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A positive correlation between plasma levels of HDL and bone mass has been reported by epidemiological studies. As scavenger receptor class B, type I (SR-BI), the gene product of Scarb1, is known to regulate HDL metabolism, we recently characterized bone metabolism in Scarb1-null mice. These mice display high femoral bone mass associated with enhanced bone formation. As gender differences have been reported in HDL metabolism and SR-BI function, we investigated gender-specific bone alterations in Scarb1-null mice by microtomography and histology. We found 16% greater relative bone volume and 39% higher bone formation rate in the vertebrae from 2-month-old Scarb1-null females. No such alteration was seen in males, indicating gender- and region-specific differences in skeletal phenotype. Total and HDL-associated cholesterol levels, as well as ACTH plasma levels, were increased in both Scarb1-null genders, the latter being concurrent to impaired corticosterone response to fasting. Plasma levels of estradiol did not differ between null and WT females, suggesting that the estrogen metabolism alteration is not relevant to the higher vertebral bone mass in female Scarb1-null mice. Constitutively, high plasma levels of leptin along with 2.5-fold increase in its expression in white adipose tissue were measured in female Scarb1-null mice only. In vitro exposure of bone marrow stromal cells to ACTH and leptin promoted osteoblast differentiation as evidenced by increased gene expression of osterix and collagen type I alpha. Our results suggest that hyperleptinemia may account for the gender-specific high bone mass seen in the vertebrae of female Scarb1-null mice., (© 2014 Society for Endocrinology.)

Scavenger receptor class B, type I (SR-BI), the Scarb1 gene product, is a receptor associated with cholesteryl ester uptake from high-density lipoproteins (HDL), which drives cholesterol movement from peripheral tissues toward the liver for excretion, and, consequently, Scarb1 null mice are prone to atherosclerosis. Because studies have linked atherosclerosis incidence with osteoporosis, we characterized the bone metabolism in these mice. Bone morphometry was assessed through microcomputed tomography and histology. Marrow stromal cells (MSCs) were used to characterize influence of endogenous SR-BI in cell functions. Total and HDL-associated cholesterol in null mice were increased by 32-60%, correlating with its role in lipoprotein metabolism. Distal metaphyses from 2- and 4-mo-old null mice showed correspondingly 46 and 37% higher bone volume fraction associated with a higher number of trabeculae. Histomorphometric analyses in 2-mo-old null male mice revealed 1.42-fold greater osteoblast surface, 1.37-fold higher percent mineralizing surface, and 1.69-fold enhanced bone formation rate. In vitro assays for MSCs from null mice revealed 37% higher proliferation rate, 48% more alkaline phosphatase activity, 70% greater mineralization potential and a 2-fold osterix (Sp7) expression, yet a 0.5-fold decrease in caveolin-1 (Cav1) expression. Selective uptake levels of HDL-associated cholesteryl oleate and estradiol were similar between MSC from wild-type and Scarb1 null mice, suggesting that its contribution to this process is not its main role in these cells. However, Scarb1 knockout stunted the HDL-dependent regulation of Cav1 genic expression. Scarb1 null mice are not prone to osteoporosis but show higher bone mass associated with enhanced bone formation.

Bone tissue is continuously remodeled by bone cells and maintenance of its mass relies on the balance between the processes of resorption and formation. We have reported the expression of numerous scavenger receptors, namely scavenger receptor (SR) class B type I and II (SR-BI and SR-BII), and CD36, in bone-forming osteoblasts but their physiological roles in bone metabolism are still unknown. To unravel the role of CD36 in bone metabolism, we determined the bone phenotype of CD36 knockout (CD36KO) mice and characterized the cell functions of osteoblasts lacking CD36. Weights of CD36KO mice were significantly lower than corresponding wild-type (WT) mice, yet no significant difference was found in femoral nor tibial length between CD36KO and WT mice. Analysis of bone architecture by micro-computed tomography revealed a low bone mass phenotype in CD36KO mice of both genders. Femoral trabecular bone from 1 to 6 month-old CD36KO mice showed lower bone volume, higher trabecular separation and reduced trabeculae number compared to WT mice; similar alterations were noticed for lumbar vertebrae. Plasma levels of osteocalcin (OCN) and N-terminal propeptide of type I procollagen (PINP), two known markers of bone formation, were significantly lower in CD36KO mice than in WT mice, whereas plasma levels of bone resorption markers were similar. Accordingly, histology highlighted lower osteoblast perimeter and reduced bone formation rate. In vitro functional characterization of bone marrow stromal cells and osteoblasts isolated from CD36KO mice showed reduced cell culture expansion and survival, lower gene expression of osteoblastic Runt-related transcription factor 2 (Runx2) and osterix (Osx), as well as bone sialoprotein (BSP) and osteocalcin (OCN). Our results indicate that CD36 is mandatory for adequate bone metabolism, playing a role in osteoblast functions ensuring adequate bone formation.

