Constitutive Androstane Receptor Agonist Research Articles

Promotion of liver growth by CAR is accompanied by Akt pathway activation and FoxM1-Nedd4-mediated repression of PTEN

Recently, we reported that treatment with the mouse agonist of the constitutive androstane receptor (CAR), 1,4-bis benzene[2-(3,5-dichloropyridyloxy)] (TCPOBOP; a well-known hepatomitogen), reduced PTEN protein levels, leading to Akt activation. Hence, the present study was performed to demonstrate the role of CAR in PTEN regulation and liver growth. Liver hyperplasia caused by CAR activation was confirmed to be mediated by a decrease in PTEN protein level and the activation of the Akt signalling pathway in the liver of mice. Treatment with the CAR agonist decreased the PTEN levels and increased Foxm1 levels, which correlate with the elevated expression of the FoxM1 target gene, Nedd4-1, an E3 ligase involved in PTEN ubiquitination, and the promotion of degradation. The increase in Nedd4-1 levels was accompanied by an increase in CAR-mediated accumulation of Foxm1 on the Nedd4-1 gene promoter. Therefore, these results provide evidence that a notable function of CAR is its liver growth promotion effect, which is accompanied by FoxM1-Nedd4-mediated repression of PTEN and Akt pathway activation.

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.

High-content analysis of constitutive androstane receptor (CAR) translocation identifies mosapride citrate as a CAR agonist that represses gluconeogenesis

The constitutive androstane receptor (CAR) plays an important role in hepatic drug metabolism and detoxification but has recently been projected as a potential drug target for metabolic disorders due to its repression of lipogenesis and gluconeogenesis. Thus, identification of physiologically-relevant CAR modulators has garnered significant interest. Here, we adapted the previously characterized human CAR (hCAR) nuclear translocation assay in human primary hepatocytes (HPH) to a high-content format and screened an FDA-approved drug library containing 978 compounds. Comparison of hCAR nuclear translocation results with the Tox21 hCAR luciferase reporter assay database in 643 shared compounds revealed significant overlap between these two assays, with approximately half of hCAR agonists also mediating nuclear translocation. Further validation of these compounds in HPH and/or using published data from literature demonstrated that hCAR translocation exhibits a higher correlation with the induction of hCAR target genes, such as CYP2B6, than the luciferase assay. In addition, some CAR antagonists which repress CYP2B6 mRNA expression in HPH, such as sorafenib, rimonabant, and CINPA1, were found to translocate hCAR to the nucleus of HPH. Notably, both the translocation assay and the luciferase assay identified mosapride citrate (MOS), a gastroprokinetic agent that is known to reduce fasting blood glucose levels in humans, as a novel hCAR activator. Further studies with MOS in HPH uncovered that MOS can repress the expression of gluconeogenic genes and decrease glucose output from hepatocytes, providing a previously unidentified liver-specific mechanism by which MOS modulates blood glucose levels.

Human constitutive androstane receptor agonist DL5016: A novel sensitizer for cyclophosphamide-based chemotherapies

The DNA alkylating prodrug cyclophosphamide (CPA), alone or in combination with other agents, is one of the most commonly used anti-cancer agents. As a prodrug, CPA is activated by cytochrome P450 2B6 (CYP2B6), which is transcriptionally regulated by the human constitutive androstane receptor (hCAR). Therefore, hCAR agonists represent novel sensitizers for CPA-based therapies. Among known hCAR agonists, compound 6-(4-chlorophenyl)imidazo-[2,1-b]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO) is the most potent and broadly utilized in biological studies. Through structural modification of CITCO, we have developed a novel compound DL5016 (32), which has an EC50 value of 0.66 μM and EMAX value of 4.9 when activating hCAR. DL5016 robustly induced the expression of hCAR target gene CYP2B6, at both the mRNA and protein levels, and caused translocation of hCAR from the cytoplasm to the nucleus in human primary hepatocytes. The effects of DL5016 were highlighted by dramatically enhancing the efficacy of CPA-based cytotoxicity to non-Hodgkin lymphoma cells.

