REGULATION OF THE HUMAN INOS GENE IN SEPSIS AND TRAUMA

败血症、创伤和损伤合并感染都会引发全身性感染 由细胞因子和其他炎症激素介导的反应释放 宿主细胞与诸如革兰氏阴性的药物相互作用后 细菌脂多糖(LPS)。在极端条件下，这种反应 可进展为细胞功能障碍和多器官衰竭，这是一个主要原因 外科危重病人的死亡率。我们的最新进展 对这种综合征的理解是基于对一氧化氮的发现 在脓毒症期间，许多组织中诱导合成一氧化氮(NO)。 回应。诱导型一氧化氮合酶(INOS)基因只是 暴露于某些细胞因子组合后在细胞中的表达 和/或内毒素。诱导的NO合成既有好处也有坏处 后果。我们已经从受刺激的人中克隆了iNOS基因 肝细胞，并发现它最大限度地表达对 脂多糖与细胞因子TNFa、IL-1b和IFNG的协同作用。我们 假设人诱导型一氧化氮合酶基因的分子调控 复杂，将涉及转录和转录后 机械装置。在这项提案中，我们将追求两个相互关联的具体 目的研究人诱导型一氧化氮合酶基因的调控表达。目标一：目标 确定转录机制和功能启动子元件 负责人类细胞因子和内毒素的诱导 Inos基因。我们将从在脂多糖中进行核模拟实验开始。 和细胞因子刺激的人肝细胞确认和量化 转录诱导。人iNOS基因启动子区域将 转基因中的分离、测序和功能特征 实验。将分析特定的DNA元素，这些元素是 内毒素和细胞因子反应性。正转录和负转录 这些因素将在凝胶位移结合分析中确定。最后， 体内可诱导启动子元件的特征将是 通过构建表达人iNOS的转基因小鼠来实现 启动子融合基因。目的II：确定转录后基因 人诱导型一氧化氮合酶基因的调控机制邮寄- 转录机制也可以通过改变基因表达来调节基因的表达 信使核糖核酸稳定性或翻译效率。我们将确定是否有内毒素或 细胞因子通过增加mRNA量增加稳态iNOS mRNA量 稳定性。翻译效率的变化对这些代理的响应 将通过脉冲追逐实验来测量。诱导型一氧化氮的抑制剂 将测试合成对信使核糖核酸稳定性和翻译的影响。 最后，如果mRNA稳定性或翻译效率的变化是 观察到，我们将分析iNOS基因的3‘-非翻译区 调节这些影响的元素。在我们完成学业后，我们 将已经表征了人类iNOS基因的分子调控 对内毒素和/或细胞因子的反应。这些信息将提供重要的 对细胞因子和其他炎症性物质如何 介体引导高调控基因在损伤和 败血症外科病人。

Sepsis, trauma, and injury complicated by infection all initiate systemic responses mediated by cytokines and other inflammatory hormones released following interaction of host cells with agents such as gram negative bacterial lipopolysaccharide (LPS). In extreme conditions, this response can progress to cell dysfunction and multiple organ failure, a major cause of mortality in critically ill surgical patients. Recent advances in our understanding of this syndrome are based on the discovery that nitric oxide (NO) synthesis is induced in a number of tissues during the septic response. The inducible nitric oxide synthase (iNOS) gene is only expressed in cells after exposure to certain combinations of cytokines and/or LPS. Induced NO synthesis can have both beneficial and detrimental consequences. We have cloned the iNOS gene from stimulated human hepatocytes and found that it is maximally expressed in response to the synergistic actions of LPS and cytokines TNFa, IL-1b, and IFNg. We hypothesize that the molecular regulation of the human iNOS gene is complex and will involve transcriptional as well as post-transcriptional mechanisms. In this proposal, we will pursue two interrelated specific aims to study the regulated expression of the human iNOS gene. AIM I: TO DEFINE THE TRANSCRIPTIONAL MECHANISMS AND FUNCTIONAL PROMOTER ELEMENTS RESPONSIBLE FOR CYTOKINE AND LIPOPOLYSACCHARIDE INDUCTION OF THE HUMAN iNOS GENE. We will begin by performing nuclear run-on experiments in LPS and cytokine-stimulated human hepatocytes to confirm and quantify transcriptional induction. The human iNOS gene promoter region will isolated, sequenced, and functionally characterized in transfection experiments. Specific DNA elements will be analyzed that are required for LPS and cytokine-responsiveness. Positive and negative transcription factors will be identified in gel shift binding assays. Finally, characterization of the inducible promoter elements in vivo will be carried out by constructing transgenic mice expressing a human iNOS promoter fusion gene. AIM II: TO DETERMINE THE POST-TRANSCRIPTIONAL MECHANISMS INVOLVED IN THE REGULATION OF THE HUMAN iNOS GENE. Post- transcriptional mechanisms can also regulate gene expression by changes in mRNA stability or translational efficiency. We will determine if LPS or cytokines increase steady state iNOS mRNA levels by increasing mRNA stability. Changes in translational efficiency in response to these agents will be measured by pulse-chase experiments. Inhibitors of induced NO synthesis will be tested for effects on mRNA stability and translation. Finally, if changes in mRNA stability or translational efficiency are observed, we will analyze the 3'-untranslated region of the iNOS gene for elements that mediate these effects. At the completion of our studies, we will have characterized the molecular regulation of the human iNOS gene in response to LPS and/or cytokines. This information will provide important insights into the mechanisms of how cytokines and other inflammatory mediators direct the expression of highly regulated genes in injured and septic surgical patients.