Differences in Gene Regulation by Dual Ligands of Nuclear Receptors Constitutive Androstane Receptor (CAR) and Pregnane X Receptor (PXR) in HepG2 Cells Stably Expressing CAR/PXR

Kanno, Yuichiro; Tanuma, Nobuaki; Yazawa, Saki; Zhao, Shuai; Inaba, Miki; Nemoto, Kiyomitsu; Inouye, Yoshio

doi:10.1124/dmd.116.070888

Cited by 7 publications

(4 citation statements)

References 26 publications

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“…Cells were allowed to reach 60–70% confluency at the time of transfection. To assess CYP450 promoter activity following treatment with nuclear receptor inhibitors (KCZ, 10 mmol/L; RA, 10 mmol/L), nuclear receptor agonists (RIF, 10 mmol/L; CITCO, 10 mmol/L) or hypoxia, HepG2 cells were transfected with 1 μg of the pGL3-CYP450 or empty vector using Lipofectamine Plus, and the obtained cells were cultured in a 5% CO 2 incubator at 37°C for 4–6 h ( Chang et al., 2014 ; Kanno et al., 2016 ).…”

Section: Methodsmentioning

confidence: 99%

Regulation of High-Altitude Hypoxia on the Transcription of CYP450 and UGT1A1 Mediated by PXR and CAR

et al. 2020

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Little is known about what roles the pregnane X receptor (PXR) and constitutive androstane receptor (CAR) play in drug metabolism in high-altitude hypoxia. Likewise, the potential interaction of nuclear receptors and drug metabolism enzymes during drug metabolism of high-altitude hypoxia is not fully understood. In this work, we investigated the effects of high-altitude hypoxia on transcriptional regulation of cytochrome P450 (CYP450) and UDP-glucuronosyltransferase 1A1 (UGT1A1) genes mediated by PXR and CAR proteins. The protein and mRNA expressions of CYP450, UGT1A1, PXR, and CAR were determined by enzyme-linked immunosorbent assay and qPCR in rats and HepG2 cell lines under hypoxia. Hypoxia potently inhibited the CYP450 isoforms, UGT1A1, PXR, and CAR protein and mRNA expression. To clarify whether PXR and CAR regulate various genes involved in drug metabolism of high-altitude hypoxia, we investigated the expression of CYP1A2, CYP2C9, CYP2E1, CYP3A4, and UGT1A1 using a dualluciferase reporter assay after treatment with Ketoconazole (KCZ) and Retinoic acid (RA), or silenced PXR and CAR gene expression. In HepG2 cells, hypoxia, KCZ, and RA inhibited CYP450 isoforms and UGT1A1 expression. Activation of PXR and CAR in cells treated with 6-(4-chlorophenyl)-imidazo (2,1-b) thiazole-5-carbaldehyde (CITCO) and rifampicin (Rif) resulted in the enhancement of CYP450 isoforms, UGT1A1, PXR, and CAR. In contrast, this effect was not observed under hypoxia. Taken together, our results suggest that hypoxia inhibits CYP1A2, CYP2C9, CYP2E1, CYP3A4, and UGT1A1 expression via the PXR and CAR regulatory pathway.

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Section: Methodsmentioning

confidence: 99%

Regulation of High-Altitude Hypoxia on the Transcription of CYP450 and UGT1A1 Mediated by PXR and CAR

et al. 2020

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“…fCAR cells, which comprise stably N-terminal FLAG-tagged CAR-expressing HepG2 cells, were previously established (Kanno et al, 2018). The HepTR/CAR cell line, derived from human hepatoma HepG2 cells, was previously established using the T-REx System (Invitrogen/Thermo Fisher Scientific, Waltham, MA), in which the expression of hCAR can be induced by Tet-treatment (Kanno et al, 2012(Kanno et al, , 2016. HepG2, fCAR, and HepTR/hCAR cells were cultured in Dulbecco's modified Eagle's medium (Wako, Osaka, Japan) containing 10% fetal bovine serum and penicillin-streptomycin at 37°C in a humidified atmosphere containing 5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

Cell Cycle and Apoptosis Regulator 1, CCAR1, Regulates Enhancer-Dependent Nuclear Receptor CAR Transactivation

