Auto-induction mechanism of aryl hydrocarbon receptor 2 (AHR2) gene by TCDD-activated AHR1 and AHR2 in the red seabream (Pagrus major)

摘要

The toxic effects of dioxins and related compounds (DRCs) are mediated by the aryl hydrocarbon receptor (AHR). Our previous study identified AHR1 and AHR2 genes from the red seabream (Pagrus major). Moreover, we found that AHR2 mRNA level was notably elevated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure in the early life stage of red seabream embryos, while AHR1 mRNA level was not altered. In this study, to investigate the regulatory mechanism of these AHR transcripts, we cloned and characterized 5′-flanking regions of AHR1 and AHR2 genes. Both of the 5′-flanking regions in these AHR genes contained 3 potential xenobiotic responsive elements (XREs). We measured the transactivation potency of the 5′-flanking regions by AHR1 and AHR2 protein using an in vitro reporter gene assay. Only AHR2-derived XREs-containing reporter plasmid showed a clear TCDD dose-dependent transactivation by AHR1 and AHR2 proteins that were transiently expressed in COS-7. This result suggests that AHR2-derived XREs have a function for AHR1- and AHR2-mediated transactivation, supporting our in ovo observation of an induction of AHR2 mRNA levels by TCDD exposure. TCDD-EC50 values for the AHR2-derived XRE transactivation by AHR1 (1.3 nM) and AHR2 (1.4 nM) were higher than in ovo TCDD-EC50 (0.30 nM) for cytochrome P450 1A (CYP1A) mRNA induction, but were closer to in ovo TCDD-EC50 (1.5 nM) for AHR2 mRNA induction. Mutations in XREs of AHR2 gene led to a decrease in luciferase induction. Electrophoretic mobility shift assay showed that XRE1, the closest XRE from the start codon in AHR2 gene is mainly responsible for the binding with TCDD-activated AHR. This suggests that TCDD-activated AHR1 and AHR2 up-regulate the AHR2 mRNA levels and this auto-induced AHR2 may amplify the signal transduction of its downstream targets including CYP1A in the red seabream.