title: Nuclear translocation of adenoviral-enhanced yellow fluorescent protein-tagged-human constitutive androstane receptor (hCAR): a novel tool for screening hCAR activators in human primary hepatocytes.
abstract: The constitutive androstane receptor [(CAR) NR1I3] is a hepatic transcription factor that controls the expression of numerous drug-metabolizing enzymes and transporters in response to xenobiotic exposures. In primary hepatocytes and intact liver, CAR resides in the cytoplasm under basal condition and translocates to the nucleus upon exposure to inducers. However, CAR spontaneously accumulates in the nucleus of immortalized cell lines and exhibits constitutive activation in the absence of activators, which makes the identification of CAR activators extremely challenging. Here, we have established an efficient screening method for determining the nuclear translocation of human (h) CAR in human primary hepatocytes (HPHs). Our results demonstrated that adenoviral-enhanced yellow fluorescent protein-tagged hCAR (Ad/EYFP-hCAR) infects HPHs with high efficiency, and the majority of Ad/EYFP-hCAR (>80%) is expressed in the cytoplasm of noninduced HPHs and is translocated to the nucleus in response to activators and antagonists of hCAR. Furthermore, 22 compounds including known hCAR activators, nonactivators, CYP2B inducers, and deactivators were evaluated in this system. Our results indicated that chemical-mediated Ad/EYFP-hCAR translocation in HPHs significantly correlated with hCAR activation and target gene induction. Compared with cell-based reporter assays in cell lines and in vitro ligand-binding assays, the established Ad/EYFP-hCAR translocation assay in HPHs exhibits apparent advantages such as sensitivity to chemical activators and responses to both direct and indirect hCAR activators. Thus, nuclear translocation of Ad/EYFP-hCAR in HPHs represents an efficient means for in vitro prediction of chemical-mediated hCAR nuclear accumulation.
authors.affiliation: Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, 20 Penn St., Baltimore, MD 21201, USA.
authors.forename: Haishan
authors.initials: H
authors.lastname: Li
authors.forename: Tao
authors.initials: T
authors.lastname: Chen
authors.forename: John
authors.initials: J
authors.lastname: Cottrell
authors.forename: Hongbing
authors.initials: H
authors.lastname: Wang
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases / Q000096:biosynthesis; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D000090702:Constitutive Androstane Receptor; D065702:Cytochrome P-450 CYP2B6; D003600:Cytosol / Q000378:metabolism; D004790:Enzyme Induction / Q000187:drug effects; D017930:Genes, Reporter; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D018613:Microscopy, Confocal; D010089:Oxidoreductases, N-Demethylating / Q000096:biosynthesis; D010957:Plasmids / Q000235:genetics; D021381:Protein Transport; D018160:Receptors, Cytoplasmic and Nuclear / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D000090702:Constitutive Androstane Receptor; D008024:Ligands; C000716327:NR1I3 protein, human; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D010089:Oxidoreductases, N-Demethylating