Roles of ATP binding cassette transporters A1 and G1, scavenger receptor BI and membrane lipid domains in cholesterol export from macrophages

PURPOSE OF REVIEW: The initial steps of reverse cholesterol transport involve export of cholesterol from peripheral cells to plasma lipoproteins for subsequent delivery to the liver. The review discusses recent developments in our understanding of how these steps occur, with particular emphasis on the macrophage, the major site of cellular cholesterol accumulation in atherosclerosis. RECENT FINDINGS: ATP binding cassette transporter (ABC) A1 exports cholesterol and phospholipid to lipid-free apolipoproteins, while ATP binding cassette transporter G1 and scavenger receptor BI export cholesterol to phospholipid-containing acceptors. ABCA1-dependent cholesterol export involves an initial interaction of apolipoprotein AI with lipid raft membrane domains, although ABCA1 and most exported cholesterol are not raft associated. ABCG1 exports cholesterol to HDL and other phospholipid-containing acceptors. These include particles generated during lipidation of apoAI by ABCA1, suggesting that the two transporters cooperate in cholesterol export. Scavenger receptor BI is atheroprotective, mediating clearance of HDL cholesterol by the liver. The relative contributions of scavenger receptor BI and ABCG to cholesterol export to HDL from macrophages is unclear and may depend on cellular cholesterol status and the cholesterol gradient between cell and acceptor. SUMMARY: The presence of distinct pathways for cholesterol efflux to lipid-free apolipoprotein AI and phospholipid-containing HDL species clarifies our understanding of reverse cholesterol transport, and provides new opportunities for its therapeutic manipulation.     

Extracellular hydrophobic regions in scavenger receptor BI play a key role in mediating HDL-cholesterol transport

The binding of high density lipoprotein (HDL) to scavenger receptor BI (SR-BI) is responsible for whole-body cholesterol disposal via reverse cholesterol transport. The extracellular domain of SR-BI is required for HDL binding and selective uptake of HDL-cholesterol. We identified six highly hydrophobic regions in this domain that may be important for receptor activity and performed site-directed mutagenesis to investigate the importance of these regions in SR-BI-mediated cholesterol transport. Non-conservative mutation of the regions encompassing V67, L140/L142, V164 or V221 reduced hydrophobicity and impaired the ability of SR-BI to bind HDL, mediate selective uptake of HDL-cholesterol, promote cholesterol efflux, and enlarge the cholesterol oxidase-sensitive pool of membrane free cholesterol. In contrast, conservative mutations at V67, V164 or V221 did not affect the hydrophobicity or these cholesterol transport activities. We conclude that the hydrophobicity of N-terminal extracellular regions of SR-BI is critical for cholesterol transport, possibly by mediating receptor-ligand and/or receptor-membrane interactions.     

Scavenger receptor BI (SR-BI) mediates free cholesterol flux independently of HDL tethering to the cell surface

In addition to its effect on high density lipoprotein (HDL) cholesteryl ester (CE) uptake, scavenger receptor BI (SR-BI) was recently reported to stimulate free cholesterol (FC) flux from Chinese hamster ovary (CHO) cells stably expressing mouse SR-BI, a novel function of SR-BI that may play a role in cholesterol removal from the vessel wall where the receptor can be found. It is possible that SR-BI stimulates flux simply by tethering acceptor HDL particles in close apposition to the cell surface thereby facilitating the movement of cholesterol between the plasma membrane and HDL. To test this, we used transiently transfected cells and compared the closely related class B scavenger receptors mouse SR-BI and rat CD36 for their ability to stimulate cholesterol efflux as both receptors bind HDL with high affinity. The results showed that, although acceptor binding to SR-BI may contribute to efflux to a modest extent, the major stimulation of FC efflux occurs independently of acceptor binding to cell surface receptors. Instead our data indicate that SR-BI mediates alterations to membrane FC domains which provoke enhanced bidirectional FC flux between cells and extracellular acceptors.     