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 HDL(3). 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.

Plasma cholesterol from low- and high-density lipoproteins (LDL and HDL) are cleared from the circulation by specific receptors that either totally degrade lipoproteins as the LDL receptor or selectively take up their cholesteryl esters (CE) like the scavenger receptor class B type I (SR-BI). The aim of the present study was to define the effect of apoC-I on the uptake of LDL and HDL(3) by HepG2 cells. In experiments conducted with exogenously added purified apoC-I, no significant effect was observed on lipoprotein-protein association and degradation; however, LDL- and HDL(3)-CE selective uptake was significantly reduced in a dose-dependent manner. This study also shows that apoC-I has the ability to associate with HepG2 cells and with LDL and HDL(3). Moreover, pre-incubation of HepG2 cells with apoC-I reduces HDL(3)-CE selective uptake and pre-incubation of LDL and HDL(3) with apoC-I decreases their CE selective uptake by HepG2 cells. Thus, apoC-I can accomplish its inhibitory effect on SR-BI activity by either binding to SR-BI or lipoproteins. We conclude that by reducing hepatic lipoprotein-CE selective uptake, apoC-I has an atherogenic character.

Oxidized LDL (OxLDL) that are positively associated with the risk of developing cardiovascular diseases are ligands of scavenger receptor-class B type I (SR-BI) and cluster of differentiation-36 (CD36) which can be found in caveolae. The contribution of these receptors in human hepatic cell is however unknown. The HepG2 cell, a human hepatic parenchymal cell model, expresses these receptors and is characterized by a very low level of caveolin-1. Our aim was to define the contribution of human CD36, SR-BI, and caveolin-1 in the metabolism of OxLDL in HepG2 cells and conversely the effects of OxLDL on the levels/localization of these receptors. By comparing mildly (M)- and heavily (H)-OxLDL metabolism between control HepG2 cells and HepG2 cells overexpressing CD36, SR-BI, or caveolin-1, we found that (1) CD36 increases M- and H-OxLDL-protein uptake; (2) SR-BI drives M-OxLDL through a degradation pathway at the expense of the cholesterol ester (CE) selective uptake pathway; (3) caveolin-1 increases M- and H-OxLDL-protein uptake and decreases CE selective uptake from M-OxLDL. Also, incubation with M- or H-OxLDL decreases the levels of SR-BI and LDL-receptor in control HepG2 cells which can be overcome by caveolin-1 expression. In addition, OxLDL move CD36 from low to high buoyant density membrane fractions, as well as caveolin-1 in cells overexpressing this protein. Thus, hepatic caveolin-1 expression has significant effects on OxLDL metabolism and on lipoprotein receptor levels., ((c) 2009 Wiley-Liss, Inc.)

Oxidized low density lipoproteins (OxLDL) are known to promote atherosclerosis, but it is only recently that OxLDL have been associated with alterations of the functions of bone-forming osteoblasts and osteoporosis. Although high density lipoproteins (HDL) are recognized for their anti-atherogenic action, there is less information about their ability to protect against osteoporosis. Therefore, we investigated the capacity of HDL3 to prevent the cell death induced by OxLDL in human osteoblastic cells. Simultaneous exposure of the cells to HDL3 and OxLDL abolished the reduction of cell viability monitored by MTT activity measurement and the induction of apoptosis determined by annexin V staining indicating that HDL3 prevent the apoptosis of osteoblasts induced by OxLDL. This protection correlated with the displacement by HDL3 of OxLDL association to osteoblasts, signifying that OxLDL binding and/or internalization are/is necessary for their cytotoxic effects. We also found that exposition of osteoblastic cells to HDL3 prior to incubation with OxLDL reduced cell death and preserved the lysosomal integrity. This protection was correlated with an increase of SR-BI expression, a modification of OxLDL metabolism with less global uptake of OxLDL and greater selective uptake of cholesterol from OxLDL. These results strongly suggest that, as for atherosclerosis, HDL may exert beneficial actions on bone metabolism.

