Hepatic lipase promotes the selective uptake of high density lipoprotein-cholesteryl esters via the scavenger receptor B1.

Hepatic lipase (HL) plays a major role in high-density lipoprotein (HDL) metabolism both as a lipolytic enzyme and as a ligand. To investigate whether HL enhances the uptake of HDL-cholesteryl ester (CE) via the newly described scavenger receptor BI (SR-BI), we measured the effects of expressing HL and SR-BI on HDL-cell association as well as uptake of 125I-labeled apoA-I and [3H]CE-HDL, by embryonal kidney 293 cells. As expected, HDL cell association and CE selective uptake were increased in SR-BI transfected cells by 2- and 4-fold, respectively, compared to controls (P < 0.001). Cells transfected with HL alone or in combination with SR-BI expressed similar amounts of HL, 20% of which was bound to cell surface proteoglycans. HL alone increased HDL cell association by 2-fold but had no effect on HDL-CE uptake in 293 cells. However, in cells expressing SR-BI, HL further enhanced the selective uptake of CE from HDL by 3-fold (P < 0.001). To determine whether the lipolytic and/or ligand function of HL are required in this process, we generated a catalytically inactive form of HL (HL-145G). Cells co-transfected with HL-145G and SR-BI increased their HDL cell association and HDL-CE selective uptake by 1.4-fold compared to cells expressing SR-BI only (P < 0.03). Heparin abolished the effect of HL-145G on SR-BI-mediated HDL-CE selective uptake.Thus, the enhanced uptake of HDL-CE by HL is mediated by both its ligand role, which requires interaction with proteoglycans, and by lipolysis with subsequent HDL particle remodeling. These results establish HL as a major modulator of SR-BI mediated selective uptake of HDL-CE.     

Expression and microvillar localization of scavenger receptor class B, type I (SR-BI) and selective cholesteryl ester uptake in Leydig cells from rat testis

This study investigates the relationship between the high density lipoprotein (HDL) receptor (scavenger receptors, SR-BI and SR-BII), selective lipoprotein-cholesteryl ester uptake, and testosterone production in Leydig cells of control, hypocholesterolemic and gonadotrophic hormone (hCG) treated rats. Leydig cells from mature control rats show poor efficiency in incorporation of labeled HDL-cholesteryl esters into testosterone, poor selective uptake of lipoprotein lipids overall, and a dramatic reduction of circulating levels of lipoproteins has no apparent effect on testosterone production or expression of intracellular enzymes synthesizing cholesterol. Leydig cells from control rats show minimal levels of SR-BI and SR-BII. However, similarly aged rats treated with hCG for several days undergo changes consistent with hormone-desensitization. Despite the resulting low levels of testosterone production, SR-BI levels are dramatically increased, Leydig cells now efficiently internalize HDL-supplied cholesteryl esters by the selective cholesterol uptake process, and various other cholesterol-sensitive genes of the cells are up-regulated. Only SR-BII expression remains negligible and unchanged throughout this period. It is of interest that Leydig cell SR-BI of hCG-treated rats is localized in surface microvilli, but is present also in an elaborate and complex channel system within the cytoplasm of the cells. In summary, Leydig cells differ from other rat steroidogenic cells in not depending on exogenous lipoprotein-cholesterol during periods of normal steroid hormone production. However, trophic hormone desensitization is accompanied by increased Leydig cell SR-BI expression and increased selective HDL-cholesteryl ester uptake, presumably in preparation for renewed testosterone production.     

The identification of intestinal scavenger receptor class B, type I (SR-BI) by expression cloning and its role in cholesterol absorption

