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: 9098706
• 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/9098706
• 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; knock-down; macrophage; member; mutant; novel; overexpression; 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）：石棉沉着症是肺纤维化的一种重要类型。肺纤维化的发展是一个复杂的过程，但人们对此知之甚少。肺泡巨噬细胞产生的 ROS（特别是 H2O2）在肺纤维化的发展中起着不可或缺的作用，因为过氧化氢酶的施用可减轻纤维化。然而，调节石棉诱导的肺泡巨噬细胞中 H2O2 生成的来源和分子机制尚不清楚。 Rac1 是 GTP 结合蛋白 Rho 家族的成员，它调节多种细胞功能，包括 H2O2 的产生。 Rac1 已被证明可以间接增加线粒体 ROS；然而，尚未描述 Rac1 直接调节巨噬细胞中线粒体 H2O2 生成的机制。我们的数据表明，Rac1 定位于从石棉肺患者获得的肺泡巨噬细胞的线粒体中，并且 Rac1 的导入需要 C 末端半胱氨酸残基 (cys-189)，该残基通过香叶基香叶基化进行修饰。香叶基香叶基转移酶抑制剂（GGTI）已用于癌症治疗，但其在预防肺纤维化方面的用途尚未得到研究。我们还发现 cys-178 对于 H2O2 的产生至关重要，因为突变体 (C178S) 降低 H2O2 水平的程度与 C189S 相似。在线粒体膜间空间 (IMS) 中，Rac1 与细胞色素 c 相互作用，细胞色素 c 通过电子转移受氧化还原状态的调节。已知具有胶原结构的巨噬细胞受体 (MARCO) 可以提供针对吸入颗粒的先天免疫防御。与正常受试者相比，从石棉沉滞症患者获得的肺泡巨噬细胞表达明显更多的 MARCO，但尚不清楚 MARCO 表达是否与线粒体氧化应激有关。 Rac1 信号传导和 PI3K/Akt 通路的相互作用可能相互正向或负向调节。尚无研究调查 PI3K/Akt 对 Rac1 介导的线粒体 H2O2 生成的调节作用。我们发现组成型活性 Akt 的过度表达会增强 Rac1 向线粒体的输入。这些观察结果表明，有一种新颖的机制途径将 MARCO、PI3K/Akt 和 Rac1 连接到线粒体中，并在线粒体中与细胞色素 c 相互作用产生 ROS，从而与肺纤维化相关。假设：Rac1 导入肺泡巨噬细胞线粒体对于产生氧化应激和介导肺纤维化至关重要，并受到 MARCO、PI3K/Akt 和香叶基香叶基化的调节。在目标 1 中，我们将研究 Rac1 的香叶基香叶基化对于线粒体输入是否是必需的，以及线粒体中 Rac1 介导的 H2O2 是否是从细胞色素 c 到 Rac1 中的 cys-178 的电子转移的继发性。在目标 2 中，我们将研究石棉是否与 MARCO 结合并调节 Rac1 输入，以及 PI3K/Akt 是否调节 Rac1 的香叶基香叶基化。在目标 3 中，我们将研究巨噬细胞 Rac1 中具有条件性 C178S 或 C189S 突变的转基因小鼠是否能够免受肺纤维化的影响，以及用 GGTI 抑制 Rac1 香叶基香叶基化是否会减弱肺纤维化的发展。