title: Functional characterization of a full length pregnane X receptor, expression in vivo, and identification of PXR alleles, in zebrafish (Danio rerio).
abstract: The pregnane X receptor (PXR) (nuclear receptor NR1I2) is a ligand activated transcription factor, mediating responses to diverse xenobiotic and endogenous chemicals. The properties of PXR in fish are not fully understood. Here we report on cloning and characterization of full-length PXR of zebrafish, Danio rerio, and pxr expression in vivo. Initial efforts gave a cDNA encoding a 430 amino acid protein identified as zebrafish pxr by phylogenetic and synteny analysis. The sequence of the cloned Pxr DNA binding domain (DBD) was highly conserved, with 74% identity to human PXR-DBD, while the ligand-binding domain (LBD) of the cloned sequence was only 44% identical to human PXR-LBD. Sequence variation among clones in the initial effort prompted sequencing of multiple clones from a single fish. There were two prominent variants, one sequence with S183, Y218 and H383 and the other with I183, C218 and N383, which we designate as alleles pxr*1 (nr1i2*1) and pxr*2 (nr1i2*2), respectively. In COS-7 cells co-transfected with a PXR-responsive reporter gene, the full-length Pxr*1 (the more common variant) was activated by known PXR agonists clotrimazole and pregnenolone 16α-carbonitrile but to a lesser extent than the full-length human PXR. Activation of full-length Pxr*1 was only 10% of that with the Pxr*1 LBD. Quantitative real time PCR analysis showed prominent expression of pxr in liver and eye, as well as brain and intestine of adult zebrafish. The pxr was expressed in heart and kidney at levels similar to that in intestine. The expression of pxr in liver was weakly induced by ligands for mammalian PXR or constitutive androstane receptor (NR1I3). The results establish a foundation for PXR studies in this vertebrate model. PXR allelic variation and the differences between the full-length PXR and the LBD in reporter assays have implications for assessing the action of PXR ligands in zebrafish.
authors.affiliation: Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA; Departamento de Bioquímica, CCB, Universidade Federal de Santa Catarina, Florianópolis, SC 88040-900, Brazil.
authors.forename: Afonso C D
authors.initials: AC
authors.lastname: Bainy
authors.forename: Akira
authors.initials: A
authors.lastname: Kubota
authors.forename: Jared V
authors.initials: JV
authors.lastname: Goldstone
authors.forename: Roger
authors.initials: R
authors.lastname: Lille-Langøy
authors.forename: Sibel I
authors.initials: SI
authors.lastname: Karchner
authors.forename: Malin C
authors.initials: MC
authors.lastname: Celander
authors.forename: Mark E
authors.initials: ME
authors.lastname: Hahn
authors.forename: Anders
authors.initials: A
authors.lastname: Goksøyr
authors.forename: John J
authors.initials: JJ
authors.lastname: Stegeman
mesh_terms: D000483:Alleles; D000818:Animals; D001921:Brain / Q000378:metabolism; D000090702:Constitutive Androstane Receptor; D005123:Eye / Q000378:metabolism; D005260:Female; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects; D023061:Gene Order; D008099:Liver / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D010634:Phenobarbital / Q000494:pharmacology; D010802:Phylogeny; D000077297:Pregnane X Receptor; D011485:Protein Binding; D011725:Pyridines / Q000494:pharmacology; D011987:Receptors, Steroid / Q000235:genetics* / Q000378:metabolism*; D014874:Water Pollutants, Chemical / Q000494:pharmacology; D015027:Zebrafish / Q000145:classification / Q000235:genetics* / Q000378:metabolism
chemical_list: D000090702:Constitutive Androstane Receptor; C000625852:NR1I2 protein, human; C000716327:NR1I3 protein, human; D000077297:Pregnane X Receptor; D011725:Pyridines; D011987:Receptors, Steroid; D014874:Water Pollutants, Chemical; C028474:1,4-bis(2-(3,5-dichloropyridyloxy))benzene; D010634:Phenobarbital
keywords: 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene; ABC transporters; ARNT2; ATP-binding cassette transporters; CAR; CLO; CYP; Cytochrome P450; DBD; DMSO; DNA binding domain; EF1α; LBD; NIF; NR; PB; PCN; PXR; Pregnane X receptor; RACE; RIF; RXR; SNPs; SXR; Steroid xenobiotic receptor; TCPOBOP; UTR; VDR; Zebrafish; aryl hydrocarbon receptor nuclear translocator; clotrimazole; constitutive androstane receptor; cytochrome P450; dimethylsulfoxide; elongation factor 1α; ligand binding domain; nifedipine; nuclear receptor; phenobarbital; pregnane X receptor; pregnenolone 16α-carbonitrile; rapid amplification of cDNA ends; retinoid X-receptor; rifampicin; single-nucleotide polymorphisms; steroid xenobiotic receptor; untranslated region; vitamin D receptor