannin for 10 min at 37°C with 1 ml of Ham's F12 containing 0.5% BSA without additions. For energy depletion, cells were washed with Hank's balanced salt solution (HBSS) either without (control) or with (energy depletion) 5 mM NaN₃ and 50 mM 2-deoxyglucose for 30 min at 37°C [17]. Labeled lipoproteins (to 5 µg/ml), with or without competitors (unlabeled HDL, AcLDL or maleyl-BSA, each to 200 µg/ml), were added and incubations were continued for 2 h at 37°C.

Cell association of fluorescent lipoproteins and flow cytometry

Cell association of DiI or alexa488 after incubation at 37°C, or cell binding after incubation at 4°C with fluorescently labeled lipoproteins were measured using either a Cytofluor 4000 fluorescence plate reader (Perkin Elmer Life Sciences) or by flow cytometry. For fluorescence detection using the fluorescence plate reader, cells were cultured in Falcon Optilux-Plus black walled, clear bottom 96 well dishes (BD Biosciences, Franklin Lakes, NJ USA). Cells were treated and incubated with labeled lipoproteins as described above. DiI fluorescence was measured using 530±25 nm excitation and 590±35 nm emission filters. Cell lysates were prepared with PBS containing 0.1% Triton X-100 and the protein content in each well was assayed. Flow cytometric analysis of DiI or alexa 488 fluorescence was as described previously [23]. The geometric mean fluorescence value of the population of cells was taken as a measure of the level of uptake (DiI or alexa 488) or eGFP-hSR-BI expression for each sample. Specific uptake was determined as the difference between total uptake (in the absence of competitors) and non-specific uptake (determined by including a 40-fold excess of either unlabeled HDL, AcLDL, or maleyl-BSA as competitors). In all cases, untransfected ldlA7 cells were included as controls. Cell sorting was performed as described above using a BD Biosciences FACSVantage instrument.

Fluorescence microscopy

Live cell confocal microscopy was performed using either a Perkin Elmer Life and Analytical Sciences Ultraview, or a Quorum Technologies Inc Wave FX spinning disc confocal microscopy system. Briefly, ldlA[eGFP-hSR-BI] cells (50,000/dish) were seeded in medium containing 3% NCLPDS and cultured for two days on glass-bottom, 35 mm dishes (Bioptics, Beaver Falls, PA). Cells were treated for 1 h with 70 µM cycloheximide and imaged at 37°C.

Immunofluorescence staining of fixed, permeabilized cells was performed as described previously [4]. For transferrin uptake, cells were cultured on poly-D-lysine coated glass slides, treated as described above and incubated at 37°C for 30 min with 50 µg/ml alexa 594-transferrin. Cells were fixed with 2.5% paraformaldehyde and stained with DAPI prior to mounting. Cells were imaged by epifluorescence microscopy using a Zeiss Axiovert 200 M wide-field inverted microscope or by confocal microscopy using a Zeiss LSM 510 laser scanning confocal microscope (Carl Zeiss Canada). Images were processed either using Axiovision or Simple PCI imaging software.

Results

Differential sensitivities of human and mouse SR-BI to protein kinase inhibitors and inhibition of endocytosis

We compared the sensitivity of human and murine SR-BI-mediated HDL lipid uptake to the phosphatidylinositol 3-kinase inhibitor wortmannin and the general protein kinase C inhibitor Ro31-8220. Both human and murine SR-BI were overexpressed in an LDL receptor deficient mutant CHO cell line, ldlA7, allowing us to monitor SR-BI-mediated lipid uptake in the absence of LDL receptor function [22], [29]. We monitored the transfer of the fluorescent lipid DiI from labeled lipoproteins into cells using a well-characterized lipid transfer assay [22]. LdlA[hSR-BI] or ldlA[mSR-BI] cells cultured in the presence of serum were treated with increasing concentrations of wortmannin for 10 min prior to and during incubation with DiI-HDL (Figure 1A and B). DiI uptake was measured using a fluorescence plate reader and normalized to cellular protein content. Wortmannin treatment of ldlA[hSR-BI] cells resulted in a dose dependent decrease in DiI uptake from DiI-HDL, to ∼50% at the highest concentrations (1 and 5 µM) tested (Figure 1A). In contrast, DiI uptake in ldlA[mSR-BI] cells was relatively insensitive to wortmannin, exhibiting only a 20% reduction at the highest wortmannin concentration (Figure 1B). Treatment of ldlA[mSR-BI] cells with increasing concentrations (0.5–10 µM) of Ro31-8220 resulted in a dose dependent inhibition of DiI uptake from DiI-HDL by ∼60% at the highest Ro31-8220 concentrations (Figure 1D). HDL-lipid uptake in ldlA[hSR-BI] cells, on the other hand, was not affected by Ro31-8220 at any concentration tested (Figure 1C). Neither wortmannin nor Ro31-8220 affected the levels of expression of hSR-BI in transfected cells (data not shown). Thus, HDL lipid uptake mediated by human and murine SR-BI in ldlA7 cells differ in their sensitivity to PI3K and PKC inhibitors. This suggests that, despite their similar ligand binding properties and primary sequences (80% identity in predicted amino acid sequences [21], [22]), human and murine SR-BI mediate HDL lipid uptake by pathways that are subject to different regulation in CHO-derived cells.

