RNA-seq of primary human CD4+ T cells treated with the LXR agonist GW3965, with and without TCR stimulation
ABSTRACT: We used RNA-sequencing to determine the transcriptional response to liver X receptor (LXR) activation in primary human CD4+ T cells, both at rest and under activation conditions. CD4+ T cells were isolated from 3 healthy donors and treated with the synthetic LXR agonist GW3965 (GW) for 24 hours. Gene expression was compared to control samples (CTRL) treated with the LXR antagonist GSK1440233. To activate the T cell antigen receptor (TCR), cells were pre-treated with CTRL/GW alone for 6 hours then transferred to plates containing anti-CD3 and anti-CD28 for a further 18 hours.
Project description:Analysis of differentially expressed genes in response to the LXR agonist GW3965 in the MCF-7, T-47D, SK-BR-3, and MDA-MB-231 breast cancer cell lines. It was previously reported that GW3965 has antiproliferative effects on these 4 different breast cancer cell lines. In the present study, we additionally determine the effects of the LXR ligand on breast cancer cells and determine their mechanism of action in reducing cell proliferation. Total RNA obtained from 4 different breast cancer cell lines (MCF-7, T-47D, SK-BR-3, MDA-MB-231) grown in culture treated with ethanol (control) or GW3965 (GW-treated, experimental) for 48 hours. Triplicates were performed.
Project description:The pharmacological manipulation of liver X receptors (LXRs) has been an attractive therapeutic strategy for atherosclerosis treatment as they control reverse cholesterol transport and inflammatory response. This study presents the development and efficacy of nanoparticles (NPs) incorporating the synthetic LXR agonist GW3965 (GW) in targeting atherosclerotic lesions. Collagen IV (Col IV) targeting ligands are employed to functionalize the NPs to improve targeting to the atherosclerotic plaque, and formulation parameters such as the length of the polyethylene glycol (PEG) coating molecules are systematically optimized. In vitro studies indicate that the GW-encapsulated NPs upregulate the LXR target genes and downregulate proinflammatory mediator in macrophages. The Col IV-targeted NPs encapsulating GW (Col IV-GW-NPs) successfully reaches atherosclerotic lesions when administered for 5 weeks to mice with preexisting lesions, substantially reducing macrophage content (?30%) compared to the PBS group, which is with greater efficacy versus nontargeting NPs encapsulating GW (GW-NPs) (?18%). In addition, mice administered the Col IV-GW-NPs do not demonstrate increased hepatic lipid biosynthesis or hyperlipidemia during the treatment period, unlike mice injected with the free GW. These findings suggest a new form of LXR-based therapeutics capable of enhanced delivery of the LXR agonist to atherosclerotic lesions without altering hepatic lipid metabolism.
Project description:This microarray is an analysis of differentially expressed genes in three pancreatic ductal adenocarcinoma cell lines treated with LXR-agonist GW 3965. We first report that GW 3965 has antiproliferative effects in three PDAC cell lines. This microarray was designed to identify key mechanisms of the antiproliferative effect of LXR agonists within pancreatic cancer cell lines. Total RNA obtained from BxPC-3, MIA-PaCa-2, and PANC-1 pancreatic cancer cells grown in culture treated GW 3965 or ethanol (vehicle control) for 72 hours.
