NO induces osteopontin,a potent trans-repressor of iNOS

NO 诱导骨桥蛋白，一种有效的 iNOS 反式阻遏蛋白

• 批准号: 6456184
• 负责人: PAUL C KUO
• 金额: $ 25.87万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6456184
• 关键词: DNA binding protein; bacterial disease; blood toxicology; electrospray ionization mass spectrometry; endotoxins; enzyme inhibitors; gene expression; gene induction /repression; gene targeting; genetic library; genetic promoter element; genetic transcription; genetically modified animals; laboratory mouse; lipopolysaccharides; macrophage; messenger RNA; nitric oxide; nitric oxide synthase; osteopontin; protein biosynthesis; reporter genes; septic shock; site directed mutagenesis; tissue /cell culture; transcription factor; transfection /expression vector

In endotoxin (LPS)-mediated sepsis, inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production alter multiple functions, including cardiac contractility, vasomotor tone, intestinal epithelial permeability, and leukocyte recruitment. While the molecular pathways which upregulate iNOS in endotoxemia have been extensively characterized, little is known of the corresponding pathways which downregulate iNOS. Utilizing both in vivo murine and in vitro murine macrophage and rat hepatocyte models of LPS stimulation, we have demonstrated that NO feedback inhibits its own synthesis by increasing gene transcription and promoter activation of osteopontin (OPN), a potent trans-repressor of iNOS expression. This negative feedback pathway of NO-dependent OPN gene transcription and protein synthesis has not been previously described. We hypothesize that transcription of OPN, a potent trans-repressor of iNOS expression, is NO-dependent in LPS-stimulated murine macrophages. In the immortalized ANA-1 murine macrophage cell line, we propose to functionally map the OPN promoter in the context of LPS stimulated NO production. Specific Aim 1. To define NO-dependent cis-acting transcriptional control regions, we will utilize OPN promoter-reporter constructs with deletion analysis and site-directed mutagenesis. Specific Aim 2. To define NO-dependent trans-acting regulation, we will isolate the NO-dependent transcription factor and its cDNA clone by using the biotin-strepavidin affinity method and screening a cDNA expression library, respectively, using the DNA recognition site as a probe. Specific Aim 3. To determine the S-nitrosylation status of our transcription factor and its effect upon DNA binding, we will use CuCl-cysteine coupled chemiluminescence. Specific Aim 4. To confirm relevancy in the LPS-stimulated macrophage, we will inhibit translation of the transcription factor mRNA with antisense techniques and alternatively, over- express the transcription factor by transient transfection. Specific Aim 5. To confirm in vivo relevancy, we will utilize a murine OPN-knockout model of endotoxemia to demonstrate lack of iNOS inhibition in the absence of OPN. Our studies will define OPN production as a unique and as yet, poorly characterized, NO- dependent transcriptional pathway which inhibits iNOS expression in the setting of endotoxemia.

在内毒素 (LPS) 介导的脓毒症中，诱导型一氧化氮合酶 (iNOS) 表达和一氧化氮 (NO) 产生改变多种功能，包括心肌收缩力、血管舒缩张力、肠上皮通透性和白细胞募集。 虽然在内毒素血症中上调 iNOS 的分子途径已得到广泛表征，但对下调 iNOS 的相应途径知之甚少。利用LPS刺激的体内小鼠和体外小鼠巨噬细胞和大鼠肝细胞模型，我们证明NO反馈通过增加基因转录和骨桥蛋白（OPN）的启动子激活来抑制其自身合成，骨桥蛋白是iNOS表达的有效反式阻遏蛋白。 这种依赖NO的OPN基因转录和蛋白质合成的负反馈途径以前从未被描述过。 我们假设 OPN（iNOS 表达的有效反式阻遏蛋白）的转录在 LPS 刺激的小鼠巨噬细胞中是 NO 依赖性的。 在永生化 ANA-1 鼠巨噬细胞系中，我们建议在 LPS 刺激 NO 产生的背景下对 OPN 启动子进行功能定位。 具体目标 1. 为了定义 NO 依赖性顺式作用转录控制区，我们将利用 OPN 启动子-报告基因构建体进行删除分析和定点诱变。 具体目标 2. 为了定义 NO 依赖性反式作用调控，我们将使用生物素-链霉亲和素亲和力方法分离 NO 依赖性转录因子及其 cDNA 克隆，并使用 DNA 识别位点作为探针筛选 cDNA 表达文库。 具体目标 3. 为了确定我们的转录因子的 S-亚硝基化状态及其对 DNA 结合的影响，我们将使用 CuCl-半胱氨酸偶联化学发光。具体目标 4. 为了确认 LPS 刺激的巨噬细胞的相关性，我们将使用反义技术抑制转录因子 mRNA 的翻译，或者通过瞬时转染过度表达转录因子。具体目标 5. 为了确认体内相关性，我们将利用内毒素血症的鼠 OPN 敲除模型来证明在不存在 OPN 的情况下缺乏 iNOS 抑制。 我们的研究将把 OPN 的产生定义为一种独特的、目前尚未充分表征的 NO 依赖性转录途径，在内毒素血症的情况下抑制 iNOS 的表达。