Constitutive Androstane Receptor Target Genes Research Articles

Regulation of hepatic xenosensor function by HNF4alpha.

Nuclear receptors such as constitutive androstane receptor (CAR), pregnane X receptor (PXR), and peroxisome proliferator-activated receptor-alpha (PPARα), and transcription factors with nuclear receptor type activity such as aryl hydrocarbon receptor (AhR) function as xenobiotic sensors. Hepatocyte nuclear factor 4alpha (HNF4α) is a highly conserved orphan nuclear receptor essential for liver function. We tested the hypothesis that HNF4α is essential for the function of these 4 major xenosensors. Wild-type (WT) and hepatocyte-specific Hnf4a null (HNF4α-KO) mice were treated with the mouse-specific activators of AhR (TCDD, 30 µg/kg), CAR (TCPOBOP, 2.5 µg/g), PXR, (PCN, 100 µg/g), and PPARα (WY-14643, 1 mg/kg). Blood and liver tissue samples were collected to study receptor activation. TCDD (AhR agonist) treatment did not affect the liver-to-body weight ratio (LW/BW) in either WT or HNF4α-KO mice. Further, TCDD activated AhR in both WT and HNF4α-KO mice, confirmed by increase in expression of AhR target genes. TCPOBOP (CAR agonist) significantly increased the LW/BW ratio and CAR target gene expression in WT mice, but not in HNF4α-KO mice. PCN (a mouse PXR agonist) significantly increased LW/BW ratio in both WT and HNF4α-KO mice however, failed to induce PXR target genes in HNF4α-KO mice. The treatment of WY-14643 (PPARα agonist) increased LW/BW ratio and PPARα target gene expression in WT mice but not in HNF4α-KO mice. Together, these data indicate that the function of CAR, PXR, and PPARα but not of AhR was disrupted in HNF4α-KO mice. These results demonstrate that HNF4α function is critical for the activation of hepatic xenosensors, which are critical for toxicological responses.

Diindoles produced from commensal microbiota metabolites function as endogenous CAR/Nr1i3 ligands

Numerous studies have demonstrated the correlation between human gut bacteria and host physiology, mediated primarily via nuclear receptors (NRs). Despite this body of work, the systematic identification and characterization of microbe-derived ligands that regulate NRs remain a considerable challenge. In this study, we discover a series of diindole molecules produced from commensal bacteria metabolites that act as specific agonists for the orphan constitutive androstane receptor (CAR). Using various biophysical analyses we show that their nanomolar affinities are comparable to those of synthetic CAR agonists, and that they can activate both rodent and human CAR orthologues, which established synthetic agonists cannot. We also find that the diindoles, diindolylmethane (DIM) and diindolylethane (DIE) selectively up-regulate bona fide CAR target genes in primary human hepatocytes and mouse liver without causing significant side effects. These findings provide new insights into the complex interplay between the gut microbiome and host physiology, as well as new tools for disease treatment.

Timosaponin AⅢ induces drug-metabolizing enzymes by activating constitutive androstane receptor (CAR) via dephosphorylation of the EGFR signaling pathway.

The current study aimed to assess the effect of timosaponin AⅢ (T-AⅢ) on drug-metabolizing enzymes during anticancer therapy. The in vivo experiments were conducted on nude and ICR mice. Following a 24-day administration of T-AⅢ, the nude mice exhibited an induction of CYP2B10, MDR1, and CYP3A11 expression in the liver tissues. In the ICR mice, the expression levels of CYP2B10 and MDR1 increased after a three-day T-AⅢ administration. The in vitro assessments with HepG2 cells revealed that T-AⅢ induced the expression of CYP2B6, MDR1, and CYP3A4, along with constitutive androstane receptor (CAR) activation. Treatment with CAR siRNA reversed the T-AⅢ-induced increases in CYP2B6 and CYP3A4 expression. Furthermore, other CAR target genes also showed a significant increase in the expression. The up-regulation of murine CAR was observed in the liver tissues of both nude and ICR mice. Subsequent findings demonstrated that T-AⅢ activated CAR by inhibiting ERK1/2 phosphorylation, with this effect being partially reversed by the ERK activator t-BHQ. Inhibition of the ERK1/2 signaling pathway was also observed in vivo. Additionally, T-AⅢinhibited the phosphorylation of EGFR at Tyr1173 and Tyr845, and suppressed EGF-induced phosphorylation of EGFR, ERK, and CAR. In the nude mice, T-AⅢ also inhibited EGFR phosphorylation. These results collectively indicate that T-AⅢ is a novel CAR activator through inhibition of the EGFR pathway.

