Development of a novel recombinant cell line for detection and characterization of Ah receptor nuclear translocation in intact cells. (August 2020)

• Record Type: Journal Article
• Authors: Hayashi, Ai; Denison, Michael S.
• Is Part Of: Toxicology in vitro. Volume 66(2020)
• Publication Date: 2020-08
• Subjects: Aryl hydrocarbon receptor -- AhR -- YFP -- Nuclear translocation 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin -- TCDD; AhR Ah receptor -- Arnt Ah receptor nuclear translocator -- AhRy AhR with a C-terminal YFP tag -- bHLH-PAS Basic-helix-loop-helix-Per-Arnt-Sim -- BNF Beta-Naphthoflavone -- DBA Dibenz(a, h)Anthracene -- DMSO Dimethylsulfoxide -- DRAQ5 Deep Red Anthroquinone 5 -- DRE Dioxin responsive element -- G418 Geneticin -- HAHs Halogenated aromatic hydrocarbons -- LMB Leptomycin B -- 3MC 3-Methylcholanthrene -- NES Nuclear export sequence -- NLS Nuclear localization sequence -- PAHs Polycyclic aromatic hydrocarbons -- TAO TAOc1BPrc1 cells -- TCDD 2, 3, 7, 8-Tetrachloridibenzo-p-dioxin -- Wt Wild-type -- wtAhR Wild-type AhR -- yAhR AhR with a N-terminal YFP tag -- YFP Yellow fluorescent protein; Toxicity testing -- In vitro -- Periodicals; Toxicology -- Periodicals; 615.9
• DOI: 10.1016/j.tiv.2020.104873
• Languages: English
• ISSNs: 0887-2333

Abstract

Abstract: The Ah receptor (AhR) is a ligand-dependent transcriptional factor that mediates the effects of structurally diverse chemicals. Ligand binding stimulates nuclear translocation of the AhR and leads to AhR DNA binding and increased gene expression. Studies of the molecular mechanisms by which ligands bind to and activate the AhR and AhR-dependent signal transduction require methods to easily examine each step of the AhR signaling pathway. While current assays can measure ligand and DNA binding in vitro and gene expression in cells, there is no simple method to monitor AhR nuclear translocation. We developed a stably transfected mouse hepatoma cell line (yAHAYc6) that expresses yellow fluorescent protein-tagged AhR (yAhR) for use in qualitative or semiquantitative assessment of nuclear/cytoplasmic distribution of yAhR in living cells by fluorescent microscopy. yAhR nuclear translocation was stimulated in a concentration- and time-dependent manner by AhR agonists and inhibited by antagonists. Inhibition of nuclear export channels by leptomycin B, resulted in increased nuclear accumulation of yAhR in the absence of added ligand, indicating endogenous nucleocytoplasmic shuttling of unliganded AhR and demonstrating the utility of these cells. This novel cell line can be used to detect and characterize AhR ligands and will facilitate mechanistic studies of AhR signaling. Highlights: A simple and rapid method is needed to monitor AhR nuclear translocation. Living cells expressing a fluorescently-tagged AhR reveal AhR nuclear translocation. AhR nuclear translocation was stimulated by agonists and inhibited by antagonists. Leptomycin B treatment revealed nucleocytoplasmic shuttling of unliganded AhR.