High density lipoprotein phospholipid composition is a major determinant of the bi-directional flux and net movement of cellular free cholesterol mediated by scavenger receptor BI

The role of high density lipoprotein (HDL) phospholipid in scavenger receptor BI (SR-BI)-mediated free cholesterol flux was examined by manipulating HDL(3) phosphatidylcholine and sphingomyelin content. Both phosphatidylcholine and sphingomyelin enrichment of HDL enhanced the net efflux of cholesterol from SR-BI-expressing COS-7 cells but by two different mechanisms. Phosphatidylcholine enrichment of HDL increased efflux, whereas sphingomyelin enrichment decreased influx of HDL cholesterol. Although similar trends were observed in control (vector-transfected) COS-7 cells, SR-BI overexpression amplified the effects of phosphatidylcholine and sphingomyelin enrichment of HDL 25- and 2.8-fold, respectively. By using both phosphatidylcholine-enriched and phospholipase A(2)-treated HDL to obtain HDL with a graded phosphatidylcholine content, we showed that SR-BI-mediated cholesterol efflux was highly correlated (r(2) = 0.985) with HDL phosphatidylcholine content. The effects of varying HDL phospholipid composition on SR-BI-mediated free cholesterol flux were not correlated with changes in either the K(d) or B(max) values for high affinity binding to SR-BI. We conclude that SR-BI-mediated free cholesterol flux is highly sensitive to HDL phospholipid composition. Thus, factors that regulate cellular SR-BI expression and the local modification of HDL phospholipid composition will have a large impact on reverse cholesterol transport.     

SR‐BI,CD36, and caveolin‐1 contribute positively to cholesterol efflux in hepatic cells

In non‐hepatic cells, scavenger receptor class B type I (SR‐BI), cluster of differentiation 36 (CD36), and caveolin‐1 were described as mediators of cholesterol efflux, the first step of reverse cholesterol transport (RCT). Stable transformants of HepG2 cells overexpressing SR‐BI, CD36, or caveolin‐1 were generated, as well as cells overexpressing both caveolin‐1 and SR‐BI or caveolin‐1 and CD36 in order to address the effect of caveolin‐1 on both receptor activities. These cells were analyzed for their ability to efflux cholesterol to HDL₃. Our results show that overexpressing SR‐BI, CD36, or caveolin‐1 increases cholesterol efflux by 106, 92, and 48%, respectively. Moreover, the dual overexpressions of caveolin‐1 and SR‐BI or caveolin‐1 and CD36 lead to a more prominent increase in cholesterol efflux. Studies were also conducted with primary cultures of SR‐BI knockout (KO), CD36 KO, and SR‐BI/CD36 double‐KO (dKO) mice. SR‐BI KO and SR‐BI/CD36 dKO hepatic cells show 41 and 56% less cholesterol efflux, respectively, than normal hepatic cells. No significant difference was observed between the efflux of normal and CD36 KO cells. The difference between the role of human and murine CD36 correlated with the absence of CD36 dimers in mouse caveolae/rafts. Overall, our results show that SR‐BI is clearly involved in cholesterol efflux in mouse and human hepatic cells, while CD36 plays a significant role in human cells. Copyright © 2010 John Wiley & Sons, Ltd.     

The role of the high-density lipoprotein receptor SR-BI in cholesterol metabolism

The HDL receptor scavenger receptor class B type I (SR-BI), which mediates selective HDL cholesterol uptake, plays a role in murine HDL metabolism, reverse cholesterol transport and whole-body cholesterol homeostasis. SR-BI is found in the liver, where its expression is regulated by estrogen, dietary cholesterol and fat, and controls murine plasma HDL cholesterol levels and bile cholesterol secretion. SR-BI is also highly expressed in rodent steroidogenic cells, where it facilitates cholesterol uptake for storage or steroid hormone synthesis and where its expression is regulated by trophic hormones. The detailed mechanism(s) underlying SR-BI-mediated selective cholesterol uptake have not yet been elucidated. Further analysis of the molecular and cellular bases of SR-BI regulation and function should provide new insights into the physiology and pathophysiology of cholesterol metabolism.     

