Constitutive Androstane Receptor Ligand Research Articles

Filling the Blank Space: Branched 4-Nonylphenol Isomers Are Responsible for Robust Constitutive Androstane Receptor (CAR) Activation by Nonylphenol.

4-Nonylphenol (4-NP), a para-substituted phenolic compound with a straight or branched carbon chain, is a ubiquitous environmental pollutant and food contaminant. 4-NP, particularly the branched form, has been identified as an endocrine disruptor (ED) with potent activities on estrogen receptors. Constitutive Androstane Receptor (CAR) is another crucial nuclear receptor that regulates hepatic lipid, glucose, and steroid metabolism and is involved in the ED mechanism of action. An NP mixture has been described as an extremely potent activator of both human and rodent CAR. However, detailed mechanistic aspects of CAR activation by 4-NP are enigmatic, and it is not known if 4-NP can directly interact with the CAR ligand binding domain (LBD). Here, we examined interactions of individual branched (22NP, 33NP, and 353NP) and linear 4-NPs with CAR variants using molecular dynamics (MD) simulations, cellular experiments with various CAR expression constructs, recombinant CAR LBD in a TR-FRET assay, or a differentiated HepaRG hepatocyte cellular model. Our results demonstrate that branched 4-NPs display more stable poses to activate both wild-type CAR1 and CAR3 variant LBDs in MD simulations. Consistently, branched 4-NPs activated CAR3 and CAR1 LBD more efficiently than linear 4-NP. Furthermore, in HepaRG cells, we observed that all 4-NPs upregulated CYP2B6 mRNA, a relevant hallmark for CAR activation. This is the first study to provide detailed insights into the direct interaction between individual 4-NPs and human CAR-LBD, as well as its dominant variant CAR3. The work could contribute to the safer use of individual 4-NPs in many areas of industry.

Steatosis and Metabolic Disorders Associated with Synergistic Activation of the CAR/RXR Heterodimer by Pesticides.

The nuclear receptor, constitutive androstane receptor (CAR), which forms a heterodimer with the retinoid X receptor (RXR), was initially reported as a transcription factor that regulates hepatic genes involved in detoxication and energy metabolism. Different studies have shown that CAR activation results in metabolic disorders, including non-alcoholic fatty liver disease, by activating lipogenesis in the liver. Our objective was to determine whether synergistic activations of the CAR/RXR heterodimer could occur in vivo as described in vitro by other authors, and to assess the metabolic consequences. For this purpose, six pesticides, ligands of CAR, were selected, and Tri-butyl-tin (TBT) was used as an RXR agonist. In mice, CAR's synergic activation was induced by dieldrin associated with TBT, and combined effects were induced by propiconazole, bifenox, boscalid, and bupirimate. Moreover, a steatosis, characterized by increased triglycerides, was observed when TBT was combined with dieldrin, propiconazole, bifenox, boscalid, and bupirimate. Metabolic disruption appeared in the form of increased cholesterol and lowered free fatty acid plasma levels. An in-depth analysis revealed increased expression of genes involved in lipid synthesis and lipid import. These results contribute to the growing understanding of how environmental contaminants can influence nuclear receptor activity and associated health risks.

Constitutive androstane receptor-responsive elements for mouse Cyp1a2 transcriptional activation induced by constitutive androstane receptor ligands

The mouse cytochrome P450 1A2 (Cyp1a2) gene is one of the constitutive androstane receptor (CAR, NR1I3) activator-inducible genes, and the regions involved in induction were examined herein. A reporter gene assay indicated the involvement of the −0.2-kb region in the induction of transcriptional activation by the mouse CAR agonist ligand 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP). Some putative nuclear receptor-binding elements were identified in this region, and mutations in the elements located at −160/-155 or −153/-148 abolished this induction. An electrophoretic mobility shift assay demonstrated that a fragment comprised of three elements was capable of binding to the CAR/retinoid X receptor alpha (RXRα) heterodimer. The three elements comprise the two elements indicated above and one located at −146/-141. A chromatin immunoprecipitation assay confirmed CAR binding to the region including these elements in chromatin after treatment with TCPOBOP. These results indicate that mouse Cyp1a2 is the direct target of CAR, and binding of the CAR/RXRα heterodimer to the newly identified region in the promoter may be involved in transcriptional activation. Binding motifs were estimated as ER1 (everted repeat with a spacing of 1 nucleotide, −160/-155 and −153/-148) and ER8 (everted repeat with a spacing of 8 nucleotides, formed with −160/-155 and −146/-141).

