POST-TRANSCRIPTIONAL REGULATION OF INFLAMMATORY MEDIATORS

描述（由申请人提供）：炎症是响应于创伤性、感染性、毒性或自身免疫性损伤而在宿主内发生的一系列事件的集合。炎症失调与关节炎、阿尔茨海默病和脓毒性休克等人类疾病有关。炎症反应的范围和持续时间由称为炎症介质的宿主产物决定，其精确的空间和时间表达对于建立适当的炎症反应至关重要。虽然在理解微生物产物对这些介质的转录调控方面取得了重大进展，但对转录后调控机制知之甚少。我们最近发现了一个MyD 88依赖性途径，稳定干扰素γ诱导的细胞因子和趋化因子mRNA。通过使用携带来自TNF的3 '-非翻译区（UTR）的富含AU（ARE）序列的荧光素酶报告基因构建体来鉴定该途径的几个组分成员，包括MLK 3和MKK 3。在这里，我们建议使用类似的报告构建为基础的屏幕，以确定额外的身份不明的因素参与转录后调节炎症介质。该项目的目标是定义和探索含ARE的mRNA稳定的机制。我们的工作假设是，某些信号分子的过表达可以通过增强其mRNA的稳定性来上调细胞内报告蛋白的表达。我们将：（1）在白细胞cDNA文库中筛选能够激活荧光素酶报告基因的基因，所述荧光素酶报告基因在其3 '-UTR处携带来自TNF的ARE，（2）确定所鉴定的候选基因影响炎性介质的mRNA稳定性的分子基础，和（3）通过测试缺乏这些基因的小鼠中的炎性应答来评估一种候选基因在体外和体内的作用。本研究的长期目标是深入了解炎症性疾病的病理基础，如生理基因调控机制的紊乱所解释的，并帮助开发新的治疗策略。

DESCRIPTION (provided by applicant): Inflammation is the collective array of events within the host that occur in response to traumatic, infectious, toxic or autoimmune injury. Dysregulated inflammation is associated with human diseases such as arthritis, Alzheimer's disease and septic shock. The scope and duration of inflammatory reactions are dictated by host products called inflammatory mediators, whose precise spatial and temporal expression is critical in mounting an appropriate inflmammatory response. While significant progress has been made in understanding the transcriptional regulation of these mediators by microbial products, much less is known about post-transcriptional regulatory mechanisms. We recently discovered a MyD88-dependent pathway that stabilized interferon-gamma-induced cytokine and chemokine mRNAs. Several component members of this pathway, including MLK3 and MKK3, were identified by using a luciferase reporter construct bearing an AU-rich (ARE) sequence from 3'-untranslated region (UTR) of TNF. Here, we propose to identify additional unidentified factors involved in the post-transcriptional regulation of inflammatory mediators using a similar reporter construct-based screen. The goal of this project is to define and explore mechanisms underlying the stabilization of ARE-containing mRNAs. Our working hypothesis is that overexpression of certain signaling molecules can up-regulate reporter protein expression inside cells by enhancing its mRNA stability. We will: (1) screen a leukocyte cDNA library for genes capable of activating a luciferase reporter carrying ARE from TNF at its 3'-UTR, (2) define the molecular basis by which identified candidate genes affect the mRNA stability of inflammatory mediators, and (3) evaluate the role of one candidate gene in vitro and in vivo by testing inflammatory responses in mice deficient for these genes. The long term goal of this study is to gain insight into the pathological basis of inflammatory diseases as explained by derangements of physiological gene regulatory mechanisms and aid in the development of novel therapeutic strategies.