3 ′-Methoxy-4 ′-nitroflavone, a reported aryl hydrocarbon receptor antagonist, enhances Cyp1a1 transcription by a dioxin responsive element-dependent mechanism

Abstract

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor, regulating expression of a group of specific genes including cytochrome P450 1A1 (Cyp1a1). Stably transfected luciferase with dioxin responsive elements (DRE) in its promoter region has been commonly used as a reporter gene to study the mechanism of AhR signaling and compare potencies of TCDD and related compounds. However, how these two genes might respond to structurally diverse AhR ligands was unknown. This study investigates their expression in the same cells in response to TCDD, the most potent agonist, and 3 ′M4 ′NF, a reported potent antagonist. Our data suggest that these two compounds appear to play different roles in regulating these genes. While TCDD enhanced transcription of both genes, 3 ′M4 ′NF induced the endogenous Cyp1a1, but not the reporter gene. Mechanistic studies indicated that the increase in induction of CYP1A1 protein by 3 ′M4 ′NF was mediated by AhR-dependent transcriptional activation. Further analysis of the Cyp1a1 promoter sequence did not reveal any 3 ′M4 ′NF-specific responsive elements other than DREs. Rather, the interaction between the 3 ′M4 ′NF-bound receptor complex and DREs was confirmed by the observation that a single nucleotide mutation in DRE core sequences obliterated AhR enhancer activity in response to both TCDD and 3 ′M4 ′NF. Together these data suggest that 3 ′M4 ′NF, a weak AhR agonist, activates the AhR to recognize and interact with the same DREs as TCDD. However, depending on its concentration as well as the promoter context of a particular gene, the ability of 3 ′M4 ′NF to act as an AhR antagonist or agonist may appear different for various genes.