Mechanisms of HuR Overexpression in Asthmatic Endotypes

摘要 尽管进行了广泛的研究，哮喘仍然是一种难以治疗的疾病，经济成本令人震惊。 不同哮喘内型的发病机制尚不清楚。RNA对转录后基因的调控 结合蛋白(RBPs)和microRNAs(MiRNAs)越来越被认为是重要的调控机制 促炎症基因，但研究不足。限制性商业惯例，如HUR(Eavl1)、Tristetraprolin(TTP)家族 与富含AU的元件(ARE)结合的成员，在调节mRNA的稳定性和 关键促炎基因在哮喘中的表达HUR是RBP的稳定剂，而TTP家族成员是 不稳定因素限制性商业惯例。由于稳定状态的mRNA水平和蛋白质之间的不一致，纯粹是转录 方法可能会忽略限制性商业惯例调节的关键炎症基因，如HUR和TTP。然而，大部分 免疫和炎症由限制性商业惯例和miRNAs在转录后水平上控制。多种细胞因子 CD4+T细胞中的基因受HUR和TTP家族成员之间的平衡控制。该字段不能 除非我们更好地了解限制性商业惯例是如何转录后调控促炎基因的 在不同哮喘内型中的表达。我们的长期目标是了解转录后基因 哮喘气道炎症不同内型的调节。这个应用程序的目标是我们的 追求这一目标的下一步是确定HUR和TTP家族成员如何竞争性地监管HUR 在哮喘中，它反过来控制着CD4+T细胞产生细胞因子。我们的中心假设是HUR 2型高哮喘患者的过度生产是由稳定和不稳定的限制性商业惯例之间的失衡所致 作用于Eavl1基因，影响其稳定性和翻译。我们的数据表明，HUR在 2型哮喘患者外周血中CD4+T细胞明显高于正常对照组和非2型哮喘患者。此外，我们还拥有 T细胞条件性Hur KO可显著减轻变应原诱导的小鼠肺部炎症 模特们。我们还证明，抑制HUR可以阻断哮喘CD4+T细胞Th2/Th17的分泌。 到目前为止，该领域主要关注下游的HUR靶基因。相比之下，HUR法规 并不是很好理解。这项研究的基本原理是，拟议的研究将使我们能够确定 在2型高哮喘肺脏中驱动HUR过度产生的分子机制 发炎。我们计划通过以下两个具体步骤来检验中心假设并实现这些目标 目的：1)确定Hur在呼吸道炎症中的正常表达机制；2)确定分子 2型高哮喘患者HUR失调的机制。在完成拟议的研究后， 我们的预期结果是确定HUR和TTP如何合作来正常控制HUR的表达以及如何 它们在2型高哮喘CD4+T细胞中变得功能失调。预计这些结果将产生积极的影响 对该领域的影响，因为它将进一步定义分子机制并提供发展机会 治疗哮喘的新疗法。

Abstract Despite extensive research, asthma remains a difficult to treat disease with staggering economic costs. Mechanisms in different asthmatic endotype are poorly understood. Posttranscriptional gene regulation by RNA- binding proteins (RBPs) and microRNAs (miRNAs) is increasingly recognized as important control mechanisms for pro-inflammatory genes but is understudied. RBPs, such as HuR (Elavl1), tristetraprolin (TTP) family members which bind to mRNA AU-rich elements (ARE), play critical roles in regulating mRNA stability and expression of key pro-inflammatory genes in asthma. HuR is a stabilizer RBP, whereas TTP family members are destabilizer RBPs. Due to the discordance between steady-state mRNA levels and protein, purely transcriptomic approaches may overlook critical inflammatory genes regulated by RBPs such as HuR and TTP. Yet, much of immunity and inflammation is controlled at the posttranscriptional level by RBPs and miRNAs. Many cytokine genes in CD4+ T cells are controlled by the balance between HuR and TTP family members. The field cannot move forward unless we better understand how RBPs posttranscriptionally regulate pro-inflammatory gene expression in different asthmatic endotypes. Our long-term goal is to understand posttranscriptional gene regulation in different endotypes of asthmatic airway inflammation. The objective of this application, which is our next step in pursuit of that goal, is to determine how HuR and TTP family members competitively regulate HuR expression, which in turn controls CD4+ T produced cytokines in asthma. Our central hypothesis is that HuR overproduction in type 2 high asthma is driven by an imbalance between stabilizing and destabilizing RBPs acting upon Elavl1 mRNA to affect its stability and translation. Our data indicate that HuR is over-expressed in type 2 high asthmatic CD4+ T cells, compared with normal controls and non-type 2 high. Furthermore, we have shown that conditional HuR KO in T cells significantly ameliorates allergen-induced lung inflammation in murine models. We have also demonstrated that HuR inhibition blocks Th2/Th17 secretion in asthmatic CD4+ T cells. Up to the present, the field has focused mostly on downstream HuR targets genes. In contrast, HuR regulation is not as well understood. The rationale for this research is that the proposed research will enable us to identify molecular mechanisms which drive HuR over-production in the context of type 2 high asthmatic lung inflammation. We plan to test the central hypothesis and accomplish these objectives by the following two specific aims: 1) Determine normal mechanisms of HuR expression in airway inflammation; 2) Determine molecular mechanisms underlying HuR dysregulation in type 2 high asthma. At the completion of the proposed research, our expected outcomes are to identify how HuR and TTP cooperate to normally control HuR expression and how they become dysfunctional in type 2 high asthmatic CD4+ T cells. These results are expected to have a positive impact on the field because it will further define molecular mechanisms and provide opportunities to develop novel therapies to treat asthma.