Development of a strategy to identify and evaluate direct and indirect activators of constitutive androstane receptor in rats.

Constitutive androstane receptor (CAR) is a nuclear receptor that plays a key role in drug metabolism and disposition and in the development of liver tumors in rodents. CAR is activated by ligands and indirect activators, which do not bind to the receptor but activate it through cellular signaling. In this study, we sought to identify direct and indirect activators of rat CAR (rCAR). Assessment of the influence of mutations on the transcriptional activity of rCAR identified a mutant termed rCAR-3A-G354Q that displays low constitutive activity and high ligand responsiveness. Reporter assays using the mutant were performed with compounds that increased the mRNA levels of Cyp2b1, a CAR target gene, in rat primary hepatocytes. Several compounds activated rCAR-3A-G354Q and were implicated as rCAR ligands. Since indirect CAR activators are considered to display little species differences, we then determined CYP2B6 mRNA levels in human hepatocyte-like HepaRG cells after treatment with compounds that increased Cyp2b1 mRNA levels in rat hepatocytes but did not activate rCAR-3A-G354Q. The results demonstrated six compounds as possible rCAR indirect activators. Taken together, the combined measurement of Cyp2b1 mRNA levels in rat primary hepatocytes and rCAR-3A-G354Q activation in reporter assays can be useful for evaluating rCAR activation by chemicals.

Abstract 12855: Matrin-3 Deficiency Exacerbates Hepatic Steatosis and Acute Phase Response

Introduction: Nonalcoholic fatty liver disease (NAFLD) has become the most common liver disease worldwide. NAFLD patients have an increased risk of cardiovascular disease. Matrin-3 is a DNA- and RNA-binding protein, and its mutation causes amyotrophic lateral sclerosis in humans. Among these patients, 76% have hepatic steatosis. However, the role of matrin-3 in NAFLD has not been previously studied. Methods and Results: We found that matrin-3 expression is induced in fatty livers of humans and mice. Matrin-3 liver specific knockout mice (LKO) displayed increased levels of plasma triglycerides compared with matrin-3 floxed mice, while the body weights of the two groups of mice are similar after 16-week high-fat diet. Moreover, histological staining and triglyceride assay revealed that LKO mice had a 1.57- and 1.58-fold increase in hepatic lipid area and triglyceride levels, respectively, which associate with an increased liver/body weight ratio. Transcriptome and bioinformatics analysis identified 622 differentially expressed genes in LKO livers. Among them, 142 are target genes of constitutive androstane receptor (CAR), a transcription factor controlling the expression of many genes involved in xenobiotic and lipid metabolism. We also found that the expression of CAR and many CAR target genes was reduced in LKO livers. These data suggest that matrin-3 is important in CAR expression and matrin-3 knockout impairs CAR signaling. Interestingly, matrin-3 LKO mice had increased acute phase response and levels of phosphorylated Stat3 in the livers, indicating increased liver injury and inflammation. Mechanistically, impaired CAR signaling reversed the suppression of Plin2, Plin3, and Plin5 proteins that stabilize lipid droplet and inhibit lipolysis, and it reversed the suppression of interleukin 1 receptor type 1 that activates Stat3 signaling in LKO livers. Lastly, CAR expression positively correlates with matrin-3 expression in human livers (r=0.85, P =0.0002, n=13). Conclusion: matrin-3-CAR axis is an important mechanism to maintain lipid homeostasis and limit acute phase response in fatty livers. It can be potentially targeted to ameliorate hepatic steatosis and inflammation and reduce the risk of cardiovascular disease in NAFLD patients.

Diazepam Promotes Translocation of Human Constitutive Androstane Receptor (CAR) via Direct Interaction with the Ligand-Binding Domain.

