ATP-binding cassette transporters G1 and G4 mediate cellular cholesterol efflux to high-density lipoproteins - PubMed (original) (raw)

ATP-binding cassette transporters G1 and G4 mediate cellular cholesterol efflux to high-density lipoproteins

Nan Wang et al. Proc Natl Acad Sci U S A. 2004.

Abstract

The mechanisms responsible for the inverse relationship between plasma high-density lipoprotein (HDL) levels and atherosclerotic cardiovascular disease are poorly understood. The ATP-binding cassette transporter A1 (ABCA1) mediates efflux of cellular cholesterol to lipid-poor apolipoproteins but not to HDL particles that constitute the bulk of plasma HDL. We show that two ABC transporters of unknown function, ABCG1 and ABCG4, mediate isotopic and net mass efflux of cellular cholesterol to HDL. In transfected 293 cells, ABCG1 and ABCG4 stimulate cholesterol efflux to both smaller (HDL-3) and larger (HDL-2) subclasses but not to lipid-poor apoA-I. Treatment of macrophages with an liver X receptor activator results in up-regulation of ABCG1 and increases cholesterol efflux to HDL. RNA interference reduced the expression of ABCG1 in liver X receptor-activated macrophages and caused a parallel decrease in cholesterol efflux to HDL. These studies indicate that ABCG1 and ABCG4 promote cholesterol efflux from cells to HDL. ABCG1 is highly expressed in macrophages and probably mediates cholesterol efflux from macrophage foam cells to the major HDL fractions, providing a mechanism to explain the relationship between HDL levels and atherosclerosis risk.

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Figures

Fig. 1.

Fig. 1.

LXR/RXR activation increases macrophage cholesterol efflux to HDL independent of ABCA1. (A and B) Cholesterol efflux to apoA-I (15 μg/ml protein) (A) or HDL-2 (25 μg/ml protein) (B) was determined in mouse peritoneal macrophages isolated from WT or ABCA1-/- mice. The cells were labeled with [3H]cholesterol in cell culture media plus 10% FBS for 16 h and then treated with or without 5 μM TO901317 plus 5 μM 9-_cis_-retinoic acid for 16 h followed by cholesterol efflux for 4 h.

Fig. 2.

Fig. 2.

Cells transfected with ABCG1 and ABCG4 cDNAs show increased cholesterol efflux to HDL. HEK293 cells were transiently transfected with plasmid constructs expressing ABCG transporters or control empty vector (mock), and cholesterol efflux was initiated by addition of HDL to media. (A) [3H]cholesterol efflux to HDL-2 or HDL-3 (25 μg/ml HDL protein) or media alone (control) for 4 h. (B) [3H]Cholesterol efflux to HDL-2 (25 μg/ml HDL protein) for 4 h. (C) [3H]Cholesterol efflux to HDL-2 at indicated concentrations for 4 h. (D)[3H]Cholesterol efflux to HDL-2 (25 μg/ml HDL protein) for the indicated period of time.

Fig. 3.

Fig. 3.

ABCG1 and ABCG4 expression increase cholesterol mass efflux to HDL and decrease cellular cholesterol content. (A) Free cholesterol mass in culture media determined in transfected 293 cells incubated with HDL-2 (25 μg/ml HDL protein) for 4 h. (B) Total cellular cholesterol was determined after 6 h incubation of transfected 293 cells with HDL-2 (25 μg/ml HDL protein).

Fig. 4.

Fig. 4.

ABCG1 and ABCG4 do not promote cholesterol efflux to apoA-I and do not bind HDL while promoting cholesterol efflux to HDL, LDL, and cyclodextrin. (A) HDL-2 (25 μg/ml HDL protein) and apoA-I (15 μg/ml protein) mediated cholesterol efflux during a 4-h incubation with 293 cells expressing ABCG transporters. (B) [3H]choline-containing phospholipid efflux to HDL-2 (25 μg/ml HDL protein) during a 4-h incubation with 293 cells expressing ABCG transporters. (C) Cholesterol efflux to HDL (25 μg/ml HDL protein), LDL (25 μg/ml LDL protein), or cyclodextrin (1 mM) during a 4-h incubation with 293 cells expressing ABCG transporters. (D) [125I]HDL binding to 293 cells expressing SR-BI or ABCG transporters.

Fig. 5.

Fig. 5.

Suppression of ABCG1 expression by RNAi decreases macrophage cholesterol efflux to HDL. [3H]cholesterol efflux to HDL-2 was determined by using mouse peritoneal macrophages after transfection of the cells with synthetic siRNA against ABCG1 (A and B). Two different concentrations of siRNA were used, as indicated. As a control, scrambled siRNA or siRNA against ABCA7 were used. mRNA levels of ABCG1 (B) normalized against β-actin mRNA from macrophages treated with 120 nM siRNA were determined by TaqMan real-time RT-PCR. *, P < 0.05; **, P < 0.01.

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