The molecular mechanisms of cholesterol absorption in the intestine are poorly understood. With the goal of defining candidate genes involved in these processes a fluorescence-activated cell sorter-based, retroviral-mediated expression cloning strategy has been devised. SCH354909, a fluorescent derivative of ezetimibe, a compound which blocks intestinal cholesterol absorption but whose mechanism of action is unknown, was synthesized and shown to block intestinal cholesterol absorption in rats. Pools of cDNAs prepared from rat intestinal cells enriched in enterocytes were introduced into BW5147 cells and screened for SCH354909 binding. Several independent clones were isolated and all found to encode the scavenger receptor class B, type I (SR-BI), a protein suggested by others to play a role in cholesterol absorption. SCH354909 bound to Chinese hamster ovary (CHO) cells expressing SR-BI in specific and saturable fashion and with high affinity (K(d) approximately 18 nM). Overexpression of SR-BI in CHO cells resulted in increased cholesterol uptake that was blocked by micromolar concentrations of ezetimibe. Analysis of rat intestinal sections by in situ hybridization demonstrated that SR-BI expression was restricted to enterocytes. Cholesterol absorption was determined in SR-B1 knockout mice using both an acute, 2-h, assay and a more chronic fecal dual isotope ratio method. The level of intestinal cholesterol uptake and absorption was similar to that seen in wild-type mice. When assayed in the SR-B1 knockout mice, the dose of ezetimibe required to inhibit hepatic cholesterol accumulation induced by a cholesterol-containing 'western' diet was similar to wild-type mice. Thus, the binding of ezetimibe to cells expressing SR-B1 and the functional blockade of SR-B1-mediated cholesterol absorption in vitro suggest that SR-B1 plays a role in intestinal cholesterol metabolism and the inhibitory activity of ezetimibe. In contrast studies with SR-B1 knockout mice suggest that SR-B1 is not essential for intestinal cholesterol absorption or the activity of ezetimibe.     

Elevated triglyceride content diminishes the capacity of high density lipoprotein to deliver cholesteryl esters via the scavenger receptor class B type I (SR-BI)

The selective uptake of high density lipoprotein (HDL) cholesteryl ester (CE) by the scavenger receptor class B type I (SR-BI) is well documented. However, the effect of altered HDL composition, such as occurs in hyperlipidemia, on this important process is not known. This study investigated the impact of variable CE and triglyceride (TG) content on selective uptake. CE selective uptake by Y1 and HepG2 cells was strongly affected by modification of either the CE or TG content of HDL. Importantly, TG, like CE, was selectively taken up by a dose-dependent, saturable process in these cells. As shown by ACTH up-regulation and receptor overexpression experiments, SR-BI mediated the selective uptake of both CE and TG. With in vitro modified HDLs of varying CE and TG composition, the selective uptake of CE and TG was dependent on the abundance of each lipid within the HDL particle. Furthermore, total selective uptake (CE + TG) remained constant, indicating that these lipids competed for cellular uptake. These data support a novel mechanism whereby SR-BI binds HDL and mediates the incorporation of a nonspecific portion of the HDL lipid core. In this way, TG directly affects the ability of HDL to donate CE to cells. Processes that raise the TG/CE ratio of HDL will impair the delivery of CE to cells via this receptor and may compromise the efficiency of sterol balancing pathways such as reverse cholesterol transport.     

The expression of scavenger receptor class B,type I (SR-BI) and caveolin-1 in parenchymal and nonparenchymal liver cells

The liver is the major site of cholesterol synthesis and metabolism, and the only substantive route for eliminating blood cholesterol. Scavenger receptor class B, type I (SR-BI) has been reported to be responsible for mediating the selective uptake of high-density lipoprotein cholesteryl esters (HDL-CE) in liver parenchymal cells (PC). We analysed the expression of SR-BI in isolated rat liver cells, and found the receptor to be highly expressed in liver PC at both the mRNA and protein levels. We also found SR-BI to be expressed in liver endothelial cells (LEC) and Kupffer cells (KC). SR-BI has not previously been reported to be present in LEC. CD36 mRNA was expressed in all three liver cell types. Since caveolin-1 appears to colocalize with SR-BI and CD36 in caveolae of several cell lines, the distribution and expression of caveolin-1 in the liver cells were investigated. Caveolin-1 was not detected in PC but was found in both LEC and KC. This led to the suggestion that caveolin-1 may be more important in the efflux of cholesterol than in the selective uptake of cholesterol in the liver.     

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

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.     

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

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.     

Lipid free apolipoprotein E binds to the class B Type I scavenger receptor I (SR-BI) and enhances cholesteryl ester uptake from lipoproteins

The Class B type I scavenger receptor I (SR-BI) is a physiologically relevant high density lipoprotein (HDL) receptor that can mediate selective cholesteryl ester (CE) uptake by cells. Direct interaction of apolipoprotein E (apoE) with this receptor has never been demonstrated, and its implication in CE uptake is still controversial. By using a human adrenal cell line (NCI-H295R), we have addressed the role of apoE in binding to SR-BI and in selective CE uptake from lipoproteins to cells. This cell line does not secrete apoE and SR-BI is its major HDL-binding protein. We can now provide evidence that 1) free apoE is a ligand for SR-BI, 2) apoE associated to lipids or in lipoproteins does not modulate binding or CE-selective uptake by the SR-BI pathway, and 3) the direct interaction of free apoE to SR-BI leads to an increase in CE uptake from lipoproteins of both low and high densities. We propose that this direct interaction could modify SR-BI structure in cell membranes and potentiate CE uptake.     

