Circadian regulation of the AhR signaling pathway

Abstract

Common in vitro cell culture systems are widely used to understand physiological processes and disease development. However, these have a number of limitations such as the lack of the physiological 3D tissue architecture but also regulatory mechanisms, such as the circadian rhythm. For that reason, in vitro methods need to be improved to mimic as closely as possible the in vivo situation found in humans, and the regulatory framework of toxicological relevant pathways should be studied to better understand physiological processes. Even if the circadian rhythm has been linked to various important physiological processes, the molecular mechanism of these circadian regulations remains unravelled. The circadian rhythm is characterized by internal oscillations of physiological processes with a recurring periodicity of approximately 24 h and its synchronization depends mainly on the light-dark periods of the day. On the molecular level, the circadian rhythm is driven by the recurring expression of CLOCK genes, which regulate up to 48% of all human genes and ensures the proper daytime-depended activity. The uniform circadian rhythm of all cells in a tissue is often lost in common 2D in vitro cell culture systems but can be easily restored through artificial synchronization of these cells. Subsequently, such synchronized cell culture systems lead to enhanced quantitative and qualitative cellular response with a higher human relevance. A highly toxicological relevant pathway, which might be under circadian control is the AhR (Aryl hydrocarbon Receptor) signaling pathway. In particular, in synchronized human breast cells the cellular response upon AhR-ligand binding shows a circadian pattern indicating a circadian regulation of the AhR pathway. This study describes for the first time important aspects of the mechanism behind the circadian regulation of the AhR signaling pathway. The circadian expression of AhR target genes, e.g. CYP1A1, is caused by a circadian activation of the CYP1A1 promoter but not a circadian expression of AhR itself. Upon ligand exposure, e.g. TCDD (2,3,7,8-Tetrachlordibenzodioxin), AhR translocates into the nucleus and binds not only with ARNT but also with the circadian rhythm regulator BMAL1. These interactions seem to be circadian phase-dependent, indicating a possible competition in AhR binding. Additionally, the AhR co-factor, p23, negatively modulated the circadian regulation of the AhR signaling and SP1 was identified as a novel AhR co-factor exhibiting a circadian protein expression, which possibly sustains the proper circadian regulation of the AhR pathway. Besides that, preliminary data suggest that GSK3β could be involved in the circadian regulation of the AhR pathway. Summarizing, all the findings of this thesis propose a possible mechanism for the circadian regulation of the AhR pathway and this work contributes to a better understanding of the circadian regulation of an important toxicological pathway, that might lead to the development of improved cell culture-based in vitro systems for toxicological and risk assessment strategies as well as for drug development with a higher human relevance.