Scavenger receptor class B type I is solely responsible for the selective uptake of cholesteryl esters from HDL by the liver and the adrenals in mice

Scavenger receptor class B type I (SR-BI) has been identified as a functional HDL binding protein that can mediate the selective uptake of cholesteryl ester (CE) from HDL. To quantify the in vivo role of SR-BI in the process of selective uptake, HDL was labeled with cholesteryl ether ([(3)H] CEt-HDL) and (125)I-tyramine cellobiose ([(125)I]TC-HDL) and injected into SR-BI knockout (KO) and wild-type (WT) mice. In SR-BI KO mice, the clearance of HDL-CE from the blood circulation was greatly diminished (0.043 +/- 0.004 pools/h for SR-BI KO mice vs. 0.106 +/- 0.004 pools/h for WT mice), while liver and adrenal uptake were greatly reduced. Utilization of double-labeled HDL ([(3)H]CEt and [(125)I]TC) indicated the total absence in vivo of the selective decay and liver uptake of CE from HDL in SR-BI KO mice. Parenchymal cells isolated from SR-BI KO mice showed similar association values for [(3)H]CEt and [(125)I]TC in contrast to WT cells, indicating that in parenchymal liver cells SR-BI is the only molecule exerting selective CE uptake from HDL. Thus, in vivo and in vitro, SR-BI is the sole molecule mediating the selective uptake of CE from HDL by the liver and the adrenals, making it the unique target to modulate reverse cholesterol transport.     

Cholesteryl ester transfer protein corrects dysfunctional high density lipoproteins and reduces aortic atherosclerosis in lecithin cholesterol acyltransferase transgenic mice

Expression of human lecithin cholesterol acyltransferase (LCAT) in mice (LCAT-Tg) leads to increased high density lipoprotein (HDL) cholesterol levels but paradoxically, enhanced atherosclerosis. We have hypothesized that the absence of cholesteryl ester transfer protein (CETP) in LCAT-Tg mice facilitates the accumulation of dysfunctional HDL leading to impaired reverse cholesterol transport and the development of a pro-atherogenic state. To test this hypothesis we cross-bred LCAT-Tg with CETP-Tg mice. On both regular chow and high fat, high cholesterol diets, expression of CETP in LCAT-Tg mice reduced total cholesterol (-39% and -13%, respectively; p < 0.05), reflecting a decrease in HDL cholesterol levels. CETP normalized both the plasma clearance of [(3)H]cholesteryl esters ([(3)H]CE) from HDL (fractional catabolic rate in days(-1): LCAT-Tg = 3.7 +/- 0.34, LCATxCETP-Tg = 6.1 +/- 0.16, and controls = 6.4 +/- 0.16) as well as the liver uptake of [(3)H]CE from HDL (LCAT-Tg = 36%, LCATxCETP-Tg = 65%, and controls = 63%) in LCAT-Tg mice. On the pro-atherogenic diet the mean aortic lesion area was reduced by 41% in LCATxCETP-Tg (21.2 +/- 2.0 micrometer(2) x 10(3)) compared with LCAT-Tg mice (35.7 +/- 2.0 micrometer(2) x 10(3); p < 0.001). Adenovirus-mediated expression of scavenger receptor class B (SR-BI) failed to normalize the plasma clearance and liver uptake of [(3)H]CE from LCAT-Tg HDL. Thus, the ability of SR-BI to facilitate the selective uptake of CE from LCAT-Tg HDL is impaired, indicating a potential mechanism leading to impaired reverse cholesterol transport and atherosclerosis in these animals. We conclude that CETP expression reduces atherosclerosis in LCAT-Tg mice by restoring the functional properties of LCAT-Tg mouse HDL and promoting the hepatic uptake of HDL-CE. These findings provide definitive in vivo evidence supporting the proposed anti-atherogenic role of CETP in facilitating HDL-mediated reverse cholesterol transport and demonstrate that CETP expression is beneficial in pro-atherogenic states that result from impaired reverse cholesterol transport.     

