Quantitative analysis of SR-BI-dependent HDL retroendocytosis in hepatocytes and fibroblasts

Abstract

Previous studies have suggested that HDL retroendocytosis may play a role in scavenger receptor class B type I (SR-BI)-dependent selective lipid uptake in a cellspecific manner. To investigate this possibility, we developed methods to quantitatively measure HDL uptake and resecretion in fibroblast (COS-7) and hepatocyte (HepG2) cells expressing exogenous SR-BI. Approximately 17% and 24% of HDL associated in an SR-BI-dependent manner with COS-7 and HepG2 cells, respectively, accumulates intracellularly after a 10 min incubation. To determine whether this intracellular HDL undergoes retroendocytosis, we developed a pulse-chase assay whereby internalized biotinylated 125 I-HDL 3 secreted from cells is quantitatively precipitated from cell supernatants using immobilized streptavidin. Our results show a rapid secretion of a portion of intracellular HDL from both cell types (representing 4-7% of the total cell-associated HDL) that is almost complete within 30 min (half-life z 10 min). In COS-7 cells, the calculated rate of HDL secretion (z0.5 ng HDL/mg/min) was .30-fold slower than the rate of SR-BI-dependent selective cholesteryl ester (CE) uptake (z17 ng HDL/mg/min), whereas the rate of release of HDL from the cell surface (z19 ng HDL/ mg/min) was similar to the rate of selective CE uptake. Notably, the rate of SR-BI-dependent HDL resecretion in COS-7 and HepG2 cells was similar. BLT1, a compound that inhibits selective CE uptake, does not alter the amount of SR-BI-mediated HDL retroendocytosis in COS-7 cells. From these data, we conclude that HDL retroendocytosis in COS-7 and HepG2 cells is similar and that the vast majority of SR-BI-dependent selective uptake occurs at the cell surface in both cell types.-Sun, B., E. R. M. Eckhardt, S. Shetty, D. R. van der Westhuyzen, and N. R. Webb. Quantitative analysis of SR-BI-dependent HDL retroendocytosis in hepatocytes and fibroblasts. J. Lipid Res. 2006. 47: 1700-1713.Supplementary key words selective cholesteryl ester uptake . biotinylation . scavenger receptor class B type I Abundant evidence has established that scavenger receptor class B type I (SR-BI) plays a primary role in HDL metabolism by mediating selective cholesteryl ester (CE) uptake, a process in which HDL CE is taken up without intracellular apolipoprotein degradation (1). In addition to mediating the selective uptake of HDL CE, SR-BI also plays a role in facilitating the bidirectional flux of unesterified cholesterol between cells and extracellular acceptors (2-4). SR-BI has a large extracellular loop (z400 residues), two transmembrane domains, and two short cytoplasmic tails at its N and C termini (5). SR-BI is highly expressed in liver and steroidogenic tissues, which are known to be major sites for selective lipid uptake. SR-BI is also expressed in other cells such as macrophages (6, 7), intestinal cells (8-10), and endothelial cells (11-13). Although SR-BI is best known as a physiological HDL receptor, it also binds other lipoprotein ligands, such as LDL, oxidized LDL, and acetylated LDL (1, 1...

CD36 Binds Oxidized Low Density Lipoprotein (LDL) in a Mechanism Dependent upon Fatty Acid Binding

“…Additionally, compared with our study that used high doses of FA for in vitro experiments (up to 500 M to observe the R max of Dii-oxLDL uptake), physiological FA concentrations range between 250 and 3,000 M, dependent on factors such as fasting/nutrition state and exercise (58). Lower levels of oxLDL uptake can be seen with highly sensitive oxLDL detection techniques such as radiolabeling ( 125 I-oxLDL) or high magnification/high exposure microscopy to observe oxLDL binding/uptake, coupled with serum-free medium delivery of oxLDL (15,59,60). We quantified the differences between Dii-oxLDL uptake using serum-free medium (small levels of uptake), serumcontaining medium (large levels of uptake), and increasing doses of FA in serum-free medium (increasing levels of DiioxLDL uptake, rescued to serum-containing levels), and we did not observe any unusual cell phenotype changes, in the short time frame of our observation (Fig.…”

Regulation of SR-BI-mediated selective lipid uptake in Chinese hamster ovary-derived cells by protein kinase signaling pathways

“…Similarly, SR-BI-mediated sterol uptake by hepatocytes and trafficking to the bile canalicular (apical) space does not appear to be energy dependent (17,18). Furthermore, the low selective uptake activities of SR-BII, a splice variant of SR-BI with a different carboxyterminal cytoplasmic tail, and of a mutant form of SR-BI with an endocytic sequence inserted into its carboxyterminal tail appear to be due to their increased endocytosis (19)(20)(21). On the other hand, mSR-BI does mediate the endocytosis and intracellular accumulation of a variety of other ligands, including serum amyloid a, lipopolysaccharide, and apoptotic cells (22)(23)(24)(25)(26).…”

Influence of HDL-cholesterol-elevating drugs on the in vitro activity of the HDL receptor SR-BI

“…The molecular target(s) of the BLTs has not yet been reported. BLTs are currently being used to explore the detailed mechanism underlying SR-BI's multiple activities (12,13,(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40).…”