Specific oxidized phospholipids inhibit scavenger receptor bi-mediated selective uptake of cholesteryl esters

We have recently demonstrated that specific oxidized phospholipids (oxPC(CD36)) accumulate at sites of oxidative stress in vivo such as within atherosclerotic lesions, hyperlipidemic plasma, and plasma with low high-density lipoprotein levels. oxPC(CD36) serve as high affinity ligands for the scavenger receptor CD36, mediate uptake of oxidized low density lipoprotein by macrophages, and promote a pro-thrombotic state via platelet scavenger receptor CD36. We now report that oxPC(CD36) represent ligands for another member of the scavenger receptor class B, type I (SR-BI). oxPC(CD36) prevent binding to SR-BI of its physiological ligand, high density lipoprotein, because of the close proximity of the binding sites for these two ligands on SR-BI. Furthermore, oxPC(CD36) interfere with SR-BI-mediated selective uptake of cholesteryl esters in hepatocytes. Thus, oxidative stress and accumulation of specific oxidized phospholipids in plasma may have an inhibitory effect on reverse cholesterol transport.     

Analysis of chimeric receptors shows that multiple distinct functional activities of scavenger receptor, class B, type I (SR-BI), are localized to the extracellular receptor domain

Scavenger receptor BI (SR-BI) mediates the selective uptake of high-density lipoprotein (HDL) cholesteryl ester (CE), a process by which HDL CE is taken into the cell without degradation of the HDL particle. In addition, SR-BI stimulates the bi-directional flux of free cholesterol (FC) between cells and lipoproteins, an activity that may be responsible for net cholesterol efflux from peripheral cells as well as the rapid hepatic clearance of FC from plasma HDL. SR-BI also increases cellular cholesterol mass and alters cholesterol distribution in plasma membrane domains as judged by the enhanced sensitivity of membrane cholesterol to extracellular cholesterol oxidase. In contrast, CD36, a closely related class B scavenger receptor, has none of these activities despite binding HDL with high affinity. In the present study, analyses of chimeric SR-BI/CD36 receptors and domain-deleted SR-BI have been used to test the various domains of SR-BI for functional activities related to HDL CE selective uptake, bi-directional FC flux, and the alteration of membrane cholesterol mass and distribution. The results show that each of these activities localizes to the extracellular domain of SR-BI. The N-terminal cytoplasmic tail and transmembrane domains appear to play no role in these activities other than targeting the receptor to the plasma membrane. The C-terminal tail of SR-BI is dispensable for activity as well for targeting to the plasma membrane. Thus, multiple distinct functional activities are localized to the SR-BI extracellular domain.     

Cellular and physiological roles of SR-BI, a lipoprotein receptor which mediates selective lipid uptake

High-density lipoproteins (HDL) play an important role in protection against atherosclerosis by mediating reverse cholesterol transport - the transport of excess cholesterol from peripheral tissues to the liver for disposal. SR-BI is a cell surface receptor for HDL and other lipoproteins (LDL and VLDL) and mediates the selective uptake of lipoprotein cholesterol by cells. Overexpression or genetic ablation of SR-BI in mice revealed that it plays an important role in HDL metabolism and reverse cholesterol transport and protects against atherosclerosis in mouse models of the disease. If it plays a similar role in humans then it may be an attractive target for therapeutic intervention. We will review some of the recent advances in the understanding of SR-BI's physiological role and cellular function in lipoprotein metabolism.     

