REGULATION OF THE HUMAN INOS GENE IN SEPSIS AND TRAUMA

脓毒症、创伤和感染引发由细胞因子和其他炎症介质调节的全身反应。 在极端情况下，这种反应可能会发展为多器官衰竭，这是手术患者死亡的主要原因。我们对这种综合征的理解的进展是基于发现诱导型一氧化氮合酶（iNOS）基因在脓毒症反应期间几乎在每个器官和组织中表达。 虽然NO合成在急性炎症期间具有有益作用，但是在脓毒症期间NO的过度产生可能是有害的，具有大量血管舒张和低血压。 iNOS的慢性表达与NO介导的细胞毒性有关，导致糖尿病、关节炎、神经退行性疾病和某些癌症。 我们的实验室已经克隆了人iNOS基因从奎宁刺激的人肝细胞。 然后，我们分离了人iNOS基因的启动子区，并表明对精氨酸敏感的DNA元件位于~ 4.7kb的上游。 最近，我们发现了一个新的NF-κ B增强子区域，该区域调节iNOS对TNF α或IL-1 β的转录，并显示了STAT 1 α在介导IFN γ诱导中的作用。 我们还鉴定了下调iNOS表达的细胞外信号，包括p53肿瘤抑制蛋白、类固醇、热休克、某些生长因子和NO本身。 由于人类iNOS基因受到严格的调控，我们假设有几种机制共同控制该基因的表达。 我们预测，这将包括尼古丁刺激的核因子，发挥积极或消极的控制转录，以及转录后机制，调节iNOS mRNA的稳定性和翻译效率。在这项建议中，我们将追求两个相互关联的具体目标。目的I：确定诱导和抑制人iNOS基因的转录机制和功能启动子元件。 将在转染实验中对另外的启动子区进行完全测序和表征。 将分析细胞因子反应性所需的DNA元件，最初关注NF-κ B和STAT 1 α之间的相互作用。 p53，类固醇，热休克，TGF-β和NO基因抑制的具体机制将被寻找。 将通过凝胶位移和体内足迹测定法鉴定阳性和阴性转录。 AIM II .目的：探讨人iNOS基因转录后的调控机制。 mRNA稳定性或翻译效率的转录后变化也可以调节基因表达。 将检测细胞因子对人iNOS mRNA稳定性的影响。 将通过脉冲追踪实验测量响应于这些试剂的翻译效率的变化。 将分析人iNOS基因的3 '-非翻译区中介导这些效应的元件。 在我们的研究完成后，我们将有特点的人iNOS基因的分子调控。 所获得的信息将增加我们对iNOS转录控制的理解，描述新的信号转导中的精氨酸协同作用机制，并帮助设计用于细胞因子表达相关的病理生理疾病状态的治疗策略。

Sepsis, trauma, and infection initiate systemic responses regulated by cytokines and other inflammatory mediators. In extreme conditions, this response can progress to multiple organ failure, a major cause of mortality in surgical patients. Advances in our understanding of this syndrome are based on the discovery that the inducible nitric oxide synthase (iNOS) gene is expressed in nearly every organ and tissue during the septic response. While NO synthesis has beneficial effects during acute inflammation, over-production of NO during sepsis can be detrimental with massive vasodilation and hypotension. Chronic expression of iNOS has been implicated in NO-mediated cytotoxicity leading to diabetes, arthritis, neurode generative disorders, and certain cancers. Our laboratory has cloned the human iNOS gene from cytokine-stimulated human hepatocytes. We then isolated the promoter region of the human iNOS gene and have shown that the cytokine-responsive DNA elements are located upstream from -4.7 kb. Recently we have characterized a novel NF-kappaB enhancer region that regulates iNOS transcription in response to TNFalpha or IL-1beta, and have shown a role for STAT1alpha in mediating IFNgamma induction. We have also identified extracellular signals that down-regulate iNOS expression including p53 tumor suppressor protein, steroids, heat shock, certain growth factors, and NO itself. Since the human iNOS gene is tightly regulated, we hypothesize that several mechanisms are working jointly to control the expression of this gene. We predict that this will include cytokine-stimulated nuclear factors that exert either positive or negative control over transcription, as well as post-transcriptional mechanisms that regulate iNOS mRNA stability and translational efficiency. In this proposal, we will pursue two interrelated specific aims. AIM I: TO DEFINE THE TRANSCRIPTIONAL MECHANISMS AND FUNCTIONAL PROMOTER ELEMENTS RESPONSIBLE FOR INDUCTION AND SUPPRESSION OF THE HUMAN iNOS GENE. Additional promoter regions will be fully sequenced and characterized in transfection experiments. DNA elements will be analyzed that are required for cytokine- responsiveness, with an initial focus on the interactions between NF-kappaB and STAT1alpha. Specific mechanisms for gene suppression by p53, steroids, heat shock, TGF-beta, and NO will be sought. Positive and negative transcription will be identified by gel shifts and in vivo footprinting assays. AIM II . TO DETERMINE THE POST-TRANSCRIPTIONAL MECHANISMS INVOLVED IN REGULATION OF THE HUMAN iNOS GENE. Post-transcriptional changes in mRNA stability or translational efficiency can also regulate gene expression. Cytokines will be tested for effects on human iNOS mRNA stability. Changes in translational efficiency in response to these agents will be measured by pulse-chase experiments. The 3'-untranslated region of the human iNOS gene will be analyzed for elements that mediate these effects. At the completion of our studies, we will have characterized the molecular regulation of the human iNOS gene. The information gained will increase our understanding of the control of iNOS transcription, describe novel mechanisms of cytokine-synergy in signal transduction, and help in designing therapeutic strategies for pathophysiological disease states where cytokine expression is relevant.