The class B, type I scavenger receptor promotes the selective uptake of high density lipoprotein cholesterol ethers into caveolae

The uptake of cholesterol esters from high density lipoproteins (HDLs) is characterized by the initial movement of cholesterol esters into a reversible plasma membrane pool. Cholesterol esters are subsequently internalized to a nonreversible pool. Unlike the uptake of cholesterol from low density lipoproteins, cholesterol ester uptake from HDL does not involve the internalization and degradation of the particle and is therefore termed selective. The class B, type I scavenger receptor (SR-BI) has been identified as an HDL receptor and shown to mediate selective cholesterol ester uptake. SR-BI is localized to cholesterol- and sphingomyelin-rich microdomains called caveolae. Caveolae are directly involved in cholesterol trafficking. Therefore, we tested the hypothesis that caveolae are acceptors for HDL-derived cholesterol ether (CE). Our studies demonstrate that in Chinese hamster ovary cells expressing SR-BI, >80% of the plasma membrane associated CE is present in caveolae after 7.5 min of selective cholesterol ether uptake. We also show that excess, unlabeled HDL can extract the radiolabeled CE from caveolae, demonstrating that caveolae constitute a reversible plasma membrane pool of CE. Furthermore, 50% of the caveolae-associated CE can be chased into a nonreversible pool. We conclude that caveolae are acceptors for HDL-derived cholesterol ethers, and that caveolae constitute a reversible, plasma membrane pool of cholesterol ethers.     

The cellular biology of scavenger receptor class B type I

The HDL receptor scavenger receptor class B type I plays an important role in meditating the uptake of HDL-derived cholesterol and cholesteryl ester in the liver and steroidogenic tissues. However, the mechanism by which scavenger receptor class B type I mediates selective cholesterol uptake is unclear. In hepatocytes scavenger receptor class B type I mediates the transcytosis of cholesterol into bile, appears to be expressed on both basolateral and apical membranes, and directly interacts with a PDZ domain containing protein that may modulate the activity of scavenger receptor class B type I. This suggests the involvement of scavenger receptor class B type I in higher order complexes in polarized cells. Scavenger receptor class B type I expression has been shown to alter plasma membrane cholesterol distribution and induce the formation of novel membrane structures, suggesting multiple roles for scavenger receptor class B type I in the cell. A close examination of scavenger receptor class B type I function in polarized cells may yield new insights into the mechanism of scavenger receptor class B type I-mediated HDL selective uptake and the effects of scavenger receptor class B type I on cellular cholesterol homeostasis.     

Mechanism of scavenger receptor class B type I-mediated selective uptake of cholesteryl esters from high density lipoprotein to adrenal cells.

Despite extensive studies and characterizations of the high density lipoprotein-cholesteryl ester (HDL-CE)-selective uptake pathway, the mechanisms by which the hydrophobic CE molecules are transferred from the HDL particle to the plasma membrane have remained elusive, until the discovery that scavenger receptor BI (SR-BI) plays an important role. To elucidate the molecular mechanism, we examined the quantitative relationships between the binding of HDL and the selective uptake of its CE in the murine adrenal Y1-BS1 cell line. A comparison of concentration dependences shows that half-maximal high affinity cell association of HDL occurs at 8.7 +/- 4.7 micrograms/ml and the Km of HDL-CE-selective uptake is 4.5 +/- 1.5 micrograms/ml. These values are similar, and there is a very high correlation between these two processes (r2 = 0.98), suggesting that they are linked. An examination of lipid uptake from reconstituted HDL particles of defined composition and size shows that there is a non-stoichiometric uptake of HDL lipid components, with CE being preferred over the major HDL phospholipids, phosphatidylcholine and sphingomyelin. Comparison of the rates of selective uptake of different classes of phospholipid in this system gives the ranking: phosphatidylserine > phosphatidylcholine approximately phosphatidylinositol > sphingomyelin. The rate of CE-selective uptake from donor particles is proportional to the amount of CE initially present in the particles, suggesting a mechanism in which CE moves down its concentration gradient from HDL particles docked on SR-BI into the cell plasma membrane. The activation energy for CE uptake from either HDL3 or reconstituted HDL is about 9 kcal/mol, indicating that HDL-CE uptake occurs via a non-aqueous pathway. HDL binding to SR-BI allows access of CE molecules to a "channel" formed by the receptor from which water is excluded and along which HDL-CE molecules move down their concentration gradient into the cell plasma membrane.     

