Macrophages have important roles in both lipid metabolism and inflammation and are central to the pathogenesis of atherosclerosis. The liver X receptors (LXRs) are established mediators of lipid-inducible gene expression, but their role in inflammation and immunity is unknown. We demonstrate here that LXRs and their ligands are negative regulators of macrophage inflammatory gene expression. Transcriptional profiling of lipopolysaccharide (LPS)-induced macrophages reveals reciprocal LXR-dependent regulation of genes involved in lipid metabolism and the innate immune response. In vitro, LXR ligands inhibit the expression of inflammatory mediators such as inducible nitric oxide synthase, cyclooxygenase (COX)-2 and interleukin-6 (IL-6) in response to bacterial infection or LPS stimulation. In vivo, LXR agonists reduce inflammation in a model of contact dermatitis and inhibit inflammatory gene expression in the aortas of atherosclerotic mice. These findings identify LXRs as lipid-dependent regulators of inflammatory gene expression that may serve to link lipid metabolism and immune functions in macrophages.

Previous work has implicated PPAR gamma in the regulation of CD36 expression and macrophage uptake of oxidized LDL (oxLDL). We provide evidence here that in addition to lipid uptake, PPAR gamma regulates a pathway of cholesterol efflux. PPAR gamma induces ABCA1 expression and cholesterol removal from macrophages through a transcriptional cascade mediated by the nuclear receptor LXR alpha. Ligand activation of PPAR gamma leads to primary induction of LXR alpha and to coupled induction of ABCA1. Transplantation of PPAR gamma null bone marrow into LDLR -/- mice results in a significant increase in atherosclerosis, consistent with the hypothesis that regulation of LXR alpha and ABCA1 expression is protective in vivo. Thus, we propose that PPAR gamma coordinates a complex physiologic response to oxLDL that involves particle uptake, processing, and cholesterol removal through ABCA1.

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...

The nuclear receptors LXR␣ and LXR␤ have been implicated in the control of cholesterol and fatty acid metabolism in multiple cell types. Activation of these receptors stimulates cholesterol efflux in macrophages, promotes bile acid synthesis in liver, and inhibits intestinal cholesterol absorption, actions that would collectively be expected to reduce atherosclerotic risk. However, synthetic LXR ligands have also been shown to induce lipogenesis and hypertriglyceridemia in mice, raising questions as to the net effects of these compounds on the development of cardiovascular disease. We demonstrate here that the nonsteroidal LXR agonist GW3965 has potent antiatherogenic activity in two different murine models. In LDLR ؊͞؊ mice, GW3965 reduced lesion area by 53% in males and 34% in females. A similar reduction of 47% was observed in male apoE ؊͞؊ mice. Long-term (12-week) treatment with LXR agonist had differential effects on plasma lipid profiles in LDLR ؊͞؊ and apoE ؊͞؊ mice. GW3965 induced expression of ATP-binding cassettes A1 and G1 in modified low-density lipoprotein-loaded macrophages in vitro as well as in the aortas of hyperlipidemic mice, suggesting that direct actions of LXR ligands on vascular gene expression are likely to contribute to their antiatherogenic effects. These observations provide direct evidence for an atheroprotective effect of LXR agonists and support their further evaluation as potential modulators of human cardiovascular disease.R ecent work has identified the nuclear receptors LXR␣ and LXR␤ as central regulators of lipid homeostasis. The physiologic ligands for these receptors are likely to be specific intermediates in the cholesterol biosynthetic pathway such as 24(S),25-epoxycholesterol (1-3). LXR␣ is expressed primarily in liver, intestine, adipose tissue, and macrophages, whereas LXR␤ is expressed in many cell types (4). In peripheral cells such as macrophages, LXRs seem to coordinate a physiologic response to cellular cholesterol loading. LXRs directly control transcription of several genes involved in the cholesterol efflux pathway, including ATP-binding cassette (ABC) A1 (5-8), ABCG1 (9), and apolipoprotein E (apoE) (10). In the intestine, ligand activation of LXR͞RXR heterodimers dramatically reduces dietary cholesterol absorption, an effect postulated to be mediated by ABCA1 (6).In the liver, LXRs seem to regulate both cholesterol and fatty acid metabolism. Mice carrying a targeted disruption of the Lxr␣ gene fail to induce transcription of the gene encoding cholesterol 7␣-hydroxylase (CYP7A1) in response to dietary cholesterol, implicating LXRs in the control of bile acid synthesis (11). Mice lacking LXR␣ were also observed to be deficient in expression of fatty acid synthase, steroyl-coA desaturase 1, acyl-coA carboxylase, and sterol regulatory element binding protein-1, suggesting an additional role in lipogenesis. This hypothesis was supported by the subsequent demonstration that the synthetic LXR ligand T1317 induces expression of lipogenic genes and raises plasma trigly...

