Novel ARNT-mediated Regulatory Paradigm of AhR signaling

 描述(申请人提供)：AHR是一种细胞质受体，与许多外源配体具有亲和力，其中包括卤代芳香烃，如最有效的AHR激活剂之一2，3，7，8-四氯二苯并-对二恶英(TCDD)，AHR信号转导环境污染物对身体组织和器官的有害影响。配体结合诱导AHR的核转位，并与AHR结合伙伴芳香烃受体核转位蛋白(ARNT)相互作用，从而识别特定的DNA增强子序列。最近，内源性和天然的AHR配体，如色氨酸代谢物和饮食化合物已被确定。这些配体诱导AHR介导的免疫调节作用，如控制正常T细胞分化和免疫耐受。然而，对ARNT在AHR免疫信号转导中的作用知之甚少。我们实验室和其他实验室对ARNT在AHR介导的免疫调节中的调节作用的初步研究表明，ARNT在不同的免疫信号通路中是一个不可或缺的辅助因子，包括AHR和NF-κB信号。在AHR信号转导中，ARNT通常被描述为AHR的结构性表达的、非调控的核结合伙伴。然而，我们的数据挑战了这一假设，并表明实际的监管范式更加错综复杂。例如，ArnT被表达为两种亚型，亚型1和亚型3，它们只有15个氨基酸存在于亚型1中。尽管它们的序列相似，但我们发现Arnt亚型在AHR信号转导中似乎具有相反的功能。有趣的是，亚型1中额外的15个氨基酸包括一个独特的磷酸化位点，我们在那里观察到了TCDD诱导的磷酸化。此外，我们发现Arnt异构体1的磷酸化对于RNA聚合酶II的募集和AHR/Arnt靶基因的反式激活是至关重要的。我们预测，Arnt的活性是特定细胞类型中特定异构体1的独特磷酸化和给定的异构体比率的函数，这反过来可能对调节AHR反应的幅度/结果很重要。为了验证我们的假设，我们建议1)定义一个由Arnt亚型调控AHR的全面分子框架，2)检测由Arnt亚型控制的AHR活性在淋巴瘤细胞生长中的作用，以及3)在体内表征Arnt亚型对AHR信号的调控。最终，这些研究将为基于ARNT的治疗奠定基础，例如剪接调制，作为一种操纵ARNT异构体的手段，以控制自身免疫性疾病和癌症中的AHR信号。

 DESCRIPTION (provided by applicant): AHR is a cytoplasmic receptor that has affinity for numerous xenobiotic ligands, which include halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), one of the most potent AHR activators, and AHR signaling mediates the detrimental effects of environmental contaminants on body tissues and organs. Ligand binding induces nuclear translocation of AHR and interaction with the AHR binding partner, aryl hydrocarbon receptor nuclear translocator (ARNT), allowing recognition of specific DNA enhancer sequences. More recently endogenous and natural AHR ligands, such as tryptophan metabolites and dietary compounds, have been identified. These ligands induce AHR-mediated immunomodulatory effects such as controlling normal T cell differentiation and immune tolerance. However, little is known about the role of ARNT in AHR immune signaling. Initial studies by our laboratory, and others, into the regulatory role of ARNT in AHR-mediated immunomodulation have suggested that ARNT is an integral cofactor in different immune signaling pathways, including AHR and NF-κB signaling. ARNT is often described in AHR signaling as a constitutively expressed, non-regulated, nuclear binding partner for AHR. However, our data challenge this assumption and show that the actual regulatory paradigm is more intricate. For instance, ARNT is expressed as two isoforms, isoform 1 and 3, which differ in only 15 amino acids present in isoform 1. Despite their sequence similarity, we have found that the ARNT isoforms appear to have opposing functions in AHR signaling. Intriguingly, the extra 15 amino acids in isoform 1 include a unique phosphorylation site, where we have observed TCDD-induced phosphorylation. Furthermore, we find that the phosphorylation of ARNT isoform 1 is crucial for the recruitment of RNA polymerase II and transactivation of AHR/ARNT target genes. We predict that ARNT activity is a function of both unique phosphorylation of isoform 1 and the given isoform ratio within a particular cell type, which in turn is likely important for regulating the magnitude/outcome of the AHR response. To investigate our hypothesis we propose to 1) Define a comprehensive molecular framework for AHR regulation by the ARNT isoforms, 2) Examine the role of AHR activity, as governed by the ARNT isoforms, in lymphoma cell growth, and 3) Characterize the regulation of AHR signaling by ARNT isoforms in vivo. Ultimately, these studies will lay the groundwork for ARNT-based therapies such as splice modulation as a means of manipulating the ARNT isoforms to control AHR signaling in autoimmune diseases and cancer.