Unlabelled: Lipoproteins transport many vitamins and hormones that have been shown to be necessary for bone formation. However, the metabolism of LDL and HDL3 by bone-forming osteoblastic cells remains unknown. Here we report that osteoblastic cells express scavenger receptors of class B that are implicated in the uptake of cholesterol and estradiol from LDL and HDL3., Introduction: The bone tissue is continuously remodeled, and its integrity requires a balance between osteoclastic bone resorption and osteoblastic bone formation. Recent studies have reported the importance of triglyceride-rich lipoproteins for the delivery of lipophilic vitamins necessary for normal bone metabolism. However, the ability of osteoblastic cells to process low- and high-density lipoproteins (LDL and HDL3) and the receptors involved remain unknown., Materials and Methods: Binding, competition, degradation, and selective uptake assays with LDL and HDL3 radiolabeled in their protein and lipid moieties or with [3H]estradiol were conducted on human osteoblasts (MG-63 cell line and primary cultures of human osteoblasts [hOB cells]) and on mouse osteoblasts (MC3T3-E1 cell line and primary cultures of murine osteoblasts [mOB cells]). The expression of scavenger receptors (SRs) by osteoblastic cells was determined by RT-PCR and Western immunoblotting, and cellular localization was assessed by sucrose gradient fractionation., Results: Osteoblastic cells were able to bind, internalize, and degrade HDL3 and LDL and are capable of selectively taking up cholesteryl esters (CEs) from these lipoproteins. Also, we provide evidence that osteoblastic cells express SR-BI, SR-BII, and CD36 (SR-Bs receptors) and that these receptors are localized in membrane lipid rafts or caveolin-rich membranes. The selective uptake of CE from LDL and HDL3 by osteoblastic cells was strongly inhibited by the known SR-B ligand oxidized LDL, indicating that SR-B receptors are responsible for the selective uptake. Finally, estradiol carried by LDL and HDL3 was selectively transferred to the osteoblastic cells also through SR-B receptors., Conclusions: Overall, our results suggest a novel mechanism for the routing of cholesterol and estradiol to osteoblasts involving the metabolism of LDL and HDL3 by SR-B receptors.

Cardiovascular diseases have recently been noted as potential risk factors for osteoporosis development. Although it is poorly understood how these two pathologies are related, it is a known fact that oxidized low-density lipoproteins (OxLDL) constitute potential determinants for both of them. The current study investigated the metabolism of OxLDL by osteoblasts and its effect on osteoblastic viability. The results obtained show that OxLDL are internalized but not degraded by osteoblasts while they can selectively transfer their CE to these cells. It is also demonstrated that OxLDL induce proliferation at low concentrations but cell death at high concentrations. This reduction of osteoblast viability was associated with lysosomal membrane damage caused by OxLDL as demonstrated by acridine orange relocalization. Accordingly, chloroquine, an inhibitor of lysosomal activity, accentuated cell death induced by OxLDL. Finally, we demonstrate that osteoblasts have the capacity to oxidize LDL and thereby potentially increase the local concentration of OxLDL. Overall, the current study confirms the potential role of OxLDL in the development of osteoporosis given its influence on osteoblastic viability.

The aim of this study was to quantify the abilities of mouse liver parenchymal and nonparenchymal cells with respect to (i) cholesteryl ester (CE) selective uptake from low-density lipoproteins (LDL), oxidized LDL (OxLDL), and high-density lipoprotein (HDL); and (ii) their free cholesterol efflux to HDL. The preparations of cells were incubated with lipoproteins labelled either in protein with iodine-125 or in CE with 3H-cholesterol oleate, and lipoprotein-protein and lipoprotein-CE associations were measured. The associations of LDL-protein and LDL-CE with nonparenchymal cells were 5- and 2-fold greater, respectively, than with parenchymal cells. However, in terms of CE-selective uptake (CE association minus protein association) both types of cell were equivalent. Similar results were obtained with OxLDL, but both types of cell showed higher abilities in OxLDL-CE than in LDL-CE selective uptake (on average by 3.4-fold). The association of HDL-protein with nonparenchymal cells was 3x that with parenchymal cells; however, nonparenchymal cells associated 45% less HDL-CE. Contrary to parenchymal cells, nonparenchymal cells did not show HDL-CE selective uptake activity. Thus parenchymal cells selectively take CE from the 3 types of lipoproteins, whereas nonparenchymal cells exert this function only on LDL and OxLDL. Efflux was 3.5-fold more important in nonparenchymal than in parenchymal cells.