DL5050, a Selective Agonist for the Human Constitutive Androstane Receptor

The constitutive androstane receptor (CAR) is a xenobiotic sensor governing the transcription of genes involved in drug disposition, energy homeostasis, and cell proliferation. However, currently available human CAR (hCAR) agonists are nonselective, which commonly activate hCAR along with other nuclear receptors, especially the closely related human pregnane X receptor (hPXR). Using a well-known hCAR agonist CITCO as a template, we report our efforts in the discovery of a potent and highly selective hCAR agonist. Two of the new compounds of the series, 18 and 19 (DL5050), demonstrated excellent potency and selectivity for hCAR over hPXR. DL5050 preferentially induced the expression of CYP2B6 (target of hCAR) over CYP3A4 (target of hPXR) on both the mRNA and protein levels. The selective hCAR agonist DL5050 represents a valuable tool molecule to further define the biological functions of hCAR, and may also be used as a new lead in the discovery of hCAR agonists for various therapeutic applications.

Stilbene compound trans-3,4,5,4´-tetramethoxystilbene, a potential anticancer drug, regulates constitutive androstane receptor (Car) target genes, but does not possess proliferative activity in mouse liver

The constitutive androstane receptor(CAR) activation is connected with mitogenic effects leading to liver hyperplasia and tumorigenesis in rodents. CAR activators, including phenobarbital, are considered rodent non-genotoxic carcinogens. Recently, trans-3,4,5,4´-tetramethoxystilbene(TMS), a potential anticancer drug (DMU-212), have been shown to alleviate N-nitrosodiethylamine/phenobarbital-induced liver carcinogenesis. We studied whether TMS inhibits mouse Car to protect from the PB-induced tumorigenesis. Unexpectedly, we identified TMS as a murine CAR agonist in reporter gene experiments, in mouse hepatocytes, and in C57BL/6 mice in vivo. TMS up-regulated Car target genes Cyp2b10, Cyp2c29 and Cyp2c55 mRNAs, but down-regulated expression of genes involved in gluconeogenesis and lipogenesis. TMS did not change or down-regulate genes involved in liver proliferation or apoptosis such as Mki67, Foxm1, Myc, Mcl1, Pcna, Bcl2, or Mdm2, which were up-regulated by another Car ligand TCPOBOP. TMS did not increase liver weight and had no significant effect on Ki67 and Pcna labeling indices in mouse liver in vivo. In murine hepatic AML12 cells, we confirmed a Car-independent proapoptotic effect of TMS. We conclude that TMS is a Car ligand with limited effects on hepatocyte proliferation, likely due to promoting apoptosis in mouse hepatic cells, while controlling Car target genes involved in xenobiotic and endobiotic metabolism.

Hepatocyte‐specific YAP deletion suppresses hepatocyte proliferation and hepatomegaly induced by CAR agonist, TCPOBOP (1,4‐Bis [2‐(3,5‐Dichloropyridyloxy)] benzene), in mice

TCPOBOP (1,4‐Bis [2‐(3,5‐Dichloropyridyloxy)] benzene) is a direct ligand of constitutive androstane receptor (CAR), which induces robust hepatocyte proliferation and hepatomegaly in mice without any liver injury or tissue loss. Organ size including that of liver is known to be tightly controlled by transcriptional regulator, yes‐associated protein (YAP). Recent studies have indicated potential interaction of CAR and YAP. Here, we directly investigated role of YAP in TCPOBOP‐induced proliferative response using hepatocyte‐specific YAP‐KO strategy. Hepatocyte‐specific YAP deletion was achieved by administering AAV8‐TBG‐CRE vector to YAP‐floxed mice. TCBOPOP treatment in control mice caused rapid activation and nuclear translocation of YAP along with decreased phosphorylation (inactive form) of YAP concomitant to proliferative response. YAP deletion neither altered protein expression of CAR nor its activation. However, YAP deletion significantly reduced TCPOBOP‐induced hepatomegaly and hepatocyte proliferation. TCPOBOP‐driven cell cycle activation was disrupted in YAP‐KO mice due to impaired protein and gene expression of cyclin D1 and decreased phosphorylation of retinoblastoma (Rb) protein. YAP regulated these cell cycle proteins via alerting expression of cMyc and FOXM1, two critical transcriptional regulators of TCPOBOP‐mediated hepatocyte proliferation. In conclusion, our study revealed an important role of YAP signaling in hepatomegaly and hepatocyte proliferation induced by TCPOBOP.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Expression Regulation of Hox Genes by Perfluorochemicals in Mouse Liver