Figure 1.  Effects of PI3K or PKC inhibition on HDL-lipid uptake mediated by human or mouse SR-BI. LdlA[hSR-BI] cells (Panels A and C) and ldlA[mSR-BI] cells (Panels B and D), were treated without (black bars) or with (grey bars) increasing concentrations of either wortmannin (Panels A and B) or Ro31-8220 (Panels C and D) for 10 min prior to and during incubation for 2 h at 37°C with 5 µg/ml DiI-HDL. Untransfected ldlA7 cells (white bars) were incubated with DiI-HDL in parallel. Cells were washed and fluorescence in each well was determined using a cytofluor 3000 fluorescence plate reader as described in the Methods section. DiI-uptake is expressed as relative fluorescence units normalized to the amount of protein in each well. Results are averages±standard deviations of three determinations.

The PI3K and PKC signaling pathways participate in the regulation of a variety of endocytic, phagocytic and membrane transport processes, including SR-BI dependent phagocytosis of apoptotic cells [30], endocytosis of the β-adrenergic receptor [31] and receptor recycling/recruitment to the cell surface [32], [33]. Recently, wortmannin has been shown to block insulin/serum stimulated recruitment of SR-BI to the cell surface in serum starved human hepatoma HepG2 cells and mouse 3T3-L1 adipocytes [10], [11]. Murine SR-BI mediates lipid uptake from HDL and LDL via a process that does not require endocytosis [16], [17]. However endocytosis and re-secretion may be necessary for SR-BI mediated lipid efflux [15]. We therefore compared the effects of inhibitors of endocytosis on human and murine SR-BI-mediated lipid uptake from HDL.

Hypertonic shock or potassium depletion both prevent the interaction of clathrin with adaptor proteins and reduce the formation of clathrin coated pits on the cell surface, blocking clathrin-dependent endocytosis [27], [28], [34], [35]. Both treatments also block clathrin-independent endocytosis, involving lipid rafts, although the mechanisms are not clear [28]. The cellular uptake of alexa 594-transferrin by control or treated ldlA[hSR-BI] cells was monitored as a control for the effects of these treatments on endocytosis (Figure 2A–C). Transferrin, taken up by clathrin-mediated endocytosis [35], [36], accumulates in the perinuclear endosomal recycling compartment (Figure 2A). As previously observed by others [16], [35], potassium depletion (Figure 2B) and hypertonic shock (Figure 2C) each substantially decreased perinuclear fluorescence and therefore cellular internalization of transferrin compared to control untreated cells (Figure 2A). Potassium depletion and hypertonic shock also significantly decreased hSR-BI-mediated DiI-uptake from HDL by 70 and 60%, respectively, almost to the low level of DiI-uptake in control ldlA7 cells which do not overexpress SR-BI (Figure 2F and G). In contrast, potassium depletion and hypertonic shock only slightly (and non-significantly) reduced HDL-lipid uptake by ldlA[mSR-BI] cells overexpressing murine SR-BI (Figure 2H and I), consistent with the findings of others using either DiI or ³H cholesterol to follow HDL-lipid uptake. These findings suggests that unlike murine SR-BI [16], [17], human SR-BI mediates lipid uptake from HDL by an endocytosis-sensitive pathway.