In blood circulation, low density lipoproteins (LDL) can undergo modification, such as oxidation, and become key factors in the development of atherosclerosis. Although the liver is the major organ involved in the elimination of oxidized LDL (oxLDL), the identity of the receptor(s) involved remains to be defined. Our work aims to clarify the role of the scavenger receptor class B type I (SR-BI) in the hepatic metabolism of mildly and standardly oxLDL as well as the relative contribution of parenchymal (hepatocytes) and nonparenchymal liver cells with a special emphasis on CE-selective uptake. The association of native LDL and mildly or standardly oxLDL labeled either in proteins or in cholesteryl esters (CE) was measured on primary cultures of mouse hepatocytes from normal and SR-BI knock-out (KO) mice. These in vitro assays demonstrated that hepatocytes are able to mediate CE-selective uptake from both LDL and oxLDL and that SR-BI KO hepatocytes have a 60% reduced ability to selectively take CE from LDL but not towards mildly or standardly oxLDL. When lipoproteins were injected in the mouse inferior vena cava, parenchymal and nonparenchymal liver cells accumulated more CE than proteins from native, mildly and standardly oxLDL, indicating that selective uptake of CE from these lipoproteins occurs in vivo in these two cell types. The parenchymal cells contribute near 90% of the LDL-CE selective uptake and SR-BI for 60% of this pathway. Nonparenchymal cells capture mainly standardly oxLDL while parenchymal and nonparenchymal cells equally take up mildly oxLDL. An 82% reduction of standardly oxLDL-CE selective uptake by the nonparenchymal cells of SR-BI KO mice allowed emphasizing the contribution of SR-BI in hepatic metabolism of standardly oxLDL. However, SR-BI is not responsible for mildly oxLDL metabolism. Thus, SR-BI is involved in LDL- and standardly oxLDL-CE selective uptake in parenchymal and nonparenchymal cells, respectively.

Receptors of the scavenger class B family were reported to be localized in caveolae, the cell surface microdomains rich in free cholesterol and glycosphyngolipids, which are characterized by the presence of caveolin-1. Parenchymal hepatic and hepatoma HepG2 cells express very low levels of caveolin-1. In the present study, stable transformants of HepG2 cells expressing caveolin-1 were generated to address the effect of caveolin-1 on receptor activity. Compared to normal cells, these cells show higher (125)I-bovine serum albumin (BSA) uptake and cholesterol efflux, two indicators of functional caveolae. By immunoprecipitation, cell fractionation and confocal analyses, we found that caveolin-1 is well colocalized with the cluster of differentiation-36 (CD36) and the low-density lipoprotein (LDL) receptor (LDLr) but to a lesser extent with the scavenger receptor class B type I (SR-BI) in HepG2 cells expressing caveolin-1. However, caveolin-1 expression favors the dimerization of SR-BI. Two clones of cells expressing caveolin-1 were investigated for their lipoprotein metabolism activity. Compared to normal cells, these cells show a 71-144% increase in (125)I-LDL degradation. The analysis of the cholesteryl esters (CE)-selective uptake (CE association minus protein association) revealed that the expression of caveolin-1 in HepG2 cells decreases by 59%-73% LDL-CE selective uptake and increases high-density lipoprotein (HDL)-CE selective uptake by 44%-66%. We conclude that the expression of caveolin-1 in HepG2 cells moves the balance of LDL degradation/CE selective uptake towards degradation and favors HDL-CE selective uptake. Thus, in the normal hepatic parenchymal situation where caveolin-1 is poorly expressed, LDL-CE selective uptake is the preferred pathway.

Plasma low- and high-density lipoproteins (LDL and HDL) are cleared from the circulation by specific receptors and are either totally degraded or their cholesteryl esters (CE) are selectively delivered to cells by receptors such as the scavenger receptor class B type I (SR-BI). The aim of the present study was to define the effect of apoC-II and apoC-III on the uptake of LDL and HDL by HepG2 cells. Stable transformants were obtained with sense or antisense strategies that secrete 47-294% the normal level of apoC-II or 60-200% that of apoC-III. Different levels of secreted apoC-II or apoC-III had little effect on LDL and HDL protein degradation by HepG2 cells. However, compared to controls, cells under-expressing apoC-II showed a 160% higher capacity to selectively take up HDL-CE, while cells under-expressing apoC-III demonstrated 70 and 160% higher capacity to take up CE from LDL and HDL, respectively. In experiments conducted with exogenously added apoC-II or apoC-III, no significant effect was observed on lipoprotein-protein association/degradation; however, LDL-CE and HDL-CE selective uptake was significantly reduced in a dose-dependent manner. These results indicate that apoC-II and apoC-III inhibit CE-selective uptake.