Kanno

Zhao

Yamashita

et al. 2019

Molecular Pharmacology

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The constitutive active/androstane receptor (CAR) controls genes involved in xenochemical metabolism. Although numerous cofactors have been reported to be involved in CAR-mediated transactivation, unknown and poorly defined proteins recruited by CAR have yet to be characterized. In this study, a novel CAR-interacting protein, cell cycle and apoptosis regulator 1 (CCAR1), was identified by coimmunoprecipitation analysis using human hepatocarcinoma HepG2 cells expressing FLAG epitope-tagged CAR. We demonstrated that CCAR1 can act as an enhancerdependent coactivator of CAR. First, we showed that overexpression of CCAR1 enhanced CAR-induced reporter gene activity with triplicate consensus direct repeat 4 motif (DR4-Luc), xenobiotic-responsive enhancer module (XREM)-enhancer of CYP3A4 (XREM-Luc), and phenobarbital-responsive enhancer module of UDP-glucuronosyltransferases 1A1 (UGT1A1) (gtPBREM)-enhancer of UGT1A1 (gtPBREM-Luc)-driven reporter plasmids but not PBREM-enhancer of CYP2B6 (PBREM-Luc)driven reporter activity. Furthermore, we showed that knockdown of CCAR1 suppressed CAR-induced UGT1A1 mRNA expression but did not affect CAR-induced CYP2B6 mRNA expression in HepTR/CAR and HepaRG cells. Moreover, CCAR1 could be recruited to the gtPBREM of the UGT1A1 enhancer by CAR but not to the PBREM of the CYP2B6 enhancer. Moreover, we showed that CCAR1 can act as a secondary coactivator by cooperating with the p160 family of steroid receptor coactivators (SRCs). These findings demonstrated CCAR1 to be a novel transcriptional cofactor for CAR and provided insight regarding the mechanism of CAR-mediated gene-selective transactivation. 120

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“…Despite multiple possible mechanisms for inducing P450 activities and expression, the major way is via activation of nuclear receptors, a superfamily of ligand-activated transcription factors. Among these receptors, pregnane X receptor (PXR) and the constitutive androstane receptor (CAR) have been identified as principal regulators with overlapping functions in the transcription of P450 genes involved in oxidative metabolism and elimination of xenobiotics/drugs (Moore et al, 2000;Sinz, 2013;Kanno et al, 2016;Buchman et al, 2018). It has been documented that both PXR and CAR are abundantly expressed in liver, and to a lower level in the kidney and intestine (Willson and Kliewer, 2002).…”

Section: Introductionmentioning

confidence: 99%

Activation of Pregnane X Receptor–Cytochrome P450s Axis: A Possible Reason for the Enhanced Accelerated Blood Clearance Phenomenon of PEGylated Liposomes In Vivo

Wang

et al. 2019

Drug Metab Dispos

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Recently, we reported that repeated injection of PEGylated liposomes (PEG-L) at certain intervals to the same rat lead to the disappearance of their long-circulation properties, referred to as the "accelerated blood clearance (ABC) phenomenon". Evidence from our recent studies suggested that cytochrome P450s (P450s) contribute to induction of the ABC phenomenon, a possibility that had been previously ignored. However, few details are known about the mechanism for induction of P450s. The present study was undertaken to investigate the roles in the ABC phenomenon of pregnane X receptor (PXR) and constitutive androstane receptor (CAR), the major upstream transcriptional regulators of the P450 genes, including CYP3A1, CYP2C6, and CYP1A2. The results demonstrated that expression of rat PXR and CAR was significantly increased in the ABC phenomenon and was accompanied by elevated CYP3A1, CYP2C6, and CYP1A2 levels. Further findings revealed that PXR but not CAR protein was substantially upregulated in the hepatocyte nucleus, together with marked nuclear colocalization of the PXR-retinoid X receptor alpha (RXRa) transcriptionally active heterodimer, indicating that nuclear translocation of PXR was induced in the ABC phenomenon, whereas nuclear translocation of CAR was not observed. Notably, pretreatment with the specific PXR inducer dexamethasone significantly induced accelerated systemic clearance of the subsequent injection of PEG-L, associating with increased nuclear colocalization of PXR-RXRa. These results revealed that the induction of P450s in the ABC phenomenon may be attributable largely to the activation of PXR induced by sequential injections of PEG-L, thus confirming the crucial involvement of the PXR-P450s axis in promoting the ABC phenomenon. SIGNIFICANCE STATEMENT The results of this study revealed that the induction of P450s in the ABC phenomenon may be largely attributable to the activation of PXR induced by sequential injections of PEG-L, thus confirming the crucial involvement of the PXR-P450s axis in promoting the ABC phenomenon. The data may help to extend our insights into 1) the role of P450s, which are regulated by the liver-enriched nuclear receptor PXR, in the ABC phenomenon, and 2) the therapeutic potential of targeting the PXR-P450 axis for reducing the magnitude of the ABC phenomenon in clinical practice.

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Differences in Gene Regulation by Dual Ligands of Nuclear Receptors Constitutive Androstane Receptor (CAR) and Pregnane X Receptor (PXR) in HepG2 Cells Stably Expressing CAR/PXR

Abstract: The constitutive androstane receptor (CAR) and pregnane X receptor

Cited by 7 publications

References 26 publications

Regulation of High-Altitude Hypoxia on the Transcription of CYP450 and UGT1A1 Mediated by PXR and CAR

Regulation of High-Altitude Hypoxia on the Transcription of CYP450 and UGT1A1 Mediated by PXR and CAR

Cell Cycle and Apoptosis Regulator 1, CCAR1, Regulates Enhancer-Dependent Nuclear Receptor CAR Transactivation

Activation of Pregnane X Receptor–Cytochrome P450s Axis: A Possible Reason for the Enhanced Accelerated Blood Clearance Phenomenon of PEGylated Liposomes In Vivo

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