Ionizing radiation control of inflammatory cytokine mRNA stability and expression

摘要 基因表达可以通过改变转运、翻译效率和转录水平在mRNA水平上进行调节。 与稳定除了转录速率外，mRNA的衰减速率决定了细胞质中 丰富的mRNA。因此，mRNA降解速率的调节是基因表达的重要控制点。 表情已经确定，不同的mRNA在真核细胞中显示不同的半衰期， 从许多炎性细胞因子和生长因子mRNA的几分钟到大多数其他mRNA的几个小时。 电离辐射（IR）暴露在组织中诱导急性促炎细胞因子反应， 特别是在巨噬细胞和其他免疫细胞中，病理性炎症反应是主要的 放射治疗和意外照射期间IR的并发影响。尽管对于 了解IR如何促进病理性炎症反应， 为描述其发生机制而进行的研究。本申请涉及 了解IR促进促炎细胞因子过度表达的分子机制， 关注其通过拮抗炎症细胞因子mRNA的正常快速降解来抑制炎症细胞因子mRNA的能力， 促衰变因子AUF 1活性。 目的1将使用永生化的野生型和AUF 1-/-巨噬细胞和质谱法来鉴定和鉴定 表征IR诱导型AUF 1相互作用蛋白，这些蛋白可能参与介导IR控制 炎症反应通过调节AUF 1活性。为了获得与临床环境相关的信息， 另外的研究将检查IR的多个较低剂量分数对AUF 1介导的衰减的影响。 炎性ARE-mRNA和AUF 1相互作用蛋白。然后，研究将确定蛋白质， AUF 1在单次高剂量和多次分次低剂量IR治疗中丢失或获得。体内 然后在生理相关条件下验证这些蛋白质与AUF 1的相互作用。 目的2将表征IR介导的AUF 1结合蛋白相互作用在快速免疫应答中的功能。 内源性促炎细胞因子ARE-mRNA的衰减。研究将使用永生化的野生型和 AUF 1-/-巨噬细胞以及相互作用蛋白的RNA沉默和cDNA过表达， 表征它们在IR介导的炎性细胞因子稳定和过度表达中的分子功能 mRNA。 目的3鉴定IR调控的主要内源性炎性细胞因子ARE-mRNAs 通过控制AUF 1活性。研究将在未治疗的小鼠中鉴定AUF 1活性的总体ARE-mRNA靶标。 和IR处理的永生化野生型和AUF 1-/-巨噬细胞，并确定其他细胞对巨噬细胞的影响程度。 ARE结合蛋白如TTP、KSRP和稳定蛋白HuR参与IR介导的稳定 炎性细胞因子ARE-mRNAs。 目的4将描述IR抑制炎性ARE衰减的分子机制。 通过作用于AUF 1和鉴定的关键AUF 1相互作用蛋白的mRNA。研究还将确定 IR是否通过作用于P体形成和功能和/或外泌体来阻断AUF 1功能， 核糖核酸酶细胞器样结构，参与短寿命mRNA的降解。

ABSTRACT Gene expression can be regulated at the mRNA level through alterations in transport, translational efficiency and stability. The rate of mRNA decay, in addition to the rate of transcription, determines cytoplasmic abundance of mRNAs. Regulation of mRNA decay rates is therefore an important control point in gene expression. It is well established that different mRNAs display diverse half-lives in eukaryotic cells, ranging from minutes for many inflammatory cytokine and growth factor mRNAs, to many hours for most other mRNAs. Acute pro-inflammatory cytokine responses are induced in tissues by ionizing radiation (IR) exposure, particularly in macrophages and other immune cells, and the pathological inflammatory response is a major complicating affect of IR during radiotherapy and accidental exposure. Despite the obvious importance for understanding how IR promotes a pathological inflammatory response, there has been remarkably little research conducted to characterize the mechanism by which this occurs. This application is directed to understanding the molecular mechanism by which IR promotes overexpression of pro-inflammatory cytokines, focusing on its ability to inhibit the normal rapid degradation of inflammatory cytokine mRNAs by antagonizing the activity of pro-decay factor, AUF1. Aim 1 will use immortalized wild type and AUF1-/- macrophages and mass spectrometry to identify and characterize IR-inducible AUF1 interacting proteins that are likely involved in mediating IR control of the inflammatory response through regulation of AUF1 activity. To obtain information relevant to the clinical setting, additional studies will examine the affect of multiple lower dose fractions of IR on AUF1-mediated decay of inflammatory ARE-mRNAs and AUF1-interacting proteins. Studies will then identify proteins whose binding to AUF1 is lost or gained with single high dose and multiple fractionated lower dose IR- treatment. The in vivo interaction of these proteins with AUF1 will then be verified under physiologically relevant conditions. Aim 2 will characterize the function of IR-mediated AUF1 binding protein interactions on the rapid decay of endogenous pro-inflammatory cytokine ARE-mRNAs. Studies will use immortalized wild type and AUF1-/- macrophages, as well RNA silencing and cDNA overexpression of interacting proteins, to fully characterize their molecular functions in IR-mediated stabilization and overexpression of inflammatory cytokine mRNAs. Aim 3 will identify the major endogenous inflammatory cytokine ARE-mRNAs that are regulated by IR through control of AUF1 activity. Studies will identify global ARE-mRNA targets of AUF1 activity in untreated and IR treated immortalized wild type and AUF1-/- macrophages, and determine the extent to which other ARE-binding proteins such as TTP, KSRP and stabilizing protein HuR are involved in IR-mediated stabilization of inflammatory cytokine ARE-mRNAs. Aim 4 will characterize the molecular mechanism by which IR inhibits the decay of inflammatory ARE- mRNAs by acting on AUF1 and the key AUF1 interacting proteins identified. Studies will also determine whether IR blocks AUF1 function by acting on P-body formation and function, and/or on the exosome, a ribonuclease organelle-like structure that is involved in the degradation of short-lived mRNAs.