The physiological role of murine scavenger receptor class B type I (SR-BI) was evaluated by in vivo clearances of human HDL3 and LDL in normal and SR-BI knockout (KO) mice. In normal mice, cholesteryl esters (CEs) were removed faster than proteins, indicating a selective uptake process from both HDL3 and LDL. SR-BI KO mice showed 80% losses of HDL-CE selective uptake and the complete loss of LDL-CE selective uptake in the first phase of clearance. However, the second phase was characterized by an acceleration of CE disappearance in SR-BI KO mice. Thus, SR-BI is the only murine receptor mediating HDL-CE selective uptake, whereas a SR-BI-independent pathway specific to LDL can rescue SR-BI deficiency. The analysis of LDL recovered 3 h after injection in mice from different genotypes revealed that LDLs are significantly depleted in CE (reduction from 19% to 50% of the CE/protein ratios). A smaller LDL size in comparison with that of noninjected LDL was also detectable but was more evident for LDL recovered from normal mice. All LDL preparations migrate faster than noninjected LDL on agarose-barbital gels. Thus, both SR-BI-dependent and -independent pathways lead to substantial changes in LDL.

The scavenger receptor class B, type I (SR-BI) mediates cholesteryl esters (CE) selective uptake from low density lipoprotein (LDL) and high-density lipoprotein (HDL) particles. In a number of tissues expressing caveolin, SR-BI is localized in caveolae. We show using detergent-free sucrose gradients that SR-BI is found in membrane rafts devoid of caveolin-1 in the human hepatoma HepG2 cell. Perturbation of the structure of HepG2 cell membrane rafts with cholesterol oxidase or sphingomyelinase decreased LDL-CE association due to selective uptake by 60%, while HDL3-CE selective uptake was increased 2.3-fold by cholesterol oxidase but was not affected by sphingomyelinase. Sequestration of membrane cholesterol with filipin III decreased LDL-CE selective uptake by 25%, while it had no effect on HDL3-CE selective uptake. Extraction of cell membrane cholesterol with beta-cyclodextrin increased LDL- and HDL3-CE selective uptake by 1.6-fold and 3-fold, respectively. We found that CE-selective uptake from both HDL and LDL occurs by a pathway involving retro-endocytosis in HepG2 cells. An analysis of the effect of SR-BI level on the expression of critical lipid sensor and lipid binding proteins was conducted with stable transformants of HepG2 cell overexpressing SR-BI. We found that liver-type fatty acid binding protein expression level is higher in SR-BI-overexpressing cells and that caveolin-1 and sterol response element binding protein-2 levels are reduced. Thus, in this hepatic cell model, SR-BI is associated with membrane rafts devoid of caveolin and its expression affects intracellular lipid binding and lipid sensor proteins. SR-BI-dependent LDL- and HDL-CE selective uptake are affected differently by the integrity of membrane rafts, but both occur by a retroendocytic pathway in HepG2 cells.

Low-density lipoprotein (LDL)-cholesteryl ester (CE) selective uptake has been demonstrated in nonhepatic cells overexpressing the scavenger receptor class B type I (SR-BI). The role of hepatic SR-BI toward LDL, the main carrier of plasma CE in humans, remains unclear. The aim of this study was to determine if SR-BI, expressed at its normal level, is implicated in LDL-CE selective uptake in human HepG2 hepatoma cells and mouse hepatic cells, to quantify its contribution and to determine if LDL-CE selective uptake is likely to occur in the presence of human HDL. First, antibody blocking experiments were conducted on normal HepG2 cells. SR-BI/BII antiserum inhibited (125)I-LDL and (125)I-HDL(3) binding (10 microg of protein/mL) by 45% (p < 0.05) and CE selective uptake by more than 85% (p < 0.01) for both ligands. Second, HepG2 cells were stably transfected with a eukaryotic vector expressing a 400-bp human SR-BI antisense cDNA fragment. Clone 17 (C17) has a 70% (p < 0.01) reduction in SR-BI expression. In this clone, (3)H-CE-LDL and (3)H-CE-HDL(3) association (10 microg of protein/mL) was 54 +/- 6% and 45 +/- 7% of control values, respectively, while (125)I-LDL and (125)I-HDL(3) protein association was 71 +/- 3% and 58 +/- 5% of controls, resulting in 46% and 55% (p < 0.01) decreases in LDL- and HDL(3)-CE selective uptake. Normalizing CE selective uptake for SR-BI expression reveals that SR-BI is responsible for 68% and 74% of LDL- and HDL(3)-CE selective uptake, respectively. Thus, both approaches show that, in HepG2 cells, SR-BI is responsible for 68-85% of CE selective uptake. Other pathways for selective uptake in HepG2 cells do not require CD36, as shown by anti-CD36 antibody blocking experiments, or class A scavenger receptors, as shown by the lack of competition by poly(inosinic acid). However, CD36 is a functional oxidized LDL receptor on HepG2 cells, as shown by antibody blocking experiments. Similar results for CE selective uptake were obtained with primary cultures of hepatic cells from normal (+/+), heterozygous (-/+), and homozygous (-/-) SR-BI knockout mice. Flow cytometry experiments show that SR-BI accounts for 75% of DiI-LDL uptake, the LDL receptor for 14%, and other pathways for 11%. CE selective uptake from LDL and HDL(3) is likely to occur in the liver, since unlabeled HDL (total and apoE-free HDL(3)) and LDL, when added in physiological proportions, only partially competed for LDL- and HDL(3)-CE selective uptake. In this setting, human hepatic SR-BI may be a crucial molecule in the turnover of both LDL- and HDL(3)-cholesterol.

