The Role of Human and Mouse Hepatic Scavenger Receptor Class B Type I (SR-BI) in the Selective Uptake of Low-Density Lipoprotein−Cholesteryl Esters

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Abstract
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 125I-LDL and 125I-HDL3 binding (10 μg 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, 3H-CE−LDL and 3H-CE−HDL3 association (10 μg of protein/mL) was 54 ± 6% and 45 ± 7% of control values, respectively, while 125I-LDL and 125I-HDL3 protein association was 71 ± 3% and 58 ± 5% of controls, resulting in 46% and 55% (p < 0.01) decreases in LDL− and HDL3−CE selective uptake. Normalizing CE selective uptake for SR-BI expression reveals that SR-BI is responsible for 68% and 74% of LDL− and HDL3−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 HDL3 is likely to occur in the liver, since unlabeled HDL (total and apoE-free HDL3) and LDL, when added in physiological proportions, only partially competed for LDL− and HDL3−CE selective uptake. In this setting, human hepatic SR-BI may be a crucial molecule in the turnover of both LDL− and HDL3−cholesterol.

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