Figure 2.  Effects of inhibition of endocytosis on HDL-lipid uptake by human or mouse SR-BI. LdlA[hSR-BI] or ldlA[mSR-BI] were either depleted of intracellular potassium, exposed to hypertonic sucrose, 10 µM cytochalasin D or 5 mM NaN₃ and 50 mM 2-deoxyglucose (Energy Depletion) as described in the Methods section. A-E: Alexa 594 transferrin uptake (red fluorescence) by ldlA[hSR-BI] cells was monitored as a control for endocytosis. Cell nuclei were stained with DAPI (blue fluorescence). Representative images are shown. Equivalent results were obtained for ldlA[mSR-BI] cells (not shown). Scale bars = 10 µm. Panels F–I: Cells were either depleted of potassium (panels F and H) or exposed to hypertonic sucrose (panels G and I) prior to and during incubation with 5 µg/ml of DiI-HDL for 2 h at 37°C in the absence or presence of 200 µg/ml of maleyl-BSA (a general competitor for SR-BI-mediated lipoprotein lipid uptake). Cellular uptake of DiI was measured by flow cytometry. Specific uptake of DiI was determined as the difference in the mean fluorescence values of the population of cells incubated without and with maleyl-BSA (equivalent results were obtained when unlabeled lipoproteins were used instead of maleyl-BSA). Values were normalized to control, untreated ldlA[hSR-BI] (panels F and G; black bars, set at 100% for ease of comparison) or ldlA[mSR-BI] cells (panels H and I; black bars, set at 100% for ease of comparison). Grey bars correspond to treated cells and white bars correspond to untreated control untrasnfected ldlA7 cells. Data are averages±standard deviations of triplicates. Stars (*) indicate p<0.05 for comparisons with untreated ldlA[hSR-BI] or ldlA[mSR-BI] cells by Student's t-test (Microsoft Excel).

Inhibition of endocytosis increases the efficiency of selective uptake by human SR-BI

We tested the ATP requirement of hSR-BI mediated lipid uptake from HDL by pretreatment of cells for 30 min with 0.5 mM NaN₃ and 50 mM 2-deoxyglucose (‘energy depletion’) [17]. This reduced the perinuclear accumulation of alexa 594-transferrin (Figure 2E) confirming that it reduced endocytosis. Energy depletion resulted in a slight increase in the biotinylation of hSR-BI in ldlA[hSR-BI] cells and a slight decrease in the biotinylation of mSR-BI in ldlA[mSR-BI] cells (Figure 3A, top panel) suggesting that it had opposite effects on the cell surface localization of human and murine SR-BI. Total human or murine SR-BI levels in cell lysates were not affected (Figure 3A, bottom panel). In contrast, energy depletion did not significantly affect the level of DiI-HDL binding to either ldlA[hSR-BI] or ldlA[mSR-BI] cells, measured at 4°C (Figure 3B). We measured the degree of selective lipid uptake in control and energy depleted ldlA[hSR-BI] cells by monitoring the uptake of both the protein and lipid portions of HDL simultaneously using HDL doubly labeled with alexa 488 and DiI, and 2-colour flow cytometric analysis (Figure 4). Untreated control ldlA[hSR-BI] cells were incubated in the absence or presence of excess unlabeled HDL competitor (200 µg/ml). Untreated ldlA[hSR-BI] cells exhibited high levels of DiI lipid and alexa 488 protein uptake, which were each reduced by a similar amount (3–4 fold) when a 40-fold mass excess of unlabeled HDL was included (Figure 4, panels A and B) such that the ratio of cell association of HDL-lipid/HDL-protein (Figure 4 panel C) was not affected. Cells subjected to energy depletion exhibited an approximately 60% reduction in DiI uptake (Figure 4 panel A) and a greater than 90% reduction in alexa 488 uptake (Figure 4 panel B). Thus, the ratio of DiI/alexa 488 uptake (Figure 4 panel C) increased by almost 5-fold compared to untreated control cells or to cells incubated with double labeled HDL in the presence of unlabeled HDL competitor. These data suggest that energy depletion decreased HDL-lipid uptake by human SR-BI by decreasing the internalization of HDL particles rather than by decreasing the transfer of lipids from associated HDL particles to cells. In contrast, energy depletion did not alter the efficiency of murine SR-BI-mediated HDL lipid uptake [17]. Thus, endocytosis appears to play an important role in human SR-BI-mediated HDL lipid uptake normally, and inhibition of endocytosis can result in an increase in the degree of selective HDL lipid uptake mediated by human SR-BI.

