Liver X receptors (LXRs) regulate the expression of genes involved in cholesterol and fatty acid homeostasis, including the genes for ATP-binding cassette transporter A1 (ABCA1) and sterol response element binding protein 1 (SREBP1). Loss of LXR leads to derepression of the ABCA1 gene in macrophages and the intestine, while the SREBP1c gene remains transcriptionally silent. Here we report that high-density-lipoprotein (HDL) cholesterol levels are increased in LXR-deficient mice, suggesting that derepression of ABCA1 and possibly other LXR target genes in selected tissues is sufficient to result in enhanced HDL biogenesis at the whole-body level. We provide several independent lines of evidence indicating that the repressive actions of LXRs are dependent on interactions with the nuclear receptor corepressor (NCoR) and the silencing mediator of retinoic acid and thyroid hormone receptors (SMRT). While dissociation of NCoR and SMRT results in derepression of the ABCA1 gene in macrophages, it is not sufficient for derepression of the SREBP1c gene. These findings reveal differential requirements for corepressors in the regulation of genes involved in cholesterol and fatty acid homeostasis and raise the possibility that these interactions may be exploited to develop synthetic ligands that selectively modulate LXR actions in vivo.

This article is available online at http://www.jlr.org early event in the development of atherosclerosis is the recruitment of macrophages to the subendothelial space of vessel walls and the uncontrolled uptake of oxidized or aggregated low density lipoprotein particles. Continued accumulation of oxidized or aggregated LDL by macrophages and an associated infl ammatory response leads to foam cell formation and the initiation of atherosclerosis ( 2 ).The liver X receptors LXR ␣ (NR1H3) and LXR ␤ (NR1H2), members of the nuclear hormone receptor superfamily of transcription factors, have been identifi ed as important regulators of cholesterol homeostasis in multiple cell types, including macrophages ( 3 ). Treatment of cells with oxysterols, natural LXR ligands derived from cholesterol, or synthetic LXR agonists promotes the effl ux of cholesterol by increasing expression of the ATP binding cassette transporters ABCA1 and ABCG1 and the apolipoprotein E (apoE) ( 4, 5 ). ABCA1, ABCG1, and apoE all participate in the transfer of intracellular and plasma membrane cholesterol to HDL, a process termed reverse cholesterol transport ( 6 ). Importantly, LXR agonists reduce atherosclerosis in animal models of cardiovascular disease, and upregulation of ABC transporters can be detected in the atherosclerotic lesions of treated animals ( 7,8 ). The ability of LXRs to inhibit pro-infl ammatory pathways may also contribute to the anti-atherogenic activities of these receptors ( 9 ). Previous work from our laboratory has demonstrated that LXR function in hema- The contribution of elevated cholesterol levels to the development of cardiovascular disease and atherosclerosis is well documented. Nevertheless the molecular signaling pathways that regulate cholesterol homeostasis at the blood vessel wall, particularly in response to elevated cholesterol levels, remain to be fully deciphered ( 1 ). A critical Abbreviations: ABCA1, ATP binding cassette transporter A1; ABCG1, ATP binding cassette transporter G1; apoE, apolipoprotein E; FPLC, fast-protein liquid chromatography; iNOS, inducible nitric oxide synthase; LXR, liver X receptor; MCP-1, monocycte chemotactic protein 1; NF ␤ , nuclear factor kappa ␤ ; SREBP, sterol-regulatory element binding protein; TNF-␣ , tumor necrosis factor-␣ .