A role for hepatic scavenger receptor class B, type I in decreasing high density lipoprotein levels in mice that lack phosphatidylethanolamine N-methyltransferase

Phosphatidylethanolamine N-methyltransferase (PEMT) is a liver-specific enzyme that converts phosphatidylethanolamine to phosphatidylcholine (PC). Mice that lack PEMT have reduced plasma levels of PC and cholesterol in high density lipoproteins (HDL). We have investigated the mechanism responsible for this reduction with experiments designed to distinguish between a decreased formation of HDL particles by hepatocytes or an increased hepatic uptake of HDL lipids. Therefore, we analyzed lipid efflux to apoA-I and HDL lipid uptake using primary cultured hepatocytes isolated from Pemt(+/+) and Pemt(-/-) mice. Hepatic levels of the ATP-binding cassette transporter A1 are not significantly different between Pemt genotypes. Moreover, hepatocytes isolated from Pemt(-/-) mice released cholesterol and PC into the medium as efficiently as did hepatocytes from Pemt(+/+) mice. Immunoblotting of liver homogenates showed a 1.5-fold increase in the amount of the scavenger receptor, class B, type 1 (SR-BI) in Pemt(-/-) compared with Pemt(+/+) livers. In addition, there was a 1.5-fold increase in the SR-BI-interacting protein PDZK1. Lipid uptake experiments using radiolabeled HDL particles revealed a greater uptake of [(3)H]cholesteryl ethers and [(3)H]PC by hepatocytes derived from Pemt(-/-) compared with Pemt(+/+) mice. Furthermore, we observed an increased association of [(3)H]cholesteryl ethers in livers of Pemt(-/-) compared with Pemt(+/+) mice after tail vein injection of [(3)H]HDL. These results strongly suggest that PEMT is involved in the regulation of plasma HDL levels in mice, mainly via HDL lipid uptake by SR-BI.     

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.     

Dual effects on HDL metabolism by cholesteryl ester transfer protein inhibition in HepG2 cells

Cholesteryl ester transfer protein (CETP) promotes reverse cholesterol transport via exchange of cholesteryl ester and triglyceride among lipoproteins. Here, we focused on HDL metabolism during inhibition of CETP expression by using CETP antisense oligodeoxynucleotides (ODNs) in HepG2 cells. CETP secretion was decreased by 70% in mRNA levels and by 52% in mass 20 h after ODNs against CETP were delivered to HepG2 cells. Furthermore, as a consequence of the downregulation of CETP, the expression of scavenger receptor class B type I (SR-BI), an HDL receptor, was also reduced by approximately 50% in mRNA and protein levels, whereas the apolipoprotein A-I (apoA-I) expression and secretion were increased by 30 and 92%, respectively. In a functional study, the selective uptake of (125)I-[(14)C]cholesteryl oleate-labeled HDL(3) was decreased. Cholesterol efflux to apoA-I and HDL(3) was significantly increased by 88 and 37%, respectively. Moreover, the CE levels in cells after antisense treatment were elevated by 20%, which was related to the about twofold increase of cholesterol esterification and increased acyl-CoA:cholesterol acyltransferase 1 mRNA levels. Taken together, these findings suggest that although acute suppression of CETP expression leads to an elevation in cellular cholesterol stores, apoA-I secretion, and cellular cholesterol efflux to apoA-I, the return of HDL-CE to hepatocytes via an SR-BI pathway was inhibited in vitro. Thus antisense inhibition of hepatic CETP expression manifests dual effects: namely, increased formation of HDL and suppression of catabolism of HDL-CE, probably via the SR-BI pathway.     