Figure 3.  Effects of energy depletion on human or mouse SR-BI cell surface localization and HDL binding. LdlA[hSR-BI] and ldlA[mSR-BI] cells were subjected to energy depletion or control treatment as described in the Methods section. Cells were chilled to 4°C, and either released from dishes for cell surface biotinylation (panel A) or incubated with 5 µg/ml DiI-HDL for 1 h at 4°C to monitor HDL binding (panel B). (A) Cell surface biotinylation, protein solubilization and streptavidin pull-down were performed as described in the Methods section. One half of the pull-down sample (top panel) and total cell lysate equivalent to 1/70 of the pull down sample (bottom panel) were analyzed by SDS-PAGE (10% acrylamide) and immunoblotting with an antibody that recognizes the C-terminus of SR-BI and a donkey anti-rabbit secondary antibody conjugated to horseradish peroxidase. Detection was by enhanced chemiluminescence. (B) Control (black bars) or energy depleted (grey bars) ldlA[hSR-BI] or ldlA[mSR-BI] or untreated control ldlA7 cells (white bars) were incubated with 5 µg/ml DiI-HDL for 1 h at 4°C, prior to washing and analysis of DiI-fluorescence using a fluorescence plate reader. Values are the means±standard deviations of triplicate samples and are expressed as the percent binding relative to untreated ldlA[hSR-BI] or ldlA[mSR-BI] cells.

Figure 4.  Inhibition of endocytosis increases the efficiency of selective HDL-lipid uptake mediated by hSR-BI. LdlA[hSR-BI] cells were either not treated (‘Control’ and ‘ + HDL’) or subjected to energy depletion (ED), potassium depletion, or treatment with cytochalasin D or 1 µM wortmannin (as indicated). DiI/alexa 488-double labeled HDL (5 µg/ml) was added without or with 200 µg/ml unlabeled HDL (‘ + HDL’). After a 2 h incubation at 37°C, two-color flow cytometric analysis was used to measure uptake of both DiI (Panel A – ‘Lipid Uptake’) and alexa 488 (Panel B – ‘Protein Uptake’). (C) For each sample, the ratio of DiI /alexa 488 fluorescence of the cell population was determined as a measure of the ratio of HDL lipid/protein uptake. Data are the averages±standard deviations of three independent samples.

We explored this further by testing the effects of potassium depletion or treatment with cytochalasin D or wortmannin on the level of HDL-lipid and protein uptake in ldlA[hSR-BI] cells incubated with double labeled HDL. Actin filaments are involved in multiple steps in endocytic pathways, participating in both ligand internalization and in vesicular trafficking to and from the degradative and/or recycling compartments. Cytochalasin D disrupts actin polymerization and impairs both ligand uptake and trafficking, as revealed by a reduction in perinuclear alexa594-transferrin fluorescence in treated versus control cells (Figure 2A and D). Cytochalasin D treatment of ldlA[hSR-BI] cells did not substantially affect HDL-lipid uptake (Figure 4A) but did inhibit the cell association of HDL-protein (Figure 4B), resulting in a greater than 2-fold increase in the ratio of uptake of HDL lipid/protein (Figure 4C). On the other hand, potassium depletion decreased cell association of both HDL protein (84%, Figure 4B) and to a lesser extent lipid (55% Figure 4A), and resulted in an approximately 2.5-fold increase in the ratio of uptake of HDL lipid/HDL protein (Figure 4C). Therefore, depletion of intracellular potassium or disruption of the actin cytoskeleton each resulted in an increased degree of selective lipid uptake mediated by hSR-BI, although they differed in their effects on HDL protein and lipid uptake. Treatment of cells with wortmannin resulted in similar reductions in both HDL-lipid and HDL-protein uptake (Figure 4A and B), without altering the degree of selective lipid uptake from HDL (Figure 4C). This suggests that wortmannin-mediated the inhibition of both endocytic and non-endocytic pathways.