The constitutive androstane receptor (CAR) is the essential regulator of genes involved both in xenobiotic and endobiotic metabolism. Diazepam has been shown as a potent stimulator of CAR nuclear translocation and is assumed as an indirect CAR activator not interacting with the CAR cavity. In this study, we sought to determine if diazepam is a ligand directly interacting with the CAR ligand binding domain (LBD) and if it regulates its target genes in a therapeutically relevant concentration. We used different CAR constructs in translocation and luciferase reporter assays, recombinant CAR-LBD in a TR-FRET assay, and target genes induction studied in primary human hepatocytes (PHHs), HepaRG cells, and in CAR humanized mice. We also used in silico docking and CAR-LBD mutants to characterize the interaction of diazepam and its metabolites with the CAR cavity. Diazepam and its metabolites such as nordazepam, temazepam, and oxazepam are activators of CAR+Ala in translocation and two-hybrid assays and fit the CAR cavity in docking experiments. In gene reporter assays with CAR3 and in the TR-FRET assay, only diazepam significantly interacts with CAR-LBD. Diazepam also promotes up-regulation of CYP2B6 in PHHs and in HepaRG cells. However, in humanized CAR mice, diazepam significantly induces neither CYP2B6 nor Cyp2b10 genes nor does it regulate critical genes involved in glucose and lipids metabolism and liver proliferation. Thus, we demonstrate that diazepam interacts with human CAR-LBD as a weak ligand, but it does not significantly affect expression of tested CAR target genes in CAR humanized mice.

Perfluorooctanoic acid activates multiple nuclear receptor pathways and skews expression of genes regulating cholesterol homeostasis in liver of humanized PPARα mice fed an American diet

Humans are exposed to per- and polyfluoroalkyl substances (PFAS) in their drinking water, food, air, dust, and by direct use of consumer products. Increased concentrations of serum total cholesterol and low density lipoprotein cholesterol are among the endpoints best supported by epidemiology. The objectives of this study were to generate a new model for examining PFAS-induced dyslipidemia and to conduct molecular studies to better define mechanism(s) of action. We tested the hypothesis that perfluorooctanoic acid (PFOA) exposure at a human-relevant level dysregulates expression of genes controlling cholesterol homeostasis in livers of mice expressing human PPARα (hPPARα). Female and male hPPARα and PPARα null mice were fed a diet based on the “What we eat in America” analysis and exposed to PFOA in drinking water (8 μM) for 6 weeks. This resulted in a serum PFOA concentration of 48 μg/ml. PFOA increased liver mass, which was associated with histologically-evident lipid accumulation. Pooled analyses of serum lipoprotein cholesterol suggest that PFOA increased serum cholesterol, particularly in male mice. PFOA induced PPARα and constitutive androstane receptor target gene expression in liver. Expression of genes in four pathways regulating cholesterol homeostasis were also measured. PFOA decreased expression of Hmgcr in a PPARα-dependent manner. PFOA decreased expression of Ldlr and Cyp7a1 in a PPARα-independent manner. Apob expression was not changed. Sex differences were evident. This novel study design (hPPARα mice, American diet, long term exposure) generated new insight on the effects of PFOA on cholesterol regulation in the liver and the role of hPPARα.

Overexpression of the constitutive androstane receptor and shaken 3D-culturing increase biotransformation and oxidative phosphorylation and sensitivity to mitochondrial amiodarone toxicity of HepaRG cells

The liver cell line HepaRG is one of the preferred sources of human hepatocytes for in vitro applications. However, mitochondrial energy metabolism is relatively low, which affects hepatic functionality and sensitivity to hepatotoxins. Culturing in a bioartificial liver (BAL) system with high oxygen, medium perfusion, low substrate stiffness, and 3D conformation increases HepaRG functionality and mitochondrial activity compared to conventional monolayer culturing. In addition, drug metabolism has been improved by overexpression of the constitutive androstane receptor (CAR), a regulator of drug and energy metabolism in the new HepaRG-CAR line. Here, we investigated the effect of BAL culturing on the HepaRG-CAR line by applying a simple and downscaled BAL culture procedure based on shaking 3D cultures, named Bal-in-a-dish (BALIAD).We compared monolayer and BALIAD cultures of HepaRG and HepaRG-CAR cells. CAR overexpression and BALIAD culturing synergistically or additively increased transcript levels of CAR and three of the seven tested CAR target genes in biotransformation. Additionally, Cytochrome P450 3A4 activity was 35-fold increased. The mitochondrial energy metabolism was enhanced; lactate production and glucose consumption switched into lactate elimination and glucose production. BALIAD culturing alone reduced glycogen content and increased oxygen consumption and mitochondrial content. Both CAR overexpression and BALIAD culturing decreased mitochondrial superoxide levels. HepaRG-CAR BALIADs were most sensitive to mitochondrial toxicity induced by the hepatotoxin amiodarone, as indicated by oxygen consumption and mitochondrial superoxide accumulation.These data show that BALIAD culturing of HepaRG-CAR cells induces high mitochondrial energy metabolism and xenobiotic metabolism, increasing its potential for drug toxicity studies.

