Differences in Transactivation between Rat CYP3A1 and Human CYP3A4 Genes by Human Pregnane X Receptor

Takada, Tomonari; Ogino, Makoto; Miyata, Masaaki; Shimada, M.; Nagata, Kiyoshi; Yamazoe, Yasushi

doi:10.2133/dmpk.19.103

Cited by 16 publications

(17 citation statements)

References 28 publications

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“…‫,ءء‬ P Ͻ 0.01, significantly different from the corresponding vehicle-treated mice based on one-way ANOVA followed by Tukey's post hoc test. vious results, however, have raised a possibility that an unidentified cis-element(s), which probably exists in the distal promoter sequence, is involved in the PXR-mediated transcriptional activation of the CYP3A4 gene (Takada et al, 2004). In the present study, we have indicated the localization of a novel rifampicin-response region in the distal promoter of the CYP3A4 gene and identified a distinct PXR response element, termed eNR3A4, to which hPXR binds as a heterodimer with hRXR␣.…”

Section: Discussionmentioning

confidence: 56%

“…The luciferase reporter plasmid, pGL3-Basic, was purchased from Promega (Madison, WI). Preparation of the chimeric CYP3A4 luciferase reporter gene constructs, including , and pCYP3A4-362, was described previously (Takada et al, 2004).…”

Section: Methodsmentioning

confidence: 99%

“…In the report, the simultaneous mutation of both elements, however, did not completely abolish the rifampicin-mediated reporter gene activation. In addition, our previous study has shown that these two PXR response elements are not enough for the maximal rifampicininduced transcriptional activation of the CYP3A4 gene (Takada et al, 2004). These data have prompted us to investigate the possibility that an unidentified cis-element(s) exists in the CYP3A4 promoter, especially for the rifampicininduced transactivation of the CYP3A4 gene.…”

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confidence: 99%

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Unveiling a New Essential Cis Element for the Transactivation of theCYP3A4Gene by Xenobiotics

Toriyabe¹,

Nagata²,

Takada³

et al. 2008

Mol Pharmacol

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Pregnane X receptor (PXR) has been shown to form a heterodimer with retinoid X receptor ␣ (RXR␣) and to bind to the distal nuclear receptor-binding element 1 and an everted repeat separated by six nucleotides in the proximal promoter of the CYP3A4 gene. In the present study, a new rifampicin-responsive region, located at Ϫ7.6 kilobases upstream from the transcription initiation site, has been identified using reporter assays in HepG2 cells. This region contains a cluster of possible nuclear receptor-binding half-sites, AG(G/T)TCA-like sequence. Of these putative half-sites, we focused six half-sites and termed them ␣-half-sites. Introduction of a mutation into either an ␣ or ␤ half-site of CYP3A4 reporter genes almost completely diminished the rifampicin-induced transcription. In electrophoretic mobility shift assays, PXR/RXR␣ heterodimer bound to the direct repeat separated by four nucleotides (DR4) formed with ␣ and ␤ half-sites. HepG2-based transactivation assays with the reporter gene constructs with or without mutations in the PXR binding element(s) demonstrated that this DR4 motif is essential for the transcriptional activation not only by rifampicin but also by various human PXR activators. In addition, reporter assays performed in human hepatocytes and mice with adenoviruses expressing luciferase derived from various CYP3A4 reporter genes and that expressing human PXR supported the results of experiments in HepG2 cells. These results suggest the obligatory role of the newly identified direct repeat separated by four nucleotides-type PXR binding element of the CYP3A4 gene for xenobiotic induction of CYP3A4.

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Section: Discussionmentioning

confidence: 56%

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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Unveiling a New Essential Cis Element for the Transactivation of theCYP3A4Gene by Xenobiotics

Toriyabe¹,

Nagata²,

Takada³

et al. 2008

Mol Pharmacol

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“…Expression vectors for human CAR (pCR3/hCAR) and human RXR␣ (pGEM-3Z/ hRXR␣) were kindly provided by Dr. Masahiko Negishi (National Institutes of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC). The human PXR expression vector (pCMV4/hPXR) was previously constructed (Takada et al, 2004). Human PXR cDNA digested from pCMV4/hPXR was subcloned into the pTNT vector (Promega, Madison, WI) for in vitro transcription/ translation.…”

Section: Methodsmentioning

confidence: 99%

Induction of Human CYP2A6 Is Mediated by the Pregnane X Receptor with Peroxisome Proliferator-Activated Receptor-γ Coactivator 1α

