IMPACT OF AH RECEPTOR DEGRADATION IN VIVO AND IN VITRO

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The Ah-receptor (AHR) is a ligand activated transcription factor that belongs to the growing family of basic-helix-loop-helix/PER-ARNT-SIM (bHLH/PAS) proteins. AHR-mediated signaling has been extensively investigated in numerous model systems in an attempt to define the components of the pathway, understand protein and DNA interactions and define specific changes in gene expression that may impact human health. There has been considerably less emphasis placed on the fate of the AHR and ARNT proteins following ligand exposure, the duration of gene regulation by AHR agonists and the mechanism involved in turning the signaling pathway off. The mechanism involved in turning off AHR-- mediated signaling and the regulation of this pathway are especially critical with respect to halogenated aromatic compounds that are not readily metabolized or cleared from the body. Therefore, the focus of this proposal is the detailed molecular analysis of AHR degradation and the implications of this process on endogenous and exogenous response to AHR agonists in vitro and in transgenic animals. Specifically, the central hypothesis is that ligand-mediated degradation of AHR protein attenuates AHR-mediated signaling. Three specific aims are proposed to test this hypothesis. (1) Determine the impact of AHR degradation on the magnitude and duration of AHR-dependent and independent gene regulation. (2) Determine the pathway responsible for degradation of AHR and characterize its regulation. (3) Generate and characterize a transgenic mouse model that does not degrade the AHR. The generation of novel cell lines and transgenic animals that do not degrade the AHR will provide models that will help in assessing (i) endogenous signaling of the AHR pathway, (ii) response to HAHs, (iii) subcellular localization of component proteins of the AHR pathway, (iv) dose-response relationships to biologically relevant endpoints, (v) interactions with other signaling pathways (i.e. hypoxia) and (vi) the consequence of prolonged activation of the AHR at genetic loci. These models are essential correlates to Ahr-/- mice and other AHR-signaling transgenics that can be used in combination to amplify signals that might not be detected in the other models or in wild type animals. From the health risk perspective, the consequence of AHR degradation must be considered in development of comprehensive models of human and animal health risk for AHR-agonists since it is clear that AHR-mediated gene regulation has to be attenuated at some level and this aspect of AHR signaling may in fact be contributory to the biological effects of HAHs in certain tissues.