Apolipoprotein A-II modulates the binding and selective lipid uptake of reconstituted high density lipoprotein by scavenger receptor BI

High density lipoprotein (HDL) represents a mixture of particles containing either apoA-I and apoA-II (LpA-I/A-II) or apoA-I without apoA-II (LpA-I). Differences in the function and metabolism of LpA-I and LpA-I/A-II have been reported, and studies in transgenic mice have suggested that apoA-II is pro-atherogenic in contrast to anti-atherogenic apoA-I. The molecular basis for these observations is unclear. The scavenger receptor BI (SR-BI) is an HDL receptor that plays a key role in HDL metabolism. In this study we investigated the abilities of apoA-I and apoA-II to mediate SR-BI-specific binding and selective uptake of cholesterol ester using reconstituted HDLs (rHDLs) that were homogeneous in size and apolipoprotein content. Particles were labeled in the protein (with (125)I) and in the lipid (with [(3)H]cholesterol ether) components and SR-BI-specific events were analyzed in SR-BI-transfected Chinese hamster ovary cells. At 1 microg/ml apolipoprotein, SR-BI-mediated cell association of palmitoyloleoylphosphatidylcholine-containing AI-rHDL was significantly greater (3-fold) than that of AI/AII-rHDL, with a lower K(d) and a higher B(max) for AI-rHDL as compared with AI/AII-rHDL. Unexpectedly, selective cholesterol ester uptake from AI/AII-rHDL was not compromised compared with AI-rHDL, despite decreased binding. The efficiency of selective cholesterol ester uptake in terms of SR-BI-associated rHDL was 4-5-fold greater for AI/AII-rHDL than AI-rHDL. These results are consistent with a two-step mechanism in which SR-BI binds ligand and then mediates selective cholesterol ester uptake with an efficiency dependent on the composition of the ligand. ApoA-II decreases binding but increases selective uptake. These findings show that apoA-II can exert a significant influence on selective cholesterol ester uptake by SR-BI and may consequently influence the metabolism and function of HDL, as well as the pathway of reverse cholesterol transport.     

Scavenger receptor BI and ATP-binding cassette transporter A1 in reverse cholesterol transport and atherosclerosis

PURPOSE OF REVIEW: The appearance of scavenger receptor class B type I (SR-BI) and ATP-binding cassette transporter A1 (ABCA1) in macrophages and liver implicates these transporters in different stages of reverse cholesterol transport. This review focuses on the role of SR-BI and ABCA1 in reverse cholesterol transport in the context of atherosclerotic lesion development. RECENT FINDINGS: Recent studies indicate that hepatic expression of ABCA1 and SR-BI is important for the generation of nascent HDL and the delivery of HDL cholesteryl esters to the liver, respectively. Although macrophage SR-BI and ABCA1 do not contribute significantly to circulating HDL levels, the perpetual cycle of HDL lipidation and delipidation by the liver ensures the availability of acceptors for cholesterol efflux that maintain cholesterol homeostasis in arterial macrophages, thereby reducing atherogenesis. In addition to its established role in the selective uptake of HDL cholesteryl esters, there is now evidence that hepatic SR-BI facilitates postprandial lipid metabolism, and that hepatic secretion of VLDL is dependent on ABCA1-mediated nascent HDL formation. Thus, remnant and HDL metabolism are more intimately intertwined in hepatic lipid metabolism than has previously been appreciated. SUMMARY: Recent advances in the understanding of the role of ABCA1 and SR-BI in HDL metabolism and their atheroprotective properties indicate the significant potential of modulating ABCA1 and SR-BI expression in both arterial wall macrophages and the liver for the treatment of atherosclerotic coronary artery disease.     

Scavenger receptor class B,type 1 facilitates cellular fatty acid uptake

SR-B1 belongs to the class B scavenger receptor, or CD36 super family. SR-B1 and CD36 share an affinity for a wide array of ligands. Although they exhibit similar ligand binding specificity, SR-B1 and CD36 have some very specific lipid transport functions. Whereas SR-B1 primarily facilitates the selective delivery of cholesteryl esters (CEs) and cholesterol from HDL particles to the liver and non-placental steroidogenic tissues, as well as participating in cholesterol efflux from cells, CD36 primarily mediates the uptake of long-chain fatty acids in high fatty acid-requiring organs such as the heart, skeletal muscle and adipose tissue. However, CD36 also mediates cholesterol efflux and facilitates selective lipoprotein-CE delivery, although less efficiently than SR-B1. Interestingly, the ability or efficiency of SR-B1 to mediate fatty acid uptake has not been reported. In this paper, using overexpression and siRNA-mediated knockdown of SR-B1, we show that SR-B1 possesses the ability to facilitate fatty acid uptake. Moreover, this function is not blocked by BLT-1, a specific chemical inhibitor of HDL-CE uptake activity of SR-B1, nor by sulfo-N-succinimidyl oleate, which inhibits fatty acid uptake by CD36. Attenuated fatty acid uptake was also observed in primary adipocytes isolated from SR-B1 knockout mice. In conclusion, facilitation of fatty acid uptake is an additional function that is mediated by SR-B1.     