Lymphocyte Specific Protein‐1 suppresses TCPOBOP induced hepatocellular proliferation

Lymphocyte specific protein‐1 (LSP‐1), an f‐actin binding protein expressed in hematopoietic cells as well as hepatocytes, is the gene with the most copy number variations detected in human HCC. Our previous work has demonstrated that LSP‐1 suppresses hepatocellular proliferation following partial hepatectomy as well as in primary hepatocyte cultures. LSP‐1 acts as a scaffold for the Ras/MEK/ERK MAP kinase pathway and along with kinase suppressor of Ras (KSR) inhibits ERK phosphorylation and thus its activation preventing downstream signaling to induce cellular proliferation and migration. To determine if LSP‐1 functions to attenuate hepatocellular growth in models which induce proliferation without loss of tissue or liver injury, we aimed to explore if LSP‐1 can modulate TCPOBOP induced hepatocellular proliferation and hepatomegaly. TCPOBOP (1,4‐Bis [2‐(3,5‐Dichloropyridyloxy)] benzene), a constitutive androstane receptor (CAR) agonist, has been shown to induce hepatocellular hyperplasia through a CAR dependent mechanism. Since LSP‐1 has been shown to suppress hepatocyte proliferation during liver regeneration following partial hepatectomy and in culture, we hypothesized that LSP‐1 will attenuate TCPOBOP driven hepatocellular proliferation. In this study, we administered TCPOBOP to both a global LSP1 knockout mouse model as well as a hepatocyte specific LSP‐1 transgenic (TG) mouse model. Livers were harvested at various time point after TCPOBOP gavage and proliferation was measured using Ki67 and PCNA immunohistochemistry as well as liver to body weight ratios. Increased LSP‐1 expression in the transgenic livers caused decreased Ki67 staining on day 2 after TCPOBOP administration as well as no measurable change in liver to body weight ratios as compared to baseline. However, by day 5, the TG liver to body weight ratios increased above those of the WT but the percentage of Ki67 positive hepatocytes was decreased in the TG as compared to the wild type controls. Loss of LSP‐1 expression led to a statistically significant difference in the liver to body weight ratios between the KO and WT mice on day 2 after TCPOBOP administration as well as increased hepatocellular proliferation on both days 2 and 5 after gavage in the KO livers. CAR target genes and downstream signaling pathways that are modulated by LSP‐1 expression will be analyzed.ConclusionLSP‐1 attenuates TCPOBOP induced hepatocellular proliferation and loss of expression leads to increased hyperplasia supporting the role of LSP‐1 as a growth suppressor.Support or Funding InformationSupported by the Department of Defense grant CA160119, the University of Pittsburgh Menten Endowment, and the Cleveland Foundation Morningstar Fund.

Identification of novel agonists by high-throughput screening and molecular modelling of human constitutive androstane receptor isoform 3.

Prediction of drug interactions, based on the induction of drug disposition, calls for the identification of chemicals, which activate xenosensing nuclear receptors. Constitutive androstane receptor (CAR) is one of the major human xenosensors; however, the constitutive activity of its reference variant CAR1 in immortalized cell lines complicates the identification of agonists. The exclusively ligand-dependent isoform CAR3 represents an obvious alternative for screening of CAR agonists. As CAR3 is even more abundant in human liver than CAR1, identification of its agonists is also of pharmacological value in its own right. We here established a cellular high-throughput screening assay for CAR3 to identify ligands of this isoform and to analyse its suitability for identifying CAR ligands in general. Proof-of-concept screening of 2054 drug-like compounds at 10µM resulted in the identification of novel CAR3 agonists. The CAR3 assay proved to detect the previously described CAR1 ligands in the screened libraries. However, we failed to detect CAR3-selective compounds, as the four novel agonists, which were selected for further investigations, all proved to activate CAR1 in different cellular and in vitro assays. In primary human hepatocytes, the compounds preferentially induced the expression of the prototypical CAR target gene CYP2B6. Failure to identify CAR3-selective compounds was investigated by molecular modelling, which showed that the isoform-specific insertion of five amino acids did not impact on the ligand binding pocket but only on heterodimerization with retinoid X receptor. In conclusion, we demonstrate here the usability of CAR3 for screening compound libraries for the presence of CAR agonists.