Itoh¹,

Nakajima²,

Higashi³

et al. 2006

J Pharmacol Exp Ther

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CYP2A6 plays important roles in the metabolism of nicotine and some clinically used drugs. Interindividual variability in the CYP2A6 expression level in human liver might be caused by an inducible property, but the molecular mechanism of induction is unclear. Rifampicin, phenobarbital, -(3,4-dichlorobenzyl)oxime, which are activators of pregnane X receptor (PXR) and constitutive androstane receptor (CAR), induced CYP2A6 mRNA in human hepatocytes. We identified three direct repeat separated by four nucleotides (DR4)-like elements at Ϫ6698, Ϫ5476, and Ϫ4618 in the CYP2A6 gene, to which PXR and CAR could bind after dimerization with retinoid X receptor (RXR)-␣. In luciferase assays, overexpression of PXR or CAR could not activate the transcriptional activity of CYP2A6 promoter constructs (Ϫ6754 to Ϫ1) in HepG2 cells. Cotransfection of hepatocyte nuclear factor-4␣ did not affect the transcriptional activities in the absence or presence of PXR or CAR. Interestingly, cotransfection of peroxisome proliferator-activated receptor-␥ coactivator 1␣ (PGC-1␣) as well as PXR significantly enhanced the transcriptional activity (3.9-fold of control). By the deletion of a possible suppresser region (Ϫ4533 to Ϫ185), the effects of PXR/PGC-1␣ on the transcriptional activity were increased (6.9-fold of control). Deletion or mutation analyses revealed that two DR4-like elements at Ϫ5476 and Ϫ4618 are essential for transactivation by PXR/PGC-1␣. Chromatin immunoprecipitation assay revealed that PXR and PGC-1␣ bind to CYP2A6 chromatin. In conclusion, we found that CYP2A6 is induced via PXR and PGC-1␣ through the DR4-like element at the distal response region. This is the first study to report the molecular mechanism of the induction of CYP2A6.

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“…Mutated reporter plasmids were constructed by the QuikChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA) using pGL3-MRP3-Acc65 as a template. Preparation of the human pregnane X receptor (hPXR) expression plasmid was previously described (Takada et al, 2004). All plasmids were sequenced to confirm their integrity.…”

Section: Methodsmentioning

confidence: 99%

Activation of p38 Mitogen-Activated Protein Kinase by Clotrimazole Induces Multidrug Resistance-Associated Protein 3 Activation through a Novel Transcriptional Element

Sasaki

Inami

Numata

et al. 2016

Journal of Pharmacology and Experimental Therapeutics

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Multidrug resistance-associated protein 3 (MRP3) is a basolaterally localized transporter in the liver and contributes to the transport of various metabolites such as conjugates of endogenous compounds and drugs from hepatocytes. MRP3 expression in the human liver is low under normal physiologic conditions but is induced by drug treatment. Although several studies have identified a region necessary for the basal transcription of MRP3, no region that responds to drugs has been reported. To identify the xenobiotic-responsive elements of MRP3, we constructed a luciferase reporter plasmid containing the MRP3 59-flanking region up to 210 kb upstream from the transcription start site. Among typical nuclear receptor ligands, clotrimazole dramatically enhanced MRP3 reporter activity in HepG2 cells, whereas rifampicin had no effect. We then conducted MRP3 reporter assays with deletion or mutation constructs to identify a clotrimazole-responsive element. The element was located approximately 26.8 kb upstream from the MRP3 transcription start site. Overexpression of the pregnane X receptor did not enhance clotrimazole-mediated transcription. We found that clotrimazole was toxic to HepG2 cells and we therefore investigated whether mitogen-activated protein kinase (MAPK) activation is involved in the transactivation of MRP3 by clotrimazole. p38 MAPK inhibitor SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole] suppressed MRP3 mRNA expression induced by clotrimazole, whereas c-Jun N-terminal kinase inhibitor SP600125 (1,9-pyrazoloanthrone) and extracellular signal-regulated kinase inhibitor PD98059 [2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one] did not. Phosphorylated p38 MAPK was detected in HepG2 cells treated with clotrimazole. These results suggest that activation of the p38 MAPK pathway induces the transcriptional activation of MRP3.

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Differences in Transactivation between Rat CYP3A1 and Human CYP3A4 Genes by Human Pregnane X Receptor

Cited by 16 publications

References 28 publications

Unveiling a New Essential Cis Element for the Transactivation of theCYP3A4Gene by Xenobiotics

Unveiling a New Essential Cis Element for the Transactivation of theCYP3A4Gene by Xenobiotics

Induction of Human CYP2A6 Is Mediated by the Pregnane X Receptor with Peroxisome Proliferator-Activated Receptor-γ Coactivator 1α

Activation of p38 Mitogen-Activated Protein Kinase by Clotrimazole Induces Multidrug Resistance-Associated Protein 3 Activation through a Novel Transcriptional Element

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