Human PXR Variants and Their Differential Effects on the Regulation of Human Udp-Glucuronosyltransferase Gene Expression

Gardner-Stephen, Dione; Heydel, Jean‐Marie; Goyal, Amit; Lü, Yuan; Xie, Wen; Lindblom, Tim; Mackenzie, Peter I.; Radominska‐Pandya, Anna

doi:10.1124/dmd.32.3.340

Cited by 140 publications

(92 citation statements)

References 27 publications

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“…6B). Recently, Gardner-Stephen et al [19] using RT-PCR based approaches have shown three variants of PXR that are expressed in liver and intestinal mucosa. Hence, using PXR antiserum we also investigated the expression of PXR variants in a liver cell line (HepG2) and intestinal adenocarcinoma cell line (COLO) by Western blot analysis.…”

Section: Detection Of Pxr In Transfected Mammalian Cells In Culturementioning

confidence: 99%

Purification of full-length human Pregnane and Xenobiotic Receptor: polyclonal antibody preparation for immunological characterization

et al. 2005

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Pregnane and Xenobiotic Receptor (PXR; or Steroid and Xenobiotic Receptor, SXR), a new member of the nuclear receptor superfamily, is thought to modulate a network of genes that are involved in xenobiotic metabolism and elimination. To further explore the role of PXR in body's homeostatic mechanisms, we for the first time, report successful prokaryotic expression and purification of full-length PXR and preparation of polyclonal antibody against the whole protein. The full-length cDNA encoding a 434 amino acids protein was sub-cloned into prokaryotic expression vector, pET-30b and transformed into E. coli BL21(DE3) cells for efficient over expression. The inclusion body fraction, containing the expressed recombinant protein, was purified first by solubilizing in sarcosine extraction buffer and then by affinity column chromatography using Ni-NTA His-Bind matrix. The efficacy of anti-PXR antibody was confirmed by immunocytology, Western blot analysis, EMSA and immunohistochemistry. The antibody obtained was capable of detecting human and mouse PXR with high specificity and sensitivity. Immunofluorescence staining of COS-1 cells transfected with human or mouse PXR showed a clear nuclear localization. Results from immunohistochemistry showed that level of PXR in liver sections is immunologically detectable in the nuclei. Similar to exogenously transfected PXR, Western blot analysis of cell extract from HepG2 and COLO320DM cells revealed a major protein band for endogenous PXR having the expected molecular weight of 50 kDa. Relevance of other immunodetectable bands with reference to PXR isoforms and current testimony are evaluated. Advantages of antibody raised against full-length PXR protein for functional characterization of receptor is discussed and its application for clinical purposes is envisaged.

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Section: Detection Of Pxr In Transfected Mammalian Cells In Culturementioning

confidence: 99%

Purification of full-length human Pregnane and Xenobiotic Receptor: polyclonal antibody preparation for immunological characterization

et al. 2005

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“…However, due to the use of heterologous promoters, these models do not provide bona fide levels of expression of PXR or CAR in all tissues of the mouse. Furthermore, splice variants of hPXR and hCAR have recently been described, some of which have been shown to have functional activity (19)(20)(21)(22)(23). None of these splice variants are expressed by mouse models in which pure cDNAs are used.…”

Section: Introductionmentioning

confidence: 99%

A novel panel of mouse models to evaluate the role of human pregnane X receptor and constitutive androstane receptor in drug response

Scheer¹,

Ross²,

Rode³

et al. 2008

J. Clin. Invest.

133

114

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The pregnane X receptor (PXR) and the constitutive androstane receptor (CAR) are closely related orphan nuclear hormone receptors that play a critical role as xenobiotic sensors in mammals. Both receptors regulate the expression of genes involved in the biotransformation of chemicals in a ligand-dependent manner. As the ligand specificity of PXR and CAR have diverged between species, the prediction of in vivo PXR and CAR interactions with a drug are difficult to extrapolate from animals to humans. We report the development of what we believe are novel PXR-and CAR-humanized mice, generated using a knockin strategy, and Pxr-and Car-KO mice as well as a panel of mice including all possible combinations of these genetic alterations. The expression of human CAR and PXR was in the predicted tissues at physiological levels, and splice variants of both human receptors were expressed. The panel of mice will allow the dissection of the crosstalk between PXR and CAR in the response to different drugs. To demonstrate the utility of this panel of mice, we used the mice to show that the in vivo induction of Cyp3a11 and Cyp2b10 by phenobarbital was only mediated by CAR, although this compound is described as a PXR and CAR activator in vitro. This panel of mouse models is a useful tool to evaluate the roles of CAR and PXR in drug bioavailability, toxicity, and efficacy in humans.

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“…[13][14][15][16][17][18] However, studies of UGTs in human hepatocytes are limited to only a few investigations. Assessments of UGT induction by several human UGT substrates has demonstrated that the primary human hepatocyte model is highly suitable for the investigation of UGT regulation.…”

Section: Introductionmentioning

confidence: 99%

Human UGT1A8 and UGT1A10 mRNA Are Expressed In Primary Human Hepatocytes

Li¹,

Bratton²,

Radominska‐Pandya³

2007

Drug Metabolism and Pharmacokinetics

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SummaryIt is widely believed that the UGT1A isoforms, UGT1A8 and -1A10, are expressed exclusively in extrahepatic tissues. In this work, human primary hepatocytes from six donors were analyzed for UGT1A8 and -1A10 mRNA expression by semi-quantitative RT-PCR. New primers to amplify UGT1A8 mRNA were designed and found to differ from those previously published. We demonstrated that UGT1A8 and -1A10 mRNA are expressed in hepatocytes. Although basal UGT mRNA levels were detected in untreated hepatocytes, significant up-regulation of the levels of mRNA for these isoforms were seen after treatment with 3-methylcholanthrene (3-MC) and rifampicin (Rif). RT-PCR products for all UGTs were sequenced and unambiguously identified as matching the corresponding cDNA. The discovery of these isoforms in hepatocytes is a novel discovery and will stimulate studies on the potential role for these isoforms in hepatic detoxification.

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Human PXR Variants and Their Differential Effects on the Regulation of Human Udp-Glucuronosyltransferase Gene Expression

Cited by 140 publications

References 27 publications

Purification of full-length human Pregnane and Xenobiotic Receptor: polyclonal antibody preparation for immunological characterization

Purification of full-length human Pregnane and Xenobiotic Receptor: polyclonal antibody preparation for immunological characterization

A novel panel of mouse models to evaluate the role of human pregnane X receptor and constitutive androstane receptor in drug response

Human UGT1A8 and UGT1A10 mRNA Are Expressed In Primary Human Hepatocytes

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