Fluorescent protein tagged-hSR-BI is functional

To visualize the dynamics of the distribution of hSR-BI in live cells, we generated human SR-BI tagged on its N-terminus with either eGFP or mRFP. EGFP-tagged human SR-BI exhibited lower mobility than either untagged human or mouse SR-BI upon SDS-PAGE and immunoblotting (Figure 5A), consistent with increased size of the polypeptide resulting from the eGFP-fusion. After incubation with 5 µg/ml DiI-HDL, ldlA[eGFP-hSR-BI] cells (overexpressing eGFP-hSR-BI) exhibited ∼20-fold greater HDL-lipid uptake than either untransfected ldlA7 cells (Figure 5B) or ldlA[eGFP-hSR-BI] cells that had not been incubated with DiI-HDL (not shown). Similar results were obtained using cells expressing mRFP-tagged hSR-BI (not shown). Therefore, fluorescent protein tagged hSR-BI was functional.

Figure 5.  Expression and activity of eGFP-tagged human SR-BI in transfected ldlA7 cells. (A) LdlA[mSR-BI] (lane 1) and ldlA7 cells (lanes 2, 3 and 5) were cultured in medium containing 5% FBS. LdlA7 cells were either not transfected (lane 2) or transfected with hSR-BI (lane 3) or eGFP-hSR-BI expression plasmids (lane 5). After 3 days, cell lysates were prepared and analyzed by SDS-PAGE and immunoblotting for SR-BI. Lane 4 is empty. (B) Cells transfected with the eGFP-hSR-BI expression plasmid were subjected to G418 selection and cell sorting as described in Materials and Methods, to generate ldlA[eGFP-hSR-BI] cells that stably overexpress eGFP-hSR-BI. Untransfected ldlA7 (grey histogram) and ldlA[eGFP-hSR-BI] (black histogram) cells were incubated with 5 µg/ml DiI-HDL for 2 h at 37°C. DiI uptake was measured by flow cytometry as described in the Methods section. Representative histograms are shown.

EGFP-hSR-BI and mRFP-hSR-BI exhibited similar distributions in stably expressing cells. They localized to the cell surface at regions of cell-cell contact (Figure 6A, B and G–J) and in tether-like extensions between cells (Figure 6A, G, H). EGFP- and mRFP fluorescence was also detected in punctate vesicle-like structures within cells (Figure 6A, B and G–J). Similar patterns of SR-BI localization were detected by immunofluorescence in permeabilized ldlA[mSR-BI] (Figure 6C) [4] and ldlA[hSR-BI] cells (data not shown). The mRFP-hSR-BI-positive, punctate, vesicle-like structures observed in ldlA[mRFP-hSR-BI] cells did not co-localize with the lysosomal markers cathepsin A or saposin A or with eGFP-clathrin light chain at 37°C in ldlA7 cells stably expressing both (Figure 7).

Figure 6.  Localization and dynamics of eGFP- hSR-BI. LdlA[eGFP-hSR-BI], ldlA[mRFP-hSR-BI] and ldlA[mSR-BI] cells were cultured on poly-D-lysine-coated glass coverslips fixed to 35 mm culture dishes. Cells were fixed with 2.5% paraformaldehyde and either visualized directly (panels A and B) or after permeabilization and indirect immunofluorescence using anti-SR-BI⁴⁹⁵ antibody and alexa 488-anti-rabbit secondary antibody (panel C) using wide-field fluorescence microscopy with standard rhodamine and FITC filter sets. Panels D–F correspond to DIC images of the cells shown in A–C. Scale bars = 10 µm. (G–J) Live-cell confocal imaging of ldlA[eGFP-hSR-BI] cells was performed as described in the Methods section. Panel G on the left shows three adjacent ldlA[eGFP-hSR-BI] cells. Scale bar = 10 µm. The white boxes represent regions that are magnified in panels H–J. Panel H shows a time series at 50 sec intervals, demonstrating eGFP-hSR-BI dynamics associated with a tether between two adjacent cells. The first panel (time 0 sec) is cropped and magnified from panel G. The arrow serves as a reference marker to illustrate the leftward motion of eGFP-hSR-BI fluorescence along the tether. Scale bar = 5 µm. This series of images corresponds to Supplementary Video 1 – online version only. Panel I shows images at 10 sec intervals, beginning at time 115 sec relative to the image in panel G. They illustrate the motion of an eGFP-hSR-BI-positive puncta (small box) towards the center of the cell. The arrow indicates an eGFP-hSR-BI-positive structure that is static within the time-frame of the images. Scale bar = 5 µm. This series of images corresponds to Supplementary Video 2 – online version only. Panel J shows a series of images taken at 5 sec intervals and focusing on the region of cell-cell contact between two adjacent cells. The cell on the upper left in this panel corresponds to the cell in the lower right in panels H and I (see panel G). Scale bar = 2.5 µm. The arrow indicates the position at which an SR-BI positive puncta, apparently derived from the cell surface, appears. This corresponds to Supplementary Video 3 – online version only.

