NITRIC OXIDE (NO) DEPENDENT ACTIVATION OF INOS IN LATE P

P 晚期 INOS 的一氧化氮 (NO) 依赖性激活

• 批准号: 6700441
• 负责人: Walter Keith Jones
• 金额: $ 2.24万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6700441
• 关键词: cardiovascular pharmacology; cytoprotection; enzyme activity; enzyme induction /repression; gene targeting; genetic transcription; genetically modified animals; immunocytochemistry; isozymes; laboratory mouse; myocardial infarct sizing; myocardial ischemia /hypoxia; myocardium; nitric oxide; nitric oxide synthase; nuclear factor kappa beta; polymerase chain reaction; protein kinase C; southern blotting; western blottings

The overall objective of this proposal is to investigate the molecular mechanisms underlying the late phase of preconditioning (PC). We will attempt to develop a unifying pathogenic paradigm applicable both to ischemia-induced late PC and to NO donor-induced late PC. our fundamental hypothesis is that, in both cases, the central cellular adaptation is responsible for late PC is the transcriptional up- regulation of the iNOS gene. We propose that transcription of iNOS is mediated by the coordinated activation of NF-kappaB and other transcription factors, which is triggered by NC via a signal transduction cascade that includes PKC, tyrosine kinases, and IkappaB kinase (IKK). Two different forms of late PC (PC induced by ischemic stress and PC induced pharmacologically with NO releasing agents) will be systematically examined. Unequivocal evidence for or against the involvement of specific genes encoding transcription and signaling factors will be provided by the use of gene targeted and transgenic mice in a well-established murine model of late PC. A broad multi- disciplinary approach will be used that will combine diverse techniques (integrative physiology, molecular biology, protein chemistry, cell biology, gene targeting and transgenesis) and will integrate genetic information at the molecular level with physiological information at the whole animal level. The effects of PC upon NOS mRNA, protein, enzymatic activity, and cellular distribution will be systematically defined for all three isoforms (eNOS, iNOS, and nNOS), providing for the first time a thorough characterization of these changes in the mouse. The signaling mechanisms that control iNOS expression will be interrogated by determining the effect of pharmacologic inhibitors of NOS, PKC, tyrosine kinases, and NF-kappaB on infarct size and by correlating these effects with their effects on iNOS transcription. For the first time, the effect of PC on the phosphorylation activity of the IKK complex (which controls NF-kappaB) will be examined. The role of NF-kappaB in late PC will be conclusively established by targeted gene ablation of three specific NF- kappaB subunits (p50, Rel-B, c-rel) and by genetic blockade of the IKK/IkappaB/NF-kappaB signaling pathway. Two novel transdominant NF- kappaB mutants (IkappaBalpha/S32A, S36A and IKK-beta/k44A) will be used to determine the role of IKK and IkappaBalpha in iNOS up-regulation. The specific modulatory proteins that govern iNOS gene expression during late PC will be systematically identified by targeted genetic ablation of the transcription factors known to bind to the iNOS promoter (IRF-1, TNFalpha, STAT1, CREB, AP-1, IL-2, IL-6). This proposal should provide important new insights into the molecular mechanisms of late PC and into the role of NO and iNOS in cardiovascular pathophysiology in general Elucidation of the mechanism of late PC should facilitate the development of novel pharmacological and/or gene therapeutic strategies that duplicate its powerful cardioprotective effects.

该建议的总体目标是研究预处理（PC）后期的分子机制。我们将尝试开发一个统一的致病模式适用于缺血诱导的晚期PC和NO供体诱导的晚期PC。我们的基本假设是，在这两种情况下，中枢细胞的适应是负责晚期PC是转录上调的iNOS基因。我们认为iNOS的转录是由NF-κ B和其他转录因子的协同激活介导的，而NF-κ B和其他转录因子的协同激活是由NC通过包括PKC、酪氨酸激酶和IkappaB激酶（IKK）的信号转导级联反应触发的。将系统地检查两种不同形式的晚期PC（由缺血应激诱导的PC和用NO释放剂诱导的PC）。通过在成熟的晚期PC小鼠模型中使用基因靶向和转基因小鼠，将提供支持或反对参与编码转录和信号传导因子的特定基因的明确证据。将采用广泛的多学科方法，将联合收割机各种技术（综合生理学、分子生物学、蛋白质化学、细胞生物学、基因靶向和转基因）结合起来，并将分子水平的遗传信息与整个动物水平的生理信息结合起来。PC对NOS mRNA，蛋白质，酶活性和细胞分布的影响将被系统地定义为所有三种亚型（eNOS，iNOS和nNOS），首次提供了这些变化在小鼠中的彻底表征。控制iNOS表达的信号传导机制将通过确定NOS、PKC、酪氨酸激酶和NF-κ B的药理学抑制剂对梗死面积的影响以及通过将这些影响与它们对iNOS转录的影响相关联来探究。首次检测PC对IKK复合物（控制NF-κ B）磷酸化活性的影响。NF-κ B在晚期PC中的作用将通过三种特异性NF-κ B亚基（p50、Rel-B、c-rel）的靶向基因消除和IKK/IkappaB/NF-κ B信号传导途径的遗传阻断来最终确定。两种新的反式显性NF-κ B突变体（IkappaB α/S32 A、S36 A和IKK-β/k44 A）将用于确定IKK和IkappaB α在iNOS上调中的作用。将通过已知与iNOS启动子结合的转录因子（IRF-1、TNF α、STAT 1、CREB、AP-1、IL-2、IL-6）的靶向基因消融，系统地鉴定晚期PC期间控制iNOS基因表达的特异性调节蛋白。该提案应为晚期PC的分子机制以及NO和iNOS在心血管病理生理学中的作用提供重要的新见解。晚期PC机制的阐明应促进新型药理学和/或基因治疗策略的开发，以复制其强大的心脏保护作用。