A central enzyme in the pathway of de novo lipogenesis, fatty acid synthase (FAS) 1 catalyzes all of the steps in the conversion of malonyl-CoA to palmitate. Expression of the FAS gene is controlled primarily at the level of transcription and is responsive to both hormonal and nutritional signals (1, 2). Previous work has shown that sterol regulatory element-binding proteins (SREBPs) play a critical role in the transcriptional regulation of a number of genes in the lipogenic pathway, including FAS, steroyl-CoA desaturase (SCD-1), and acetyl-CoA carboxylase (ACC) (3-8). Three SREBP isoforms have been described: SREBP-1a and Ϫ1c (also called ADD1), which are derived from the same gene through alternative splicing, and SREBP-2, which is encoded by a separate gene (9, 10). Although their transcriptional targets overlap significantly, studies suggest that SREBP-1 preferentially activates genes involved in lipogenesis, whereas SREBP-2 preferentially activates genes in the cholesterol biosynthetic pathway (11-14). SREBPs have been shown to regulate FAS expression through direct interaction with the FAS promoter at multiple sites (7, 15). Overexpression of nuclear SREBP-1 is sufficient to induce expression of the FAS gene in cultured cells as well as transgenic mice (5,8). Recent work has also implicated the nuclear receptors LXR␣ and LXR␤ in the control of lipogenesis. Both LXRs bind to DNA and regulate transcription of target genes in a heterodimeric complex with RXR (16). Although early studies on LXRs focused on their role in cholesterol metabolism, mice carrying a targeted disruption in the LXR␣ gene were noted to be deficient in expression of FAS, SCD-1, ACC, and SREBP-1, consistent with a role in lipogenesis as well (17). Further support for this idea came with the observation that the administration of the synthetic LXR ligand T1317 to mice triggers induction of the lipogenic pathway and raises plasma triglyceride levels (18). The demonstration that the SREBP-1c promoter is a direct target for regulation by LXR/RXR heterodimers provided a straightforward explanation for the ability of LXR ligands to induce hepatic lipogenesis (19,20). Until now, the effects of LXR activation on the expression of lipogenic genes, including FAS, have been presumed to be entirely indirect.We demonstrate here that the FAS promoter is a direct target for regulation by the LXR/RXR heterodimer as well as SREBPs. This novel mechanism for the regulation of FAS expression and lipogenesis by LXRs has implications for the development of LXR agonists as modulators of human lipid metabolism. EXPERIMENTAL PROCEDURESReagents and Plasmids-Expression plasmids for RXR␣ and LXR␣, and nuclear SREBP-1a, -1c, and -2 have been described (21,22). GW3965 (23) and T0901317 (18) were provided by Timothy M. Willson (GlaxoSmithKline). Ligands were dissolved in Me 2 SO prior to use in cell culture. The Ϫ1594, Ϫ700, Ϫ150, and Ϫ135 rat FAS promoter luciferase reporter constructs were described previously (3). Mutations were