Project description:Gene expression: Identification of primary target genes of liver X receptor (LXR) in an immune-related cellular model (THP-1 cells) to study, in conjunction with LXR binding data from ChIP-seq, the genome-wide mechanisms of transcriptional regulation by LXR. ChIP-Seq: We performed ChIP-seq in macrophage-type PMA-differentiated THP-1 cells after stimulation with the potent synthetic LXR ligand T0901317 (T09). As a reference we performed microarray gene expression analysis in the same cellular model. We identified in total 1357 LXR binding locations on chromatin (FDR < 1%), of which 526 were observed after T09 treatment. De novo analysis of LXR site sequences identified DR4-type binding sites as major motif. gene expression: THP-1 cells were treated for 4 h with 1 µM T09 or vehicle (DMSO) ChIP-Seq: PMA-differentiated THP-1 cells were treated for 60 min with 1 µM T09 or vehicle (DMSO)
Project description:Activation of the transcription factor liver X receptor (LXR) has beneficial effects on macrophage lipid metabolism and inflammation, making it a potential candidate for therapeutic targeting in cardiometabolic disease. While small molecule delivery via nanomedicine has promising applications for a number of chronic diseases, questions remain as to how nanoparticle formulation might be tailored to suit different tissue microenvironments and aid in drug delivery. In the current study, we aimed to compare the in vitro drug delivering capability of three nanoparticle (NP) formulations encapsulating the LXR activator, GW-3965. We observed little difference in the base characteristics of standard PLGA-PEG NP when compared to two redox-active polymeric NP formulations, which we called redox-responsive (RR)1 and RR2. Moreover, we also observed similar uptake of these NP into primary mouse macrophages. We used the transcript and protein expression of the cholesterol efflux protein and LXR target ATP-binding cassette A1 (ABCA1) as a readout of GW-3956-induced LXR activation. Following an initial acute uptake period that was meant to mimic circulating exposure in vivo, we determined that although the induction of transcript expression was similar between NPs, treatment with the redox-sensitive RR1 NPs resulted in a higher level of ABCA1 protein. Our results suggest that NP formulations responsive to cellular cues may be an effective tool for targeted and disease-specific drug release.
Project description:Lxr-/- mice and WT mice were fed with a western diet and a control diet; Transcriptomic analysis of total RNA samples originated from latero-dorsal prostates was performed as described below between the four conditions: Wild type Mice +/+ on normal diet, Wild type Mice +/+ on western diet, lxr KO Mice-/- on normal diet, lxr KO Mice-/- on western diet. 4 samples will be pooled for each condition: WT +/+ base diet WT +/+ western diet, LXR -/- base diet LXR -/- western diet. Analysis of diet effects: 1. On WT mice: WT Mice +/+ normal diet vs WT Mice +/+ western diet. Wild type base diet RNA pool will be compared to Wild type western diet RNA pool by performing a direct comparison on an Agilent array using the 2-color approach. Dye flips will be prepared for a total of 2 hybridizations. 2. On KO mice: LXR mice -/- base diet vs LXR mice -/- western diet. LXR KO base diet RNA pool will be compared to LXR KO western diet RNA pool by performing a direct comparison on an Agilent array using the 2-color approach. Dye flips will be prepared for a total of 2 hybridizations. Analysis of genotype effects: 1. Under western diet condition: WT +/+ western diet vs LXR -/- western diet. Wild type western diet RNA pool will be compared to LXR KO western diet RNA pool by performing a direct comparison on an Agilent array using the 2-color approach. Dye flips will be prepared for a total of 2 hybridizations. 2. Under base diet condition: WT +/+ base diet vs LXR -/- base diet Wild type base diet RNA pool will be compared to LXR KO base diet RNA pool by performing a direct comparison on an Agilent array using the 2-color approach. Dye flips will be prepared for a total of 2 hybridizations.
Project description:The liver X receptors (LXRs) are nuclear receptors that form permissive heterodimers with retinoid X receptor (RXR) and are important regulators of lipid metabolism in the liver. We have recently shown that RXR agonist-induced hypertriglyceridemia and hepatic steatosis in mice is dependent on LXR and correlates with an LXR-dependent hepatic induction of lipogenic genes. To further investigate the role of RXR and LXR in the regulation of hepatic gene expression, we have mapped the ligand-regulated genome-wide binding of these factors in mouse liver. We find that the RXR agonist bexarotene primarily increases the genomic binding of RXR, whereas the LXR agonist T0901317 greatly increases both LXR and RXR binding. Functional annotation of putative direct LXR target genes revealed a significant association with classical LXR-regulated pathways as well as PPAR signaling pathways, and subsequent ChIP-seq mapping of PPARα binding demonstrated binding of PPARα to 71-88% of the identified LXR:RXR binding sites. Sequence analysis of shared binding regions combined with sequential ChIP on selected sites indicate that LXR:RXR and PPARα:RXR bind to degenerate response elements in a mutually exclusive manner. Together our findings suggest extensive and unexpected cross-talk between hepatic LXR and PPARα at the level of binding to shared genomic sites LXR, RXR, PPARalpha and RNA Polymerase II ChIP-seq on livers from female C57BL/6 wild-type and/or LXRα/β-deficient mice (13 weeks of age, n=1) treated by oral gavage once daily for 14 days with the RXR agonist bexarotene (100 mg/kg body weight [mpk], in 1% carboxymethylcellulose), the LXR agonist T0901317 (T09, 30 mpk) or vehicle alone.