Lymphocyte-Specific Protein-1 Suppresses Xenobiotic-Induced Constitutive Androstane Receptor and Subsequent Yes-Associated Protein–Activated Hepatocyte Proliferation

Activation of constitutive androstane receptor (CAR) transcription factor by xenobiotics promotes hepatocellular proliferation, promotes hypertrophy without liver injury, and induces drug metabolism genes. Previous work demonstrated that lymphocyte-specific protein-1 (LSP1), an F-actin binding protein and gene involved in human hepatocellular carcinoma, suppresses hepatocellular proliferation after partial hepatectomy. The current study investigated the role of LSP1 in liver enlargement induced by chemical mitogens, a regenerative process independent of tissue loss. 1,4-Bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), a direct CAR ligand and strong chemical mitogen, was administered to global Lsp1 knockout and hepatocyte-specific Lsp1 transgenic (TG) mice and measured cell proliferation, hypertrophy, and expression of CAR-dependent drug metabolism genes. TG livers displayed a significant decrease in Ki-67 labeling and liver/body weight ratios compared with wild type on day 2. Surprisingly, this was reversed by day 5, due to hepatocyte hypertrophy. There was no difference in CAR-regulated drug metabolism genes between wild type and TG. TG livers displayed increased Yes-associated protein (YAP) phosphorylation, decreased nuclear YAP, and direct interaction between LSP1 and YAP, suggesting LSP1 suppresses TCPOBOP-driven hepatocellular proliferation, but not hepatocyte volume, through YAP. Conversely, loss of LSP1 led to increased hepatocellular proliferation on days 2, 5, and 7. LSP1 selectively suppresses CAR-induced hepatocellular proliferation, but not drug metabolism, through the interaction of LSP1 with YAP, supporting the role of LSP1 as a selective growth suppressor.

Off-target lipid metabolism disruption by the mouse constitutive androstane receptor ligand TCPOBOP in humanized mice

The constitutive androstane receptor (CAR) controls xenobiotic clearance, regulates liver glucose, lipid metabolism, and energy homeostasis. These functions have been mainly discovered using the prototypical mouse-specific CAR ligand TCPOBOP in wild-type or CAR null mice. However, TCPOBOP is reported to result in some off-target metabolic effects in CAR null mice.In this study, we compared the metabolic effects of TCPOBOP using lipidomic, transcriptomic, and proteomic analyses in wild-type and humanized CAR-PXR-CYP3A4/3A7 mice. In the model, human CAR retains its constitutive activity in metabolism regulation; however, it is not activated by TCPOBOB.Notably, we observed that TCPOBOP affected lipid homeostasis by elevating serum and liver triglyceride levels and promoted hepatocyte hypertrophy in humanized CAR mice. Hepatic lipidomic analysis revealed a significant accumulation of triglycerides and decrease of its metabolites in humanized CAR mice. RNA-seq analysis has shown divergent gene expression levels in wild-type and humanized CAR mice. Gene expression regulation in humanized mice is mainly involved in lipid metabolic processes and in the PPAR, leptin, thyroid, and circadian clock pathways. In contrast, CAR activation by TCPOBOP in wild-type mice reduced liver and plasma triglyceride levels and induced a typical transcriptomic proliferative response in the liver.In summary, we identified TCPOBOP as a disruptor of lipid metabolism in humanized CAR mice. The divergent effects of TCPOBOP in humanized mice in comparison with the prototypical CAR-mediated response in WT mice warrant the use of appropriate model ligands and humanized animal models during the testing of endocrine disruption and the characterization of adverse outcome pathways.

Activation of the Constitutive Androstane Receptor Inhibits Leukocyte Adhesiveness to Dysfunctional Endothelium.

