Cholesterol-sensing liver X receptors stimulate Th2-driven allergic eosinophilic asthma in mice

Introduction: Liver X receptors (LXRs) are nuclear receptors that function as cholesterol sensors and regulate cholesterol homeostasis. High cholesterol has been recognized as a risk factor in asthma; however, the mechanism of this linkage is not known. Methods: To explore the importance of cholesterol homeostasis for asthma, we investigated the contribution of LXR activity in an ovalbumin- and a house dust mite-driven eosinophilic asthma mouse model. Results: In both models, airway inflammation, airway hyper-reactivity, and goblet cell hyperplasia were reduced in mice deficient for both LXR and LXR isoforms (LXR-/--/-) as compared to wild-type mice. Inversely, treatment with the LXR agonist GW3965 showed increased eosinophilic airway inflammation. LXR activity contributed to airway inflammation through promotion of type 2 cytokine production as LXR-/--/- mice showed strongly reduced protein levels of IL-5 and IL-13 in the lungs as well as reduced expression of these cytokines by CD4(+) lung cells and lung-draining lymph node cells. In line herewith, LXR activation resulted in increased type 2 cytokine production by the lung-draining lymph node cells. Conclusions: In conclusion, our study demonstrates that the cholesterol regulator LXR acts as a positive regulator of eosinophilic asthma in mice, contributing to airway inflammation through regulation of type 2 cytokine production

Human NCU-G1 can function as a transcription factor and as a nuclear receptor co-activator

<p>Abstract</p> <p>Background</p> <p>Novel, uncharacterised proteins represent a challenge in biochemistry and molecular biology. In this report we present an initial functional characterization of human kidney predominant protein, NCU-G1.</p> <p>Results</p> <p>NCU-G1 was found to be a highly conserved nuclear protein rich in proline with a molecular weight of approximately 44 kDa. It is localized on chromosome 1 and consists of 6 exons. Analysis of the amino acid sequence revealed no known transcription activation domains or DNA binding regions, however, four nuclear receptor boxes (LXXLL), and four SH3-interaction motives in addition to numerous potential phosphorylation sites were found. Two nuclear export signals were identified, but no nuclear localization signal. In man, NCU-G1 was found to be widely expressed at the mRNA level with especially high levels detected in prostate, liver and kidney. Electrophoretic mobility shift analysis showed specific binding of NCU-G1 to an oligonucleotide representing the footprint 1 element of the human cellular retinol-binding protein 1 gene promoter. NCU-G1 was found to activate transcription from this promoter and required presence of the footprint 1 element. In transiently transfected Drosophila Schneider S2 cells, we demonstrated that NCU-G1 functions as a co-activator for ligand-activated PPAR-alpha, resulting in an increased expression of a CAT reporter gene under control of the peroxisome proliferator-activated receptor-alpha responsive acyl-CoA oxidase promoter.</p> <p>Conclusion</p> <p>We propose that NCU-G1 is a dual-function protein capable of functioning as a transcription factor as well as a nuclear receptor co-activator.</p

Genome-wide landscape of liver X receptor chromatin binding and gene regulation in human macrophages

<p>Abstract</p> <p>Background</p> <p>The liver X receptors (LXRs) are oxysterol sensing nuclear receptors with multiple effects on metabolism and immune cells. However, the complete genome-wide cistrome of LXR in cells of human origin has not yet been provided.</p> <p>Results</p> <p>We performed ChIP-seq in phorbol myristate acetate-differentiated THP-1 cells (macrophage-type) after stimulation with the potent synthetic LXR ligand T0901317 (T09). Microarray gene expression analysis was performed in the same cellular model. We identified 1357 genome-wide LXR locations (FDR < 1%), of which 526 were observed after T09 treatment. <it>De novo </it>analysis of LXR binding sequences identified a DR4-type element as the major motif. On mRNA level T09 up-regulated 1258 genes and repressed 455 genes. Our results show that LXR actions are focused on 112 genomic regions that contain up to 11 T09 target genes per region under the control of highly stringent LXR binding sites with individual constellations for each region. We could confirm that LXR controls lipid metabolism and transport and observed a strong association with apoptosis-related functions.</p> <p>Conclusions</p> <p>This first report on genome-wide binding of LXR in a human cell line provides new insights into the transcriptional network of LXR and its target genes with their link to physiological processes, such as apoptosis.</p> <p>The gene expression microarray and sequence data have been submitted collectively to the NCBI Gene Expression Omnibus <url>http://www.ncbi.nlm.nih.gov/geo</url> under accession number GSE28319.</p