Scavenger receptor class B type I-mediated uptake of serum cholesterol is essential for optimal adrenal glucocorticoid production

Impaired scavenger receptor class B type I (SR-BI)-mediated uptake of HDL-cholesterol esters (HDL-CE) induces adrenal insufficiency in mice. Humans contain an alternative route of HDL-CE clearance, namely through the transfer by cholesteryl ester transfer protein (CETP) to apolipoprotein B lipoproteins for subsequent uptake via the LDL receptor. In this study, we determined whether CETP can compensate for loss of adrenal SR-BI. Transgenic expression of human CETP (CETP Tg) in SR-BI knockout (KO) mice increased adrenal HDL-CE clearance from 33–58% of the control value. SR-BI KO/CETP Tg and SR-BI KO mice displayed adrenal hypertrophy due to equally high plasma adrenocorticotropic hormone levels. Adrenal cholesterol levels and plasma corticosterone levels were 38–52% decreased in SR-BI KO mice with and without CETP expression. SR-BI KO/CETP Tg mice also failed to increase their corticosterone level after lipopolysaccharide challenge, leading to an identical >4-fold increased tumor necrosis factor-α response compared with controls. These data indicate that uptake of CE via other routes than SR-BI is not sufficient to generate the cholesterol pool needed for optimal adrenal steroidogenesis. In conclusion, we have shown that CETP-mediated transfer of HDL-CE is not able to reverse adrenal insufficiency in SR-BI knockout mice. Thus, SR-BI-mediated uptake of serum cholesterol is essential for optimal adrenal function.     

Hepatic overexpression of cholesteryl ester hydrolase enhances cholesterol elimination and in vivo reverse cholesterol transport

Neutral cholesteryl ester hydrolase (CEH)-mediated hydrolysis of cellular cholesteryl esters (CEs) is required not only to generate free cholesterol (FC) for efflux from macrophages but also to release FC from lipoprotein-delivered CE in the liver for bile acid synthesis or direct secretion into the bile. We hypothesized that hepatic expression of CEH would regulate the hydrolysis of lipoprotein-derived CE and enhance reverse cholesterol transport (RCT). Adenoviral-mediated CEH overexpression led to a significant increase in bile acid output. To assess the role of hepatic CEH in promoting flux of cholesterol from macrophages to feces, cholesterol-loaded and [(3)H]cholesterol-labeled J774 macrophages were injected intraperitoneally into mice and the appearance of [(3)H]cholesterol in gallbladder bile and feces over 48 h was quantified. Mice overexpressing CEH had significantly higher [(3)H]cholesterol radiolabel in bile and feces, and it was associated with bile acids. This CEH-mediated increased movement of [(3)H]cholesterol from macrophages to bile acids and feces was significantly attenuated in SR-BI(-/-) mice. These studies demonstrate that similar to macrophage CEH that rate-limits the first step, hepatic CEH regulates the last step of RCT by promoting the flux of cholesterol entering the liver via SR-BI and increasing hepatic bile acid output.     

Selective uptake of HDL cholesteryl esters and cholesterol efflux from mouse peritoneal macrophages independent of SR-BI

Scavenger receptor class B type I (SR-BI) mediates the selective uptake of HDL cholesteryl esters (CEs) and facilitates the efflux of unesterified cholesterol. SR-BI expression in macrophages presumably plays a role in atherosclerosis. The role of SR-BI for selective CE uptake and cholesterol efflux in macrophages was explored. Macrophages and HDL originated from wild-type (WT) or SR-BI knockout (KO; homozygous) mice. For uptake, macrophages were incubated in medium containing 125I-/3H-labeled HDL. For lipid removal, [3H]cholesterol efflux was analyzed using HDL as acceptor. Selective uptake of HDL CE ([3H]cholesteryl oleyl ether - 125I-tyramine cellobiose) was similar in WT and SR-BI KO macrophages. Radiolabeled SR-BI KO-HDL yielded a lower rate of selective uptake compared with WT-HDL in WT and SR-BI KO macrophages. Cholesterol efflux was similar in WT and SR-BI KO cells using HDL as acceptor. SR-BI KO-HDL more efficiently promoted cholesterol removal compared with WT-HDL from both types of macrophages. Macrophages selectively take up HDL CE independently of SR-BI. Additionally, in macrophages, there is substantial cholesterol efflux that is not mediated by SR-BI. Therefore, SR-BI-independent mechanisms mediate selective CE uptake and cholesterol removal. SR-BI KO-HDL is an inferior donor for selective CE uptake compared with WT-HDL, whereas SR-BI KO-HDL more efficiently promotes cholesterol efflux.     