Low density lipoprotein (LDL) can follow either a holoparticle uptake pathway, initiated by the LDL receptor (LDLr), and be completely degraded, or it can deliver its cholesteryl esters (CE) selectively to HepG2 cells. Although high density lipoprotein-CE selective uptake has been shown to be linked to cell cholesterol homeostasis in nonhepatic cells, there is no available information on the effect of LDL-CE selective uptake on hepatic cell cholesterol homeostasis. In order to define the role of the LDL-CE selective uptake pathway in hepatic cell cholesterol homeostasis, we used a cellular model that expresses constitutively a LDLr antisense mRNA and that shows LDLr activity at 31% the normal level (HepG2-all cells). The addition of a specific antibody anti-LDLr (IgG-C7) reduces LDL protein degradation (LDLr activity) to 7%. This cellular model therefore reflects, above all, LDL-CE selective uptake activity when incubated with LDL. The inactivation of LDLr reduces LDL-protein association by 78% and LDL-CE association by only 43%. The LDL-CE selective uptake was not reduced by the inactivation of LDLr. The activities of the various enzymes involved in cell cholesterol homeostasis were measured in normal and LDLr-deficient cells during incubation in the absence or presence of LDL as a cholesterol source. Essentially, 3-hydroxy-3-methylglutaryl coenzyme A reductase and acyl coenzyme A:cholesterol acyltransferase (ACAT) activities responded to LDL in LDLr-deficient cells as well as in normal HepG2 cells. Inhibition of lysosomal hydrolysis with chloroquine abolished the effect measured on ACAT activity in the presence of LDL, suggesting that CE of LDL, but not free cholesterol, maintains cell cholesterol homeostasis. Thus, in HepG2 cells, when LDLr function is virtually abolished, LDL-CE selective uptake is coupled to cell cholesterol homeostasis.

Class B scavenger receptors (SR-Bs) interact with native, acetylated and oxidized low-density lipoprotein (LDL, AcLDL and OxLDL), high-density lipoprotein (HDL3) and maleylated BSA (M-BSA). The aim of this study was to analyze the catabolism of CD36- and LIMPII-analogous-1 (CLA-1), the human orthologue for the scavenger receptor class B type I (SR-BI), and CD36 ligands in HepG2 (human hepatoma) cells. Saturation binding experiments revealed moderate-affinity binding sites for all the SR-B ligands tested with dissociation constants ranging from 20 to 30 microg.mL-1. Competition binding studies at 4 degrees C showed that HDL and modified and native LDL share common binding site(s), as OxLDL competed for the binding of 125I-LDL and 125I-HDL3 and vice versa, and that only M-BSA and LDL may have distinct binding sites. Degradation/association ratios for SR-B ligands show that LDL is very efficiently degraded, while M-BSA and HDL3 are poorly degraded. The modified LDL degradation/association ratio is equivalent to 60% of the LDL degradation ratio, but is three times higher than that of HDL3. All lipoproteins were good cholesteryl ester (CE) donors to HepG2 cells, as a 3.6-4.7-fold CE-selective uptake ([3H]CE association/125I-protein association) was measured. M-BSA efficiently competed for the CE-selective uptake of LDL-, OxLDL-, AcLDL- and HDL3-CE. All other lipoproteins tested were also good competitors with some minor variations. Hydrolysis of [3H]CE-lipoproteins in the presence of chloroquine demonstrated that modified and native LDL-CE were mainly hydrolyzed in lysosomes, whereas HDL3-CE was hydrolyzed in both lysosomal and extralysosomal compartments. Inhibition of the selective uptake of CE from HDL and native modified LDL by SR-B ligands clearly suggests that CLA-1 and/or CD36 are involved at least partially in this process in HepG2 cells.