Polyunsaturated fatty acids up-regulate hepatic scavenger receptor B1 (SR-BI) expression and HDL cholesteryl ester uptake in the hamster.

Diets rich in polyunsaturated fatty acids lower plasma HDL cholesterol concentrations when compared to diets rich in saturated fatty acids. We investigated the mechanistic basis for this effect in the hamster and sought to determine whether reduced plasma HDL cholesterol concentrations resulting from a high polyunsaturated fat diet are associated with a decrease in reverse cholesterol transport. Animals were fed semisynthetic diets enriched with polyunsaturated or saturated fatty acids for 6 weeks. We then determined the effect of these diets on the following parameters: 1) hepatic scavenger receptor B1 (SR-BI) mRNA and protein levels, 2) the rate of hepatic HDL cholesteryl ester uptake, and 3) the rate of cholesterol acquisition by the extrahepatic tissues (from de novo synthesis, LDL and HDL) as a measure of the rate of reverse cholesterol transport. Compared to saturated fatty acids, dietary polyunsaturated fatty acids up-regulated hepatic SR-BI expression by approximately 50% and increased HDL cholesteryl ester transport to the liver; as a consequence, plasma HDL cholesteryl ester concentrations were reduced. Although dietary polyunsaturated fatty acids increased hepatic HDL cholesteryl ester uptake and lowered plasma HDL cholesterol concentrations, there was no change in the cholesterol content or in the rate of cholesterol acquisition (via de novo synthesis and lipoprotein uptake) by the extrahepatic tissues.These studies indicate that substitution of polyunsaturated for saturated fatty acids in the diet increases SR-BI expression and lowers plasma HDL cholesteryl ester concentrations but does not affect reverse cholesterol transport.     

Sterol regulation of scavenger receptor class B type I in macrophages

Scavenger receptor class B type I (SR-BI) is expressed in macrophages, but its role in sterol trafficking in these cells remains controversial. We examined the effect of sterol loading on SR-BI expression in human monocytes/macrophages, mouse peritoneal macrophages, and a cultured mouse macrophage cell line (J774 cells). Sterol loading using either acetylated LDL or 25-hydroxycholesterol resulted in a time- and concentration-dependent decrease in SR-BI protein and mRNA levels. Treatment of lipid-loaded J774 cells with cyclodextrin or HDL to promote cellular sterol efflux was associated with an increase in SR-BI expression. Studies were performed to determine if the sterol-associated downregulation of SR-BI in macrophages was mediated by either sterol regulatory element binding proteins (SREBPs) or the liver X receptor (LXR). Expression of constitutively active SREBPs failed to alter the expression of a luciferase reporter placed downstream of a 2556 bp 5' flanking sequence from the mouse SR-BI gene. Reduction in SR-BI expression was also seen in sterol-loaded peritoneal macrophages from mice expressing no LXRalpha and LXRbeta. We conclude that SR-BI levels in macrophages are responsive to changes in intracellular sterol content and that these sterol-associated changes are not mediated by LXR and are unlikely to be mediated by an SREBP pathway.     

The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus

We discovered that the hepatitis C virus (HCV) envelope glycoprotein E2 binds to human hepatoma cell lines independently of the previously proposed HCV receptor CD81. Comparative binding studies using recombinant E2 from the most prevalent 1a and 1b genotypes revealed that E2 recognition by hepatoma cells is independent from the viral isolate, while E2-CD81 interaction is isolate specific. Binding of soluble E2 to human hepatoma cells was impaired by deletion of the hypervariable region 1 (HVR1), but the wild-type phenotype was recovered by introducing a compensatory mutation reported previously to rescue infectivity of an HVR1-deleted HCV infectious clone. We have identified the receptor responsible for E2 binding to human hepatic cells as the human scavenger receptor class B type I (SR-BI). E2-SR-BI interaction is very selective since neither mouse SR-BI nor the closely related human scavenger receptor CD36, were able to bind E2. Finally, E2 recognition by SR-BI was competed out in an isolate-specific manner both on the hepatoma cell line and on the human SR-BI-transfected cell line by an anti-HVR1 monoclonal antibody.