Remodeling of HDL by CETP in vivo and by CETP and hepatic lipase in vitro results in enhanced uptake of HDL CE by cells expressing scavenger receptor B-I.

The transport of HDL cholesteryl esters (CE) from plasma to the liver involves a direct uptake pathway, mediated by hepatic scavenger receptor B-I (SR-BI), and an indirect pathway, involving the exchange of HDL CE for triglycerides (TG) of TG-rich lipoproteins by cholesteryl ester transfer protein (CETP). We carried out HDL CE turnover studies in mice expressing human CETP and/or human lecithin:cholesterol acyltransferase (LCAT) transgenes on a background of human apoA-I expression. The fractional clearance of HDL CE by the liver was delayed by LCAT transgene, while the CETP transgene increased it. However, there was no incremental transfer of HDL CE radioactivity to the TG-rich lipoprotein fraction in mice expressing CETP, suggesting increased direct removal of HDL CE in the liver. To evaluate the possibility that this might be mediated by SR-BI, HDL isolated from plasma of the different groups of transgenic mice was incubated with SR-BI transfected or control CHO cells. HDL isolated from mice expressing CETP showed a 2- to 4-fold increase in SR-BI-mediated HDL CE uptake, compared to HDL from mice lacking CETP. The addition of pure CETP to HDL in cell culture did not lead to increased selective uptake of HDL CE by cells. However, when human HDL was enriched with TG by incubation with TG-rich lipoproteins in the presence of CETP, then treated with hepatic lipase, there was a significant enhancement of HDL CE uptake. Thus, the remodeling of human HDL by CETP, involving CE;-TG interchange, followed by the action of hepatic lipase (HL), leads to the enhanced uptake of HDL CE by cellular SR-BI.These observations suggest that in animals such as humans in which both the selective uptake and CETP pathways are active, the two pathways could operate in a synergistic fashion to enhance reverse cholesterol transport.     

Selective uptake from LDL is stimulated by unsaturated fatty acids and modulated by cholesterol content in the plasma membrane: role of plasma membrane composition in regulating non-SR-BI-mediated selective lipid transfer

We previously reported that unsaturated fatty acids stimulated low-density lipoprotein (LDL) particle uptake in J774 macrophages by increasing LDL receptor activity. Since free fatty acids (FFA) also change plasma membrane properties, a putative cholesteryl ester (CE) acceptor for selective uptake (SU), we questioned the ability of FFA to modulate SU from LDL. Using [(3)H]cholesteryl ether/(125)I-LDL to trace CE core and whole particle uptake, we found that oleic acid and eicosapentaenoic acid, but not saturated stearic acid, increased SU by 30% over control levels. An ACAT inhibitor, Dup128, abolished FFA effects on SU, indicating that increased SU by FFA was secondary to changes in cell-free cholesterol (FC). Consistent with these observations, ACAT inhibition increased cell FC and reduced LDL SU by half. The important role of plasma membrane composition was further demonstrated in that beta-cyclodextrin- (beta-CD-) mediated FC removal from the plasma membrane increased SU from LDL and was further stimulated by U18666A, a compound that inhibits FC transport between lysosomes and the plasma membrane. In contrast, cholesterol-saturated beta-CD markedly reduced LDL SU. In contrast to LDL SU, oleic acid, ACAT inhibition, U18666A, or beta-CD had no effects on HDL SU. Moreover, HDL SU was inhibited by antimouse SR-BI antibody by more than 50% but had little effect on LDL SU. In C57BL/6 mice fed a high fat diet, plasma FFA levels increased, and SU accounted for an almost 4-fold increased proportion of total cholesterol delivery to the arterial wall. Taken together, these data suggest that LDL SU is mediated by pathways independent of SR-BI and is influenced by plasma membrane FC content. Moreover, in conditions where elevated plasma FFA occur, SU from LDL can be an important mechanism for cholesterol delivery in vivo.     

