Phenobarbital-Responsive Nuclear Translocation of the Receptor CAR in Induction of the CYP2B Gene

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. DOI: 10.1128/MCB.19.9.6318
Source: PubMed


The constitutively active receptor (CAR) transactivates a distal enhancer called the phenobarbital (PB)-responsive enhancer module (PBREM) found in PB-inducible
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
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.


Available from: Igor N Zelko
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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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    • "The constitutive androstane receptor (CAR, nuclear receptor subfamily 1, group I, member 3 [NR1I3]) and pregnane X receptor (PXR, NR1I2) are predominantly expressed in the liver and gut, and have been identified as xenobiotic-sensing transcription factors that regulate the gene expression of phase I, II, and III metabolic enzymes and transporters that are involved in the metabolism and elimination of endogenous and exogenous substances, such as bilirubin, steroid hormones, and xenobiotics (Timsit and Negishi, 2007). CAR localizes to the cytoplasm of normal mouse hepatocytes without stimuli such as drug treatments, translocates to the nucleus in response to CAR activators including phenobarbital (Kawamoto et al., 1999), subsequently forms a heterodimer with the retinoid X receptor (RXR), and transactivates its target genes (Honkakoski et al., 1998). The threonine-38 of human CAR plays a central role in the initial step of CAR activation; the phosphorylation of threonine-38 by protein kinase C retains CAR in the cytoplasm, and its dephosphorylation by protein phosphatase (PP) 2A causes CAR to translocate to the nucleus (Yoshinari et al., 2003; Mutoh et al., 2009). "
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    ABSTRACT: The human pregnane X receptor (hPXR) is recognized as a xenobiotic-sensing nuclear receptor that transcriptionally regulates the gene expression of drug-metabolizing enzymes and transporters. The present study elucidated the mechanism by which the localization of hPXR was regulated through threonine-290. A phosphomimetic mutation at threonine-290 (T290D) retained hPXR in the cytoplasm of HepG2, HuH6, and SW480 cells in vitro and the mouse liver in vivo even after the treatment with rifampicin, while a phosphodeficient mutation (T290A) translocated from the cytoplasm to the nucleus as the wild-type hPXR. The amount of the unphosphorylated wild-type YFP-hPXR, but not the T290A mutant increased on Phos-tag gels in response to stimulations with rifampicin and cyclin-dependent kinase 2 inhibitor roscovitine, and a marked increase was observed in the unphosphorylated levels of the T290A mutant in non-treated cells. The Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93 and transfection with anti-CaMKII small interfering RNA (siRNA) enhanced the unphosphorylated levels of the wild-type protein. CaMKII directly phosphorylated the threonine-290 of hPXR, while the T290A mutant conferred resistance to CaMKII. The protein phosphatase (PP) inhibitor okadaic acid (100 nM) and transfection with anti-PP1 siRNA, but not anti-PP2A siRNA led to the reduced expression of CYP3A4 mRNA. Following the rifampicin and roscovitine stimulations, PP1 was recruited to the wild-type hPXR, but not the T290A mutant. These results suggest that phosphorylation at threonine-290 by CaMKII may impair the function of hPXR by repressing its translocation to the nucleus, and dephosphorylation by PP1 is necessary for the xenobiotic-dependent nuclear translocation of hPXR.
    Drug metabolism and disposition: the biological fate of chemicals 07/2014; 42(10). DOI:10.1124/dmd.114.059139 · 3.25 Impact Factor
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