Figure 7.  mRFP-tagged hSR-BI does not co-localize with lysosomal markers or with clathrin. LdlA[mRFP-hSR-BI] cells (panels A–F) and ldlA7 cells stably expressing both eGFP-clathrin and mRF-hSR-BI (panels G–I) were cultured on glass cover slips and fixed. Cells were permeabilized and immunostained for lysosomal markers cathepsin A (panel A) or saposin A (panel D). Alexa 488 labeled secondary antibody was used for detection prior to wide-field epifluorescence microscopy using a Zeiss Axiovert 200 M inverted microscope. Alternatively, cells were imaged directly (panels G–I) using a Zeiss LSM510 laser scanning confocal microscope. A, D and G: Alexa 488 or eGFP fluorescence. B, E and H: mRFP fluorescence. C, F and I: merged color images in which green corresponds to cathepsin A (panel C), saposin A (panel F) or eGFP-clathrin (panel I) and red corresponds to mRFP-hSR-BI. Scale bars = 10 µm. Representative images are shown.

Live-cell confocal imaging revealed the dynamics of the SR-BI containing structures in ldlA[eGFP-hSR-BI] cells. For these experiments, cells were treated with cycloheximide for 1 hr to block new protein synthesis prior to imaging at 37°C. EGFP-hSR-BI in cell surface extensions between cells appeared to be most concentrated at either one or both ends of the extension and to exhibit motion towards one of the cells (Figure 6G and H and Supplementary Video 1 – online version only). SR-BI at the cell surface at regions of cell-cell contact appeared to localize in smaller extensions between cells, diffusely along the cell periphery and in punctae within cells, adjacent to the periphery (Figure 6G–J). EGFP-hSR-BI in the smaller extensions exhibited a similar mobility to that in the longer extensions, although it was difficult to determine direction (Figure 6H and Supplementary Video 1 – online version only). SR-BI at the cell periphery in regions of cell-cell contact was also dynamic (Figure 6I and J and Supplementary Videos 2 and 3 – online version only). EGFP-hSR-BI was also present in punctae within cells that appeared to represent vesicles. These were highly dynamic, moving both radially towards the center of cells as well as circumferentially (Figure 6I and Supplementary Video 2 – online version only). Some of these appeared to originate at or adjacent to the cell periphery at regions of cell-cell contact (Figure 6I and J and Supplementary Videos 2 and 3 – online version only). These observations are consistent with evidence that SR-BI undergoes continuous internalization from and recycling back to the cell surface [9], [12], [14], and suggests that at least some of these punctate structures represent hSR-BI-containing vesicles either derived from or destined for the cell surface.

Mutations in human SR-BI's C-terminus do not alter its sensitivity to inhibition of endocytosis

