IMPACT OF AH RECEPTOR DEGRADATION IN VIVO AND IN VITRO

AH受体(AHR)是一种配体激活的转录因子，属于碱性-螺旋-环-螺旋/Per-Arnt-SIM(bHLH/PAS)蛋白家族。AHR介导的信号转导已经在许多模型系统中得到了广泛的研究，试图定义该途径的组成部分，了解蛋白质和DNA的相互作用，并确定可能影响人类健康的基因表达的特定变化。人们对AHR和Arnt蛋白在配体暴露后的命运、AHR激动剂对基因调控的持续时间以及关闭信号通路的机制的关注要少得多。对于不容易从体内代谢或清除的卤代芳香化合物来说，关闭AHR介导的信号转导和这一途径的调节机制尤其关键。因此，这项建议的重点是详细的AHR降解的分子分析，以及这一过程在体外和转基因动物中对AHR激动剂的内源性和外源性反应的影响。具体地说，中心假设是配体介导的AHR蛋白的降解减弱了AHR介导的信号转导。为了检验这一假说，本文提出了三个具体目标。(1)确定AHR降解对AHR依赖和独立基因调控的幅度和持续时间的影响。(2)确定AHR降解的途径，并对其进行调控。(3)建立和鉴定不降解AHR的转基因小鼠模型。不降解AHR的新细胞系和转基因动物的产生将提供有助于评估(I)AHR途径的内源性信号转导，(Ii)对HAHS的反应，(Iii)AHR途径组成蛋白的亚细胞定位，(Iv)与生物相关终点的剂量-反应关系，(V)与其他信号途径的相互作用(即低氧)和(Vi)AHR在遗传位点长期激活的后果的模型。这些模型与Ahr-/-小鼠和其他AHR信号转基因密切相关，它们可以结合使用来放大可能在其他模型或野生动物中检测不到的信号。从健康风险的角度来看，在开发AHR激动剂的人和动物健康风险的综合模型时，必须考虑AHR降解的后果，因为很明显，AHR介导的基因调控必须在一定程度上减弱，而AHR信号的这一方面实际上可能有助于某些组织中HAHS的生物学效应。

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.