TCPOBOP-Induced Hepatomegaly and Hepatocyte Proliferation are Attenuated by Combined Disruption of MET and EGFR Signaling.

TCPOBOP (1,4-Bis [2-(3,5-Dichloropyridyloxy)] benzene) is a constitutive androstane receptor (CAR) agonist that induces robust hepatocyte proliferation and hepatomegaly without any liver injury or tissue loss. TCPOBOP-induced direct hyperplasia has been considered to be CAR-dependent with no evidence of involvement of cytokines or growth factor signaling. Receptor tyrosine kinases (RTKs), MET and epidermal growth factor receptor (EGFR), are known to play a critical role in liver regeneration after partial hepatectomy, but their role in TCPOBOP-induced direct hyperplasia, not yet explored, is investigated in the current study. Disruption of the RTK-mediated signaling was achieved using MET knockout (KO) mice along with Canertinib treatment for EGFR inhibition. Combined elimination of MET and EGFR signaling [MET KO + EGFR inhibitor (EGFRi)], but not individual disruption, dramatically reduced TCPOBOP-induced hepatomegaly and hepatocyte proliferation. TCPOBOP-driven CAR activation was not altered in [MET KO + EGFRi] mice, as measured by nuclear CAR translocation and analysis of typical CAR target genes. However, TCPOBOP-induced cell cycle activation was impaired in [MET KO + EGFRi] mice due to defective induction of cyclins, which regulate cell cycle initiation and progression. TCPOBOP-driven induction of FOXM1, a key transcriptional regulator of cell cycle progression during TCPOBOP-mediated hepatocyte proliferation, was greatly attenuated in [MET KO + EGFRi] mice. Interestingly, TCPOBOP treatment caused transient decline in hepatocyte nuclear factor 4 alpha expression concomitant to proliferative response; this was not seen in [MET KO + EGFRi] mice. Transcriptomic profiling revealed the vast majority (~40%) of TCPOBOP-dependent genes primarily related to proliferative response, but not to drug metabolism, were differentially expressed in [MET KO + EGFRi] mice. Conclusion: Taken together, combined disruption of EGFR and MET signaling lead to dramatic impairment of TCPOBOP-induced proliferative response without altering CAR activation.

Teriflunomide Is an Indirect Human Constitutive Androstane Receptor (CAR) Activator Interacting With Epidermal Growth Factor (EGF) Signaling.

The constitutive androstane receptor (CAR) is a nuclear receptor involved mainly in xenobiotic and endobiotic metabolism regulation. CAR is activated directly by its ligands via the ligand binding domain (LBD) or indirectly by inhibition of the epidermal growth factor (EGF) signaling. We found that leflunomide (LEF) and its main metabolite teriflunomide (TER), both used for autoimmune diseases treatment, induce the prototype CAR target gene CYP2B6 in primary human hepatocytes. As TER was discovered to be an EGF receptor antagonist, we sought to determine if TER is an indirect activator of CAR. In primary human hepatocytes and in differentiated HepaRG cells, we found that LEF and TER up-regulate CAR target genes CYP2B6 and CYP3A4 mRNAs and enzymatic activities. TER stimulated CAR+A mutant translocation into the nucleus but neither LEF nor TER activated the CAR LBD, CAR3 variant or pregnane X receptor (PXR) in gene reporter assays. Interestingly, TER significantly up-regulated CAR mRNA expression, a result which could be a consequence of both EGF receptor and ELK-1 transcription factor inhibition by TER or by TER-mediated activation of glucocorticoid receptor (GR), an upstream hormonal regulator of CAR. We can conclude that TER is a novel indirect CAR activator which through EGF inhibition and GR activation controls both detoxification and some intermediary metabolism genes.

Mouse Hepatomas with Ha-ras and B-raf Mutations Differ in Mitogen-Activated Protein Kinase Signaling and Response to Constitutive Androstane Receptor Activation.

Nuclear receptors mediate the hepatic induction of drug-metabolizing enzymes by xenobiotics. Not much is known about enzyme induction in liver tumors. Here, we treated tumor-bearing mice with phenobarbital, an activator of the constitutive androstane receptor (CAR), to analyze the response of chemically induced Ha-ras- and B-raf-mutated mouse liver adenoma to CAR activation in vivo. Both tumor subpopulations possess almost identical gene expression profiles. CAR target gene induction in the tumors was studied at the mRNA and protein levels, and a reverse-phase protein microarray approach was chosen to characterize important signaling cascades. CAR target gene induction was pronounced in B-raf-mutated but not in Ha-ras-mutated tumors. Phosphoproteomic profiling revealed that phosphorylation-activated extracellular signal-regulated kinase (ERK) 1/2 was more abundant in Ha-ras-mutated than in B-raf-mutated tumors. ERK activation in tumor tissue was negatively correlated with CAR target induction. ERK activation is known to inhibit CAR-dependent transcription. In summary, profound differences exist between the two closely related tumor subpopulations with respect to the activation of mitogenic signaling cascades, and these dissimilarities might explain the differences in xenobiotic induction of CAR target genes.

