Estrogen receptor-hijacking by dioxin-like 3,3′4,4′,5-pentachlorobiphenyl (PCB126) in salmon hepatocytes involves both receptor activation and receptor protein stability

Abstract

Several hypotheses have been proposed explaining the interactions between estrogen receptor (ER) and aryl hydrocarbon receptor (AhR) signaling pathways in both fish and mammalian systems. In both piscine and mammalian systems, ligand-activated AhR may recruit basal ER (i.e. hijack) in the absence of ER ligand and bind to the estrogen responsive elements (ERE) to activate ER-responsive genes. We have evaluated, the roles of receptor activation and receptor-protein stability on dioxin-like [3,3′4,4′,5-pentachlorobiphenyl: PCB 126] mediated ER-hijacking in a salmon in vitro system. Primary salmon hepatocytes were exposed to PCB126 (1, 10 and 50nM) with or without an ER-antagonist (ICI), putative AhR inhibitor (3′,4′-dimethoxyflavone; DMF) or protein synthesis inhibitor (cycloheximide; CHX). Hepatocytes were exposed for 6, 12 and 24h. The expression of genes and proteins involved in ER (ERα, ERβ and vitellogenin) and AhR (CYP1A1, AhR-repressor, AhR2-isotypes and cofactors) pathways were analysed using qPCR and immunochemical methods. PCB126 induced transcripts of ER and AhR signalling pathways that were variably influenced by protein synthesis and receptor inhibitors. CHX stimulated a coordinated recruitment of the proteasome complex, resulting in the ubiquitination and degradation of ER and AhR isoforms and downstream protein products. Interestingly, DMF produced differential effects on the AhR signalling pathway, in the presence or absence of PCB126. Overall, ER-hijacking by dioxin-like compounds and subsequent activation of ER responsive genes involves both receptor activation/deactivation and receptor-protein degradation/destabilization (stability). Given that the Per–AhR/Arnt–Sim homology sequence of transcription factors usually associate with each other to form heterodimers and bind the XRE or ERE sequences in the promoter regions of target genes to regulate their expression, the complete mechanism of interactions between dioxin-like and estrogenic compounds in vertebrate systems may require additional characterization.