The aryl hydrocarbon receptor-mediated disruption of vitellogenin synthesis in the fish liver: Cross-talk between AHR- and ERα-signalling pathways

BACKGROUND: In the fish liver, the synthesis of egg yolk protein precursor vitellogenin (VTG) is under control of the estrogen receptor alpha (ERalpha). Environmental contaminants such as 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) are suspected to have antiestrogenic effects. The aryl hydrocarbon receptor (AHR) is the initial cellular target for TCDD and related compounds. The AHR is a ligand-activated transcription factor that stimulates the expression of the genes encoding xenobiotic metabolizing enzymes, such as cytochrome P450 1A (CYP1A). In this study, the effects of activation of AHR on the hepatic expression of VTG and ERalpha genes, in primary cultured salmon hepatocytes, have been investigated. RESULTS: The expression of the genes encoding VTG and ERalpha were strongly induced by 17beta-estradiol (E2). However, the expression of VTG was disrupted by exposure of the cells to TCDD while CYP1A expression was enhanced. The effect of TCDD on VTG and CYP1A expression was annulled by the AHR-inhibitor alpha-naphthoflavone. Furthermore, exposure of the cells to TCDD abolished E2-induced accumulation of ERalpha mRNA. The AHR-mediated inhibitory effects on the expression of the VTG and ERalpha genes may occur at transcriptional and/or post-transcriptional levels. Nuclear run-off experiments revealed that simultaneous exposure of the cells to E2 and TCDD strongly inhibited the initiation of transcription of the VTG and ERalpha genes. In addition, inhibition of RNA synthesis by actinomycin D treatment showed that post-transcriptional levels of VTG and ERalpha mRNAs were not significantly altered upon treatment of the cells with TCDD. These results suggested that activation of AHR may inhibit the transactivation capacity of the ERalpha. Further, electrophoretic mobility shift assays using nuclear extracts prepared from cells treated for one or two hours with E2, alone or in mixture with TCDD, showed a strong reduction in the DNA binding activities upon TCDD treatment. These results also suggested that activation of the AHR signalling pathway caused a marked decrease in the number of the nuclear ERalpha or that activated AHR blocked the ability of ERalpha to bind to its target DNA sequence. Finally, our results from Northern hybridizations indicated that E2 treatment of the cells did not cause any significant effect on the TCDD-induced levels of CYP1A mRNA. CONCLUSION: In fish hepatocytes E2 induces ERalpha and VTG gene expression. The presence of dioxin (TCDD) abolishes this induction, probably through the action of AHR in complex with AHR nuclear translocator, and possibly by direct interference with the auto-regulatory transcriptional loop of ERalpha. Furthermore, E2 does not interfere with TCDD induced CYP1A gene expression, suggesting that cross-talk between the ERalpha- and AHR-signalling pathways is unidirectional.