Genome wide binding and transcriptome analysis of human FXR in primary human hepatocytes [HTS]
ABSTRACT: We report the genome-wide profiling of FXR binding by ChIP-seq from GW4064 or DMSO treated primary human hepatocytes. We reported altered RNA expression profiles in primary human hepatocypes upon GW4064 treatment compared to DMSO control by RNA-seq. We also reported the altered RNA expression profiles in livers from WT C57BL/6J mice upon GW4064 treatment compared to vehicle control. Primary human hepatocytes were treated with 5uM GW4064 or DMSO control, 1 hour later, cells were fixed and collected for chromatin isolation. 24 hours post treatment, cells were isolated for RNA isolation. This submission represents HTS component of study.
Project description:We report the genome-wide profiling of FXR binding by ChIP-seq from GW4064 or DMSO treated primary human hepatocytes. We reported altered RNA expression profiles in primary human hepatocypes upon GW4064 treatment compared to DMSO control by RNA-seq. We also reported the altered RNA expression profiles in livers from WT C57BL/6J mice upon GW4064 treatment compared to vehicle control. Primary human hepatocytes were treated with 5uM GW4064 or DMSO control, 1 hour later, cells were fixed and collected for chromatin isolation. 24 hours post treatment, cells were isolated for RNA isolation. This submission represents HTS component of study.
Project description:<h4>Background & aims</h4>Farnesoid X receptor (FXR, NR1H4) is a ligand-activated transcription factor, belonging to the nuclear receptor superfamily. FXR is highly expressed in the liver and is essential in regulating bile acid homeostasis. FXR deficiency is implicated in numerous liver diseases and mice with modulation of FXR have been used as animal models to study liver physiology and pathology. We have reported genome-wide binding of FXR in mice by chromatin immunoprecipitation - deep sequencing (ChIP-seq), with results indicating that FXR may be involved in regulating diverse pathways in liver. However, limited information exists for the functions of human FXR and the suitability of using murine models to study human FXR functions.<h4>Methods</h4>In the current study, we performed ChIP-seq in primary human hepatocytes (PHHs) treated with a synthetic FXR agonist, GW4064 or DMSO control. In parallel, RNA deep sequencing (RNA-seq) and RNA microarray were performed for GW4064 or control treated PHHs and wild type mouse livers, respectively.<h4>Results</h4>ChIP-seq showed similar profiles of genome-wide FXR binding in humans and mice in terms of motif analysis and pathway prediction. However, RNA-seq and microarray showed more different transcriptome profiles between PHHs and mouse livers upon GW4064 treatment.<h4>Conclusions</h4>In summary, we have established genome-wide human FXR binding and transcriptome profiles. These results will aid in determining the human FXR functions, as well as judging to what level the mouse models could be used to study human FXR functions.
Project description:The bile acid (BA)-sensing nuclear receptor, farnesoid X receptor (FXR), regulates postprandial metabolic responses, including inhibition of BA synthesis, by inducing the intestinal hormone, fibroblast growth factor (FGF)15 (FGF19 in human). In this study, we tested a novel hypothesis that FXR not only induces intestinal FGF15 but also primes the liver for effectively responding to the signal by transcriptional induction of the obligate coreceptor for FGF15, ?-Klotho (?KL). Activation of FXR by a synthetic agonist, GW4064, in mice increased occupancy of FXR and its DNA-binding partner, retinoid X receptor-?, at FGF15-signaling component genes, particularly ?KL, and induced expression of these genes. Interestingly, mRNA levels of Fgfr4, the FGF15 receptor, were not increased by GW4064, but protein levels increased as a result of ?KL-dependent increased protein stability. Both FGF receptor 4 and ?KL protein levels were substantially decreased in FXR-knockout (KO) mice, and FGF19 signaling, monitored by phosphorylated ERK, was blunted in FXR-KO mice, FXR-KO mouse hepatocytes, and FXR-down-regulated human hepatocytes. Overexpression of ?KL in FXR-lacking hepatocytes partially restored FGF19 signaling and inhibition by FGF19 of Cyp7a1, which encodes the rate-limiting BA biosynthetic enzyme. In mice, transient inductions of intestinal Fgf15 and hepatic ?KL were temporally correlated after GW4064 treatment, and pretreatment of hepatocytes with GW4064 before FGF19 treatment enhanced FGF19 signaling, which was abolished by transcriptional inhibition or ?KL down-regulation. This study identifies FXR as a gut-liver metabolic coordinator for FGF15/19 action that orchestrates transient induction of hepatic ?KL and intestinal Fgf15/19 in a temporally correlated manner.
