Regulation of NF-Kappa B

描述(申请人提供)：慢性炎症在许多疾病的发生和发展中起主要作用。炎症过程的主要参与者和介体之一是一个被称为核因子-βB的转录因子家族。我们建议研究炎症及其对疾病的后果的分子机制。我们计划进行：对核因子-βB途径的新介体进行全基因组RNAi筛选。通过利用shRNA慢病毒文库和高通量筛选(HTS)技术，我们已经鉴定了30个在外界因素刺激下能够影响核因子-βB活性的激酶。为了深入了解调节核因子-βB途径活性的完全有效的蛋白激酶的生物学特性，我们将对参与调控核因子-βB途径的候选蛋白进行广泛的生化和生物学分析。糖皮质激素受体对核因子？B的抑制：新分子机制的鉴定。糖皮质激素是最强的抗炎药之一，也是最常见的抑制炎症的治疗形式之一；然而，长期使用可能会出现许多负面副作用。我们已经确定P53是一个候选基因，其产物是糖皮质激素诱导抑制NF-B活性所必需的。我们还确定了糖皮质激素诱导的抑制所需的大约25个激酶。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一种主要代谢缺陷，与各种疾病的风险增加有关。巨噬细胞通过分泌多种致炎趋化因子和细胞因子，是炎症反应的重要调节器。我们假设，在造血细胞(包括巨噬细胞)中敲除TLR4信号将减少肥胖引起的巨噬细胞浸润和炎症增加，从而防止体内的胰岛素抵抗。我们将通过生物学或使用拮抗剂来击倒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，各种可用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。我们建议研究炎症在局部炎症和肿瘤周围炎症环境中的作用。具体地说，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：我们已经发现，作为核因子-βB途径中的关键激酶，IKK2还可以通过磷酸化DNA损伤反应中的两个丝氨酸(S362、S366)来调节P53的稳定性，从而通过2-TrCP依赖的途径导致P53的降解。这一结果将两条主要的信号通路--通常是拮抗的我们将产生产生更稳定形式的p53的小鼠，并研究它可能如何影响肿瘤的结果。综上所述，我们建议在未来5年内进行一系列广泛的实验，以了解核因子-β的调节，更重要的是它在炎症和疾病中的作用。 与公共健康相关：慢性炎症是许多疾病的核心，如癌症、糖尿病、阿尔茨海默氏症和衰老。我们建议通过研究精液分子核因子-βB在诱导炎症过程中的调节来研究炎症过程的分子机制。

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. PUBLIC HEALTH RELEVANCE: Chronic inflammation is central to many diseases, like cancer, diabetes, Alzheimer's and aging. We propose to study the molecular mechanism of inflammatory processes by studying the regulation of NF-?B a seminal molecule in the induction of inflammation.