Homeobox (Hox) genes encode homeodomain proteins, which play important roles in the development and morphological diversification of plants and animals. Perfluorochemicals (PFCs), which are the persistent environmental pollutants and frequently detected in human blood, can interfere with animal development. PFCs produce numerous hepatic adverse effects, such as increased peroxisomes, hepatomegaly and altered lipid metabolism, primarily via activation of peroxisome proliferator‐activated receptors (PPARs) and/or constitutive androstane receptor (CAR). No study has reported that PFCs can regulate the expression of Hox genes. This study was designed to determine the expression regulation of Hox and paraHox genes by perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), and perfluorodecanoic acid (PFDA) in mouse liver. A single dose of 0.1 mmole/kg PFOA, PFNA and PFDA was intraperitoneally administered to adult male wild‐type, and age‐matched male PPARα‐null and male CAR‐null mice (n=6/treatment). Four days later, mouse livers were collected and processed. Messenger RNA expression of Hox genes (including Hox‐a to ‐d subfamily members) and paraHox genes (including Gsx, Pdx, and Cdx gene family members) were determined. Our results showed that PFOA, PFNA, and PFDA all induced mRNA expression of Hoxb7, c5, d10 and Zeb2 in wild‐type mouse liver, but not in PPARα‐null mouse liver. We further showed that PFNA induced mRNA expression of Hoxa5, b7, c5, c8, and d10 in livers of wild‐type and CAR‐null mice, but not in PPARα‐null mouse liver, indicating a PPARα‐dependent mechanism. Furthermore, knocking‐out of PPARα or CAR in mouse liver decreased constitutive mRNA expression of Hoxc5. Wy‐14643, a classical PPARα agonist, induced mRNA expression of Hoxc5, but not of Hoxa5, b7 or d10 in wild‐type mouse liver. 1,4‐bis‐[2‐(3,5,‐dichloropyridyloxy)] benzene (TCPOBOP), a classical CAR agonist, also induced mRNA expression of Hoxc5, but not of Hoxa5, b7 or d10 in wild‐type mouse liver. Messenger RNA expression of studied paraHox genes were not apparently altered by PFCs in mouse liver. In conclusion, perfluorochemicals induced mRNA expression of certain Hox genes such as Hoxc5, maybe via activation of PPARα and/or CAR.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Activation of constitutive androstane receptor inhibits intestinal CFTR-mediated chloride transport