To explore whether the carboxy terminal cytoplasmic domain of hSR-BI was required for its endocytosis-dependent HDL lipid uptake, we generated eGFP-tagged point or deletion mutants targeting this region and compared their expression and activity to eGFP-tagged wild type hSR-BI. The sequence Y⁴⁷¹LFW in the cytoplasmic C-terminus of human SR-BI has the appearance of a potential endocytic motif of the form YXXφ (where X is any amino acid and φ is hydrophobic) [37]. This is conserved in bovine, pig and rabbit SR-BI sequences, but those of mouse, rat and hamster contain F in place of Y⁴⁷¹[21], [22], [29], [38–41]. To test if this contributed to the requirement for endocytosis for hSR-BI-mediated HDL lipid uptake, we generated point mutants in which Y⁴⁷¹ was changed to either F (to mimic mouse SR-BI) or D (to mimic potential Y-phosphorylation [37]). We also generated a deletion mutant lacking the entire C-terminal cytoplasmic domain (hSR-BI-ΔC) as described by others [42]. LdlA7 cells stably expressing either eGFP-hSR-BI, eGFP-hSR-BI-Y⁴⁷¹F, eGFP-hSR-BI-Y⁴⁷¹D or eGFP-hSR-BI-ΔC were treated with or without energy depletion. Immunoblotting with antibodies specific for either GFP or the C-terminus of SR-BI (Figure 8A) and eGFP fluorescence levels (not shown) revealed that eGFP-hSR-BI and the Y⁴⁷¹D and Y⁴⁷¹F mutants were expressed at similar levels, whereas the eGFP-hSR-BI-ΔC mutant appeared to be expressed at a higher level. Energy depletion did not affect the level of eGFP-hSR-BI expression (Figure 8A). Cells were incubated with DiI-HDL for 2 h, prior to analysis of both DiI and GFP fluorescence by flow cytometry. Figure 8B shows the effects of either energy depletion or hypertonic shock on HDL-lipid uptake expressed as a percentage of the level of DiI uptake in control untreated cells. Both energy depletion and exposure to hypertonic sucrose reduced DiI-uptake from DiI-HDL to similar extents in cells expressing either wild type, Y⁴⁷¹F/D-mutant hSR-BI, or hSR-BI lacking its C-terminal cytoplasmic tail. Therefore, neither mutation of Y⁴⁷¹ to F or D, nor deletion of the entire C-terminal cytoplasmic region of hSR-BI affected the dependence of HDL lipid uptake on endocytosis.

Figure 8.  Mutations in the C-terminal cytoplasmic domain do not affect the dependence of hSR-BI mediated lipid uptake on endocytosis. LdlA7 cells were transfected with eGFP-hSR-BI (wild type, WT), eGFP-hSR-BI-Y⁴⁷¹F, eGFP-hSR-BI-Y⁴⁷¹D and eGFP-hSR-BI-ΔC expression plasmids and subjected to G418 selection. (A) Detergent extracts of control and energy depleted cells were prepared and proteins were subjected to SDS-PAGE and immunoblotting using antibodies specific for GFP (top panel), SR-BI-C terminal peptide (middle panel) and β-actin (loading control, bottom panel). Equal amounts of protein were applied to each lane. (B) Cells subjected to energy depletion or exposed to hypertonic sucrose (grey bars), control untreated cells (black bars) or untransfected ldlA7 cells (white bar) were incubated with DiI-HDL and DiI uptake and GFP-fluorescence were measured as described in the Methods section. The level of DiI uptake is expressed as the percentage of control, untreated cells and data represent the average±range of two independent experiments.

Discussion

Our data suggests that human SR-BI can mediate both the endocytic uptake and selective uptake of HDL lipids in a manner that is sensitive to the PI3K inhibitor wortmannin but not the PKC inhibitor Ro31-8220. SR-BI mediates the internalization of a variety of its ligands including serum amyloid A, bacterial lipopolysaccharide, bacteria and apoptotic cells [30], [43–46]. It also mediates the internalization and re-secretion of HDL [12], [14–16]. We have found that endocytosis is necessary for human SR-BI-mediated HDL-lipid uptake in CHO-derived ldlA7 cells overexpressing human SR-BI. In contrast, murine SR-BI mediates HDL-lipid uptake via a pathway that is not affected by inhibition of endocytosis [16], [17] but is sensitive to the PKC inhibitor Ro31-8220 [23].

We have used the fluorescent lipid, DiI, for these studies as a convenient marker of HDL lipid uptake [22]. The endocytosis step is not a characteristic of DiI uptake, nor does it appear to be a characteristic of the cell type used in these studies since murine SR-BI expressed in the same cell type mediated uptake of the same lipid (DiI) from HDL in an endocytosis-independent manner (Figure 2 and [16]). Instead, our findings suggest that the pathway by which HDL lipids are taken up is determined by the characteristics of the receptor (human versus mouse SR-BI). Disruption of endocytosis by depletion of ATP or potassium, or by disruption of actin polymerization resulted in more pronounced decreases in the cell association of HDL protein than lipid, resulting in an apparent increase in the degree of selective lipid uptake mediated by hSR-BI. These results suggest that human SR-BI can mediate HDL lipid uptake by both endocytic and selective lipid uptake pathways, and that endocytosis contributes substantially to total lipid uptake mediated by this receptor. The increased degree of selective lipid uptake (ratio of HDL lipid/protein taken up) resulting from inhibition of endocytosis suggests that endocytosis is a dynamic regulator of SR-BI's selective lipid uptake activity.