Project description:BACKGROUND:Excessive dietary fat intake induces lipid deposition and contributes to the progress of nonalcoholic fatty liver disease (NAFLD). However, the underlying mechanisms are still unclear. METHODS:Yellow catfish were given two experimental diets with dietary lipid levels of 11.3 and 15.4%, respectively, for 56?days, and the contents of triglyceride (TG), nonesterified free fatty acids (NEFA) and bile acid (BA), RNA-seq, enzymatic activities and mRNA expression were deteremined in the liver tissues. Hepatocytes from yellow catfish liver tissues were isolated and cultured. Fatty acids (FA) (palmitic acid: OA, oleic acid =1:1), pathway inhibitors (MA, autophagy inhibitor; guggulsterone, FXR inhibitor) and agonist (rapamyicn, autophagy agonist; GW4064, FXR agonist) were used to incubate the cells. TG and NEFA contents, ultrastructural observation, autophagic vesicles and intracellular LD,apoptosis,western blot and Co-IP, and Immunofluorescence analysis, enzymatic activities and Q-PCR were decided. RESULTS:Using RNA sequencing, we found that high fat diets induced changes in expression of many genes associated with the pathways of lipid metabolism and autophagy. The mRNA profiles of the differentially expressed genes (DEG) indicated that high dietary fat-induced lipid deposition was predominantly influenced by the inhibition of autophagy. Using primary hepatocytes, we found that fatty acids (FA) suppressed autophagy, which in turn reduced cellular free FA level by decreasing triglyceride (TG) breakdown. Moreover, our study indicated that farnesoid X receptor (FXR)-cyclic AMP-responsive element-binding protein (CREB) axis was the pivotal physiological switch regulating FA-induced changes of autophagy and lipid metabolism, which represented cellular defenses against FA-induced lipotoxicity. CONCLUSION:This discovery may provide new targets for treating pathological changes involved in the dysfunction of autophagy and metabolism, including NAFLD. Video Abstract.
Project description:To assess the effect of farnesoid X receptor (FXR), a bile acid nuclear receptor, on renal proximal tubular cells, primary cultured mouse kidney proximal tubular cells were treated with GW4064 (a FXR agonist) or DMSO (as controls) overnight. Analysis of gene expression in the proximal tubular cells by whole genome microarrays indicated that FXR activation induced genes involved in fatty acid degradation and oxidation reduction. Among them, genes involved in glutathione metabolism were mostly induced. Here we describe in details the contents and quality controls for the gene expression and related results associated with the data uploaded to Gene Expression Omnibus (accession number GSE70296).
Project description:The farnesoid X receptor (Fxr) controls bile acid homeostasis by coordinately regulating the expression of synthesizing enzymes (Cyp7a1, Cyp8b1), conjugating enzymes (Bal, Baat) and transporters in the ileum (Asbt, Ost?/?) and liver (Ntcp, Bsep, Ost?). Transcriptional regulation by Fxr can be direct, or through the ileal Fgf15/FGF19 and hepatic Shp pathways. Circulating bile acids are increased during pregnancy due to hormone-mediated disruption of Fxr signaling. While this adaptation enhances lipid absorption, elevated bile acids may predispose women to develop maternal cholestasis. The objective of this study was to determine whether short-term treatment of pregnant mice with GW4064 (a potent FXR agonist) restores Fxr signaling to the level observed in virgin mice. Plasma, liver and ilea were collected from virgin and pregnant mice administered vehicle or GW4064 by oral gavage. Treatment of pregnant mice with GW4064 induced ileal Fgf15, Shp and Ost?/? mRNAs, and restored hepatic Shp, Bal, Ntcp, and Bsep back to vehicle-treated virgin levels. Pregnant mice exhibited 2.5-fold increase in Cyp7a1 mRNA compared to virgin controls, which was reduced by GW4064. Similarly treatment of mouse primary hepatocytes with plasma isolated from pregnant mice induced Cyp7a1 mRNA by nearly 3-fold as compared to virgin plasma, which could be attenuated by co-treatment with either GW4064 or recombinant FGF19 protein. Collectively, these data reveal that repressed activity of intestinal and hepatic Fxr in pregnancy, as previously demonstrated, may be restored by pharmacological activation. This study provides the basis for a novel approach to restore bile acid homeostasis in patients with maternal cholestasis.