Streptococcal serum opacity factor increases the rate of hepatocyte uptake of human plasma high-density lipoprotein cholesterol

Serum opacity factor (SOF), a virulence determinant of Streptococcus pyogenes, converts plasma high-density lipoproteins (HDL) to three distinct species: lipid-free apolipoprotein (apo) A-I, neo HDL, a small discoidal HDL-like particle, and a large cholesteryl ester-rich microemulsion (CERM) that contains the cholesterol esters (CE) of up to ～400000 HDL particles and apo E as its major protein. Similar SOF reaction products are obtained with HDL, total plasma lipoproteins, and whole plasma. We hypothesized that hepatic uptake of CERM-CE via multiple apo E-dependent receptors would be faster than that of HDL-CE. We tested our hypothesis using human hepatoma cells and lipoprotein receptor-specific Chinese hamster ovary (CHO) cells. The uptake of [(3)H]CE by HepG2 and Huh7 cells from HDL after SOF treatment, which transfers >90% of HDL-CE to CERM, was 2.4 and 4.5 times faster, respectively, than from control HDL. CERM-[(3)H]CE uptake was inhibited by LDL and HDL, suggestive of uptake by both the LDL receptor (LDL-R) and scavenger receptor class B type I (SR-BI). Studies in CHO cells specifically expressing LDL-R and SR-BI confirmed CERM-[(3)H]CE uptake by both receptors. RAP and heparin inhibit CERM-[(3)H]CE but not HDL-[(3)H]CE uptake, thereby implicating LRP-1 and cell surface proteoglycans in this process. These data demonstrate that SOF treatment of HDL increases the rate of CE uptake via multiple hepatic apo E receptors. In so doing, SOF might increase the level of hepatic disposal of plasma cholesterol in a way that is therapeutically useful.     

Scavenger receptor class B, type I-mediated uptake of various lipids into cells. Influence of the nature of the donor particle interaction with the receptor

Scavenger receptor (SR)-BI is the first molecularly defined receptor for high density lipoprotein (HDL) and it can mediate the selective uptake of cholesteryl ester into cells. To elucidate the molecular mechanisms by which SR-BI facilitates lipid uptake, we examined the connection between lipid donor particle binding and lipid uptake using kidney COS-7 cells transiently transfected with SR-BI. We systematically compared the uptake of [(3)H]cholesteryl oleoyl ether (CE) and [(14)C]sphingomyelin (SM) from apolipoprotein (apo) A-I-containing reconstituted HDL (rHDL) particles and apo-free lipid donor particles. Although both types of lipid donor could bind to SR-BI, only apo-containing lipid donors exhibited preferential delivery of CE over SM (i.e. nonstoichiometric lipid uptake). In contrast, apo-free lipid donor particles (phospholipid unilamellar vesicles, lipid emulsion particles) gave rise to stoichiometric lipid uptake due to interaction with SR-BI. This apparent whole particle uptake was not due to endocytosis, but rather fusion of the lipid components of the lipid donor with the cell plasma membrane; this process is perhaps mediated by a fusogenic motif in the extracellular domain of SR-BI. The interaction of apoA-I with SR-BI not only prevents fusion of the lipid donor with the plasma membrane but also allows the optimal selective lipid uptake. A comparison of rHDL particles containing apoA-I and apoE-3 showed that while both particles bound equally well to SR-BI, the apoA-I particle gave approximately 2-fold greater CE selective uptake. Catabolism of all major HDL lipids can occur via SR-BI with the relative selective uptake rate constants for CE, free cholesterol, triglycerides (triolein), and phosphatidylcholine being 1, 1.6, 0.7, and 0.2, respectively. It follows that a putative nonpolar channel created by SR-BI between the bound HDL particle and the cell plasma membrane is better able to accommodate the uptake of neutral lipids (e.g. cholesterol) relative to polar phospholipids.     

