Article

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

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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    • "Consistent with its initial designation, CAR is constitutively activated in immortalized cell lines and spontaneously accumulated in the nucleus without xenobiotic stimulation [23] [24]. In contrast, CAR is sequestered in the cytoplasm of the more physiologically relevant primary hepatocytes before activation [25]. In primary hepatocytes, CAR forms a cytoplasmic multiprotein complex with a number of chaperone proteins and only translocates to the nucleus upon chemical-stimulated activation [26]. "
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    ABSTRACT: The constitutive androstane receptor (CAR) modulates the transcription of numerous genes involving drug metabolism, energy homeostasis, and cell proliferation. Most functions of CAR however were defined from animal studies. Given the known species difference of CAR and the significant cross-talk between CAR and the pregnane X receptor (PXR), it is extremely difficult to decipher the exact role of human CAR (hCAR) in gene regulation, relying predominantly on pharmacological manipulations. Here, utilizing a newly generated hCAR-knockout (KO) HepaRG cell line, we carried out RNA-seq analysis of the global transcriptomes in wild-type (WT) and hCAR-KO HepaRG cells treated with CITCO, a selective hCAR agonist, phenobarbital (PB), a dual activator of hCAR and hPXR, or vehicle control. Real-time PCR assays in separate experiments were used to validate RNA-seq findings. Our results indicate that genes encoding drug-metabolizing enzymes are among the main clusters altered by both CITCO and PB. Specifically, CITCO significantly changed the expression of 135 genes in an hCAR-dependent manner, while PB altered the expression of 227 genes in WT cells of which 94 were simultaneously modulated in both cell lines reflecting dual effects of PB on hCAR/PXR. Notably, we found that many genes promoting cell proliferation and tumorigenesis were up-regulated in hCAR-KO cells, suggesting that hCAR may play an important role in cell growth that differs from mouse CAR. Together, our results reveal both novel and known targets of hCAR and support the role of hCAR in maintaining the homeostasis of metabolism and cell proliferation in the liver. Copyright © 2015. Published by Elsevier Inc.
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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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    • "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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