LXR␣ is a nuclear receptor that has previously been shown to regulate the metabolic conversion of cholesterol to bile acids. Here we define a role for this transcription factor in the control of cellular cholesterol efflux. We demonstrate that retroviral expression of LXR␣ in NIH 3T3 fibroblasts or RAW264.7 macrophages and͞or treatment of these cells with oxysterol ligands of LXR results in 7-to 30-fold induction of the mRNA encoding the putative cholesterol͞phospholipid transporter ATP-binding cassette (ABC)A1. In contrast, induction of ABCA1 mRNA in response to oxysterols is attenuated in cells that constitutively express dominant-negative forms of LXR␣ or LXR␤ that lack the AF2 transcriptional activation domain. We further demonstrate that expression of LXR␣ in NIH 3T3 fibroblasts and͞or treatment of these cells with oxysterols is sufficient to stimulate cholesterol efflux to extracellular apolipoprotein AI. The ability of oxysterol ligands of LXR to stimulate efflux is dramatically reduced in Tangier fibroblasts, which carry a loss of function mutation in the ABCA1 gene. Taken together, these results indicate that cellular cholesterol efflux is controlled, at least in part, at the level of transcription by a nuclear receptor-signaling pathway. They suggest a model in which activation of LXRs by oxysterols in response to cellular sterol loading leads to induction of the ABCA1 transporter and the stimulation of lipid efflux to extracellular acceptors. These findings have important implications for our understanding of mammalian cholesterol homeostasis and suggest new opportunities for pharmacological regulation of cellular lipid metabolism.H uman and murine macrophages express cell surface receptors that bind and then rapidly internalize oxidized or modified (e.g., acetylated) low density lipoprotein (LDL) (1). This rapid and unregulated uptake of the cholesterol-rich LDL results in the formation of intracellular cholesteryl ester inclusions that give the macrophages a foamy appearance (2). Such cholesteryl ester-rich foam cells have been identified in both early and advanced atherosclerotic lesions of humans and animals and are thought to play a critical role in the development of the disease (3, 4). Thus, the differential expression of specific genes in lipid loaded macrophages is likely to play an important role in the pathology of atherosclerosis.Recent studies have identified two genes, ATP-binding cassette (ABC)A1 (5) and ABCG1 (6, 7), whose mRNAs are induced 7-to Ͼ100-fold in lipid-loaded macrophages. In earlier publications, ABCA1 was referred to as ABC1, whereas ABCG1 was referred to as either ABC8 (murine) or white (human). ABCA1 and ABCG1 are members of the ATP binding cassette (ABC) superfamily of transporter proteins that bind and hydrolyze ATP to provide energy for transmembrane transport (8-10). The ABCA1 gene encodes a full transporter protein containing 12 putative transmembrane domains and two ABCs (11), whereas the ABCG1 gene encodes a half transporter protein containing six putative transme...