Selective uptake of cholesteryl esters (CE) from lipoproteins by cells has been extensively studied with high density lipoproteins (HDL). It is only recently that such a mechanism has been attributed to intermediate and low density lipoproteins (IDL and LDL). Here, we compare the association of proteins and CE from very low density lipoproteins (VLDL), IDL, LDL and HDL3 to HepG2 cells. These lipoproteins were either labelled in proteins with 125I or in CE with 3H-cholesteryl oleate. We show that, at any lipoprotein concentration, protein association to the cells is significantly smaller for IDL, LDL, and HDL3 than CE association, but not for VLDL. At a concentration of 20 microg lipoprotein/mL, these associations reveal CE-selective uptake in the order of 2-, 4-, and 11-fold for IDL, LDL, and HDL3, respectively. These studies reveal that LDL and HDL3 are good selective donors of CE to HepG2 cells, while IDL is a poor donor and VLDL is not a donor. A significant inverse correlation (r2 = 0.973) was found between the total lipid/protein ratios of the four classes of lipoproteins and the extent of CE-selective uptake by HepG2 cells. The fate of 3H-CE of the two best CE donors (LDL and HDL3) was followed in HepG2 cells after 3 h of incubation. Cells were shown to hydrolyze approximately 25% of the 3H-CE of both lipoproteins. However, when the cells were treated with 100 microM of chloroquine, a lysosomotropic agent, 85 and 40% of 3H-CE hydrolysis was lost for LDL and HDL3, respectively. The fate of LDL and HDL3-CE in HepG2 cells deficient in LDL-receptor was found to be the same, indicating that the portion of CE hydrolysis sensitive to chloroquine is not significantly linked to LDL-receptor activity. Thus, in HepG2 cells, the magnitude of CE-selective uptake is inversely correlated with the total lipid/protein ratios of the lipoproteins and CE-selective uptake from the two best CE donors (LDL and HDL3) appears to follow different pathways.

Low-density lipoproteins (LDL) are taken up by both LDL receptor (LDLr)-dependent and -independent pathways. In order to determine the importance of these pathways in the activity of the various enzymes that are important in maintaining the cellular cholesterol level in hepatic cells, we created HepG2 cells expressing lower levels of LDLr. Thus HepG2 cells were transfected with a constitutive expression vector (pRc/CMV) containing a fragment of LDLr cDNA inserted in an antisense manner. Stable transformants were obtained that showed significant reductions of 42, 72 and 85% of LDLr protein levels compared with the control, as demonstrated by immunoblotting and confirmed by the LDL binding assay. The best inactivation was achieved with the construct containing the first 0.7 kb of LDLr cDNA. Incubating the different HepG2 cell subtypes with LDL showed similar association of apolipoprotein B (apo B) or cholesteryl esters from LDL with the cells, indicating that the LDLr deficiency did not significantly affect LDL uptake by the cell. However, apoB degradation was reduced significantly by 71-82% in the most LDLr-deficient HepG2 cells. We also found that 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCoA red) activity is significantly increased by 32-35% in HepG2 cells expressing very low levels of LDLr that also demonstrate a significant decrease of 20% in acyl-CoA:cholesterol acyltransferase (ACAT) activity. However, these effects are moderate compared with those observed when cells were incubated in lipoprotein-depleted medium, where a >900% increase in HMGCoA red activity and a loss of 60% of ACAT activity was observed. Thus, in HepG2 cells, different levels of LDLr affect LDL-apoB degradation, but have very little effect on LDL association, HMGCoA red and ACAT activities, revealing that LDLr is more important in the clearance of LDL-apoB than in HepG2 cell cholesterol homoeostasis, a role that should be attributable to both LDLr-dependent and -independent pathways.

The study described in this paper shows that 125I-labelled low-density lipoproteins (LDL) interact with high- and low-affinity binding sites on human hepatoma (HepG2) cells. The former site is the LDL receptor and the latter is the lipoprotein-binding site (LBS). The association of 125I-labelled LDL and [3H]cholesteryl ethers-LDL with HepG2 cells revealed a 4-fold selective uptake of cholesteryl esters (CE) in a 4 h incubation period, which correlated with the depletion of CE mass in LDL. This selective uptake was not observed when the cells were incubated in the presence of a 100-fold excess of high-density lipoprotein 3, conditions where only the LDL receptor is being monitored. Also, no reduction in uptake was observed in the presence of IgG-C7, an anti-(LDL receptor) monoclonal antibody. Both findings indicate that the selective uptake occurs through the LBS and that the LBS contributes more to the entry of CE from LDL into the cell than does the LDL receptor. The fates of CE entering the cell via the LDL receptor and the LBS were also followed. To achieve this, LDL were labelled with [3H]cholesteryl oleate and the hydrolysis of [3H]cholesteryl oleate was monitored. The results indicated that 45% of the CE were hydrolysed after a 4 h incubation period, irrespective of the site of entry. Chloroquine (100 microM) was shown to inhibit hydrolysis, indicating that lysosomal enzymes were responsible for the hydrolysis of LDL-CE, whichever pathway was used. Thus our results reveal, for the first time, that the mass of CE entering the cell via the LBS is substantial and that hydrolysis of CE is by lysosomal enzyme activity. Overall, this suggests that the LBS has significant physiological importance.