Constitutive androstane receptor (CAR) belonging to the nuclear receptor superfamily plays an important role in the xenobiotic metabolism and disposition. It has been reported that CAR regulates the expression of the ATP-binding cassette (ABC) transporters in the intestine, such as multidrug resistance protein 1 (MDR1) and multidrug resistance-associated protein 2/3 (MRP2 and MRP3). In this study, we investigated the role of CAR in the regulation of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated chloride transport in T84 human colonic epithelial cells and mouse intestinal tissues. Treatments of T84 cell monolayers with specific CAR agonists (CITCO and phenytoin at concentrations of 1 μM and 5 μM, respectively) for 24 h decreased transepithelial Cl− secretion in response to cAMP-dependent agonist. This inhibition was abolished by coincubation of CITCO with a CAR antagonist, CINPA1. We confirmed that an inhibitory effect of CAR agonists was not due to their cytotoxicity. Basolateral membrane permeabilization experiments also revealed that activation of CAR decreased apical Cl− current stimulated by both CPT-cAMP and genistein (a direct CFTR activator). Such activation also reduced both mRNA and protein expression of CFTR. Furthermore, CITCO decreased cholera toxin (CT)-induced Cl− secretion across T84 cell monolayers. In ICR mice, administration of TCPOBOP (3 mg/kgBW), a murine-specific CAR agonist, for 7 days produced significant decreases in CFTR mRNA and protein expressions in intestinal tissues. Interestingly, TCPOBOP also inhibited CT-induced intestinal fluid accumulation in mice. This is the first evidence showing that CFTR was downregulated by CAR activation in the intestine. Our findings suggest that CAR has potential as a new drug target for treatment of condition with hyperactivity/ hyperfunction of CFTR especially secretory diarrheas.

Regulation of Hepatic Long Noncoding RNAs by Pregnane X Receptor and Constitutive Androstane Receptor Agonists in Mouse Liver.

Altered expression of long noncoding RNAs (lncRNAs) by environmental chemicals modulates the expression of xenobiotic biotransformation-related genes and may serve as therapeutic targets and novel biomarkers of exposure. The pregnane X receptor (PXR/NR1I2) is a critical xenobiotic-sensing nuclear receptor that regulates the expression of many drug-processing genes, and it has similar target-gene profiles and DNA-binding motifs with another xenobiotic-sensing nuclear receptor, namely, constitutive andronstrane receptor (CAR/Nr1i3). To test our hypothesis that lncRNAs are regulated by PXR in concert with protein-coding genes (PCGs) and to compare the PXR-targeted lncRNAs with CAR-targeted lncRNAs, RNA-Seq was performed from livers of adult male C57BL/6 mice treated with corn oil, the PXR agonist PCN, or the CAR agonist 1, 4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP). Among 125,680 known lncRNAs, 3843 were expressed in liver, and 193 were differentially regulated by PXR (among which 40% were also regulated by CAR). Most PXR- or CAR-regulated lncRNAs were mapped to the introns and 3'-untranslated regions (UTRs) of PCGs, as well as intergenic regions. Combining the RNA-Seq data with a published PXR chromatin immunoprecipitation coupled with high-throughput sequencing; cytochrome P450 (P450; ChIP-Seq) data set, we identified 774 expressed lncRNAs with direct PXR-DNA binding sites, and 26.8% of differentially expressed lncRNAs had changes in PXR-DNA binding after PCN exposure. De novo motif analysis identified colocalization of PXR with liver receptor homolog (LRH-1), which regulates bile acid synthesis after PCN exposure. There was limited overlap of PXR binding with an epigenetic mark for transcriptional activation (histone-H3K4-di-methylation, H3K4me2) but no overlap with epigenetic marks for transcriptional silencing [H3 lysine 27 tri-methylation (H3K27me3) and DNA methylation]. Among differentially expressed lncRNAs, 264 were in proximity of PCGs, and the lncRNA-PCG pairs displayed a high coregulatory pattern by PXR and CAR activation. This study was among the first to demonstrate that lncRNAs are regulated by PXR and CAR activation and that they may be important regulators of PCGs involved in xenobiotic metabolism.

Constitutive Androstane Receptor Differentially Regulates Bile Acid Homeostasis in Mouse Models of Intrahepatic Cholestasis.