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Recent studies have identified the liver X receptors (LXR␣ and LXR␤) as important regulators of cholesterol and lipid metabolism. Although originally identified as liver-enriched transcription factors, LXRs are also expressed in skeletal muscle, a tissue that accounts for ϳ40% of human total body weight and is the major site of glucose utilization and fatty acid oxidation. Nevertheless, no studies have yet addressed the functional role of LXRs in muscle. In this work we utilize a combination of in vivo and in vitro analysis to demonstrate that LXRs can functionally regulate genes involved in cholesterol metabolism in skeletal muscle. Furthermore we show that treatment of muscle cells in vitro with synthetic agonists of LXR increases the efflux of intracellular cholesterol to extracellular acceptors such as high density lipoprotein, thus identifying this tissue as a potential important regulator of reverse cholesterol transport and high density lipoprotein levels. Additionally we demonstrate that LXR␣ and a subset of LXR target genes are induced during myogenesis, suggesting a role for LXR-dependent signaling in the differentiation process.Disorders of cholesterol and lipid metabolism are associated with cardiovascular disease, obesity, diabetes, and hypertension. Not surprisingly organisms have developed exquisite regulatory networks that ensure lipid homeostasis is maintained by controlling dietary intake, de novo synthesis, transport, and catabolism. For instance, numerous studies over the past five years have identified members of the nuclear hormone receptor superfamily of ligand-dependent transcription factors as important regulators of the genes involved cholesterol and lipid metabolism (3). In particular, the peroxisome proliferator-activated receptors (PPAR␣, 1 ␤/␦, and ␥), the farnesoid X receptor, and the liver X receptors (LXR␣, and LXR␤) are transcription factors whose activity can be controlled by the direct binding of fatty acids (PPARs and LXRs) and cholesterol derivatives (LXRs and farnesoid X receptor). Thus, these transcription factors are poised to sense changes in the intracellular concentrations of lipids and cholesterol and to regulate cellular metabolism accordingly (3,4). Recently several studies have demonstrated that the LXRs play a dynamic role in the regulation of genes involved in cholesterol and fatty acid metabolism. LXRs bind to DNA as obligate heterodimers with retinoid X receptors and directly bind cholesterol metabolites and fatty acids (5, 6). Interestingly cholesterol derivatives and fatty acids have opposing effects on LXR transcriptional activity. Oxysterols including 24(S), 25-epoxycholesterol, 22(R)-hydroxycholesterol, and 24(S)-hydroxycholesterol are activators of LXR and increase transcription of genes involved in sterol transport including the ATP binding cassette transporters ABCA1, ABCG1, ABCG5, and ABCG8 and the apolipoprotein apoE (7-11). The importance of these LXR target genes to sterol metabolism has recently been highlighted by linkage of ABCA1 to Tangier disea...

Azepino[4,5-b]indoles have been identified as potent agonists of the farnesoid X receptor (FXR). In vitro and in vivo optimization has led to the discovery of 6m (XL335, WAY-362450) as a potent, selective, and orally bioavailable FXR agonist (EC(50) = 4 nM, Eff = 149%). Oral administration of 6m to LDLR(-/-) mice results in lowering of cholesterol and triglycerides. Chronic administration in an atherosclerosis model results in significant reduction in aortic arch lesions.

