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: 6390420
• 负责人: Walter Keith Jones
• 金额: $ 27.95万
• 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/6390420
• 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.

本提案的总体目标是研究预适应（PC）后期的分子机制。我们将尝试建立一个统一的致病范式，适用于缺血诱导的晚期PC和一氧化氮供体诱导的晚期PC。我们的基本假设是，在这两种情况下，负责晚期PC的中心细胞适应是iNOS基因的转录上调。我们认为iNOS的转录是由NF-kappaB和其他转录因子的协同激活介导的，而NC通过包括PKC、酪氨酸激酶和IkappaB激酶（IKK）在内的信号转导级联触发。两种不同形式的晚期PC（缺血应激诱导的PC和NO释放剂诱导的PC）将被系统地检查。支持或反对编码转录和信号因子的特定基因参与的明确证据将通过在成熟的晚期PC小鼠模型中使用基因靶向和转基因小鼠来提供。将采用广泛的多学科方法，结合多种技术（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），并将分子水平的遗传信息与整个动物水平的生理信息相结合。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），首次提供小鼠这些变化的全面表征。通过确定NOS、PKC、酪氨酸激酶和nf - κ b药物抑制剂对梗死面积的影响，并将这些影响与它们对iNOS转录的影响联系起来，研究控制iNOS表达的信号机制。我们将首次研究PC对IKK复合物（控制NF-kappaB）磷酸化活性的影响。NF-kappaB在晚期PC中的作用将通过靶向NF-kappaB三个特定亚基（p50, Rel-B, c-rel）的基因消融和IKK/IkappaB/NF-kappaB信号通路的遗传阻断来最终确定。两个新的跨显性NF- kappaB突变体（IkappaBalpha/S32A， S36A和IKK-beta/k44A）将被用来确定IKK和IkappaBalpha在iNOS上调中的作用。通过靶向基因消融已知与iNOS启动子结合的转录因子（IRF-1、TNFalpha、STAT1、CREB、AP-1、IL-2、IL-6），系统地鉴定出在PC晚期控制iNOS基因表达的特定调节蛋白。这一建议将为晚期PC的分子机制以及NO和iNOS在心血管病理生理中的作用提供重要的新见解，阐明晚期PC的机制将有助于开发新的药理学和/或基因治疗策略，以复制其强大的心脏保护作用。