Bile acid (BA) homeostasis is tightly regulated by multiple transcription factors, including farnesoid X receptor (FXR) and small heterodimer partner (SHP). We previously reported that loss of the FXR/SHP axis causes severe intrahepatic cholestasis, similar to human progressive familial intrahepatic cholestasis type 5 (PFIC5). In this study, we found that constitutive androstane receptor (CAR) is endogenously activated in Fxr:Shp double knockout (DKO) mice. To test the hypothesis that CAR activation protects DKO mice from further liver damage, we generated Fxr;Shp;Car triple knockout (TKO) mice. In TKO mice, residual adenosine triphosphate (ATP) binding cassette, subfamily B member 11 (ABCB11; alias bile salt export pump [BSEP]) function and fecal BA excretion are completely impaired, resulting in severe hepatic and biliary damage due to excess BA overload. In addition, we discovered that pharmacologic CAR activation has different effects on intrahepatic cholestasis of different etiologies. In DKO mice, CAR agonist 1,4‐bis[2‐(3,5‐dichloropyridyloxy)]benzene (TCPOBOP; here on TC) treatment attenuated cholestatic liver injury, as expected. However, in the PFIC2 model Bsep knockout (BKO) mice, TC treatment exhibited opposite effects that reflect increased BA accumulation and liver injury. These contrasting results may be linked to differential regulation of systemic cholesterol homeostasis in DKO and BKO livers. TC treatment selectively up‐regulated hepatic cholesterol levels in BKO mice, supporting de novo BA synthesis. Conclusion: CAR activation in DKO mice is generally protective against cholestatic liver injury in these mice, which model PFIC5, but not in the PFIC2 model BKO mice. Our results emphasize the importance of the genetic and physiologic background when implementing targeted therapies to treat intrahepatic cholestasis.

Pharmacological Activation of PXR and CAR Downregulates Distinct Bile Acid-Metabolizing Intestinal Bacteria and Alters Bile Acid Homeostasis.

The gut microbiome regulates important host metabolic pathways including xenobiotic metabolism and intermediary metabolism, such as the conversion of primary bile acids (BAs) into secondary BAs. The nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are well-known regulators for xenobiotic biotransformation in liver. However, little is known regarding the potential effects of PXR and CAR on the composition and function of the gut microbiome. To test our hypothesis that activation of PXR and CAR regulates gut microbiota and secondary BA synthesis, 9-week-old male conventional and germ-free mice were orally gavaged with corn oil, PXR agonist PCN (75 mg/kg), or CAR agonist TCPOBOP (3 mg/kg) once daily for 4 days. PCN and TCPOBOP decreased two taxa in the Bifidobacterium genus, which corresponded with decreased gene abundance of the BA-deconjugating enzyme bile salt hydrolase. In liver and small intestinal content of germ-free mice, there was a TCPOBOP-mediated increase in total, primary, and conjugated BAs corresponding with increased Cyp7a1 mRNA. Bifidobacterium, Dorea, Peptociccaceae, Anaeroplasma, and Ruminococcus positively correlated with T-UDCA in LIC, but negatively correlated with T-CDCA in serum. In conclusion, PXR and CAR activation downregulates BA-metabolizing bacteria in the intestine and modulates BA homeostasis in a gut microbiota-dependent manner.

Constitutive androstane receptor and pregnane X receptor cooperatively ameliorate DSS-induced colitis

BackgroundNuclear receptor pregnane X receptor (PXR) was shown to be protective in case of dextran sulfate sodium (DSS)-induced colitis. Constitutive androstane receptor (CAR) belongs to the same nuclear receptor subfamily with PXR. The roles of both receptors in DSS-induced colitis were evaluated. MethodsWild-type, Car-null, Pxr-null, and Car/Pxr-null mice were treated with a CAR/PXR agonist or vehicle and administered 2.5% DSS in the drinking water. The typical clinical symptoms, histological scoring, proinflammatory cytokine, and apoptosis were analyzed. ResultsMice treated with the PXR agonist pregnenolone-16α-carbonitrile (PCN) were protected from DSS-induced colitis, as in a previous study. Mice treated with the CAR agonist, 4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) were also protected from DSS-induced colitis. Interestingly, the protective effects of PCN in the Car-null mice and those of TCPOBOP in the Pxr-null mice both decreased. PCN or TCPOBOP pretreatment significantly decreased the macrophage and monocyte infiltration in DSS-induced colitis. PXR and CAR agonists reduced the mRNA expression of several proinflammatory cytokines in a PXR- and CAR-dependent manner, respectively. CAR inhibited apoptosis by inducing Gadd45b. PXR inhibited TNF-α and IL-1b and CAR induced Gadd45b in in vitro cell analyses. ConclusionsWe showed that CAR and PXR cooperatively ameliorate DSS-induced colitis. PXR and CAR protected against DSS-induced colitis by inhibiting proinflammatory cytokines and apoptosis, respectively.

