Role of P53 in LITAF Process

P53 在 LITAF 过程中的作用

• 批准号: 7654785
• 负责人: XIAOREN TANG
• 金额: $ 20.31万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-05 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7654785
• 关键词: Address; Atherosclerosis; Binding Sites; Biological Assay; Blood Vessels; CCL2 gene; Cell Culture Techniques; Cells; Chronic; Chronic Disease; Complex; Crohn' s disease; Cytokine Gene; DNA; DNA-Binding Proteins; DNase-I Footprinting; Data; Defense Mechanisms; Development; Disease; Down-Regulation; EMSA; Eicosanoids; Gene Expression; Genes; Human; Immune; In Vitro; Inflammation; Inflammatory; Inflammatory Bowel Diseases; Injection of therapeutic agent; Injury; Insulin-Dependent Diabetes Mellitus; Interleukin-1; Interleukin-6; Laboratories; Lead; Length; Leukocytes; Light; Link; Lipopolysaccharides; Malignant Neoplasms; Manuscripts; Mediating; Mediator of activation protein; Mus; Mutate; Mutation; Necrosis; Null Lymphocytes; Pattern; Peptides; Periodontal Diseases; Play; Process; Production; Protein p53; Regulation; Rheumatoid Arthritis; Role; Sepsis; Series; Site; Structure; TNF gene; TP53 gene; Techniques; Testing; Tissues; Transcriptional Regulation; Transfection; Tumor Necrosis Factor-alpha; Ulcerative Colitis; Vascular Endothelial Growth Factors; Work; animal data; carcinogenesis; cytokine; design; granulocyte; human TNF protein; human disease; in vitro testing; in vivo; inhibitor/antagonist; macrophage; monocyte; mutant; novel; novel therapeutic intervention; promoter; public health relevance; reactive oxygen intermediate; repaired; response; serial analysis of gene expression; synthetic peptide; transcription factor

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.

描述（由申请人提供）：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（例如TNF、IL-1、IL-6、MCP-1、VEGF）、类二十烷酸和活性氧中间体的介体级联激活附近的炎性细胞，例如巨噬细胞和粒细胞。炎症期间细胞因子的调节是特别关键的，因为细胞因子失调涉及几种疾病（例如I型糖尿病、动脉粥样硬化、类风湿性关节炎、克罗恩病）。1998年，我们的实验室发现了一种新的转录因子，脂多糖诱导的TNF-α因子（LITAF），它有助于TNF和其他促炎细胞因子基因的转录调节。单独地，使用基因表达的系列分析鉴定了一系列p53诱导的基因。在这些基因中鉴定出LITAF。我们的初步数据指向p53蛋白-DNA结合所需的人LITAF启动子内的特定位点。瞬时转染p53突变DNA构建体，称为p53 LFB 164，或其相应的合成肽，降低LITAF启动子活性和下调LITAF表达。此外，将该肽引入人单核细胞导致包括TNF-1在内的9种细胞因子的下调。由于TNF-1和LITAF在人类炎性疾病（即，牙周病、炎性肠病、克罗恩病）中起重要作用，因此阐明连接p53和LITAF介导的炎性过程的机制可能导致用于这些或其他炎性疾病的新的治疗方法。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LITAF启动子DNA（pMLFP）的缺失和一系列pcMp 53突变DNA。为了检查小鼠LITAF启动子活性是否受小鼠p53调节，将p53缺失细胞与pMLFP（或其缺失构建体）和pcMp 53（或其突变构建体）共转染。此外，将进行EMSA和DNA酶I足迹法以确定与LITAF启动子相互作用的p53的特定区域。在目标2中，我们将充分表征小鼠p53短肽，其作为抑制剂下调LITAF介导的体外和体内炎症细胞因子产生，以响应脂多糖（LPS）刺激。将合成小鼠p53短肽，并将其转染或与DNA共转染到p53缺失细胞中，用于肽依赖性启动子测定。此外，将肽递送至暴露于LPS的细胞中，并分析培养物上清液的细胞因子表达。最后，在LPS注射后不久，用p53肽静脉内处理小鼠，并充分分析其对全身LPS处理的影响。来自这些动物研究的数据将用于验证我们的体外观察结果，并使我们能够设计药理学方法。公共卫生相关性：各种慢性疾病和癌症发展的一个共同组成部分是炎症过程。目前的工作建议调查p53和炎症之间的一种新的联系，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发展中起着关键作用，因此了解p53与炎症过程之间的联系可能会揭示慢性炎症与癌症发生之间已建立但尚未完全理解的联系。