It has recently been demonstrated that the reduced selective uptake activity of murine SR-BII, a splice variant of SR-BI with a different C-terminal cytoplasmic domain may be the result of its increased endocytosis [47]. The insertion of a di-leucine endocytic motif derived from SR-BII's C-terminal cytoplasmic domain into the corresponding region of murine SR-BI resulted in its increased endocytosis and decreased mSR-BI-mediated selective uptake activity [47]. This provides further support for the idea that SR-BI's selective uptake activity may be subject to regulation by modulating its endocytosis. SR-BI contains two short cytoplasmic regions at the extreme N and C-termini of ∼9 and 45 amino acid residues, respectively. We tested if the C-terminal cytoplasmic region was involved in the endocytosis-dependent HDL-lipid uptake mediated by hSR-BI. We analyzed hSR-BI mutants that either lacked the cytoplasmic C-terminus [42] or contained Y⁴⁷¹F or Y⁴⁷¹D point mutations. None of these mutations affected the sensitivity of hSR-BI-mediated HDL lipid uptake to inhibition of endocytosis (Figure 8B). Therefore the endocytosis-dependence of HDL lipid uptake by hSR-BI is not conferred by its C-terminal cytoplasmic tail.

These findings have potentially important implications for understanding the role of human SR-BI in selective lipid uptake and its regulation in vivo. Our data suggest that, at least in CHO-derived cells, human SR-BI mediated HDL lipid uptake involves endocytosis, possibly of HDL mediated by hSR-BI. Inhibition of hSR-BI-mediated HDL endocytosis results in increased efficiency of selective lipid uptake. This suggests that the process of endocytosis is a dynamic regulator of SR-BI's selective lipid uptake activity, possibly diverting HDL-derived lipids to different intracellular pools, although this remains to be tested. Identification of the features of the hSR-BI molecule that influence the endocytic versus selective lipid uptake pathway will allow for a better understanding of the regulation of these opposing pathways and potentially provide insight into means of regulating SR-BI's selective lipid uptake activity.

Legends to Supplementary Videos (online version only)

Supplementary Video 1. Live cell confocal imaging of two adjacent ldlA[eGFP-hSR-BI] cells with an SR-BI-positive tether-like extension between them as well as smaller extensions above an area of apparently direct cell-cell contact. This video corresponds to the sequence of images shown in Figure 6H. Images were captured every 5 sec over a total of 205 sec and are played back at 2 frames/sec, representing 10×normal speed.

Supplementary Video 2. Live cell confocal imaging of a different region of the same two adjacent ldlA[eGFP-hSR-BI] cells as that shown in Supplemental Video 1. Shown is the region of direct contact between the two cells, exhibiting dynamic SR-BI localization. SR-BI localizes diffusely at the region of cell contact, to punctae that appear to be at and just below the cell surface and to punctae within the cells. An SR-BI-positive puncta adjacent to the cell surface can be seen migrating towards the interior of the cell on the left (see also the corresponding series of panels in Figure 6I). Images were captured every 5 sec over a total of 205 sec and are played back at 2 frames/sec, representing 10×normal speed.

Supplementary Video 3. Live cell confocal imaging of two adjacent ldlA[eGFP-hSR-BI] cells. The cell on the upper left in this video is the same as the cell on the lower right in Supplementary Video 2. Shown is a region of direct contact between the two cells, exhibiting dynamic SR-BI localization. This video corresponds to a 205 sec sequence encompassing the series of images in Figure 6J. The video shows two instances of eGFP-SR-BI fluorescence accumulating in what appear to be vesicles derived from the cell surface. Images were captured every 5 sec over a total of 205 sec and are played back at 2 frames/sec, representing 10×normal speed.

Acknowledgements

We thank E. Roediger for expert technical assistance. FACS was performed by H. Liang in the McMaster University Flow Cytometry Facility. We thank J. Daniel for helpful discussions, and M. Krieger, R. Tsien, S. Grinstein and J.H, Keen for generously providing reagents. This research was supported by funding from the Heart and Stroke Foundation of Ontario and the Natural Sciences and Engineering Research Council of Canada to BLT. BLT is a Heart and Stroke Foundation of Canada New Investigator and RT is a Canadian Institutes of Health Research New Investigator.