Role of the pregnane X receptor in binge ethanol-induced steatosis and hepatotoxicity.

The pregnane X receptor (PXR, NR1I2) is a xenobiotic-sensing nuclear receptor that defends against toxic agents. We have shown that PXR promotes chronic ethanol (EtOH)-induced steatosis. Therefore, we examined the role of PXR in binge EtOH-induced hepatotoxicity. Male wild type (WT) and Pxr-null mice were orally administered three binge doses of EtOH (4.5 g/kg, every 12 hours) and euthanized four hours after the final dose. Pxr-null mice displayed higher basal mRNA levels of hepatic lipogenic transcription factor sterol regulatory element binding protein 1c (Srebp-1c) and its target stearoyl-CoA desaturase 1 (Scd1) and the lipid peroxide detoxifying aldo-keto reductase 1b7 (Akr1b7) and higher protein levels of EtOH-metabolizing alcohol dehydrogenase 1 (ADH1). In both genotypes, binge EtOH-induced triglyceride accumulation was associated with inhibition of fatty acid β-oxidation and upregulation of Srebp-1c- regulated lipogenic genes and hepatic CYP2E1 protein. Unexpectedly, gene expression of Cyp2b10, a constitutive androstane receptor target gene, implicated in EtOH hepatotoxicity, was PXR-dependent upregulated by binge EtOH. Also, PXR-dependent was the binge EtOH-induced inhibition of hepatic Akr1b8 mRNA, and protein levels of aldehyde dehydrogenase (ALDH) 1A1 and anti-apoptotic Bcl-2, but increased pro-apoptotic Bax protein expression, leading to increases in residual EtOH concentration and the cellular oxidative stress marker, malondialdehyde. In contrast, Pxr-null mice displayed increased Akr1b7 gene and ADH1 protein expression and hypertriglyceridemia following binge EtOH exposure. Taken together, this study demonstrates that PXR ablation prevents EtOH induced upregulation of Cyp2b10 and that PXR potentiates binge EtOH-induced oxidative stress and inhibition of EtOH catabolism, but protects against alcoholic hyperlipidemia.

Modulation of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) activation by ursolic acid (UA) attenuates rifampin-isoniazid cytotoxicity

BackgroundInteractions between transcriptional inducers of cytochrome P450 (CYP450) enzymes and therapeutic drugs may be prevented by antagonizing the activation of a nuclear receptor (NR), pregnane X receptor (PXR, NR1I2), thus improving therapeutic efficacy. PurposeIn the present study, we aim to identify that ursolic acid (UA), a widely distributed pentacyclic triterpene, may act as an effective antagonist of PXR and its sister NR receptor, constitutive androstane receptor (CAR, NR1I3). MethodsThe hepatocellular carcinoma cell line, HepG2, was used to evaluate the promoter activity of PXR and CAR target genes, CYP3A4 and CYP2B6, respectively. Catalytic activities, mRNA, and protein expression of CYP3A4 and CYP2B6 were evaluated in a differentiated HepaRG cell line. Coregulation of PXR with coregulators on CYP3A4 promoter response elements was also been characterized. ResultsTransient transfection assays showed that UA effectively attenuated CYP3A4 and CYP2B6 promoter activities mediated by rifampin (RIF, human PXR agonist) and CITCO (human CAR agonist). These inhibitory effects were well correlated with the expression and catalytic activities of CYP3A4 and CYP2B6. Furthermore, the interaction of co-regulators with PXR and the transcriptional complexes in the CYP3A4 promoter activity and CYP3A4 promoter xenobiotic response element (everted repeat 6, ER6), respectively, were disrupted in the presence of UA. UA showed an antagonistic effect against PXR, and reversed the cytotoxic effects of isoniazid (INH) induced by RIF. Taken together, these results show that UA inhibits the transactivation effects of PXR and CAR, and reduces the expression and function of CYP3A4 and CYP2B6. ConclusionThe present study suggests that UA could be a powerful agent for reducing potentially dangerous interactions between transcriptional inducers of CYP enzymes and therapeutic drugs.

