Phenobarbital responsive nuclear translocation of the receptor CAR in induction of the CYP2B gene. Mol Cell Biol

Pharmacogenetics Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Molecular and Cellular Biology (Impact Factor: 4.78). 10/1999; 19(9):6318-22.
Source: PubMed

ABSTRACT The constitutively active receptor (CAR) transactivates a distal enhancer called the phenobarbital (PB)-responsive enhancer module (PBREM) found in PB-inducible CYP2B genes. CAR dramatically increases its binding to PBREM in livers of PB-treated mice. We have investigated the cellular mechanism of PB-induced increase of CAR binding. Western blot analyses of mouse livers revealed an extensive nuclear accumulation of CAR following PB treatment. Nuclear contents of CAR perfectly correlate with an increase of CAR binding to PBREM. PB-elicited nuclear accumulation of CAR appears to be a general step regulating the induction of CYP2B genes, since treatments with other PB-type inducers result in the same nuclear accumulation of CAR. Both immunoprecipitation and immunohistochemistry studies show cytoplasmic localization of CAR in the livers of nontreated mice, indicating that CAR translocates into nuclei following PB treatment. Nuclear translocation of CAR also occurs in mouse primary hepatocytes but not in hepatocytes treated with the protein phosphatase inhibitor okadaic acid. Thus, the CAR-mediated transactivation of PBREM in vivo becomes PB responsive through an okadaic acid-sensitive nuclear translocation process.

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Available from: Igor N Zelko, Sep 29, 2015
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    • "Promiscuous NRs bind to a wide range of different molecules and, depending on the molecule, activate the transcription of a wide range of proteins. Some of these promiscuous NRs are involved in inducing phase I-III responses following exposure to toxicants (Kliewer et al., 1998; Kawamoto et al., 1999; Wei et al., 2000; King-Jones et al., 2006; Karimullina et al., 2012). It has been hypothesized that specificity/promiscuity comes into play when examining the evolution of nuclear receptors due to natural selection. "
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    ABSTRACT: Most nuclear receptors (NRs) are ligand-dependent transcription factors crucial in homeostatic physiological responses or environmental responses. We annotated the Daphniamagna NRs and compared them to Daphniapulex and other species, primarily through phylogenetic analysis. Daphnia species contain 26 NRs spanning all seven gene subfamilies. Thirteen of the 26 receptors found in Daphnia species phylogenetically segregate into the NR1 subfamily, primarily involved in energy metabolism and resource allocation. Some of the Daphnia NRs, such as RXR, HR96, and E75 show strong conservation between D. magna and D. pulex. Other receptors, such as EcRb, THRL-11 and RARL-10 have diverged considerably and therefore may show different functions in the two species. Curiously, there is an inverse association between the number of NR splice variants and conservation of the LBD. Overall, D. pulex and D. magna possess the same NRs; however not all of the NRs demonstrate high conservation indicating the potential for a divergence of function.
    Gene 09/2014; 552(1). DOI:10.1016/j.gene.2014.09.024 · 2.14 Impact Factor
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    • "phenobarbital (PB)-like inducers via interactions with DR4 motifs (Kawamoto et al. 1999). It therefore acts as a xenobiotic-sensing nuclear receptor. "
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    ABSTRACT: Antagonizing the action of the pregnane X receptor (PXR) may have important clinical implications for preventing inducer-drug interactions and improving therapeutic efficacy. We identified a widely distributed isothiocyanate, allyl isothiocyanate (AITC), which acts as an effective antagonist of the nuclear receptor pregnane X receptor (PXR, NR1I2) and constitutive androstane receptor (CAR, NR1I3). HepG2 cells were used to assay reporter function, mRNA levels, and protein expression. Catalytic activities of the PXR and CAR target genes, CYP3A4 and CYP2B6, respectively, were also assessed in differentiated HepaRG cells. Protective effects of AITC on rifampin-induced cytotoxicity were observed, and transient transfection assays showed that AITC was able to effectively attenuate the agonist effects of rifampin and CITCO on human PXR and CAR activity, respectively. AITC-mediated reduction in the transcriptional activity of PXR and CAR correlated well with the suppression of CYP3A4 and CYP2B6 expression in HepG2 cells, which reflected the reduced catalytic activities of both of these genes following AITC treatment in differentiated HepaRG cells. Furthermore, AITC disrupts the co-regulations of PXR with several important co-regulators. Furthermore, the antagonist effect of AITC against PXR was found in HepaRG cells upon addition of acetaminophen (APAP) and amiodarone, indicating that AITC protects cells from drug-induced cytotoxicity. Taken together, our results show that AITC inhibits the transactivation effects of PXR and CAR and reduces the expression and function of CYP3A4 and CYP2B6. Additionally, AITC reversed the cytotoxic effects of APAP and amiodarone induced by PXR ligand. Results from this study suggest that AITC could be a powerful agent for reducing potentially dangerous interactions between transcriptional inducers of CYP enzymes and therapeutic drugs.
    Archive für Toxikologie 07/2014; 89(1). DOI:10.1007/s00204-014-1230-x · 5.98 Impact Factor
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    • "Our current results demonstrate that in HPH, four of the ten compounds of interest are potent inducers of CYP2B6, the prototypical target gene of hCAR, despite all of them exhibiting potent activation of hCAR1 + A in HepG2 cells. In agreement with this observation, all four compounds efficiently translocated Ad/EYFP-hCAR from cytoplasm to the nucleus of HPH, the initial and essential step of CAR activation (Kawamoto et al., 1999). Although it is out of the scope of this manuscript, we speculate that extensive metabolism in HPH may contribute to the lack of CYP2B6 induction by the other six compounds. "
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    ABSTRACT: Objective: Accumulating evidence suggests that activation of mouse constitutive androstane receptor (mCAR) alleviates type 2 diabetes and obesity by inhibiting hepatic gluconeogenesis, lipogenesis, and fatty acid synthesis. However, the role of human (h) CAR in energy metabolism is largely unknown. The present study aims to investigate the effects of selective hCAR activators on hepatic energy metabolism in human primary hepatocytes (HPH). Methods: Ligand-based structure-activity models were used for virtual screening of the Specs database ( followed by biological validation in cell-based luciferase assays. The effects of two novel hCAR activators (UM104 and UM145) on hepatic energy metabolism were evaluated in HPH. Results: Real-time PCR and Western blotting analyses reveal that activation of hCAR by UM104 and UM145 significantly repressed the expression of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, two pivotal gluconeogenic enzymes, while exerting negligible effects on the expression of genes associated with lipogenesis and fatty acid synthesis. Functional experiments show that UM104 and UM145 markedly inhibit hepatic synthesis of glucose but not triglycerides in HPH. In contrast, activation of mCAR by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, a selective mCAR activator, repressed the expression of genes associated with gluconeogenesis, lipogenesis, and fatty acid synthesis in mouse primary hepatocytes, which were consistent with previous observations in mouse model in vivo. Conclusion: Our findings uncover an important species difference between hCAR and mCAR in hepatic energy metabolism, where hCAR selectively inhibits gluconeogenesis without suppressing fatty acid synthesis. Implications: Such species selectivity should be considered when exploring CAR as a potential therapeutic target for metabolic disorders.
    Toxicology and Applied Pharmacology 05/2014; 279(1). DOI:10.1016/j.taap.2014.05.009 · 3.71 Impact Factor
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