In the case of PXR, only one AF-2 is present in the LBD domain.
The total zfPXR coding sequence (M1-T430) was isolated from the ZFL cells extracts by RT-PCR with primers containing XhoI and KpnI restriction enzyme sites (). The full-length sequence of the zfPXR was then integrated in the pSG5-puromycin plasmid between XhoI and KpnI sites. Primers were then designed to amplify the ligand binding domain (LBD) of the zfPXR (M111-T430). The LBD sequence of the zfPXR was then integrated in the pSG5-Gal4(DNA-binding domain (DBD))-puromycin plasmid between XhoI and SacI enzyme restriction sites.
The schematic of PXR nuclear receptor activation is shown in .
P450 gene induction by structurally diverse xenochemicals: central role of nuclear receptors CAR, PXR, and PPAR.
The PGL4.24-6xPXRE reporter plasmid () is a kind gift from Anke Lange and Charles Tyler. The PXRE6-TATA-luciferase-hygromycin was constructed by adding the hygromycin resistance in this plasmid.
Xenobiotics bind to the LBD of the PXR after they enter the cell.
INDIGO社の核内受容体ルシフェラーゼレポーターアッセイでは、核内受容体の種類により、１） 全長の核内受容体を導入したアッセイ系と、２） 酵母の転写因子Gal4を用いたハイブリッド型核内受容体を導入したアッセイ系の2種類を構築しています。
The cell lines used in this study are listed in . The HG5LN, HG5LN Gal4-hPXR and HG5LN Gal4-zfPXR cell lines were previously described (). Briefly, HeLa cells stably transfected with the GAL4RE5-βGlobin-Luc-SVNeo plasmid (HG5LN cell line) (). HG5LN cells were stably transfected with the pSG5-GAL4(DBD)-hPXR(LBD)-puro or pSG5-GAL4(DBD)-zfPXR(LBD)-puro plasmids. HG5LN Gal4-hPXR and Gal4-zfPXR cell lines were selected for their inducibility in presence of SR12813 3 μM and clotrimazole 1 μM, respectively.
Signaling cascades play a role in regulating PXR activity.
The ZFL zfPXR cells were obtained by stable cotransfection of ZFL cells with the pSG5-zfPXR-puromycin and PXRE6-TATA-luciferase-hygromycin plasmids and selected for their inducibility in presence of clotrimazole 1 μM.
HG5LN cells were cultured in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (D-MEM/F-12) containing phenol red and 1 g/L glucose and supplemented with 5% fetal bovine serum, 100 units/mL of penicillin and 100 µg/mL of streptomycin supplemented with 1 mg/mL geneticin in a 5% CO2 humidified atmosphere at 37°C. HG5LN GAL4-hPXR and GAL4-zfPXR were cultured in the same culture medium supplemented with 0.5 µg/mL puromycin.
PXR-Mediated Regulation of Other Target Genes
ZFL zfPXR cells were cultured in Leibovitz’s L-15, D-MEM, Ham’s F-12 medium (LDF medium) containing 50% L-15, 35% D-MEM, and 15% F-12, 5% FCS with 0.15 g/L sodium bicarbonate, 15 mM HEPES buffer, 0.01 mg/mL insulin, 50 ng/mL EGF, 100 units/mL of penicillin, 100 µg/mL of streptomycin supplemented with 0.5 µg/mL puromycin and 0.25 mg/mL hygromycin B in a humidified atmosphere at 28°C.
The ability of chemicals to modulate the hPXR and zfPXR was investigated in HG5LN, HG5LN-hPXR, HG5LN-zfPXR, and ZFL-zfPXR cell lines after exposure to serial dilutions of the compounds and measurement of luciferase activity. Exposure of the HG5LN cells which do not express PXR allows to check the specificity of the PXR transactivation in HG5LN-hPXR and HG5LN-zfPXR cell lines. Briefly, cells were seeded in 96-well white opaque flat bottom microplates at 0.5 105 cells per well in culture medium. After 24h of growing at 37°C (HG5LN, HG5LN-hPXR and HG5LN-zfPXR cell lines) or 28°C (ZFL-zfPXR cell line), the culture medium was removed and replaced by DMEM-F12 medium without phenol red (Gibco 21041-025) supplemented with 100 units/mL of penicillin, 100 µg/mL of streptomycin and dextran-coated charcoal-treated fetal calf serum DCC-FCS (5%) (test medium) for HG5LN-hPXR and HG5LN-zfPXR cell lines, and LDF medium supplemented with 100 units/mL of penicillin, 100 µg/mL of streptomycin and dextran-coated charcoal-treated fetal calf serum DCC-FCS (5%) for the ZFL-zfPXR cell line. Cells were then exposed to a concentration range of the compounds by using an automated workstation (Biomek 3000, Beckman Coulter). The final concentration of DMSO in the well never exceeds 0.1% (v/v). After 16h of exposure, medium was removed and 50 µL of test medium containing 0.3 mM D-luciferin were added per well. After 5 min, the production of light was assessed in living cells using microplate reader (MicroBeta, PerkinElmer SAS, Courtabœuf, France).
