Asbestosis is regulated by Rac1-mediated mitochondrial H2O2 levels

石棉沉滞症受 Rac1 介导的线粒体 H2O2 水平调节

• 批准号: 9060666
• 负责人: A BRENT CARTER
• 金额: $ 37.12万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9060666
• 关键词: Alveolar Macrophages; Asbestos; Asbestosis; Attenuated; Binding; Binding Proteins; Breathing; C-terminal; Cell physiology; Cells; Cicatrix; Complex; Coupled; Cysteine; Data; Development; Electron Transport; Family; Fibrosis; Generations; Goals; Guanosine; Hydrogen Peroxide; Immune; Link; Lung; Lung diseases; Mediating; Mitochondria; Molecular; Mutation; Oxidation-Reduction; Oxidative Stress; Oxygen; Pathway interactions; Patients; Play; Process; Production; Pulmonary Fibrosis; Regulation; Research; Role; Secondary to; Signal Transduction; Small Interfering RNA; Source; Structure; Structure of parenchyma of lung; Therapeutic; Transgenic Mice; cancer therapy; catalase; cell type; cytochrome c; fighting; geranylgeranylation; in vivo; inhibitor/antagonist; macrophage; member; mutant; novel; particle; prevent; receptor; rho; rho GTP-Binding Proteins; tripolyphosphate

DESCRIPTION (provided by applicant): Asbestosis is an important type of pulmonary fibrosis. The development of pulmonary fibrosis is a complex process that is poorly understood. The generation of ROS, particularly H2O2, from alveolar macrophages plays an integral role in the development of pulmonary fibrosis as administration of catalase attenuates fibrosis. However, the source and molecular mechanism(s) that regulate asbestos-induced H2O2 generation in alveolar macrophages is not known. Rac1 is a member of the Rho family of GTP-binding proteins, and it regulates several cellular functions, including H2O2 production. Rac1 has been shown to indirectly increase mitochondrial ROS; however, no mechanism(s) by which Rac1 directly modulates mitochondrial H2O2 generation in macrophages have been described. Our data demonstrate that Rac1 is localized in the mitochondria of alveolar macrophages obtained from asbestosis patients and that Rac1 import required the C-terminal cysteine residue (cys-189), which is modified by geranylgeranylation. Geranylgeranyltransferase inhibitors (GGTI) have been used in cancer therapy, but their use in preventing pulmonary fibrosis has not been investigated. We also found that cys-178 is critical for H2O2 generation as a mutant (C178S) attenuates H2O2 levels to a similar degree as C189S. In the mitochondrial intermembrane space (IMS), Rac1 interacts with cytochrome c, which is regulated by its redox state via transfer of electrons. The macrophage receptor with collagenous structure (MARCO) is known to provide innate immune defense against inhaled particles. Alveolar macrophages obtained from asbestosis patients express significantly more MARCO compared to normal subjects, but MARCO expression is not known to be linked to mitochondrial oxidative stress. The interaction of Rac1 signaling and the PI3K/Akt pathway may positively or negatively modulate each other. No studies have investigated PI3K/Akt in the regulation of Rac1-mediated mitochondrial H2O2 generation. We found that over expression of a constitutive active Akt enhances Rac1 import into mitochondria. These observations suggest a novel, mechanistic pathway linking MARCO, PI3K/Akt, and Rac1 import into the mitochondria where it interacts with cytochrome c to generate ROS, which is coupled to pulmonary fibrosis. Hypothesis: the import of Rac1 into the mitochondria in alveolar macrophages is pivotal in generating oxidative stress and mediating pulmonary fibrosis and is modulated by MARCO, PI3K/Akt, and geranylgeranylation. In Aim 1, we will investigate if geranylgeranylation of Rac1 is necessary for mitochondrial import and if Rac1-mediated H2O2 in mitochondria is secondary to electron transfer from cytochrome c to cys-178 in Rac1. In Aim 2, we will investigate if asbestos binds on MARCO and modulates Rac1 import and if PI3K/Akt regulate geranylgeranylation of Rac1. In Aim 3, we will investigate if transgenic mice with a conditional C178S or C189S mutation in macrophage Rac1 are protected from pulmonary fibrosis and if inhibition of Rac1 geranylgeranylation with GGTI attenuates the development of pulmonary fibrosis.

描述（由申请人提供）：石棉病是肺纤维化的重要类型。肺纤维化的发展是一个复杂的过程，知之甚少。从肺泡巨噬细胞中产生的ROS，尤其是H2O2的产生在肺纤维化的发展中起着不可或缺的作用，因为过氧化氢酶的给药可减弱纤维化。然而，尚不清楚调节石棉诱导的H2O2产生的源和分子机制尚不清楚。 Rac1是RHO GTP结合蛋白家族的成员，它调节了几种细胞功能，包括H2O2产生。 Rac1已显示可间接增加线粒体ROS；但是，尚未描述Rac1直接调节巨噬细胞中线粒体H2O2产生的机制。我们的数据表明，RAC1位于从石棉患者获得的肺泡巨噬细胞的线粒体中，Rac1进口需要C末端半胱氨酸残基（CYS-189），这是通过天甘烷基果仁化修饰的。黄烷基凝血酶转移酶抑制剂（GGTI）已用于癌症治疗，但尚未研究它们在预防肺纤维化方面的使用。我们还发现，Cys-178对于H2O2作为突变体（C178S）的生成至关重要。在线粒体间膜间空间（IMS）中，Rac1与细胞色素C相互作用，该CytoChrome C通过其氧化还原态通过电子转移来调节。已知具有胶原结构（MARCO）的巨噬细胞受体可提供对吸入颗粒的先天免疫防御。与正常受试者相比，从石棉患者获得的肺泡巨噬细胞表现出明显更多的Marco，但Marco表达与线粒体氧化应激无关。 RAC1信号传导和PI3K/AKT途径的相互作用可能会呈正面或负面调节。没有研究研究RAC1介导的线粒体H2O2生成的PI3K/AKT。我们发现，本构主动AKT的表达会增强RAC1进口到线粒体中。这些观察结果表明，将Marco，PI3K/AKT和Rac1与线粒体相互作用的新型机械途径与细胞色素C相互作用以产生ROS，从而与肺纤维化耦合。假设：Rac1在肺泡巨噬细胞中将Rac1的进口在产生氧化应激和介导肺纤维化方面至关重要，并且由MARCO，PI3K/AKT和黄甘甘烷基化调节。在AIM 1中，我们将研究RAC1的黄烷基凝聚力是否对于线粒体进口是必需的，以及线粒体中Rac1介导的H2O2是否继发于从细胞色素C到RAC1中Cys-178的电子转移。在AIM 2中，我们将调查石棉是否与Marco结合并调节Rac1的进口，以及PI3K/AKT是否调节Rac1的黄烷基凝聚酰化。在AIM 3中，我们将研究巨噬细胞Rac1中具有条件C178S或C189S突变的转基因小鼠免受肺纤维化的保护，以及GGTI对RAC1黄烷基化的抑制是否会减轻肺纤维化的发展。