出版社：American Society for Biochemistry and Molecular Biology
摘要：Previous studies have suggested that HDL retroendocytosis mayplay a role in scavenger receptor class B type I (SR-BI)-dependentselective lipid uptake in a cell-specific manner. To investigatethis possibility, we developed methods to quantitatively measureHDL 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 withCOS-7 and HepG2 cells, respectively, accumulates intracellularlyafter a 10 min incubation. To determine whether this intracellularHDL undergoes retroendocytosis, we developed a pulse-chase assaywhereby internalized biotinylated 125I-HDL3 secreted from cellsis quantitatively precipitated from cell supernatants usingimmobilized streptavidin. Our results show a rapid secretionof a portion of intracellular HDL from both cell types (representing4–7% of the total cell-associated HDL) that is almostcomplete within 30 min (half-life 10 min). In COS-7 cells,the calculated rate of HDL secretion (0.5 ng HDL/mg/min) was>30-fold slower than the rate of SR-BI-dependent selectivecholesteryl ester (CE) uptake (17 ng HDL/mg/min), whereas therate of release of HDL from the cell surface (19 ng HDL/mg/min)was similar to the rate of selective CE uptake. Notably, therate of SR-BI-dependent HDL resecretion in COS-7 and HepG2 cellswas similar. BLT1, a compound that inhibits selective CE uptake,does not alter the amount of SR-BI-mediated HDL retroendocytosisin COS-7 cells. From these data, we conclude that HDL retroendocytosisin COS-7 and HepG2 cells is similar and that the vast majorityof SR-BI-dependent selective uptake occurs at the cell surfacein both cell types.Supplementary key words selective cholesteryl ester uptake • biotinylation • scavenger receptor class B type I
Abbreviations: CE, cholesteryl ester; MFI, mean fluorescence intensity; MOI, multiplicity of infection; SR-BI, scavenger receptor class B type I
10 min). In COS-7 cells, the calculated rate of HDL secretion (