This article is available online at http://www.jlr.org early event in the development of atherosclerosis is the recruitment of macrophages to the subendothelial space of vessel walls and the uncontrolled uptake of oxidized or aggregated low density lipoprotein particles. Continued accumulation of oxidized or aggregated LDL by macrophages and an associated infl ammatory response leads to foam cell formation and the initiation of atherosclerosis ( 2 ).The liver X receptors LXR ␣ (NR1H3) and LXR ␤ (NR1H2), members of the nuclear hormone receptor superfamily of transcription factors, have been identifi ed as important regulators of cholesterol homeostasis in multiple cell types, including macrophages ( 3 ). Treatment of cells with oxysterols, natural LXR ligands derived from cholesterol, or synthetic LXR agonists promotes the effl ux of cholesterol by increasing expression of the ATP binding cassette transporters ABCA1 and ABCG1 and the apolipoprotein E (apoE) ( 4, 5 ). ABCA1, ABCG1, and apoE all participate in the transfer of intracellular and plasma membrane cholesterol to HDL, a process termed reverse cholesterol transport ( 6 ). Importantly, LXR agonists reduce atherosclerosis in animal models of cardiovascular disease, and upregulation of ABC transporters can be detected in the atherosclerotic lesions of treated animals ( 7,8 ). The ability of LXRs to inhibit pro-infl ammatory pathways may also contribute to the anti-atherogenic activities of these receptors ( 9 ). Previous work from our laboratory has demonstrated that LXR function in hema- The contribution of elevated cholesterol levels to the development of cardiovascular disease and atherosclerosis is well documented. Nevertheless the molecular signaling pathways that regulate cholesterol homeostasis at the blood vessel wall, particularly in response to elevated cholesterol levels, remain to be fully deciphered ( 1 ). A critical Abbreviations: ABCA1, ATP binding cassette transporter A1; ABCG1, ATP binding cassette transporter G1; apoE, apolipoprotein E; FPLC, fast-protein liquid chromatography; iNOS, inducible nitric oxide synthase; LXR, liver X receptor; MCP-1, monocycte chemotactic protein 1; NF ␤ , nuclear factor kappa ␤ ; SREBP, sterol-regulatory element binding protein; TNF-␣ , tumor necrosis factor-␣ .

Gene delivery via murine-based recombinant retroviral vectors is currently widely used in gene therapy clinical trials. The vectors are engineered to be replication defective by replacing the structural and nonstructural genes of a cloned infectious retrovirus with a therapeutic gene of interest. The retroviral particles are currently generated in packaging cell lines, which supply all retroviral proteins in trans. Recombination between short homologous regions of the retroviral vector and packaging cell line elements can theoretically generate replication-competent retrovirus (RCR) and hence the Food and Drug Administration (FDA) requires the monitoring of clinical trial subjects for the presence of RCR. Sensitive polymerase chain reaction (PCR) assays have been used for the detection of murine leukemia virus (MLV) nucleotide sequences in peripheral blood mononuclear cells (PBMCs). A novel serological enzyme-linked immunosorbent assay (ELISA) for the detection of anti-MLV specific immunoglobulin (Ig) has been developed to be used as an alternative to the PCR assay. Both assays were used to monitor human immunodeficiency virus (HIV)-positive clinical trial subjects who had received multiple injections of HIV-IT (V), a retroviral vector encoding HIV-1 IIIBenv/rev. Western blot analysis and an in vitro vector neutralization assay were used to characterize further a subset of serum samples tested by ELISA. Results show no evidence of RCR infection in clinical trial subjects. PCR and ELISA assays are discussed in terms of their advantages and limitations as routine screening assays for RCR. The PCR assay is our current choice for monitoring clinical trial subjects receiving direct administration of vector, and the ELISA is our choice for those receiving ex vivo treatment regimens.

Genomic clones encoding two isozymes of aspartate aminotransferase (AAT) were isolated from an alfalfa genomic library and their DNA sequences were determined. The AAT1 gene contains 12 exons that encode a cytosolic protein expressed at similar levels in roots, stems and nodules. In nodules, the amount of AAT1 mRNA was similar at all stages of development, and was slightly reduced in nodules incapable of fixing nitrogen. The AAT1 mRNA is polyadenylated at multiple sites differing by more than 250 bp. The AAT2 gene contains 11 exons, with 5 introns located in positions identical to those found in animal AAT genes, and encodes a plastid-localized isozyme. The AAT2 mRNA is polyadenylated at a very limited range of sites. The transit peptide of AAT2 is encoded by the first two and part of the third exon. AAT2 mRNA is much more abundant in nodules than in other organs, and increases dramatically during the course of nodule development. Unlike AAT1, expression of AAT2 is significantly reduced in nodules incapable of fixing nitrogen. Phylogenetic analysis of deduced AAT proteins revealed 4 separate but related groups of AAT proteins; the animal cytosolic AATs, the plant cytosolic AATs, the plant plastid AATs, and the mitochondrial AATs.