Brief Overview of Physiological Functions of PXR
Transcriptional analysis of the orphan nuclear receptor constitutive androstane receptor (NR1I3) gene promoter: identification of a distal glucocorticoid response element.
pesticides, steroids, reporter cell lines, human PXR, zebrafish PXR
The aim of this study is to better characterize the cross-species differences between human and zebrafish in the modulation and the function of the PXR. To this end, we first screened a set of chemically and structurally various compounds (steroids and pesticides) using human stable reporter gene cell lines based on chimeric (Gal4-PXR) receptors. We then confirmed the zfPXR potency in a zebrafish reporter cell line. Finally, we investigated the structural basis of the differences in PXR modulation between human and fish applying homology docking models.
PXR has also been considered in tumor progression [].
(1) passive diffusion of a ligand into cells; (2) binding to PXR that forms a cytoplasmic complex with chaperones (Hsp90), CCRP, and SMRT.
Mathematical Models of PXR Activation and PXR-Induced Gene Expression
In this study, we evaluated the ability of PXR prototypical ligands, 21 steroids and 80 pesticides (), to modulate both zfPXR and hPXR. The chemicals were first tested for non-specific modulation of luciferase expression on the HG5LN parental cell line, which contains the same reporter gene as HG5LN-GAL4-PXRs cells, but lacks Gal4-PXRs (data not shown). Then the chemicals were tested on the HG5LN GAL4-hPXR and GAL4-zfPXR at concentrations that were not able to inhibit or activate luciferase expression in the HG5LN reporter cell line.
Models of PXR-Mediated Regulation of CYP3A Enzymes
The binding to a ligand promotes release of these proteins from PXR; (3) cytoplasm-nuclear translocation; (4) binding of coactivators and other nuclear receptors (such as RXRα) to form a transcriptional complex; (5) binding of the complex to PXR response elements in target genes’ promoter regions; (6) recruitment of transcription machinery factors and mRNA synthesis; (7) export of mRNA into cytosol and translation.
We do not have models that consider additional PXR ligands.
HNF4α, hepatocyte nuclear factor 4α, a nuclear receptor that coactivates human PXR (hPXR); PGC1α, Peroxisome proliferator-activated receptor gamma coactivator 1-alpha, a coactivator of PXR; PolII, RNA polymerase II; TBP, TATA-binding protein; TFII, transcription factor II D. "> ]. ">
Xenosensors CAR and PXR at work: Impact on statin metabolism.
The activity of the chemicals on hPXR was compared to the activity of the reference PXR agonist SR12813. This compound fully activated hPXR with an EC50 of 0.16 µM while it was not active in the HG5LN Gal-zfPXR cells ( and ). Rifampicin, another well-characterized hPXR full agonist, was also found exclusively active on hPXR with an EC50 of 0.44 µM (). For zfPXR, the activity of the chemicals was compared to the activity of the reference ligand clotrimazole. This compound activated both the HG5LN Gal-zfPXR and Gal-hPXR cell lines with the half-maximal effective concentration (EC50) values of 0.03 µM and 1.0 μM respectively ( and ). SPA70 () and econazole nitrate were used as reference antagonists for hPXR and zfPXR, respectively. SPA70 inhibited only hPXR, with an IC50 of 0.41 µM ( and ). Econazole nitrate inhibited luciferase activity in HG5LN Gal-zfPXR whereas it activated it in HG5LN Gal-hPXR cell lines. The EC50 for hPXR was 12 μM and the IC50 for zfPXR was 2.8 μM ( and ). Finally, pregnenolone 16α-carbonitrile, a mouse PXR agonist, partially activated hPXR whereas it did not activate zfPXR. Dexamethasone, another mouse PXR agonist, had no effect on hPXR and zfPXR.
2.1 A crystal structure of human PXR in complex with the St.
One of the major target genes regulated by PXR is the cytochrome P450 enzyme (CYP3A4), which is the most important human drug-metabolizing enzyme.
Biology of PXR: Role in drug-hormone interactions.
In addition, PXR is supposed to be involved both in basal and/or inducible expression of many other CYPs, such as CYP2B6, CYP2C8, 2C9 and 2C19, CYP3A5, CYP3A7, and CYP2A6.
Induction of PXR-mediated metabolism by beta-carotene.
Reduction in cytochrome P-450 enzyme expression is associated with repression of CAR (constitutive androstane receptor) and PXR (pregnane X receptor) in mouse liver during the acute phase response.