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.
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.
Rhainds
David
D
Département des Sciences Biologiques, Université du Québec à Montréal, Montréal, Québec, Canada H3C 3P8. david.rhainds@internet.uqam.ca
Brodeur
Mathieu
M
Lapointe
Jany
J
Charpentier
Daniel
D
Falstrault
Louise
L
Brissette
Louise
L
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism; D001667:Binding, Competitive; D018955:CD36 Antigens / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D002788:Cholesterol Esters / Q000378:metabolism*; D008078:Cholesterol, LDL / Q000378:metabolism*; D005434:Flow Cytometry; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007456:Iodine Isotopes; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D008565:Membrane Proteins; D051379:Mice; D008815:Mice, Inbred Strains; D018345:Mice, Knockout; D011971:Receptors, Immunologic; D018110:Receptors, Lipoprotein; D051116:Receptors, Scavenger; D051117:Scavenger Receptors, Class A; D051122:Scavenger Receptors, Class B; D014316:Tritium; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D018955:CD36 Antigens; D002788:Cholesterol Esters; D008078:Cholesterol, LDL; D007456:Iodine Isotopes; D008077:Lipoproteins, LDL; D008565:Membrane Proteins; D011971:Receptors, Immunologic; D018110:Receptors, Lipoprotein; D051116:Receptors, Scavenger; C495124:SCARB1 protein, human; C454739:Scarb1 protein, mouse; D051117:Scavenger Receptors, Class A; D051122:Scavenger Receptors, Class B; D014316:Tritium