Integrated miRNA regulation of Nox4 and cellular redox state in vascular disease

血管疾病中 Nox4 和细胞氧化还原状态的整合 miRNA 调节

• 批准号: 9316697
• 负责人: FRANCIS J MILLER
• 金额: $ 29.99万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-18 至 2020-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9316697
• 关键词: Apoptosis; Biological; Blood Vessels; Cardiovascular Diseases; Carotid Arteries; Cell Differentiation process; Cell physiology; Cells; Cellular biology; Couples; Data; Development; Differentiation Antigens; Disease; Disulfides; Endoplasmic Reticulum; Event; Foundations; Future; Gene Expression; Genes; Genetic Transcription; Glutathione; Goals; Homeostasis; Human; Hydrogen Peroxide; Hyperplasia; Individual; Inflammation; Injury; Ion Channel; Malignant Neoplasms; Mediating; Messenger RNA; MicroRNAs; Mitochondria; Molecular; Muscle Cells; NADPH Oxidase; Organelles; Oxidants; Oxidases; Oxidation-Reduction; Oxidative Stress; Phenotype; Pilot Projects; Protein Isoforms; Proteins; RNA Splicing; Reaction; Reactive Oxygen Species; Receptor Protein-Tyrosine Kinases; Regulation; Reporting; Research; Second Messenger Systems; Signal Transduction; Signal Transduction Pathway; Smooth Muscle Myocytes; Sulfhydryl Compounds; Sum; System; Testing; Vascular Diseases; Vascular Smooth Muscle; Work; in vivo; innovation; insight; novel; oxidation; programs; public health relevance; response; response to injury; senescence; therapeutic target

 DESCRIPTION (provided by applicant): It is the integration of both redox signaling and the intracellular redox state that determines cellular response by coordinating multiple signaling networks. The long-term goal of our research program is to understand how NADPH oxidases can be manipulated to treat cardiovascular diseases. We have reported that Nox4 NADPH oxidase modifies diverse cellular responses. However, the molecular mechanisms by which Nox4 regulate cellular functions are poorly understood. Nox4 is expressed in cellular organelles whose functions are susceptible to changes in the redox potential. In contrast to the other Nox homologs, it is unlikely that Nox4-derived reactive oxygen species function as second messengers since 1) Nox4 is constitutively active; 2) activation of Nox4 is primarily regulated by its expression level; and 3) the reaction between thiols and H2O2 (the primary product of Nox4) is too slow to be of biological relevance. In pilot studies, we made the novel observations that 1) microRNA-9 (miR-9) is a novel regulator of Nox4 expression; 2) miR-25 induces expression of miR-9; 3) miR-9 elicits changes in Nox4 mRNA splicing; 4) miR-25 and miR-9 levels increase following vascular injury; and 5) changes in Nox4 levels modify the cellular glutathione redox potential and protein thiol status. We therefore hypothesize that miRNA-mediated changes in Nox4 expression dynamically regulate the cellular redox state and coordinate the expression of genes implicated in the development of vascular disease. Aim 1 will test the hypothesis that miR-induced miR expression mediates changes in Nox4 levels, cellular localization, and SMC activation. We will first determine the mechanism by which both miR-25 and miR-9 are required for decreasing Nox4 protein levels. We will then test the hypothesis that miR-9 alters the splice isoform profile and subcellular distribution of Nox4. Next, we will explore the mechanism by which miR-25 induces miR-9 expression. Finally, we will determine whether miR-9 modifies vascular smooth muscle cell (SMC) differentiation and the vascular response to injury. Aim 2 will test the hypothesis that Nox4 dynamically regulates the cellular redox state and transcriptional activity in cultured SMC and in the vessel wall. Proposed studies will determine the effect of changes in Nox4 expression on cellular thiol-disulfide status, the redox potential in different cellular compartments, and on the transcription of redox-sensitive genes. In vivo studies will explore how changes in SMC Nox4 modify vascular redox state and neointimal hyperplasia. This proposal is innovative in that we introduce a new paradigm whereby Nox4 acts as a redox rheostat, establishing the thiol oxidation state of cellular proteins to coordinate signaling event. These data may provide a unifying mechanism for the diverse and ambiguous functions of Nox4 across multiple biologic systems. Our data will be the first to provide evidence that one miRNA is indirectly induced by another miRNA. Finally, our findings will provide crucial insights into whether Nox4, miR-9, and miR-25 are therapeutic targets in vascular disease.

 描述（由适用提供）：通过协调多个信号网络来确定细胞响应的氧化还原信号和细胞内氧化还原状态的整合。我们的研究计划的长期目标是了解如何操纵NADPH氧化物以治疗心血管疾病。我们报告说，NOX4 NADPH氧化物会改变各种细胞反应。但是，NOX4调节细胞功能的分子机制知之甚少。 NOX4在细胞细胞器中表达，其功能易受氧化还原电位变化的影响。与其他NOX同源物相比，NOX4衍生的活性氧不可能作为第二使者起作用，因为1）NOX4具有组成性活性。 2）NOX4的激活主要受其表达水平的调节； 3）硫醇和H2O2（NOX4的主要产物）之间的反应太慢，无法具有生物学相关性。在试点研究中，我们对1）进行了新的观察。 MicroRNA-9（miR-9）是NOX4表达的新型调节剂。 2）miR-25影响miR-9的表达； 3）miR-9引起NOX4 mRNA剪接的变化； 4）血管损伤后miR-25和miR-9水平升高； 5）NOX4水平的变化改变了细胞谷胱甘肽氧化还原潜力和蛋白硫醇状态。因此，我们假设miRNA介导的NOX4表达的变化动态调节细胞氧化还原状态，并协调在血管疾病发展中实现的基因的表达。 AIM 1将检验以下假设：miR诱导的miR表达介导NOX4水平，细胞定位和SMC激活的变化。我们将首先确定miR-25和miR-9降低NOX4蛋白水平所必需的机制。然后，我们将测试miR-9改变NOX4的剪接同工型和亚细胞分布的假设。接下来，我们将探讨miR-25诱导miR-9表达的机制。最后，我们将确定miR-9是否修饰了血管平滑肌细胞（SMC）分化以及对损伤的血管反应。 AIM 2将检验以下假设：NOX4动态调节培养的SMC和血管壁中的细胞氧化还原态和转录活性。拟议的研究将确定NOX4表达变化对细胞硫醇二硫化物状态的影响，不同细胞室中的氧化还原电位以及对氧化还原敏感基因的转录。体内研究将探讨SMC NOX4的变化如何修饰血管氧化还原状态和新内膜增生。该提议具有创新性，因为我们引入了一种新的范式，NOX4充当氧化还原的晶状体，建立了细胞蛋白的硫醇氧化物状态以协调信号传导事件。这些数据可以为NOX4在多个生物系统中的潜水员和模棱两可的功能提供统一的机制。我们的数据将是第一个提供证据，表明另一个miRNA是由另一个miRNA间接诱导的。最后，我们的发现将为NOX4，miR-9和miR-25是血管疾病的热靶标提供至关重要的见解。