Leukocyte cell recruitment into the vascular subendothelium constitutes an early event in the atherogenic process. As the effect of the constitutive androstane receptor (CAR) on leukocyte recruitment and endothelial dysfunction is poorly understood, this study investigated whether the role of CAR activation can affect this response and the underlying mechanisms involved. Under physiological flow conditions, TNFα-induced endothelial adhesion of human leukocyte cells was concentration-dependently inhibited by preincubation of human umbilical arterial endothelial cells with the selective human CAR ligand CITCO. CAR agonism also prevented TNFα induced VCAM-1 expression, as well as MCP-1/CCL-2 and RANTES/CCL-5 release in endothelial cells. Suppression of CAR expression with a small interfering RNA abrogated the inhibitory effects of CITCO on these responses. Furthermore, CITCO increased interaction of CAR with Retinoid X Receptor (RXR) and reduced TNFα-induced p38-MAPK/NF-κB activation. In vivo, using intravital microscopy in the mouse cremasteric microcirculation treatment with the selective mouse CAR ligand TCPOBOP inhibited TNFα-induced leukocyte rolling flux, adhesion, and emigration and decreased VCAM-1 in endothelium. These results reveal that CAR agonists can inhibit the initial inflammatory response that precedes the atherogenic process by targeting different steps in the leukocyte recruitment cascade. Therefore, CAR agonists may constitute a new therapeutic tool in controlling cardiovascular disease-associated inflammatory processes.

A high-throughput cell-based gaussia luciferase reporter assay for measurement of CYP1A1, CYP2B6, and CYP3A4 induction

The induction of cytochrome P450s can result in reduced drug efficacy and lead to potential drug–drug interactions. The xenoreceptors—aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), and pregnane X receptor (PXR)—play key roles in CYP induction by xenobiotics. In order to be able to rapidly screen for the induction of three enzymes (CYP1A1, CYP2B6, and CYP3A4), we generated a stable AhR-responsive HepG2 cell line, a stable CAR-responsive HepG2 cell line, and a stable PXR-responsive HepG2 cell line. To validate these stable xenoreceptor-responsive HepG2 cell lines, we evaluated the induction of the different Gaussia reporter activities, as well as the mRNA and protein expression levels of endogenous CYPs in response to different inducers. The induction of luciferase activity in the stable xenoreceptor-responsive HepG2 cell lines by specific inducers occurred in a concentration dependent manner. There was a positive correlation between the induction of luciferase activities and the induction endogenous CYP mRNA expression levels. These xenoreceptor-responsive HepG2 cell lines were further validated with known CYP1A1, CYP2B6, and CYP3A4 inducers. These stable xenoreceptor-responsive HepG2 cell lines may be used in preclinical research for the rapid and sensitive detection of AhR, CAR, and PXR ligands that induce CYP450 isoforms.

Transporter Gene Regulation in Sandwich Cultured Human Hepatocytes Through the Activation of Constitutive Androstane Receptor (CAR) or Aryl Hydrocarbon Receptor (AhR).

The induction potentials of ligand-activated nuclear receptors on metabolizing enzyme genes are routinely tested for new chemical entities. However, regulations of drug transporter genes by the nuclear receptor ligands are underappreciated, especially in differentiated human hepatocyte cultures. In this study, gene induction by the ligands of constitutive androstane receptor (CAR) and aryl hydrocarbon receptor (AhR) was characterized in sandwich-cultured human hepatocytes (SCHH) from multiple donors. The cells were treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), omeprazole (OP), 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO) and phenobarbital (PB) for three days. RNA samples were analyzed by qRT-PCR method. As expected, CITCO, the direct activator, and PB, the indirect activator of CAR, induced CYP3A4 (31 and 40-fold), CYP2B6 (24 and 28-fold) and UGT1A1 (2.9 and 4.2-fold), respectively. Conversely, TCDD and OP, the activators of AhR, induced CYP1A1 (38 and 37-fold), and UGT1A1 (4.3 and 5.0-fold), respectively. In addition, OP but not TCDD induced CY3A4 by about 61-fold. Twenty-four hepatic drug transporter genes were characterized, and of those, SLC51B was induced the most by PB and OP by about 3.3 and 6.5 fold, respectively. Marginal inductions (about 2-fold) of SLC47A1 and SLCO4C1 genes by PB, and ABCG2 gene by TCDD were observed. In contrast, SLC10A1 gene was suppressed about 2-fold by TCDD and CITCO. While clinical relevance of SLC51B gene induction or SLC10A1 gene suppression warrants further investigation, the results verified that the assessment of transporter gene inductions are not required for new drug entities, when a drug does not remarkably induce metabolizing enzyme genes by CAR and AhR activation.

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.

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.

