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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.

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.

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.