Ligand-dependent interactions of the Ah receptor with coactivators in a mammalian two-hybrid assay.

Abstract

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.

• Authors: Shu Zhang, Craig Rowlands, Stephen Safe
• Journal: Toxicology and applied pharmacology (Toxicol Appl Pharmacol) Vol. 227 Issue 2 Pg. 196-206 (Mar 01 2008) ISSN: 0041-008X [Print] United States
• PMID: 18048071 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)

Chemical References

• Herpes Simplex Virus Protein Vmw65
• Hydrocarbons, Aromatic
• Ligands
• RNA, Messenger
• Receptors, Aryl Hydrocarbon
• Cytochrome P-450 CYP1A1

Topics

• Animals
• Blotting, Western
• Cell Line
• Chimera (genetics)
• Cytochrome P-450 CYP1A1 (biosynthesis)
• Enzyme Induction (drug effects)
• Herpes Simplex Virus Protein Vmw65 (genetics)
• Humans
• Hydrocarbons, Aromatic (pharmacology)
• Ligands
• Mice
• RNA, Messenger (biosynthesis, genetics)
• Receptors, Aryl Hydrocarbon (drug effects, genetics)
• Reverse Transcriptase Polymerase Chain Reaction
• Structure-Activity Relationship
• Transfection