To identify genes that are transcriptionally activated when human macrophages accumulate excess lipids, we employed the mRNA differential display technique using RNA isolated from human monocyte-macrophages incubated in the absence or presence of acetylated low density lipoprotein and sterols (cholesterol and 25-hydroxycholesterol). These studies identified a mRNA whose levels were highly induced in lipid-loaded macrophages. The mRNA encoded the human White protein, a member of the ATP-binding cassette (ABC) transporter superfamily of proteins. The mRNA levels of ABC8, the murine homolog of the human white gene, were also induced when a murine macrophage cell line, RAW264.7, was incubated with acetylated low density lipoprotein and sterols. Additional studies demonstrated that white/ ABC8 mRNA levels were induced by specific oxysterols that included 25-, 20(S)-, and 22(R)-hydroxycholesterol, and by a retinoid X receptor-specific ligand. Furthermore, the oxysterol-mediated induction of ABC8 expression in mouse peritoneal macrophages was dependent on the presence of the nuclear oxysterol receptors, liver X receptors (LXRs). Macrophages derived from mice lacking both LXR␣ and LXR␤ failed to up-regulate the expression of ABC8 following incubation with 22(R)-hydroxycholesterol. Oxysterol-dependent induction of white/ABC8 mRNA was blocked by actinomycin D but not by cycloheximide treatment of cells. We conclude that the white and ABC8 genes are primary response genes that are transcriptionally activated by specific oxysterols and that this induction is mediated by the LXR subfamily of nuclear hormone receptors. These data strongly support the hypothesis that white/ABC8 has a role in cellular sterol homeostasis.An early event in the development of the fatty streak in the artery wall involves the entry of circulating monocytes into the subendothelial space and their subsequent differentiation into macrophages (1, 2). In the presence of oxidized or modified forms of LDL, 1 these macrophages accumulate cytoplasmic lipid droplets that contain excess cholesteryl esters (1-3). The latter cells are termed macrophage "foam" cells, because the lipid droplets give them a characteristic foamy appearance when viewed under the microscope (1). The importance of monocyte/macrophages in the development of fatty streaks and the more advanced atherosclerotic lesions can be gauged from the effect of deletions or mutations of genes that are involved in either monocyte recruitment into the artery wall or their subsequent differentiation into macrophages. For example, mice defective in monocyte chemoattractant protein-1 (4), the monocyte chemoattractant protein-1 receptor (5), or macrophagecolony-stimulating factor (6, 7) exhibit reduced levels of atherosclerotic lesions when compared with normal mice.Relatively little is known about the alterations in gene expression that occur when macrophages accumulate excess lipids to become macrophage foam cells. In previous attempts to identify such genes, macrophages were incubated with Ac-LDL to pr...

The human ABCG1 gene encodes a member of the ATPbinding cassette (ABC) superfamily of transporter proteins and is highly induced when macrophages are incubated with oxysterols. Using mRNA from oxysterol-treated human THP-1 cells together with 5-rapid amplification of cDNA ends and polymerase chain reaction, we identified a novel ABCG1 transcript that encodes a putative protein of 786 residues containing a new amino terminus of 203 amino acids. Characterization of the genomic organization and structure of the human ABCG1 gene demonstrates that: (i) the gene consists of 23 exons spanning 98 kilobase pairs (kb) on chromosome 21q22.3, (ii) the 203 amino acids are encoded on three previously unidentified exons, 8-10, and (iii) a promoter, containing a TATA box and two liver X receptor (LXR) ␣ response elements (LXREs), is located upstream of exon 8. Northern analysis using exon-specific probes confirms that oxysterol treatment results in >10-fold induction of ABCG1 transcripts that are derived from either exons 8-23 or exons 5, 7, and 11-23. Electromobility shift assays demonstrate that LXR␣ and retinoid X receptor ␣ bind to the two LXREs in intron 7. Cells were transiently transfected with reporter luciferase constructs under the control of either (i) 9 kb of genomic DNA corresponding to intron 7 and part of exon 8 and containing either wild-type or mutant LXREs or (ii) two copies of the wild-type or mutant LXRE. In all cases, the wild-type construct was regulated in an LXR-and oxysterol-dependent manner, and this regulation was attenuated when the LXREs were mutated. In conclusion, the human ABCG1 gene contains multiple promoters, spans more than 98 kb and comprises 23 exons that give rise to alternative transcripts encoding proteins with different amino-terminal sequences. Elucidation of the various roles of different ABCG1 isoforms will be important for our understanding of mammalian cholesterol homeostasis.In the presence of oxidized, aggregated, or acetylated LDL, 1 macrophages take on a "foamy" appearance as a result of the cytoplasmic accumulation of cholesteryl ester lipid droplets (1-3). Such macrophage foam cells are found in both fatty streaks and more advanced lesions in the artery wall and appear to be important in the development of atherosclerosis (2). Recent studies have begun to explore the changes in macrophage gene expression that occur during lipid loading, on the assumption that such changes effect the development of fatty streaks and/or the stability of advanced plaques. These studies led to the identification of three mRNAs, ABCG1, ABCA1, and apoE, that are highly induced when macrophages are incubated in the presence of either modified LDL or specific oxysterols (4 -7). Induction of each mRNA requires the nuclear hormone receptor LXR (4 -6, 8, 9). ABCG1 (also referred to as human White or murine ABC8), and ABCA1 are two members of the ATP-binding cassette (ABC) transporter superfamily of proteins (10 -12). The ABC superfamily consists of membrane-bound proteins that mediate the ATP-dependent ...

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