Ligand-dependent interactions of the Ah receptor with coactivators in a mammalian two-hybrid assay.
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a high affinity ligand for the aryl hydrocarbon receptor (AhR). In this study, we investigated structure-dependent differences in activation of the AhR by a series of halogenated aromatic hydrocarbons. TCDD, 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), and 3,3',4,4',5-pentachlorobiphenyl (PCB126) induced CYP1A1-dependent activities in HEK293 human embryonic kidney, Panc1 pancreatic cancer, and Hepa1c1c7 mouse hepatoma cell lines. There was a structure-dependent difference in the efficacy of TCDF and PCB126 in HEK293 and Panc1 cells since induced CYP1A1 mRNA levels were lower than observed for the other congeners. A mammalian two-hybrid assay in cells transfected with GAL4-coactivator and AhR-VP16 chimeras was used to investigate structure-dependent interactions of these chimeras in Panc1, HEK293, and Hepa1c1c7 cells. The reporter construct pGAL4-luc contains five tandem GAL4 response elements linked to the luciferase gene and the GAL4-coactivator chimeras express several coactivators including steroid receptor coactivator 1 (SRC-1), SRC-2 and SRC-3, the mediator coactivator TRAP220, coactivator associated arginine methyl transferase 1 (CARM-1), and peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1). Results of the mammalian two-hybrid studies clearly demonstrate that activation of pGAL4-luc in cells transfected with VP-AhR and GAL4-coactivator chimeras is dependent on the structure of the HAH congener, cell context, and coactivator, suggesting that the prototypical HAH congeners used in this study exhibit selective AhR modulator activity.
Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX 77843, USA.
Shu
S
Zhang
Craig
C
Rowlands
Stephen
S
Safe
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002678:Chimera / Q000235:genetics; D019363:Cytochrome P-450 CYP1A1 / Q000096:biosynthesis; D004790:Enzyme Induction / Q000187:drug effects; D017405:Herpes Simplex Virus Protein Vmw65 / Q000235:genetics; D006801:Humans; D006841:Hydrocarbons, Aromatic / Q000494:pharmacology; D008024:Ligands; D051379:Mice; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D018336:Receptors, Aryl Hydrocarbon / Q000187:drug effects* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013329:Structure-Activity Relationship; D014162:Transfection
D017405:Herpes Simplex Virus Protein Vmw65; D006841:Hydrocarbons, Aromatic; D008024:Ligands; D012333:RNA, Messenger; D018336:Receptors, Aryl Hydrocarbon; D019363:Cytochrome P-450 CYP1A1