Anti-inflammatory nuclear receptor superfamily in multiple sclerosis patients from Sardinia and Sweden

Several nuclear hormone receptors have been associated with inflammatory reactions. Particularly, liver X receptors (LXRs) have recently been identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. LXRs are negative regulators of macrophage inflammatory gene expression. Multiple sclerosis (MS), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. Sweden belongs to the countries with a high MS incidence. In Italy, the MS incidence is lower, except on the island of Sardinia where the incidence is even higher than in Sweden. Subjects from Sardinia are ethnically more homogeneous, and differ from Swedes also regarding genetic background and environment. We studied mRNA expression of several nuclear hormone receptors in blood mononuclear cells (MNC) from female patients with untreated relapsing-remitting MS from Sassari, Sardinia, and Stockholm, Sweden. Sex- and age-matched healthy controls (HC) were from both areas. mRNA expression was evaluated by quantitative real-time PCR. We found altered mRNA expression of LXRs, estrogen receptors (ERs), and androgen receptor (AR) in MS. mRNA expression of both LXRα and LXRβ is lower in MS from Stockholm but not from Sassari. In particular, LXRα mRNA expression was significantly lower in MS from Stockholm as compared with all groups in the study including MS from Sassari. Low levels of ERα mRNA are seen in MS from both Stockholm and Sassari. The splice variant ERβcx showed significantly higher mRNA expression in MS from Sassari and Stockholm as compared with corresponding HC. In particular, ERβcx mRNA in MS from Sassari was remarkably higher as compared with all other groups in the study. Higher levels of AR mRNA are present in HC from Sassari. The findings indicate that the expression levels of anti-inflammatory nuclear receptor superfamily genes in MS appear to reflect both ethnic and environmental influences

LXRα improves myocardial glucose tolerance and reduces cardiac hypertrophy in a mouse model of obesity-induced type 2 diabetes

Aims/hypothesis Diabetic cardiomyopathy is a myocardial disease triggered by impaired insulin signalling, increased fatty acid uptake and diminished glucose utilisation. Liver X receptors (LXRs) are key transcriptional regulators of metabolic homeostasis. However, their effect in the diabetic heart is largely unknown. Methods We cloned murine Lxr alpha (also known as Nr1h3) behind the a-myosin heavy chain (alpha Mhc; also known as Myh6) promoter to create transgenic (Lxr alpha-Tg) mice and transgene-negative littermates (wild-type [WT]). A mouse model of type 2 diabetes was induced by a high-fat diet (HFD, 60% energy from fat) over 16 weeks and compared with a low-fat diet (10% energy from fat). A mouse model of type 1 diabetes was induced via streptozotocin injection over 12 weeks. Results HFD manifested comparable increases in body weight, plasma triacylglycerol and insulin resistance per OGTT in Lxr alpha-Tg and WT mice. HFD significantly increased left ventricular weight by 21% in WT hearts, but only by 5% in Lxr alpha-Tg. To elucidate metabolic effects in the heart, microPET (positron emission tomography) imaging revealed that cardiac glucose uptake was increased by 1.4-fold in WT mice on an HFD, but further augmented by 1.7-fold in Lxr alpha-Tg hearts, in part through 5' adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and restoration of glucose transporter 4 (GLUT4). By contrast, streptozotocin-induced ablation of insulin signalling diminished cardiac glucose uptake levels and caused cardiac dysfunction, indicating that insulin may be important in Lxr alpha-mediated glucose uptake. Chromatin immunoprecipitation assays identified natriuretic peptides, atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP), as potential direct targets of cardiac Lxr alpha overexpression. Conclusions/interpretation Cardiac-specific Lxr alpha overexpression ameliorates the progression of HFD-induced left ventricular hypertrophy in association with increased glucose reliance and natriuretic peptide signalling during the early phase of diabetic cardiomyopathy. These findings implicate a potential protective role for LXR in targeting metabolic disturbances underlying diabetes