Project description:Identified genes deregulated in mouse primary hepatocytes after glucagon and /or GW4064 treatment Overall design: We analyzed Mouse primary hepatocytes (MPH) using the Affymetrix MoGene 2,0ST chip. Array data was processed by Genespring software . Two technical replicates were pooled by sample. Each experiment was performed on pooled MPH providing from three mice
Project description:In our previous study, we found zebrafish embryos treated with 5uM 11,12-EET (epoxyeicosatrienoic acid) had increased stem cell marker, runx1, expression in the AGM. EET also induced ectopic runx1 expression in the tail. To systematically study how EET regulates gene expression, we performed microarray analysis on EET-treated embryos. Zebrafish whole embryos were synchronized at fertilization. Embryos were grown at 28 degree overnight. 25 embryos per group were treated with DMSO or 5uM 11,12-EET starting from 24 hpf (hour post fertilization) until 36 hpf at 28 degree. The triplicates were from three different clutches of embryos, and split into DMSO v.s. EET for each clutch. EET vs. DMSO
Project description:The purpose of the present study was to explore in liver cells the connectivity that operates between three nuclear receptors in the liver, LXR, FXR, and PPARa, all three known to act on lipid and glucose metabolism, and also on inflammation. The human cell line HepaRG was selected for its proximity to human primary hepatocytes. Global gene expression of differentiated HepaRG cells was assessed after 4 hours and 24 hours of exposure to GW3965 (LXR agonist), GW7647 (PPARα agonist), and GW4064 and CDCA (FXR synthetic and natural agonist, respectively). Overall design: The study includes 36 samples from two time points, 4h and 24h. The two time points were analyzed separately. The cell cultures received six different treatments: four ligands binding to nuclear receptors (CDCA, FXR-L, LXR-L and PPARα-L) and two control treatments (DMSO vehicle control and untreated control). There were three replicate cell cultures for each treatment. For the data analysis, DMSO and untreated control were lumped together into a single control group.
Project description:The small freshwater teleost, medaka (Oryzias latipes), has a history of usage in studies of chronic toxicity of liver and biliary system. Recent progress with this model has focused on defining the medaka hepatobiliary system. Here we investigate critical liver function and toxicity by examining the in vivo role and function of the farnesoid X receptor alpha (FXRalpha, NR1H4), a member of the nuclear receptor superfamily that plays an essential role in the regulation of bile acid homeostasis. Quantitative mRNA analysis of medaka FXRalpha demonstrates differential expression of two FXRalpha isoforms designated Fxralpha1 and Fxralpha2, in both free swimming medaka embryos with remaining yolk (eleutheroembryos, EEs) and adults. Activation of medaka Fxralpha in vivo with GW4064 (a strong FXRalpha agonist) resulted in modification of gene expression for defined FXRalpha gene targets including the bile salt export protein, small heterodimer partner, and cytochrome P450 7A1. Histological examination of medaka liver subsequent to GW4064 exposure demonstrated significant lipid accumulation, cellular and organelle alterations in both hepatocytes and biliary epithelial cells of the liver. This report of hepatobiliary injury following GW4064 exposure extends previous investigations of the intrahepatic biliary system in medaka, reveals sensitivity to toxicant exposure, and illustrates the need for added resolution in detection and interpretation of toxic responses in this vertebrate.