The connection of lateral preferences with the specificity of cardiac rhythm variability in children

Nuclear receptor pregnane X receptor (PXR) was shown to be protective in case of dextran sulfate sodium (DSS)-induced colitis. Constitutive androstane receptor (CAR) belongs to the same nuclear receptor subfamily with PXR. The roles of both receptors in DSS-induced colitis were evaluated.Wild-type, Car-null, Pxr-null, and Car/Pxr-null mice were treated with a CAR/PXR agonist or vehicle and administered 2.5% DSS in the drinking water. The typical clinical symptoms, histological scoring, proinflammatory cytokine, and apoptosis were analyzed.Mice treated with the PXR agonist pregnenolone-16α-carbonitrile (PCN) were protected from DSS-induced colitis, as in a previous study. Mice treated with the CAR agonist, 4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) were also protected from DSS-induced colitis. Interestingly, the protective effects of PCN in the Car-null mice and those of TCPOBOP in the Pxr-null mice both decreased. PCN or TCPOBOP pretreatment significantly decreased the macrophage and monocyte infiltration in DSS-induced colitis. PXR and CAR agonists reduced the mRNA expression of several proinflammatory cytokines in a PXR- and CAR-dependent manner, respectively. CAR inhibited apoptosis by inducing Gadd45b. PXR inhibited TNF-α and IL-1b and CAR induced Gadd45b in in vitro cell analyses.We showed that CAR and PXR cooperatively ameliorate DSS-induced colitis. PXR and CAR protected against DSS-induced colitis by inhibiting proinflammatory cytokines and apoptosis, respectively.

High Frequency of β-Catenin Mutations in Mouse Hepatocellular Carcinomas Induced by a Nongenotoxic Constitutive Androstane Receptor Agonist

Activation of Wnt/β-catenin signaling is frequent in human and rodent hepatocarcinogenesis. Although in mice the tumor-promoting activity of agonists of constitutive androstane receptor (CAR) occurs by selection of carcinogen-initiated cells harboring β-catenin mutations, the molecular alterations leading to hepatocellular carcinoma (HCC) development by the CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCP) in the absence of genotoxic injury are unknown. Here, we show that CAR activation per se induced HCC in mice and that 91% of them carried β-catenin point mutations or large in-frame deletions/exon skipping targeting Ctnnb1 exon 3. Point mutations in HCCs induced by TCP alone displayed different nucleotide substitutions compared with those found in HCCs from mice pretreated with diethylnitrosamine. Moreover, unlike those occurring in HCCs from diethylnitrosamine+TCP mice, they did not result in increased expression of β-catenin target genes, such as Glul, Lgr5, Rgn, Lect2, Tbx3, Axin2, and Ccnd1, or nuclear translocation of β-catenin compared with the control liver. Remarkably, in the nontumoral liver tissue, chronic CAR activation led to down-regulation of these genes and to a partial loss of glutamine synthetase-positive hepatocytes. These results show that, although chronic CAR activation per se induces HCCs carrying β-catenin mutations, it concurrently down-regulates the Wnt/β-catenin pathway in nontumoral liver. They also indicate that the relationship between CAR and β-catenin may be profoundly different between normal and neoplastic hepatocytes.