Insights From Liver-Humanized Mice on Cholesterol Lipoprotein Metabolism and LXR-Agonist Pharmacodynamics in Humans

Background and Aims Genetically modified mice have been used extensively to study human disease. However, the data gained are not always translatable to humans because of major species differences. Liver-humanized mice (LHM) are considered a promising model to study human hepatic and systemic metabolism. Therefore, we aimed to further explore their lipoprotein metabolism and to characterize key hepatic species-related, physiological differences. Approach and Results Fah(-/-), Rag2(-/-), and Il2rg(-/-) knockout mice on the nonobese diabetic (FRGN) background were repopulated with primary human hepatocytes from different donors. Cholesterol lipoprotein profiles of LHM showed a human-like pattern, characterized by a high ratio of low-density lipoprotein to high-density lipoprotein, and dependency on the human donor. This pattern was determined by a higher level of apolipoprotein B100 in circulation, as a result of lower hepatic mRNA editing and low-density lipoprotein receptor expression, and higher levels of circulating proprotein convertase subtilisin/kexin type 9. As a consequence, LHM lipoproteins bind to human aortic proteoglycans in a pattern similar to human lipoproteins. Unexpectedly, cholesteryl ester transfer protein was not required to determine the human-like cholesterol lipoprotein profile. Moreover, LHM treated with GW3965 mimicked the negative lipid outcomes of the first human trial of liver X receptor stimulation (i.e., a dramatic increase of cholesterol and triglycerides in circulation). Innovatively, LHM allowed the characterization of these effects at a molecular level. Conclusions LHM represent an interesting translatable model of human hepatic and lipoprotein metabolism. Because several metabolic parameters displayed donor dependency, LHM may also be used in studies for personalized medicine.Peer reviewe

Functional and genetic analysis in type 2 diabetes of Liver X receptor alleles – a cohort study

<p>Abstract</p> <p>Background</p> <p>Liver X receptor alpha <it>(LXRA</it>) and beta (<it>LXRB</it>) regulate glucose and lipid homeostasis in model systems but their importance in human physiology is poorly understood. This project aimed to determine whether common genetic variations in <it>LXRA </it>and <it>LXRB </it>associate with type 2 diabetes (T2D) and quantitative measures of glucose homeostasis, and, if so, reveal the underlying mechanisms.</p> <p>Methods</p> <p>Eight common single nucleotide polymorphisms in <it>LXRA </it>and <it>LXRB </it>were analyzed for association with T2D in one French cohort (N = 988 cases and 941 controls), and for association with quantitative measures reflecting glucose homeostasis in two non-diabetic population-based samples comprising N = 697 and N = 1344 adults. Investigated quantitative phenotypes included fasting plasma glucose, serum insulin, and HOMA_IRas measure of overall insulin resistance. An oral glucose tolerance test was performed in N = 1344 of adults. The two alleles of the proximal <it>LXRB </it>promoter, differing only at the SNP rs17373080, were cloned into reporter vectors and transiently transfected, whereupon allele-specific luciferase activity was measured. rs17373080 overlapped, according to <it>in silico </it>analysis, with a binding site for Nuclear factor 1 (NF1). Promoter alleles were tested for interaction with NF1 using direct DNA binding and transactivation assays.</p> <p>Results</p> <p>Genotypes at two <it>LXRB </it>promoter SNPs, rs35463555 and rs17373080, associated nominally with T2D (P values 0.047 and 0.026). No <it>LXRA </it>or <it>LXRB </it>SNP associated with quantitative measures reflecting glucose homeostasis. The rs17373080 C allele displayed higher basal transcription activity (P value < 0.05). The DNA-mobility shift assay indicated that oligonucleotides corresponding to either rs17373080 allele bound NF1 transcription factors in whole cell extracts to the same extent. Different NF1 family members showed different capacity to transactivate the <it>LXRB </it>gene promoter, but there was no difference between promoter alleles in NF1 induced transactivation activity.</p> <p>Conclusion</p> <p>Variations in the <it>LXRB </it>gene promoter may be part of the aetiology of T2D. However, the association between <it>LXRB </it>rs35463555 and rs17373080, and T2D are preliminary and needs to be investigated in additional larger cohorts. Common genetic variation in <it>LXRA </it>is unlikely to affect the risk of developing T2D or quantitative phenotypes related to glucose homeostasis.</p