SR-BI associates with ABCG1 and inhibits ABCG1-mediated cholesterol efflux from cells to high-density lipoprotein 3

Abstract

Background: The single and combined effects of scavenger receptor-BI (SR-BI), ATP-binding cassette transporter (ABC) A1 and G1 on cholesterol efflux from Chinese Hamster Ovary (CHO) cells were investigated.

Results: When apolipoproteinA-I (apoA-I) was used as an acceptor, ABCA1 overexpression led to an increase in total cholesterol (TC) in medium which is attributable to a 2-fold increase in free cholesterol (FC) content. When high-density lipoprotein 3 (HDL3) was used as an acceptor, SR-BI overexpression not only promoted FC efflux, but also promoted the uptake of cholesteryl ester (CE) into cells, resulting in no TC varieties in medium. Overexpression of ABCG1 increased both the FC and CE levels in medium. However, when apoA-I and HDL3 were both used as acceptors, coexpression of SR-BI has no effect on ABCA1-mediated increased FC and TC accumulation in medium. Interestingly, coexpression of SR-BI with ABCG1 blocked the ABCG1-mediated cholesterol efflux to HDL3, mostly by promoting the reuptake of CE from the medium. Furthermore, co-immunoprecipitation experiments revealed that SR-BI interacted with ABCG1 in BHK cells overexpressing ABCG1 and SR-BI.

Conclusions: We found SR-BI associates with ABCG1 and inhibits ABCG1-mediated cholesterol efflux from cells to HDL3.

Figure 1

The single effects of SR-BI, ABCA1 and ABCG1 on cellular cholesterol efflux. CHO cells were transiently transfected with plasmid constructs expressing SR-BI, ABCA1, ABCG1 or empty vector. 24 h after transfection, the cells were incubated with 150 μg/ml HDL3 (HDL3 chase) or 10 μg/ml apoA-I (apoA-I chase) for 24 h in Opti-MEM I Medium. Cholesterol efflux was determined by measuring the increased free cholesterol (FC, A), cholesteryl ester (CE, B), and total cholesterol (TC, C) in medium. Values are means ± SD of three independent experiments. *p < 0.05, **p < 0.01.

Figure 2

SR-BI does not affect ABCA1-mediated cellular cholesterol efflux. CHO cells were transiently transfected with SR-BI alone, ABCA1 alone, and SR-BI plus ABCA1 (co-transfection). 24 h after transfection, cholesterol efflux was initiated by the addition of HDL3 (150 μg protein/ml) and apoA-I (10 μg/ml) to medium for 24 h. The increased FC (A), CE (B) and TC (C) mass in medium were determined. Values are means ± SD of three independent experiments. * p < 0.05, ** p < 0.01. D shows the western blots of SR-BI and ABCA1 protein in vector transfected, SR-BI transfected, ABCA1 transfected and cotransfected cells.

Figure 3

SR-BI inhibits ABCG1-mediated cellular cholesterol efflux. CHO cells were transiently transfected with SR-BI alone, ABCG1 alone, and SR-BI plus ABCG1 (co-transfection). 24 h after transfection, cholesterol efflux was initiated by the addition of HDL3 (150 μg protein/ml) to medium for 24 h. The increased FC (A), CE (B) and TC (C) mass in medium were determined. Values are means ± SD of three independent experiments. * p < 0.05, ** p < 0.01. D shows the western blots of SR-BI and ABCG1 protein in vector transfected, SR-BI transfected, ABCG1 transfected and cotransfected cells.

Figure 4

Co-immunoprecipitation of SR-BI with ABCG1 in BHK cells overexpressing SR-BI and ABCG1. The cell lysates from the transfected BHK cells were immunoprecipitated with anti-ABCG1 antibody. The immunoprecipitates were then analyzed by western blot with antibody against SR-BI.