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

 描述（由申请人提供）：氧化还原信号和细胞内氧化还原状态的整合通过协调多个信号网络来决定细胞反应。我们研究计划的长期目标是了解如何操纵NADPH氧化酶来治疗心血管疾病。我们已经报道了Nox 4 NADPH氧化酶修饰多种细胞反应。然而，Nox4调节细胞功能的分子机制知之甚少。Nox4在细胞器中表达，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，Nox 4衍生的活性氧物质不太可能作为第二信使发挥作用，因为1）Nox 4是组成型活性的; 2）Nox 4的活化主要由其表达水平调节;和3）硫醇和H2O2（Nox 4的主要产物）之间的反应太慢而不具有生物学相关性。在初步研究中，我们发现了以下新的观察结果：1） microRNA-9（miR-9）是Nox 4表达的新型调节剂; 2）miR-25诱导miR-9的表达; 3）miR-9促进Nox 4 mRNA剪接的变化; 4）miR-25和miR-9水平在血管损伤后增加;以及5）Nox 4水平的变化改变细胞谷胱甘肽氧化还原电位和蛋白巯基状态。因此，我们推测，miRNA介导的Nox4表达的变化动态调节细胞的氧化还原状态，并协调与血管疾病的发展有关的基因的表达。目的1将检验miR诱导的miR表达介导Nox4水平、细胞定位和SMC活化变化的假设。我们将首先确定降低Nox4蛋白水平所需的miR-25和miR-9的机制。然后，我们将检验miR-9改变Nox 4的剪接异构体谱和亚细胞分布的假设。接下来，我们将探索miR-25诱导miR-9表达的机制。最后，我们将确定miR-9是否改变血管平滑肌细胞（SMC）分化和血管对损伤的反应。目的2将验证Nox 4在培养的SMC和血管壁中动态调节细胞氧化还原状态和转录活性的假设。拟议的研究将确定细胞巯基-二硫化物状态，在不同的细胞室的氧化还原电位，并对氧化还原敏感基因的转录在Nox4表达的变化的影响。体内研究将探讨SMC Nox4的变化如何改变血管氧化还原状态和新生内膜增生。这个建议是创新的，因为我们引入了一个新的范例，其中Nox4作为氧化还原变阻器，建立细胞蛋白质的巯基氧化态，以协调信号事件。这些数据可能为Nox4在多个生物系统中的多样性和模糊性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一种miRNA是由另一种miRNA间接诱导的。最后，我们的研究结果将为Nox4，miR-9和miR-25是否是血管疾病的治疗靶点提供重要的见解。

 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.