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: 6607114
• 负责人: Walter Keith Jones
• 金额: $ 34.1万
• 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/6607114
• 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.

该提案的总体目的是研究预处理后期（PC）的分子机制。我们将尝试开发一种适用于缺血引起的PC且没有供体诱导的晚期PC的统一的致病范式。我们的基本假设是，在这两种情况下，中央细胞适应性均导致PC后期是iNOS基因的转录上调节。我们提出，iNOS的转录是由NF-kappab和其他转录因子的协调激活介导的，NC通过包括PKC，酪氨酸激酶和Ikappab激酶（IKK）的信号转导级联触发。将系统检查两种不同形式的PC（由缺血性应力和PC诱导的PC诱导，没有释放剂）。在已建立的PC的公认的鼠模型中，使用靶向基因和转基因小鼠的基因和转基因小鼠的使用明确的证据或反对编码转录和信号传导因子的特定基因的明确证据。将使用一种广泛的多学科方法，该方法将结合多种技术（综合生理学，分子生物学，蛋白质化学，细胞生物学，基因靶向和转离），并将在分子水平将遗传信息与整个动物水平的生理信息整合在一起。 PC对NOS mRNA，蛋白质，酶活性和细胞分布的影响将针对所有三种同工型（ENOS，INOS和NNOS）系统定义，从而首次对小鼠中这些变化进行彻底表征。通过确定NOS，PKC，酪氨酸激酶和NF-kappab对梗塞大小的药理抑制剂的影响，通过确定控制iNOS表达的信号传导机制将受到询问，并通过将这些影响与对INOS转录的影响相关联。将首次检查PC对IKK复合物（控制NF-kappab）的磷酸化活性的影响。 NF-kappab在晚期PC中的作用将通过三个特定的NF-KAPPAB亚基（P50，REL-B，C-REL）以及IKK/IKK/IKK/IKAPAB/NF-KAPPAB信号通路的遗传阻断的靶向基因消融最终确定。将使用两个新型的跨性别nf-kappab突变体（Ikappabalpha/s32a，s36a和ikk-beta/k44a）来确定IKK和IKAPPABALPHA在INOS上调中的作用。在PC晚期期间控制INOS基因表达的特定调节蛋白将通过已知与INOS启动子结合的转录因子的靶向遗传消融系统鉴定（IRF-1，TNFALPHA，STAT1，CREB，AP-1，IL-2，IL-2，IL-6）。该提案应提供对晚期PC的分子机制的重要新见解，并为NO和INOS在心血管病理生理学中的作用，从而将后期PC机理的一般阐明，应促进新型药理和/或基因治疗策略的发展，以重复其强大的心脏保护作用。