Molecular and species-specific determinants of aryl hydrocarbon receptor transcriptional activity

The aryl hydrocarbon receptor (AHR) is a transcription factor that modulates gene expression in response to ligand binding. AHR ligands include a structurally diverse group of planar hydrophobic polycyclic organic compounds, which include dietary flavinoids such as 2-(3,4dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one (quercetin), as well as endogenous compounds like bilirubin and the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The AHR mediates most if not all of the adverse affects of TCDD exposure. Rodent models have been traditionally used in studies aimed at investigating TCDD-mediated toxicity and carcinogenesis and in the assessment of human risk. In order to enhance the accuracy of rodent-based toxicological data it is important to understand the molecular differences between human and rodent AHRs. We proposed that the hAHR would be functionally distinct from the mAHR. Therefore we attempted to identify biochemical and molecular factors that may influence interspecies differences between the mouse and human AHR. At a cellular level the hAHR also has contrasting nucleo-cytoplasmic shuttling properties compared to the mouse AHR (mAHR). This led to the hypothesis that identifying the underlying molecular mechanisms responsible for the ligand-independent nucleo-cytoplasmic shuttling of the AHR would shed light on this interspecies difference. Firstly we investigated the ability of the AHR to modulate gene expression in the absence of heterodimerization with the aryl hydrocarbon receptor nuclear translocator (ARNT). Through the use of an ARNT-binding deficient AHR mutant in cell culture based luciferase-reporter assays it was demonstrated that the AHR is unable to activate dioxin responsive element (DRE)-driven gene activity or repress estrogen receptor activity in the absence of interaction with ARNT. Compared to the mAHR the hAHR also interacts less stably with heat shock protein 90 (HSP90). The co-chaperone p23 is a component of the unliganded AHR complex that was presumed to be important to stable interaction between AHR and HSP90. In the third chapter a p23 knockout mouse was used to investigate the importance of p23 expression to AHR ligand-binding and transcriptional activity in vivo. Gene expression and ligand binding analysis revealed that p23 expression was dispensable for AHR ligand binding and AHR-regulated gene induction. Amino acid sequence identity differences exist for the AHR between and within species that is in part responsible for the contrasting inter-species and intra-species sensitivity to TCDD exposure. We show in the fourth chapter that the amino acid sequence dissimilarity in the C-terminal domains of the mAHR and hAHR may actually result in differential recruitment of LXXLL-coactivator binding motif proteins which suggests that each receptor may differentially recruit coactivators. Compared to the human AHR (hAHR) the Ahrb allele, the most commonly used allele in TCDD toxicity and carcinogenesis studies, has a 10-fold higher affinity for typical AHR ligands like TCDD. In contrast to this it is demonstrated that the hAHR shows a higher relative ligand binding affinity for certain ligands such as indirubin and quercetin in the fifth chapter. Finally, using microarray analysis we show that the hAHR and mAHR actually regulate different subsets of genes known to be associated with distinct biological pathways. Altogether these discoveries have identified a number of novel interspecies differences between the hAHR and mAHR. This research may have significant implications for model systems aimed at defining human risk associated with TCDD exposure. This study also serves to highlight the importance of utilizing transgenic hAHR mice in the investigation of AHR-mediated gene regulation and toxicity.