Project description:The Liver X Receptors (LXRs) play important roles in multiple metabolic pathways, including fatty acid, cholesterol, carbohydrate and energy metabolism. To expand the knowledge of the functions of LXR signaling during embryonic development, we performed a whole-genome microarray analysis of Lxr target genes in zebrafish larvae treated with either one of the synthetic LXR ligands T0901317 or GW3965. Assessment of the biological processes enriched by differentially expressed genes revealed a prime role for Lxr in regulating lipid metabolic processes, similarly to the function of LXR in mammals. In addition, exposure to the Lxr ligands induced changes in expression of genes in the neural retina and lens of the zebrafish eye, including the photoreceptor guanylate cyclase activators and lens gamma crystallins, suggesting a potential novel role for Lxr in modulating the transcription of genes associated with visual function in zebrafish. The regulation of expression of metabolic genes was phenotypically reflected in an increased absorption of yolk in the zebrafish larvae, and changes in the expression of genes involved in visual perception were associated with morphological alterations in the retina and lens of the developing zebrafish eye. The regulation of expression of both lipid metabolic and eye specific genes was sustained in 1 month old fish. The transcriptional networks demonstrated several conserved effects of LXR activation between zebrafish and mammals, and also identified potential novel functions of Lxr, supporting zebrafish as a promising model for investigating the role of Lxr during development. Overall design: Wild type zebrafish WIK embryos were collected after spawning. At 4 days post fertilization (dpf), zebrafish larvae were transferred to 6-well plates (pools of 30 embryos/well) in 3 mL of embryo media (E3: 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.3 mM MgSO4). Larvae at 4 dpf were treated with DMSO as vehicle control, 2 mM T0901317 (T0) or 1 mM GW3965 (GW). Final DMSO concentration in all treatments and control was 0.1%. Treatments were renewed after 24 h and larvae were collected for RNA extraction at 6 dpf. Treatments with vehicle (0.1% DMSO), T0 and GW were performed in biological quadruplicates of pools of RNA. Array corresponding to sample 33576 (T0 treatment replicate 2) was excluded from microarray data analysis.
Project description:LXR is a transcription factor. Two isoforms exist in mice (alpha and beta). They are both expressed in the liver. We examined the role of LXR by obtaining gene expression profiles from the livers of wild-type and LXR-/- mice from the same genetic background. Liver gene expression was measured from male mice (5 mice/group) of genotypes wild-type and LXR-/- on a mixed Sv129/C57Bl6J background (Repa et al. , 2000, Science, 289(5484):1524-9; Cummins et al., 2006, J Clin Invest,116(7):1902-12) fed for 7 weeks a normal diet (ND) or a Western diet (WD).
Project description:Here, we have focused on studying the link between metabolic changes driven by the differentiation into mature adipocytes of a human preadipocyte cell line (SGBS) and their regulation, through a combined experimental and computational approach. By collecting data on gene expression, PPARg, CEBPa, LXR and H3K4me3 genome-wide ChIP-seq profles and transcriptome-wide microRNA target identification for miR-27a, miR29a and miR-222, and using constraint-based modeling to estimate metabolic reaction activity, we obtained a comprehensive set of information highlighting how epigenetic, transcriptional and post-transcriptional regulation impacts the metabolic network. Illumina HT12 V3.0 microarrays: LXR ligand activation with 1 microM T0901317 for 4 h in SGBS day 10 differentiated adipocytes (6 samples, treatment vs control, in triplicate) This submission represents transcriptome component of study.