Regulation of NF-Kappa B

描述（由申请人提供）：慢性炎症在许多疾病的发生和发展中起着重要作用。炎症过程的主要参与者和介质之一是被称为NF- B的转录因子家族。我们建议研究炎症及其对疾病的影响的分子机制。我们计划进行一项全激酶范围的RNAi筛选NF-？B通路。通过使用shRNA慢病毒文库和高通量筛选（HTS）技术，我们鉴定出了30个可以影响NF-？B受外界刺激物刺激后的活性。为了深入了解完全验证的激酶的生物学特性，这些激酶可以调节NF-？B途径，我们将进行广泛的生化和生物学分析参与调节NF-？B通路。糖皮质激素受体抑制NF-？B：鉴定新的分子机制。糖皮质激素是最强的抗炎剂之一，也是抑制炎症最常见的治疗形式之一；然而，长期使用会产生许多负面副作用。我们已经确定p53是一个候选基因，其产物是诱导NF-？糖皮质激素对B活性的影响。我们还确定了在糖皮质激素诱导的抑制中需要的大约25种激酶。炎症与糖尿病：Tlr4在巨噬细胞中的作用。胰岛素抵抗是肥胖的主要代谢缺陷，与各种疾病的风险增加有关。巨噬细胞通过其分泌多种促炎趋化因子和细胞因子的能力是炎症的重要调节剂。我们假设敲除造血源性细胞（包括巨噬细胞）中的Tlr4信号将减少肥胖相关的巨噬细胞浸润和炎症的增加，并随后预防体内胰岛素抵抗。我们将通过生物途径或使用拮抗剂来抑制Tlr4，看看这些动物现在是否对胰岛素敏感。炎症和癌症：炎症和癌症之间的联系已经确立，各种各样的新工具将使我们能够从分子上剖析这种联系，从而产生治疗实体。我们建议在局部炎症和肿瘤周围炎症环境的背景下研究炎症的作用。具体来说，我们建议利用我们最近的小鼠模型在免疫能力强的小鼠中产生胶质瘤。我们将在肿瘤和周围环境中创造炎症条件，以了解其在肿瘤形成和发展中的作用。P53和IKK2：我们发现IKK2， NF-？B途径，也可以在DNA损伤反应中磷酸化两条丝氨酸（S362, S366），从而通过2- trcp依赖途径导致p53降解，从而调节p53的稳定性。这一结果将两种主要信号通路NF-？B和p53-通常是拮抗的-前者是细胞存活因子，后者促进细胞凋亡。我们将让老鼠产生一种更稳定的p53，并研究它如何影响肿瘤的结果。总之，我们建议在未来5年内进行一系列广泛的实验，以了解NF-？更重要的是它在炎症和疾病中的作用。

DESCRIPTION (provided by applicant): Chronic inflammation plays a major role in the onset and progression of many diseases. One of the major players and mediators of inflammatory processes are a family of transcription factors known as NF-?B. We are proposing to study the molecular mechanisms involved in inflammation and their consequences to disease. We plan to undertake: A kinome-wide RNAi screen for novel mediators of the NF-?B pathway. By using shRNA lentiviral library and high-throughput screening (HTS) technology we have identified 30 kinases, which can effect NF-?B activity upon stimulation by external agents. To gain insight into the biology of the fully validated kinases that modulate the activity of NF-?B pathway, we will undertake extensive biochemical and biological analysis of the top candidates involved in modulating NF-?B pathway. Glucocorticoid receptor repression of NF-?B: Identification of novel molecular mechanisms. Glucocorticoids are among the strongest anti-inflammatory agents and one of the most common forms of treatment to suppress inflammation; however many negative side effects can occur with long term use. We have identified p53 as a candidate gene whose product is required for induced suppression of NF-?B activity by glucocorticoid. We have also identified about 25 kinases that are needed in glucocorticoid induced repression. Inflammation and diabetes: Role of Tlr4 in macrophages. Insulin resistance is a major metabolic defect in obesity, and is associated with increased risk of various diseases. Macrophages are an important modulator of inflammation through their capacity to secrete a variety of proinflammatory chemokines and cytokines. We hypothesized that knock-out of Tlr4 signaling in hematopoietic-derived cells (which includes macrophages), would reduce obesity-related increases in macrophage infiltration and inflammation and subsequently prevent in vivo insulin resistance. We will knock down Tlr4 either biologically or use an antagonist and see if the animals are now insulin sensitive. Inflammation and cancer: The link between inflammation and cancer is well established and the variety of new tools available will allow us to molecularly dissect this connection, leading to therapeutic entities. We propose to study the role of inflammation in the context of local inflammation and the inflammatory milieu surrounding the tumor. Specifically we propose to take advantage of our recent mouse model to generate gliomas in immune competent mice. We will create conditions of inflammation both in the tumor and in the surrounding milieu to understand its role in tumor formation and progression. P53 and IKK2: We have found that IKK2, the key kinase in the NF-?B pathway, can also regulate the p53 stability by phosphorylating two serines (S362, S366) upon DNA damage response, which leads to p53 degradation through the2-TrCP dependent pathway. This result links the two major signal pathways NF-?B and p53-often antagonistic-the former a cell survival factor and the latter promoting apoptosis. We will generate mice generating a more stable form of p53 and study how it may affect the outcome of tumors. In summary we have proposed an extensive series of experiments in the next 5 years to understand the regulation of NF-?B and more importantly its role in inflammation and disease.