Scavenger receptor BI (SR-BI) clustered on microvillar extensions suggests that this plasma membrane domain is a way station for cholesterol trafficking between cells and high-density lipoprotein

Receptor-mediated trafficking of cholesterol between lipoproteins and cells is a fundamental biological process at the organismal and cellular levels. In contrast to the well-studied pathway of LDL receptor-mediated endocytosis, little is known about the trafficking of high-density lipoprotein (HDL) cholesterol by the HDL receptor, scavenger receptor BI (SR-BI). SR-BI mediates HDL cholesteryl ester uptake in a process in which HDL lipids are selectively transferred to the cell membrane without the uptake and degradation of the HDL particle. We report here the cell surface locale where the trafficking of HDL cholesterol occurs. Fluorescence confocal microscopy showed SR-BI in patches and small extensions of the cell surface that were distinct from sites of caveolin-1 expression. Electron microscopy showed SR-BI in patches or clusters primarily on microvillar extensions of the plasma membrane. The organization of SR-BI in this manner suggests that this microvillar domain is a way station for cholesterol trafficking between HDL and cells. The types of phospholipids in this domain are unknown, but SR-BI is not strongly associated with classical membrane rafts rich in detergent-resistant saturated phospholipids. We speculate that SR-BI is in a more fluid membrane domain that will favor rapid cholesterol flux between the membrane and HDL.     

Scavenger receptor CD36 mediates uptake of high density lipoproteins in mice and by cultured cells

The mechanisms of HDL-mediated cholesterol transport from peripheral tissues to the liver are incompletely defined. Here the function of scavenger receptor cluster of differentiation 36 (CD36) for HDL uptake by the liver was investigated. CD36 knockout (KO) mice, which were the model, have a 37% increase (P = 0.008) of plasma HDL cholesterol compared with wild-type (WT) littermates. To explore the mechanism of this increase, HDL metabolism was investigated with HDL radiolabeled in the apolipoprotein (¹²⁵I) and cholesteryl ester (CE, [³H]) moiety. Liver uptake of [³H] and ¹²⁵I from HDL decreased in CD36 KO mice and the difference, i. e. hepatic selective CE uptake ([³H]¹²⁵I), declined (–33%, P = 0.0003) in CD36 KO compared with WT mice. Hepatic HDL holo-particle uptake (¹²⁵I) decreased (–29%, P = 0.0038) in CD36 KO mice. In vitro, uptake of ¹²⁵I-/[³H]HDL by primary liver cells from WT or CD36 KO mice revealed a diminished HDL uptake in CD36-deficient hepatocytes. Adenovirus-mediated expression of CD36 in cells induced an increase in selective CE uptake from HDL and a stimulation of holo-particle internalization. In conclusion, CD36 plays a role in HDL uptake in mice and by cultured cells. A physiologic function of CD36 in HDL metabolism in vivo is suggested.     

A role for the scavenger receptor,class B type I in high density lipoprotein dependent activation of cellular signaling pathways

High density lipoprotein (HDL) levels are inversely proportional to the risk of coronary heart disease. HDL mediates various anti-atherogenic pathways including reverse cholesterol transport from cells of the arterial wall to the liver and steroidogenic tissues. In addition HDL activates various intracellular signaling events that confer atheroprotection. The HDL receptor, scavenger receptor class B type I (SR-BI) has been implicated directly and indirectly in HDL induced signaling. The aim of this review is to summarize the role of SR-BI in HDL induced signaling in the vasculature.     