Interaction of PER2 with the Constitutive Androstane Receptor Possibly Links Circadian Rhythms to Metabolism.

Period 2 (PER2) is an important factor in daily oscillations called circadian rhythms, which are emerging as one of the most important regulatory networks, responsible for homeostasis and transcriptional regulation of a number of genes. Our work shows that PER2 could act as a co-activator of the constitutive androstane receptor (CAR), a key nuclear receptor (NR) that regulates the metabolism of endobiotics and xenobiotics. Bioinformatic analysis shows that PER2 and CAR possess structural elements that could enable them to interact which was confirmed experimentally by CoIP experiment. Co-transfection of mouse hepatocarcinoma cells with plasmids overexpressing Per2 and Car increases expression of Bmal1, a potential CAR target gene, more than transfections with Car only. This is the first report indicating the interaction of PER2 and CAR.

Sex-Differential Responses of Tumor Promotion-Associated Genes and Dysregulation of Novel Long Noncoding RNAs in Constitutive Androstane Receptor-Activated Mouse Liver.

Xenobiotic agonists of constitutive androstane receptor (CAR) induce many hepatic drug metabolizing enzymes, but following prolonged exposure, promote hepatocellular carcinoma, most notably in male mouse liver. Here, we used nuclear RNA-seq to characterize global changes in the mouse liver transcriptome following exposure to the CAR-specific agonist ligand 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), including changes in novel long noncoding RNAs that may contribute to xenobiotic-induced pathophysiology. Protein-coding genes dysregulated by 3 h TCPOBOP exposure were strongly enriched in KEGG pathways of xenobiotic and drug metabolism, with stronger and more extensive gene responses observed in female than male liver. After 27 h TCPOBOP exposure, the number of responsive genes increased >8-fold in males, where the top enriched pathways and their upstream regulators expanded to include factors implicated in cell cycle dysregulation and hepatocellular carcinoma progression (cyclin-D1, oncogenes E2f, Yap, Rb, Myc, and proto-oncogenes β-catenin, FoxM1, FoxO1, all predicted to be activated by TCPOBOP in male but not female liver; and tumor suppressors p21 and p53, both predicted to be inhibited). Upstream regulators uniquely associated with 3 h TCPOBOP-exposed females include TNF/NFkB pathway members, which negatively regulate CAR-dependent proliferative responses and may contribute to the relative resistance of female liver to TCPOBOP-induced tumor promotion. These responses may be modified by the many long noncoding liver RNAs we show are dysregulated by TCPOBOP or pregnane-X-receptor agonist exposure, including lncRNAs proximal to CAR target genes Cyp2b10, Por, and Alas1. These data provide a comprehensive view of the CAR-regulated transcriptome and give insight into the mechanism of sex-biased susceptibility to CAR-dependent mouse liver tumorigenesis.

Activation of nuclear receptor CAR by an environmental pollutant perfluorooctanoic acid.

Perfluorocarboxylic acids (PFCAs) including perfluorooctanoic acid (PFOA) are environmental pollutants showing high accumulation, thermochemical stability and hepatocarcinogenicity. Peroxisome proliferator-activated receptor α is suggested to mediate their toxicities, but the precise mechanism remains unclear. Previous reports also imply a possible role of constitutive androstane receptor (CAR), a key transcription factor for the xenobiotic-induced expression of various genes involved in drug metabolism and disposition as well as hepatocarcinogenesis. Therefore, we have investigated whether PFCAs activate CAR. In wild-type but not Car-null mice, mRNA levels of Cyp2b10, a CAR target gene, were increased by PFOA treatment. PFCA treatment induced the nuclear translocation of CAR in mouse livers. Since CAR activators are divided into two types, ligand-type activators and phenobarbital-like indirect activators, we investigated whether PFCAs are CAR ligands or not using the cell-based reporter gene assay that can detect CAR ligands but not indirect activators. As results, neither PFCAs nor phenobarbital increased reporter activities. Interestingly, in mouse hepatocytes, pretreatment with the protein phosphatase inhibitor okadaic acid prevented an increase in Cyp2b10 mRNA levels induced by phenobarbital as reported, but not that by PFOA. Finally, in human hepatocyte-like HepaRG cells, PFOA treatment increased mRNA levels of CYP2B6, a CAR target gene, as did phenobarbital. Taken together, our present results suggest that PFCAs including PFOA are indirect activators of mouse and human CAR and that the mechanism might be different from that for phenobarbital. The results imply a role of CAR in the hepatotoxicity of PFCAs.

