Role of P53 in LITAF Process

描述(申请人提供)：炎症是一种复杂但必不可少的先天免疫防御机制。在损伤部位，包括细胞因子(如肿瘤坏死因子、IL-1、IL-6、单核细胞趋化蛋白-1、血管内皮生长因子)、二十烷基类化合物和活性氧中间体在内的一系列介质激活了附近的炎症细胞，如巨噬细胞和粒细胞。炎症过程中细胞因子的调节尤其重要，因为细胞因子失调与几种疾病(如I型糖尿病、动脉粥样硬化、类风湿性关节炎、克罗恩病)有关。1998年，我们实验室发现了一种新的转录因子--脂多糖诱导的肿瘤坏死因子-α因子(LITAF)，它参与了对肿瘤坏死因子和其他促炎细胞因子基因的转录调控。分别利用基因表达序列分析鉴定了一系列P53诱导的基因。在这些基因中发现了LITAF。我们的初步数据指向人类LITAF启动子中的一个特定位置，这是p53蛋白与DNA结合所必需的。瞬时转染名为p53LFB164的p53突变DNA构建体或其相应的合成肽，可降低LITAF启动子的活性并下调LITAF的表达。此外，将该多肽引入人单核细胞，可下调包括肿瘤坏死因子-1在内的9种细胞因子的表达。由于肿瘤坏死因子-1和LITAF在人类炎症性疾病(如牙周病、炎症性肠病、克罗恩病)中都发挥着重要作用，阐明P53和LITAF介导的炎症过程的机制可能会为这些或其他炎症性疾病提供新的治疗方法。为了解决这一重要问题，提出了2个目标：在目标1中，将构建缺失LITAF启动子DNA(PMLFP)和一系列pcMp53突变的DNA。为了检测小鼠LITAF启动子的活性是否受小鼠p53的调节，将pMLFP(或其缺失构建体)和pcMp53(或其突变构建体)共转染p53缺失的细胞。此外，EMSA和DNase I足迹将被用来确定P53与LITAF启动子相互作用的特定区域。在目标2中，我们将充分研究小鼠P53短肽的功能，它作为一种抑制因子，在体外和体内下调LITAF介导的炎症细胞因子的产生，以响应脂多糖(LPS)的刺激。合成小鼠P53短肽，并将其与DNA共转染入P53缺失细胞，用于多肽依赖启动子的检测。此外，多肽将被输送到脂多糖暴露的细胞中，并分析培养上清液中细胞因子的表达。最后，在注射内毒素后不久，将小鼠静脉注射P53多肽，并充分分析其对全身内毒素治疗的影响。这些动物研究的数据将被用来验证我们的体外观察，并使我们能够设计药物治疗方法。与公共卫生的相关性：在各种慢性病和癌症的发展中，一个共同的组成部分是炎症过程。目前的工作建议调查P53和炎症之间的新联系，并测试一种潜在的新的干预方法来控制P53相关的炎症过程。此外，由于P53的丢失或失活在癌症的发展中起着关键作用，了解P53和炎症过程之间的联系可能有助于揭示慢性炎症和癌症发生之间已建立但尚未完全了解的联系。

DESCRIPTION (provided by applicant): Inflammation is a complex but essential innate immune defense mechanism. At the site of injury, a cascade of mediators, including cytokines (e.g. TNF, IL-1, IL-6, MCP-1, VEGF), eicosanoids, and reactive oxygen intermediates activate nearby inflammatory cells such as macrophages and granulocytes. The regulation of cytokines during inflammation is particularly critical as cytokine dysregulation is implicated in several diseases (e.g. type I diabetes, atherosclerosis, rheumatoid arthritis, Crohn's disease). In 1998 our laboratory discovered a new transcription factor, Lipopolysaccharide-Induced TNF-Alpha Factor (LITAF), which contributes to the transcriptional regulation of TNF and other proinflammatory cytokine genes. Separately, using serial analysis of gene expression a series of p53-induced genes were identified. LITAF was identified among these genes. Our preliminary data point to a specific site within the human LITAF promoter required for p53 protein-DNA binding. Transient transfection of either a p53 mutant DNA construct, called p53LFB164, or its corresponding synthetic peptide, reduced LITAF promoter activity and down-regulated LITAF expression. Furthermore, introduction of this peptide into human monocytes resulted in down-regulation of 9 cytokines including TNF-1. Since both TNF-1 and LITAF play important roles in human inflammatory diseases (i.e. Periodontal Disease, inflammatory bowel disease, Crohn's disease), clarification of the mechanism linking p53 and LITAF-mediated inflammatory processes may lead to novel therapeutic approaches for these or other inflammatory diseases. To address this important issue 2 aims are proposed: In Aim 1, deletions of LITAF promoter DNA (pMLFP) and a series of pcMp53 mutated DNAs will be constructed. To examine whether mouse LITAF promoter activity is regulated by mouse p53, p53-null cells will be co-transfected with pMLFP (or its deletion constructs) and pcMp53 (or its mutation constructs). Furthermore, EMSA and DNase I footprinting will be performed to determine the specific regions of p53 which interacts with the LITAF promoter. In Aim 2, we will fully characterize the mouse p53 short peptide that functions as an inhibitor to down-regulate LITAF-mediated in vitro and in vivo inflammatory cytokine production in response to lipopolysaccharide (LPS) stimulation. Mouse p53 short peptide will be synthesized and transfected or cotransfected with DNAs into p53-null cells for peptide-dependent promoter assays. In addition, peptides will be delivered into LPS-exposed cells, and the culture supernatants analyzed for cytokine expression. Finally, shortly after LPS injection, mice will be treated intravenously with p53 peptide and its effect on systemic LPS treatment will be fully analyzed. The data from these animal studies will be used to validate our in vitro observations and enable us to design pharmacotherapeutic approaches. Public Health Relevance: A common component in various chronic diseases and cancer development is the inflammatory process. The present work proposes to investigate a novel link between p53 and inflammation, and to test a potentially novel interventional approach to control p53-associated inflammatory processes. In addition, since loss or inactivation of p53 plays a key role in cancer development, understanding a link between p53 and the inflammatory process might shed light onto the established but incompletely understood connection between chronic inflammation and carcinogenesis.