Nitric Oxide Signaling in Hypoxia and Immunity in Drosophila

描述（由申请人提供）：缺氧是最重要的健康问题。它负责心肌梗死和中风引起的缺血损伤，并在限制肿瘤生长和减弱重要化疗的作用方面发挥核心作用。虽然我们对缺氧诱导因子（Hypoxia Inducible Factor， HIF）在缺氧条件下调控转录的机制有了一定的了解，但对缺氧的其他反应模式仍知之甚少。线粒体是细胞中主要的氧气消耗者，它如何适应氧气供应的不足，它如何向细胞的其他部分发出信号，要求适应这些不足？线粒体产生的活性氧被认为是将氧应激传递给细胞的责任，但信号产生、转导和作用的确切途径尚未建立。认识到问题的复杂性和问题的基本性质，似乎模式生物中强大的遗传学可以做出重大贡献。我们发现一氧化氮（NO）介导果蝇对缺氧的即时反应，包括胚胎在可逆的假死状态下的停止。我们致力于开发一个强大的和大大简化的实验环境，在这个环境中我们可以解剖no信号传导机制。我们发现NO调节果蝇的先天免疫反应，揭示了缺氧与免疫的联系，并为我们提供了我们所寻求的简化。免疫报告信号一氧化氮的作用，对缺氧的反应可以在培养细胞中重现，可适应强大的RNAi筛选。在本研究中，我们将结合体内研究和细胞培养模型分析来研究no介导的信号传导。体内研究将探讨感染如何诱导NOS，以及NO信号如何参与免疫反应。在果蝇S2细胞中，我们将研究缺氧诱导的NO信号的基础，该信号似乎起源于线粒体，并将追踪该信号在传递到细胞质时的转化和运输。最后，我们将定义线粒体感知缺氧并在线粒体内发出剧烈变化信号的基因和途径，以及线粒体变化与线粒体发出的信号之间的耦合，这些信号对细胞的生存和人类的福祉产生巨大影响。这些研究将使我们对缺氧反应的主要组成部分有一个机制上的了解，有助于理解对缺氧耐受性的生物多样性，并为我们提供操纵缺氧敏感性的方法，以潜在地对重要的健康问题有益。最常见的危及生命的健康问题，心脏梗死和中风，通过中断氧气供应造成损害，但我们对由此产生的急性缺氧的生物学反应知之甚少。我们将剖析对缺氧的反应机制在一个强大的模式遗传系统，果蝇。我们的发现有可能影响心脏梗死和中风的治疗，并可能对维持器官移植和癌症治疗等不同领域产生影响。

DESCRIPTION (provided by applicant): Hypoxia figures importantly in the biggest of health issues. It is responsible for the damage caused by the ischemia accompanying cardiac infarct and stroke, and it plays a central role in limiting tumor growth as well as blunting the actions of important chemotherapies. While we have a mechanistic understanding of hypoxic regulation of transcription by Hypoxia Inducible Factor (HIF), other modes of response to oxygen deprivation are still poorly understood. How does the mitochondrion, the major consumer of oxygen in the cell, adjust to shortfalls in oxygen supply, and how does it signal to the rest of the cell, demanding accommodations to these shortfalls? Reactive oxygen species produced by mitochondria have been ascribed the responsibility of communicating oxygen stress to the cell, but precise pathways of signal generation, transduction and action have not been established. Recognizing the complexity of the issues and the fundamental nature of the questions, it seems that the powerful genetics available in model organisms could make a major contribution. We found that nitric oxide (NO) mediates immediate responses to hypoxia in Drosophila, including an arrest of embryos in a reversible state of suspended animation. We have devoted ourselves to the development of a powerful and greatly simplified experimental context in which we can dissect the NO-signaling mechanisms. Our finding that NO regulates innate immune responses in Drosophila revealed a hypoxia-immunity connection, and gave us the simplification we sought. Immune reporters signal the action of NO, and the response to hypoxia can be recapitulated in cultured cells amenable to powerful RNAi screens. In this proposal, we will combine in vivo studies and analysis of cell culture models to investigate NO-mediated signaling. In vivo studies will probe how infection induces NOS, and how NO signaling contributes to the immune response. In Drosophila S2 cells, we will examine the basis of hypoxia-induced NO signaling that appears to originate in the mitochondria, and will trace the transformation and transport of this signal as it is conveyed to the cytoplasm. Finally, we will define the genes and pathways by which mitochondria sense hypoxia and signal the dramatic changes within the mitochondria, and will the coupling between mitochondrial changes and the signals emanating from the mitochondria that have such huge impacts on survival of cells and the well-being of man. These studies will give us a mechanistic understanding of a major component of the hypoxia response, contribute to the understanding of the biological diversity in tolerance to hypoxia, and give us approaches to manipulate hypoxia sensitivity to potentially benefit issues of important health concern. PUBLIC HEALTH RELEVANCE The most common life-threatening health problems, cardiac infarct and stroke, cause damage by interrupting oxygen supply, yet we understand little about the biological responses to the resulting acute hypoxia. We will dissect the mechanisms of response to hypoxia in a powerful model genetic system, Drosophila. Our findings have the potential to influence treatment of cardiac infarct and stroke, and could have an impact on areas as diverse as sustaining organs for transplantation, and cancer therapies.