The CAR agonist TCPOBOP inhibits lipogenesis and promotes fibrosis in the mammary gland of adolescent female mice

Constitutive androstane receptor (CAR) is a nuclear receptor that not only regulates drug-metabolizing enzymes but also influences energy metabolism. TC, 1, 4-bis [2-(3, 5-dichloropyridyloxy)] benzene (TCPOBOP) has been shown to inhibit lipogenesis in the liver and adipose tissues. The mammary gland is mainly composed of fat pads and duct systems in adolescent female mice. Here, activation of CAR by TC reduces the mammary gland weight, blocks lipid accumulation by inhibiting lipogenesis and gluconeogenesis, and accelerates collagen formation and fibrosis in the mammary fat pad of adolescent female mice. This information provides a reference for CAR activation, which may affect mammary gland development in adolescent females.

Epidermal Growth Factor Represses Constitutive Androstane Receptor Expression in Primary Human Hepatocytes and Favors Regulation by Pregnane X Receptor.

Growth factors have key roles in liver physiology and pathology, particularly by promoting cell proliferation and growth. Recently, it has been shown that in mouse hepatocytes, epidermal growth factor receptor (EGFR) plays a crucial role in the activation of the xenosensor constitutive androstane receptor (CAR) by the antiepileptic drug phenobarbital. Due to the species selectivity of CAR signaling, here we investigated epidermal growth factor (EGF) role in CAR signaling in primary human hepatocytes. Primary human hepatocytes were incubated with CITCO, a human CAR agonist, or with phenobarbital, an indirect CAR activator, in the presence or absence of EGF. CAR-dependent gene expression modulation and PXR involvement in these responses were assessed upon siRNA-based silencing of the genes that encode CAR and PXR. EGF significantly reduced CAR expression and prevented gene induction by CITCO and, to a lower extent, by phenobarbital. In the absence of EGF, phenobarbital and CITCO modulated the expression of 144 and 111 genes, respectively, in primary human hepatocytes. Among these genes, only 15 were regulated by CITCO and one by phenobarbital in a CAR-dependent manner. Conversely, in the presence of EGF, CITCO and phenobarbital modulated gene expression only in a CAR-independent and PXR-dependent manner. Overall, our findings suggest that in primary human hepatocytes, EGF suppresses specifically CAR signaling mainly through transcriptional regulation and drives the xenobiotic response toward a pregnane X receptor (PXR)-mediated mechanism.

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.

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.

Constitutive androstane receptor regulates the intestinal mucosal response to injury.

The pathogenesis of the inflammatory bowel diseases (IBD), comprising Crohn's disease (CD) and ulcerative colitis (UC), involves aberrant interactions between a genetically susceptible individual, their microbiota and environmental factors. Alterations in xenobiotic receptor expression and function are associated with increased risk for IBD. Here, we have assessed the role of the constitutive androstane receptor (CAR), a xenobiotic receptor closely related to the pregnane X receptor, in the regulation of intestinal mucosal homeostasis. CAR expression was assessed in intestinal mucosal biopsies obtained from CD and UC patients, and in C57/Bl6 mice exposed to dextran sulphate sodium (DSS; 3.5% w/v in drinking water) to evoke intestinal inflammation and tissue damage. CAR-deficient mice were exposed to DSS and mucosal healing assessed. Modulation of wound healing by CAR was assessed in vitro. The therapeutic potential of CAR activation was evaluated, using 3,3',5,5'-tetrachloro-1,4-bis(pyridyloxy)benzene (TCPOBOP), a selective rodent CAR agonist. CAR expression was reduced in CD and UC samples, compared with expression in healthy controls. This was reproduced in our DSS studies, where CAR expression was reduced in colitic mice. CAR-deficient mice exhibited reduced healing following DSS exposure. In vitro, CAR activation accelerated intestinal epithelial wound healing by enhancing cell migration. Lastly, treating mice with TCPOBOP, following induction of colitis, enhanced mucosal healing. Our results support the notion that xenobiotic sensing is altered during intestinal inflammation, and suggest that CAR activation may prove effective in enhancing mucosal healing in patients with IBD.