Changes in plasma membrane properties and phosphatidylcholine subspecies of insect Sf9 cells due to expression of scavenger receptor class B, type I, and CD36

In mammalian cells scavenger receptor class B, type I (SR-BI), mediates the selective uptake of high density lipoprotein (HDL) cholesteryl ester into hepatic and steroidogenic cells. In addition, SR-BI has a variety of effects on plasma membrane properties including stimulation of the bidirectional flux of free cholesterol (FC) between cells and HDL and changes in the organization of plasma membrane FC as indicated by increased susceptibility to exogenous cholesterol oxidase. Recent studies in SR-BI-deficient mice and in SR-BI-expressing Sf9 insect cells showed that SR-BI has significant effects on plasma membrane ultrastructure. The present study was designed to test the range of SR-BI effects in Sf9 insect cells that typically have very low cholesterol content and a different phospholipid profile compared with mammalian cells. The results showed that, as in mammalian cells, SR-BI expression increased HDL cholesteryl ester selective uptake, cellular cholesterol mass, FC efflux to HDL, and the sensitivity of membrane FC to cholesterol oxidase. These activities were diminished or absent upon expression of the related scavenger receptor CD36. Thus, SR-BI has fundamental effects on cholesterol flux and membrane properties that occur in cells of evolutionarily divergent origins. Profiling of phospholipid species by electrospray ionization mass spectrometry showed that scavenger receptor expression led to the accumulation of phosphatidylcholine species with longer mono- or polyunsaturated acyl chains. These changes would be expected to decrease phosphatidylcholine/cholesterol interactions and thereby enhance cholesterol desorption from the membrane. Scavenger receptor-mediated changes in membrane phosphatidylcholine may contribute to the increased flux of cholesterol and other lipids elicited by these receptors.     

Susceptibility to murine cholesterol gallstone formation is not affected by partial disruption of the HDL receptor SR-BI

High density lipoprotein (HDL) promotes reverse cholesterol transport from peripheral tissues to the liver where its cholesterol is secreted preferentially into bile. The scavenger receptor class B type I (SR-BI) is believed to play a pivotal role in unloading HDL cholesterol and its ester to hepatocytes. Here, using male SR-BI "att" mice with a dysfunctional mutation in the Sr-b1 promoter, we studied whether approximately 50% of normal SR-BI expression influences gallstone susceptibility in these mice fed a lithogenic diet containing 1% cholesterol, 0.5% cholic acid and 15% butterfat. Our results showed that the disruption of SR-BI expression reduced cholesterol secretion by 37% in the chow-fed state and 10% on the lithogenic diet, and while delaying incidence slightly, did not influence cumulative susceptibility to cholesterol gallstones. The lithogenic diet induced marked increases in biliary cholesterol and phospholipid secretion rates but not of bile salts. Basal expression of hepatic SR-BI protein was dissimilar in both wild-type and SR-BI mice, and remained unaltered in response to the lithogenic diet. By two independent dual isotope methods, intestinal cholesterol absorption was unimpaired by attenuation of the SR-BI which also displays low-density expression on small intestinal enterocytes. We conclude that although HDL cholesterol is a principal source of biliary cholesterol in the basal state, uptake of cholesterol from chylomicron remnants appears to be the major contributor to biliary cholesterol hypersecretion during diet-induced cholelithogenesis in the mouse.     