Impact of vitamin A on high-density lipoprotein-cholesterol and scavenger receptor class BI in the obese rat

Objective: Scavenger receptor class BI (SR‐BI), authentic high‐density lipoprotein (HDL) receptors expressed in liver, are known to play an important role in HDL‐cholesterol (C) metabolism and reverse cholesterol transport. Interestingly, obese rats of WNIN/Ob strain have abnormally elevated levels of serum HDL‐C compared with their lean counterparts. Based on the well‐established role of SR‐B1 in HDL‐C metabolism, it was hypothesized that these obese rats may have an underexpression of hepatic SR‐B1 receptors. In view of the significant role of vitamin A in energy expenditure and obesity, we also tested whether vitamin A supplementation can correct abnormal HDL‐C metabolism. Research Methods and Procedures: To test this hypothesis, 7‐month‐old male lean and obese rats of WNIN/Ob strain were divided into two groups; each group was subdivided into two subgroups consisting of six lean and six obese rats and received diets containing either 2.6 or 129 mg vitamin A/kg diet for 2 months. Results: At the end, obese rats receiving normal levels of vitamin A diet showed high serum HDL‐C and lower hepatic SR‐BI expression levels compared with lean counterparts. Furthermore, chronic dietary vitamin A supplementation resulted in overexpression of hepatic SR‐BI receptors (protein and gene) with concomitant reduction in serum HDL‐C levels in obese rats. Discussion: Thus, our observations highlight the role of vitamin A in reverse cholesterol transport through up‐regulation of hepatic SR‐BI receptors and, thereby, HDL‐C homeostasis in obese rats of WNIN/Ob strain.     

Scavenger receptor class B Type I (SR-BI) assembles into detergent-sensitive dimers and tetramers

High density lipoproteins (HDL) are protective against cardiovascular disease due to their important role in the reverse cholesterol transport (RCT) pathway. The selective transfer of cholesteryl ester (CE) from the HDL core to cells, the last step in RCT, is mediated by scavenger receptor class B type I (SR-BI). SR-BI is a heavily glycosylated cell surface receptor that is highly expressed in the liver, ovaries, testes and adrenal glands, where selective uptake of HDL-CE is most prevalent. Previous studies have shown that SR-BI oligomerizes with itself in steroidogenic tissues as well as in diverse cell lines. In the present study, we provide further evidence for the homo-oligomerization of SR-BI. We show by FPLC and blue native PAGE that SR-BI forms complexes whose sizes suggest the formation of monomers, dimers, and tetramers. Interestingly, homo-oligomerization occurs even with the absence of SR-BI's C-terminal cytoplasmic domain. Finally, we report that an inhibitor of SR-BI-mediated cholesterol transport, BLT-1, and mutations in the putative leucine zipper region of SR-BI have profound effects on SR-BI function, however, they do not affect receptor self-association. These observations indicate that SR-BI homo-oligomerization occurs even when the receptor is non-functional.     

The farnesoid X-receptor is an essential regulator of cholesterol homeostasis

To address the importance of the farnesoid X-receptor (FXR; NR1H4) for normal cholesterol homeostasis, we evaluated the major pathways of cholesterol metabolism in the FXR-deficient (-/-) mouse model. Compared with wild-type, FXR(-/-) mice have increased plasma high density lipoprotein (HDL) cholesterol and a markedly reduced rate of plasma HDL cholesterol ester clearance. Concomitantly, FXR(-/-) mice exhibit reduced expression of hepatic genes involved in reverse cholesterol transport, most notably, that for scavenger receptor BI. FXR(-/-) mice also have increased: (i) plasma non-HDL cholesterol and triglyceride levels, (ii) apolipoprotein B-containing lipoprotein synthesis, and (iii) intestinal cholesterol absorption. Surprisingly, biliary cholesterol elimination was increased in FXR(-/-) mice, despite decreased expression of hepatic genes thought to be involved in this process. These data demonstrate that FXR is a critical regulator of normal cholesterol metabolism and that genetic changes affecting FXR function have the potential to be pro-atherogenic.