The control of lipid and glucose metabolism is closely linked. The nuclear receptors liver X receptor (LXR)␣ and LXR␤ have been implicated in gene expression linked to lipid homeostasis; however, their role in glucose metabolism is not clear. We demonstrate here that the synthetic LXR agonist GW3965 improves glucose tolerance in a murine model of diet-induced obesity and insulin resistance. Analysis of gene expression in LXR agonist-treated mice reveals coordinate regulation of genes involved in glucose metabolism in liver and adipose tissue. In the liver, activation of LXR led to the suppression of the gluconeogenic program including down-regulation of peroxisome proliferator-activated receptor ␥ coactivator-1␣ (PGC-1), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase expression. Inhibition of gluconeogenic genes was accompanied by an induction in expression of glucokinase, which promotes hepatic glucose utilization. In adipose tissue, activation of LXR led to the transcriptional induction of the insulin-sensitive glucose transporter, GLUT4. We show that the GLUT4 promoter is a direct transcriptional target for the LXR͞retinoid X receptor heterodimer and that the ability of LXR ligands to induce GLUT4 expression is abolished in LXR null cells and animals. Consistent with their effects on GLUT4 expression, LXR agonists promote glucose uptake in 3T3-L1 adipocytes in vitro. Thus, activation of LXR alters the expression of genes in liver and adipose tissue that collectively would be expected to limit hepatic glucose output and improve peripheral glucose uptake. These results outline a role for LXRs in the coordination of lipid and glucose metabolism.L iver X receptor (LXR)␣ and LXR␤ have emerged as important regulators of lipid and lipoprotein metabolism. The LXRs are activated by physiological concentrations of oxidized derivatives of cholesterol such as 22(R)-hydroxycholesterol, 27-hydroxycholesterol, and 24(S),25-epoxycholesterol (1-3). LXR␣ is expressed at particularly high levels in liver, adipose tissue, and macrophages, whereas LXR␤ is expressed ubiquitously. These ligand-activated transcription factors form obligate heterodimers with the retinoid X receptor (RXR) and regulate the expression of target genes containing LXR response elements (LXREs). All the LXREs identified thus far are DR-4 hormone response elements (direct repeat of the consensus AGGTCA separated by four nucleotides) (4).To date, more than a dozen LXR target genes have been identified (5). In the liver, LXRs regulate expression of a number of proteins involved in cholesterol and fatty acid metabolism, including CYP7A and sterol regulatory binding element protein 1c (SREBP-1c) (6, 7). In macrophages and other peripheral cells, LXRs have been implicated in the reverse cholesterol transport pathway. LXRs control the transcription of several genes involved in cellular cholesterol efflux including ATP-binding cassette (ABC)A1, ABCG1, and apolipoprotein E (8-11). LXRs also seem to influence lipoprotein metabolism through the c...

Recent studies have identified the liver X receptors (LXR␣ and LXR␤) as important regulators of cholesterol metabolism and transport. LXRs control transcription of genes critical to a range of biological functions including regulation of high density lipoprotein cholesterol metabolism, hepatic cholesterol catabolism, and intestinal sterol absorption. Although LXR activity has been proposed to be critical for physiologic lipid metabolism and transport, direct evidence linking LXR signaling pathways to the pathogenesis of cardiovascular disease has yet to be established. In this study bone marrow transplantations were used to selectively eliminate macrophage LXR expression in the context of murine models of atherosclerosis. Our results demonstrate that LXRs are endogenous inhibitors of atherogenesis. Additionally, elimination of LXR activity in bone marrow-derived cells mimics many aspects of Tangier disease, a human high density lipoprotein deficiency, including aberrant regulation of cholesterol transporter expression, lipid accumulation in macrophages, splenomegaly, and increased atherosclerosis. These results identify LXRs as targets for intervention in cardiovascular disease.

Apolipoprotein E (apoE) secreted by macrophages in the artery wall exerts an important protective effect against the development of atherosclerosis, presumably through its ability to promote lipid efflux. Previous studies have shown that increases in cellular free cholesterol levels stimulate apoE transcription in macrophages and adipocytes; however, the molecular basis for this regulation is unknown. Recently, Taylor and colleagues [Shih, S. J., Allan, C., Grehan, S., Tse, E., Moran, C. & Taylor, J. M. (2000) J. Biol. Chem. 275, 31567-31572] identified two enhancers from the human apoE gene, termed multienhancer 1 (ME.1) and multienhancer 2 (ME.2), that direct macrophage- and adipose-specific expression in transgenic mice. We demonstrate here that the nuclear receptors LXRalpha and LXRbeta and their oxysterol ligands are key regulators of apoE expression in both macrophages and adipose tissue. We show that LXR/RXR heterodimers regulate apoE transcription directly, through interaction with a conserved LXR response element present in both ME.1 and ME.2. Moreover, we demonstrate that the ability of oxysterols and synthetic ligands to regulate apoE expression in adipose tissue and peritoneal macrophages is reduced in Lxralpha-/- or Lxrbeta-/- mice and abolished in double knockouts. Basal expression of apoE is not compromised in Lxr null mice, however, indicating that LXRs mediate lipid-inducible rather than tissue-specific expression of this gene. Together with our previous work, these findings support a central role for LXR signaling pathways in the control of macrophage cholesterol efflux through the coordinate regulation of apoE, ABCA1, and ABCG1 expression.