SR-BI inhibits ABCG1-stimulated net cholesterol efflux from cells to plasma HDL

This study compares the roles of ABCG1 and scavenger receptor class B type I (SR-BI) singly or together in promoting net cellular cholesterol efflux to plasma HDL containing active LCAT. In transfected cells, SR-BI promoted free cholesterol efflux to HDL, but this was offset by an increased uptake of HDL cholesteryl ester (CE) into cells, resulting in no net efflux. Coexpression of SR-BI with ABCG1 inhibited the ABCG1-mediated net cholesterol efflux to HDL, apparently by promoting the reuptake of CE from medium. However, ABCG1-mediated cholesterol efflux was not altered in cholesterol-loaded, SR-BI-deficient (SR-BI(-/-)) macrophages. Briefly cultured macrophages collected from SR-BI(-/-) mice loaded with acetylated LDL in the peritoneal cavity did exhibit reduced efflux to HDL. However, this was attributable to reduced expression of ABCG1 and ABCA1, likely reflecting increased macrophage cholesterol efflux to apolipoprotein E-enriched HDL during loading in SR-BI(-/-) mice. In conclusion, cellular SR-BI does not promote net cholesterol efflux from cells to plasma HDL containing active LCAT as a result of the reuptake of HDL-CE into cells. Previous findings of increased atherosclerosis in mice transplanted with SR-BI(-/-) bone marrow probably cannot be explained by a defect in macrophage cholesterol efflux.     

Oxidized low density lipoprotein decreases macrophage expression of scavenger receptor B-I

Scavenger receptor class B type I (SR-BI) has recently been identified as a high density lipoprotein (HDL) receptor that mediates bidirectional flux of cholesterol across the plasma membrane. We have previously demonstrated that oxidized low density lipoprotein (OxLDL) will increase expression of another class B scavenger receptor, CD36 (Han, J., Hajjar, D. P., Febbraio, M., and Nicholson, A. C. (1997) J. Biol. Chem. 272, 21654-21659). In studies reported herein, we evaluated the effects of OxLDL on expression of SR-BI in macrophages to determine how exposure to this modified lipoprotein could alter SR-BI expression and cellular lipid flux. OxLDL decreased SR-BI expression in a dose- and time-dependent manner. Incubation with OxLDL had no effect on the membrane distribution of SB-BI, and it decreased expression of both cytosolic and membrane protein. Consistent with its effect on SR-BI protein expression, OxLDL decreased SR-BI mRNA in a dose-dependent manner. The ability of OxLDL to decrease SR-BI expression was dependent on the degree of LDL oxidation. OxLDL decreased both [(14)C]cholesteryl oleate/HDL uptake and efflux of [(14)C]cholesterol to HDL in a time-dependent manner. Incubation of macrophages with 7-ketocholesterol, but not free cholesterol, also inhibited expression of SR-BI. Finally, we demonstrate that the effect of OxLDL on SR-BI is dependent on the differentiation state of the monocyte/macrophage. These results imply that in addition to its effect in inducing foam cell formation in macrophages through increased uptake of oxidized lipids, OxLDL may also enhance foam cell formation by altering SR-BI-mediated lipid flux across the cell membrane.     

Scavenger receptor class B type I-mediated cholesteryl ester-selective uptake and efflux of unesterified cholesterol. Influence of high density lipoprotein size and structure

