Asbestosis is regulated by Rac1-mediated mitochondrial H2O2 levels

描述（由申请人提供）：石棉肺是一种重要的肺纤维化类型。肺纤维化的发展是一个复杂的过程，人们对其了解甚少。肺泡巨噬细胞产生ROS，特别是H2O2，在肺纤维化的发展中起着不可或缺的作用，因为过氧化氢酶可以减轻纤维化。然而，石棉诱导肺泡巨噬细胞产生H2O2的调控来源和分子机制尚不清楚。Rac1是gtp结合蛋白Rho家族的一员，它调节多种细胞功能，包括H2O2的产生。Rac1已被证明间接增加线粒体ROS；然而，Rac1直接调节巨噬细胞线粒体H2O2生成的机制尚未被描述。我们的数据表明，Rac1定位于石棉沉滞症患者肺泡巨噬细胞的线粒体中，并且Rac1的输入需要c端半胱氨酸残基（cys-189），该残基通过香叶酰基酰化修饰。香叶基香叶基转移酶抑制剂（GGTI）已被用于癌症治疗，但其在预防肺纤维化方面的应用尚未被研究。我们还发现，cys-178对H2O2的产生至关重要，因为突变体（C178S）对H2O2水平的衰减程度与C189S相似。在线粒体膜间空间（IMS）中，Rac1与细胞色素c相互作用，并通过电子转移受其氧化还原状态的调节。具有胶原结构的巨噬细胞受体（MARCO）已知对吸入颗粒提供先天免疫防御。与正常受试者相比，石棉沉滞患者肺泡巨噬细胞表达的MARCO明显更多，但MARCO表达与线粒体氧化应激无关。Rac1信号与PI3K/Akt通路的相互作用可能是正向或负向的相互调节。没有研究研究PI3K/Akt在rac1介导的线粒体H2O2生成中的调控作用。我们发现，过表达组成型活性Akt可促进Rac1进入线粒体。这些观察结果表明，一种新的机制途径将MARCO、PI3K/Akt和Rac1导入线粒体，并与细胞色素c相互作用产生ROS，这与肺纤维化有关。假设：在肺泡巨噬细胞中，Rac1进入线粒体是产生氧化应激和介导肺纤维化的关键，并受MARCO、PI3K/Akt和香叶酰化的调节。在Aim 1中，我们将研究Rac1的香叶酰化对线粒体导入是否必要，以及Rac1介导的线粒体H2O2是否继发于Rac1中细胞色素c向cys-178的电子转移。在Aim 2中，我们将研究石棉是否结合在MARCO上并调节Rac1的导入，以及PI3K/Akt是否调节Rac1的香gerylgeranyation。在Aim 3中，我们将研究巨噬细胞Rac1中具有条件C178S或C189S突变的转基因小鼠是否能保护其免受肺纤维化，以及GGTI抑制Rac1香叶酰香叶酰化是否能减轻肺纤维化的发展。

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.