Xenobiotic Nuclear Receptors Pregnane X Receptor and Constitutive Androstane Receptor Regulate Antiretroviral Drug Efflux Transporters at the Blood-Testis Barrier

Poor antiretroviral drug (ARV) penetration in the testes could be due, in part, to the presence of ATP-binding cassette (ABC) membrane-associated drug efflux transporters such as P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance-associated proteins (MRPs) expressed at the blood-testis barrier (BTB). The functional expression of these transporters is known to be regulated by ligand-activated nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) in various tissues. This study aimed to investigate in vitro and ex vivo the role of PXR and CAR in the regulation of ABC transporters at the BTB. Both PXR and CAR proteins were expressed in human testicular tissue and in mouse TM4 Sertoli cells (an in vitro cell line model of the BTB). In addition, we demonstrated an upregulation of P-gp, Bcrp, and Mrp4 mRNA and protein expression, after exposure to PXR or CAR ligands in TM4 cells. Small interfering RNA downregulation of PXR or CAR attenuated the expression of these transporters, suggesting the direct involvement of these nuclear receptors in regulating P-gp, Bcrp, and Mrp4 in this system. In an ex vivo study using freshly isolated mouse seminiferous tubules, we found that exposure to PXR or CAR ligands, including ARVs, significantly increased P-gp expression and function. Together, our data suggest that ABC transporters could be regulated at the BTB during chronic treatment with ARVs that can serve as ligands for PXR and CAR, which could in turn further limit testicular ARV concentrations.

Agonistic effects of diverse xenobiotics on the constitutive androstane receptor as detected in a recombinant yeast-cell assay

The constitutive androstane receptor (CAR) is a nuclear receptor and transcription factor regulating proteins involved in xenobiotic metabolism. Agonist activation of the CAR can trigger metabolic activation and toxification as well as detoxification and clearance; accordingly, xenobiotic substances acting as CAR ligands may pose a threat to human and animal health. We used yeast cells transduced with the human CAR and the response pathway to measure the CAR-agonistic activities of 549 synthetic or natural compounds: 216 of the tested compounds exhibited CAR-agonistic effects. Eighty-four percent of CAR-activating compounds were aromatic compounds, and >65% of these active compounds were aromatic hydrocarbons, bisphenols, monoalkyl phenols, phthalates, styrene dimers, diphenyl ethers, organochlorines, and organophosphates. The ten most potent compounds were 4-tert-octylphenol (4tOP; reference substance), 4-nonylphenol, diethylstilbestrol, benzyl n-butyl phthalate, 2-(4-hydroxyphenyl)-2,4,4-trimethylchroman, o,p′-DDT, methoxychlor, di-n-propyl phthalate, hexestrol, and octachlorostyrene. The activities of these nine non-reference compounds exceeded 10% of the 4tOP activity. Analysis of para-monoalkyl phenols suggests that branching of the alkyl group and chlorination at the ortho position raises potency. This study provides critical information for identifying the potential of CAR-mediated toxic hazards and for understanding the relevant mechanism.

Rosuvastatin and Atorvastatin Are Ligands of the Human Constitutive Androstane Receptor/Retinoid X Receptor α Complex.

Statins are well known lipid lowering agents that inhibit the enzyme 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase. They also activate drug metabolism but their exact receptor-mediated action has not been proven so far. We tested whether atorvastatin and rosuvastatin are direct ligands of human constitutive androstane receptor (CAR). We measured binding activities of atorvastatin and rosuvastatin to the human constitutive androstane receptor/retinoid X receptor α ligand-binding domain (CAR/RXRα-LBD) heterodimer with surface plasmon resonance (SPR). Additionally, three-dimensional models of CAR/RXRα-LBD were constructed by ligand-based and structure-based in silico modeling. Experiments and computational modeling show that atorvastatin and rosuvastatin bind to the human CAR/RXRα-LBD heterodimer, suggesting both can modulate the activity of CAR through direct interaction with the LBD of this receptor. We confirm that atorvastatin and rosuvastatin are direct ligands of CAR. The clinical consequences of CAR activation by statins are in their potential drug-drug interactions, and changes in glucose and energy metabolism.