RNA-Seq reveals common and unique PXR- and CAR-target gene signatures in the mouse liver transcriptome

The pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are well-known xenobiotic-sensing nuclear receptors with overlapping functions. However, there lacks a quantitative characterization to distinguish between the PXR and CAR target genes and signaling pathways in the liver. The present study performed a transcriptomic comparison of the PXR- and CAR-targets using RNA-Seq in livers of adult wild-type mice that were treated with the prototypical PXR ligand PCN (200mg/kg, i.p. once daily for 4days in corn oil) or the prototypical CAR ligand TCPOBOP (3mg/kg, i.p., once daily for 4days in corn oil). At the given doses, TCPOBOP differentially regulated many more genes (2125) than PCN (212), and 147 of the same genes were differentially regulated by both chemicals. As expected, the top pathways differentially regulated by both PCN and TCPOBOP were involved in xenobiotic metabolism, and they also up-regulated genes involved in retinoid metabolism, but down-regulated genes involved in inflammation and iron homeostasis. Regarding unique pathways, PXR activation appeared to overlap with the aryl hydrocarbon receptor signaling, whereas CAR activation appeared to overlap with the farnesoid X receptor signaling, acute-phase response, and mitochondrial dysfunction. The mRNAs of differentially regulated drug-processing genes (DPGs) partitioned into three patterns, namely TCPOBOP-induced, PCN-induced, as well as TCPOBOP-suppressed gene clusters. The cumulative mRNAs of the differentially regulated DPGs, phase-I and -II enzymes, as well as efflux transporters were all up-regulated by both PCN and TCPOBOPOP, whereas the cumulative mRNAs of the uptake transporters were down-regulated only by TCPOBOP. The absolute mRNA abundance in control and receptor-activated conditions was examined in each DPG category to predict the contribution of specific DPG genes in the PXR/CAR-mediated pharmacokinetic responses. The preferable differential regulation by TCPOBOP in the entire hepatic transcriptome correlated with a marked change in the expression of many DNA and histone epigenetic modifiers. In conclusion, the present study has revealed known and novel, as well as common and unique targets of PXR and CAR in mouse liver following pharmacological activation using their prototypical ligands. Results from this study will further support the role of these receptors in regulating the homeostasis of xenobiotic and intermediary metabolism in the liver, and aid in distinguishing between PXR and CAR signaling at various physiological and pathophysiological conditions. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie

Regulation of mouse liver chromatin accessibility and associated early gene responses by CAR

Many xenobiotics are metabolized in mammalian liver by pathways regulated by constitutive androstane receptor (CAR). Here, we identify early gene targets of mouse liver CAR and investigate their associated, CAR‐induced changes in local chromatin accessibility and transcription factor (TF) recruitment. Genes induced or repressed by the CAR agonist TCPOBOP were identified by RNA‐Seq after 3 or 27 hr. Chromatin sites that dynamically open or close following CAR activation (changes in DNase I hypersensitive sites, ΔDHS) were identified by DNase‐Seq. Genes dysregulated in 3 hr TCPOBOP‐treated mouse liver were enriched in KEGG pathways for retinol and drug metabolism (DAVID analysis; p<E‐15). IPA identified activated CAR and its agonists as the top predicted upstream regulators of these gene responses. After 27 hr TCPOBOP treatment, many more genes were identified and the predicted upstream regulators expanded to include cyclin D and vitamin D, indicating significant secondary transcriptional regulation. DNase‐Seq identified ~3,600 ΔDHS that were opened or closed by 3 hr TCPOBOP treatment. These ΔDHS were not randomly distributed: mapping to their putative gene targets (i.e., nearest gene within 10 kb) revealed enrichment (12.4‐fold, p<E‐45) of the TCPOBOP‐induced DHS for genes inducible by CAR. Thus, chromatin opening is associated with activation of nearby CAR target genes. Genomic sequences in the 3 hr TCPOBOP‐inducible DHS regions were enriched for TF binding motifs for CAR and for CCAAT‐enhancer binding protein (CEBP), suggesting that CEBPs cooperate with CAR to open chromatin and up regulate target gene expression. Studies designed to test this hypothesis are in progress and will be reported. Grant support: NIH ES024421.