The analysis of the association of (125)I-LDL and [(3)H]cholesteryl ethers (CEt)-LDL with HepG2 cells revealed a selective uptake of cholesteryl esters (CE) of the LDL, as in the order of three-fold more CE were associated with the cells than LDL-proteins for an incubation of 4 h. To determine if a trans-signalling pathway is involved in this selective uptake, HepG2 cells were pre-treated for 2 h with either a Protein Kinase A activator [8-(4-chlorophenylthioadenosine 3'-5' cyclic monophosphate (CPT-cAMP)] or a Protein Kinase C activator [phorbol 12-myristate 13-acetate (PMA)]. We found that CPT-cAMP had a minimal effect, while PMA was able to significantly increase the selective uptake of the CE of LDL. Indeed, upon a 2 h pre-incubation of HepG2 cells with PMA at a concentration of 160 microM, an increase of more than 3-fold in CE selective uptake was registered and was shown to occur by the lipoprotein binding sites (LBS) of HepG2 cells. Also, an incubation of the cells with 100 nM calphostin C, an inhibitor of protein kinase C, decreased the selective uptake by 41%. The effect of PMA is not abolished by either cycloheximide or actinomycin D. However, cycloheximide was shown to potentiate the effect of PMA on the LBS activity, suggesting that a protein which synthesis is affected by cycloheximide is involved in maintaining the LBS activity low. Our results show that the HepG2 cell activity of CE selective uptake parallels the activity of Protein Kinase C and suggest that the LBS could be a G-protein linked receptor.

We have recently shown by the analysis of the association to HepG2 cells of human intermediate density lipoproteins (IDL) either labeled in cholesteryl ester (CE) with [3H]cholesteryl ethers (CEt) or in proteins by iodine-125 that the CE of IDL are selectively taken up by these cells. Our results also revealed that the addition of a sufficient quantity of HDL3 abolishes the binding of IDL to the 'Lipoprotein binding site' (LBS) and also the CE-selective uptake. This suggested that the LBS mediates this uptake (Brissette and Falstrault (1992) Biochim. Biophys. Acta. 1165, 84-92). This study was undertaken to analyze further the mechanism of the selective uptake of the CE of IDL by HepG2 cells to determine if the LBS is directly or indirectly involved. We show that the different labeling had no effect on the binding affinity of IDL to HepG2 cells. To verify that the apolipoprotein moiety of HDL3 was responsible for the abolishment of the CE selective uptake, we have studied the effect of free apoA-I and apoA-II on the association of IDL. Our results demonstrate that apoA-I and apoA-II are approximately 10-times better than HDL3 or apoA-I liposomes in abolishing the selective uptake of the CE from IDL. We also show that this correlates with a more efficient reduction of the binding of 125I-IDL to HepG2 cells by free apoA-I compared to apoA-I associated with lipids. Thus free apoA-I interfere with the binding of IDL to the LBS and free apolipoproteins have a better capacity to saturate the LBS than lipoproteins. Also, we found no evidence for the transfer of CE from the labeled IDL to HDL3 or to apolipoproteins used to abolish the interaction of IDL to the LBS. Thus our results indicate that the LBS is directly responsible for the selective uptake of CE.

The binding of human intermediate density lipoproteins (IDL) to HepG2 cells was studied. We found that human 125I-IDL interact with a binding site of high-affinity (Kd 0.74 micrograms/ml, Bmax 0.049 micrograms/mg cell protein) and a binding site of lower affinity (Kd 86.8 micrograms/ml; Bmax 0.53 micrograms/mg cell protein). The high-affinity binding sites show characteristics of LDL-receptors since they interact with IDL and low-density lipoproteins (LDL) and are calcium dependent. The low-affinity binding sites are calcium-independent and interact with IDL, LDL, high density lipoproteins-3 (HDL3), apolipoprotein (apo) E-liposomes, apoCs-liposomes, apoA-I-liposomes but not with liposomes containing albumin or erythrocyte membrane proteins. Therefore, HepG2 cells have on their surface a binding site that resembles or is identical to the lipoprotein binding site (LBS) that we found on rat liver membranes (Brissette and Noël (1986) J. Biol. Chem. 261, 6847-6852). Internalization, degradation and cholesterol ester selective uptake were determined in the presence or in the absence of a sufficient amount of human HDL3 to abolish the interaction of IDL to the LBS in order to obtain information on the function of this site. Our results suggest that the LBS participates in the internalization of IDL but not in their degradation and that it is responsible for the selective uptake of cholesterol esters of IDL.