Scavenger receptor (SR)-BI catalyzes the selective uptake of cholesteryl ester (CE) from high density lipoprotein (HDL) by a two-step process that involves the following: 1) binding of HDL to the receptor and 2) diffusion of the CE molecules into the cell plasma membrane. We examined the effects of the size of discoidal HDL particles containing wild-type (WT) apoA-I on selective uptake of CE and efflux of cellular free (unesterified) cholesterol (FC) from COS-7 cells expressing SR-BI to determine the following: 1) the influence of apoA-I conformation on the lipid transfer process, and 2) the contribution of receptor binding-dependent processes to the overall efflux of cellular FC. Large (10 nm diameter) reconstituted HDL bound to SR-BI better (B(max) approximately 420 versus 220 ng of apoA-I/mg cell protein), delivered more CE, and promoted more FC efflux than small ( approximately 8 nm) particles. When normalized to the number of reconstituted HDL particles bound to the receptor, the efficiencies of either CE uptake or FC efflux with these particles were the same indicating that altering the conformation of WT apoA-I modulates binding to the receptor (step 1) but does not change the efficiency of the subsequent lipid transfer (step 2); this implies that binding induces an optimal alignment of the WT apoA-I.SR-BI complex so that the efficiency of lipid transfer is always the same. FC efflux to HDL is affected both by binding of HDL to SR-BI and by the ability of the receptor to perturb the packing of FC molecules in the cell plasma membrane.     

Extracellular disulfide bonds support scavenger receptor class B type I-mediated cholesterol transport

Scavenger receptor class B type I (SR-BI) binds high-density lipoprotein (HDL) and mediates the selective uptake of cholesteryl esters (CE). Although the extracellular domain of SR-BI is critical for function, the structural characteristics of this region remain elusive. Using sulfhydryl labeling strategies, we report the novel finding that all six cysteine (Cys) residues in the extracellular domain of SR-BI are involved in disulfide bond formation that is intramolecular by nature. We hypothesized that an SR-BI conformation stabilized by extracellular disulfide bonds is a prerequisite for SR-BI-mediated cholesterol transport. Thus, single-Cys mutant SR-BI receptors (C251S-, C280S-, C321S-, C323S-, C334S-, and C384S-SR-BI), as well as Cys-less SR-BI, a mutant SR-BI receptor void of all Cys residues, were created, and plasma membrane localization was confirmed. Functional assays revealed that C280S-, C321S-, C323S-, and C334S-SR-BI and Cys-less SR-BI mutant receptors displayed weakened HDL binding and subsequent selective uptake of HDL-CE. However, only C323S-SR-BI and Cys-less SR-BI were unable to mediate wild-type levels of efflux of free cholesterol (FC) to HDL. None of the Cys mutations disrupted SR-BI's ability to redistribute plasma membrane FC. Taken together, the intramolecular disulfide bonds in the extracellular domain of SR-BI appear to maintain the receptor in a conformation integral to its cholesterol transport functions.     

Impaired capacity of acute-phase high density lipoprotein particles to deliver cholesteryl ester to the human HUH-7 hepatoma cell line

The major role of native high density lipoprotein (HDL) is to carry cholesterol from peripheral tissues to the liver for bile excretion. As acute-phase (AP)-HDL has a decreased ability for cellular cholesterol efflux but an increased capacity for cholesteryl ester (CE) delivery to peripheral tissues, the interaction of AP-HDL with human hepatoma cells was studied. Binding studies to HUH-7 cells revealed saturable binding properties for HDL and AP-HDL at 4 degrees C. At 37 degrees C, specific cell-association of (125)I- and [1,2,6,7-(3)H]-cholesteryl palmitate ([(3)H]CE)-labeled lipoprotein particles was 2.2- and 1.6-fold higher for HDL indicating that total CE delivery was significantly (P<0.05) higher for HDL in comparison to AP-HDL. In parallel, selective CE uptake (the difference between total lipid uptake and holoparticle uptake) from AP-HDL was decreased compared with HDL. The fact that the capacity for cellular cholesterol efflux from HUH-7 cells is slightly impaired by AP-HDL (compared with HDL) is of support that scavenger receptor class B, type I (SR-BI), the only receptor so far known to mediate bi-directional lipid flux, might be involved in altered HUH-7 cholesterol hemostasis by AP-HDL. Our in vitro findings suggest that HDL and AP-HDL interact differently with cells of hepatic origin resulting in decreased hepatic cholesterol removal from the circulation during the AP reaction.