Age‐ and species‐specific activation of CAR on bile acid homeostasis in mice

Pharmacological activation of the constitutive androstane receptor (CAR) has been shown to protect against bile acid (BA)‐induced liver injury in adults (Beilke et al., 2009). During development, targeting CAR by agonists has been suggested to be one of the ultimate goals in the management and treatment of total parenteral nutrition related cholestasis in newborns (Hendaus, 2013). However, very little is known regarding the age‐ and species‐specific effects of CAR activation on BA homeostasis during development. The goal of this study was to use RNA sequencing and BA metabolomics to investigate the effects of CAR activation on the expression of genes involved in BA homeostasis and the BA profiles in 5‐ and 60‐day‐old wild‐type (WT) and humanized CAR transgenic (hCAR‐TG) mice. Male mice were i.p. administered corn oil or a species‐appropriate CAR ligand (TCPOBOP at 3mg/kg for mCAR, and CITCO at 30mg/kg for hCAR) once daily for 4 days. In control mice of both genotypes, from newborns to adulthood, there was a marked decrease in total serum BAs likely due to the maturation of the enterohepatic system. Specifically, serum total primary BAs and conjugated BAs were down‐regulated with age in both WT and hCAR‐TG mice. Conversely, there was a marked increase in serum total secondary BAs and unconjugated BAs, likely due to an ontogenic increase in intestinal bacteria for BA‐dehydroxylation and deconjugation. Following mCAR‐activation, there was an increase in serum total BAs, primary BAs, secondary BAs, and conjugated BAs at both Day 5 and 60. However, the increase in serum BAs was not observed following hCAR activation. In fact, serum cholic acid was decreased by CITCO in 60‐day‐old hCAR‐TG mice. Regarding hepatic BA synthesis, Cyp7a1 mRNA was minimally influenced by CAR activation regardless of the age or species. Conversely, the regulation of Cyp8b1 mRNA seems age‐specific, because it was up‐regulated by CAR of both species at Day 5, but was down‐regulated at Day 60. The regulation of many other BA‐synthetic enzymes at mRNA level was mCAR‐specific, evidenced by an induction of Akr1d1, but a reduction of Amacr, Hsd17b4, Hsd3b7, and Scp2 at both ages only by activation of mCAR but not hCAR. Age‐ and/or species‐specific regulatory effects of CAR activation were also observed for genes involved in BA conjugation and transport. However, several BA‐processing genes appeared to be “universal targets” of CAR of both species and at both ages, including a consistent up‐regulation of Cyp39a1, Akr1c14, Mrp3, and Mrp4. However, in general, mCAR activation produced greater degree of induction as compared to hCAR activation at the same age. In conclusion, the present study suggests that hCAR activation has less effects on serum BA metabolites as compared to mCAR, whereas mCAR and hCAR activation produce both age‐ and species‐specific effects on the hepatic expression of genes involved in BA homeostasis. Understanding the age‐ and species‐differences in the pharmacodynamics of CAR activation is essential in precision medicine for pediatric patients.Support or Funding InformationGrace Liejun Guo glg48@eohsi.rutgers.edu

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.

2-(3-Methoxyphenyl)quinazoline Derivatives: A New Class of Direct Constitutive Androstane Receptor (CAR) Agonists.

Constitutive androstane receptor (CAR) is a key regulator of xenobiotic and endobiotic metabolism. Together with pregnane X (PXR) and aryl hydrocarbon (AHR) receptors, it is referred to as "xenobiotic receptor". The unique properties of human CAR, such as its high constitutive activity, both direct (ligand-binding domain-dependent) and indirect activation have hindered the discovery of direct selective human CAR ligands. Herein, we report a novel class of direct human CAR agonists in a group of 2-(3-methoxyphenyl)quinazoline derivatives. The compounds are even more potent activators of human CAR than is prototype 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO). The three most potent ligands are at the same time extremely potent activators of the other xenobiotic or hormonal receptors, namely PXR, AHR, and vitamin D receptor, which regulate major xenobiotic-metabolizing enzymes and efflux transporters. Thus, the novel CAR ligands can be also considered as constituting the first class of potent pan-xenobiotic receptor ligands that can serve as potential antidotes boosting overall metabolic elimination of xenobiotic or toxic compounds.

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

Activation of Constitutive Androstane Receptor (CAR) in Mice Results in Maintained Biliary Excretion of Bile Acids Despite a Marked Decrease of Bile Acids in Liver.

Activation of Constitutive Androstane Receptor (CAR) protects against bile acid (BA)-induced liver injury. This study was performed to determine the effect of CAR activation on bile flow, BA profile, as well as expression of BA synthesis and transport genes. Synthetic CAR ligand 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) was administered to mice for 4 days. BAs were quantified by UPLC-MS/MS (ultraperformance liquid chromatography-tandem mass spectrometry). CAR activation decreases total BAs in livers of male (49%) and female mice (26%), largely attributable to decreases of the 12α-hydroxylated BA taurocholic acid (T-CA) (males (M) 65%, females (F) 45%). Bile flow in both sexes was increased by CAR activation, and the increases were BA-independent. CAR activation did not alter biliary excretion of total BAs, but overall BA composition changed. Excretion of muricholic (6-hydroxylated) BAs was increased in males (101%), and the 12α-OH proportion of biliary BAs was decreased in both males (37%) and females (28%). The decrease of T-CA in livers of males and females correlates with the decreased mRNA of the sterol 12α-hydroxylase Cyp8b1 in males (71%) and females (54%). As a response to restore BAs to physiologic concentrations in liver, mRNA of Cyp7a1 is upregulated following TCPOBOP (males 185%, females 132%). In ilea, mRNA of the negative feedback regulator Fgf15 was unaltered by CAR activation, indicating biliary BA excretion was sufficient to maintain concentrations of total BAs in the small intestine. In summary, the effects of CAR activation on BAs in male and female mice are quite similar, with a marked decrease in the major BA T-CA in the liver.

PXR- and CAR-mediated herbal effect on human diseases

The pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are two members of the nuclear receptor superfamily that regulate a broad range of genes involved in drug metabolism and transport. A variety of naturally occurring compounds present in herbal medicines were identified as ligands of PXR and CAR. Recently, accumulative evidences have revealed the PXR- and CAR-mediated herbal effect against multiple human diseases, including inflammatory bowel disease (IBD), cholestatic liver disease, and jaundice. The current review summarized the recent progress in identifying the expanding libraries of herbal medicine as ligands for PXR and CAR. Moreover, the potential for herbal medicines as promising therapeutic agents which were mainly regulated through PXR/CAR signaling pathways was also discussed. The discovery of herbal medicines as modulators of PXR and CAR, and their PXR- and CAR-mediated effect on human diseases will provide a basis for rational drug design, and eventually be explored as a novel therapeutic approach against human diseases. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie.

Identification of Candidate Target <i>Cyp</i> Genes for microRNAs Whose Expression Is Altered by PCN and TCPOBOP, Representative Ligands of PXR and CAR

MicroRNAs (miRNAs) are small non-coding RNAs that are involved in mRNA post-transcriptional regulation. The deregulation of miRNAs affects the expression of drug-metabolizing enzymes, drug transporters, and nuclear receptors, all of which are important in regulating drug metabolism. miRNA expression can be altered by several endogenous or exogenous agents, such as steroid hormones, carcinogens, and therapeutic drugs. However, it is unclear whether hepatic miRNA expression is regulated by nuclear receptors, such as pregnane X receptor (PXR) and constitutive androstane receptor (CAR), which are indispensable for the expression of the CYPs. Here we investigated the effects of the mouse PXR and CAR ligands pregnenolone-16α-carbonitrile (PCN) and 1,4-bis[(3,5-dichloropyridin-2-yl)oxy]benzene (TCPOBOP) on hepatic miRNA expression in mice. We found that the expression of 9 miRNAs was increased (>2-fold) and of 4 miRNAs was decreased (>50%) in response to PCN, while TCPOBOP treatment led to the up-regulation of 8 miRNAs and down-regulation of 6 miRNAs. Using several miRNA target prediction algorithms, we found that the predicted target genes included several lesser known Cyp genes (Cyp1a1, Cyp1b1, Cyp2b10, Cyp2c38, Cyp2u1, Cyp4a12a/b, Cyp4v3, Cyp17a1, Cyp39a1, and Cyp51). We analyzed the expression of these genes in response to PCN and TCPOBOP and found changes in their mRNA levels, some of which were negatively correlated with the expression of their corresponding miRNAs, suggesting that miRNAs may play a role in regulating Cyp enzyme expression. Further studies will be required to fully elucidate the miRNA regulatory mechanisms that contribute to modulating CYP expression.