Asbestosis is regulated by Rac1-mediated mitochondrial H2O2 levels

描述（由申请人提供）：石棉肺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，人们对其了解甚少。肺泡巨噬细胞产生的ROS，特别是H2 O2，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减轻了纤维化。然而，在肺泡巨噬细胞中调节石棉诱导的H2 O2产生的来源和分子机制尚不清楚。Rac 1是GTP结合蛋白Rho家族的成员，它调节多种细胞功能，包括H2 O2的产生。Rac 1已被证明间接增加线粒体ROS;然而，没有Rac 1直接调节巨噬细胞中线粒体H2 O2产生的机制。我们的数据表明，Rac 1是本地化的肺泡巨噬细胞从石棉肺患者的线粒体和Rac 1进口需要的C-末端半胱氨酸残基（cys-189），这是由geranylgeranylation修改。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中的用途尚未研究。我们还发现cys-178对于H2 O2的产生至关重要，因为突变体（C178 S）将H2 O2水平减弱到与C189 S相似的程度。在线粒体膜间隙（IMS）中，Rac 1与细胞色素c相互作用，细胞色素c通过电子转移受其氧化还原状态调节。已知具有胶原结构的巨噬细胞受体（MARCO）提供针对吸入颗粒的先天免疫防御。与正常受试者相比，从石棉肺患者获得的肺泡巨噬细胞表达显著更多的MARCO，但MARCO表达与线粒体氧化应激无关。Rac 1信号通路与PI 3 K/Akt信号通路的相互作用可能是正性或负性的。没有研究调查PI 3 K/Akt在Rac 1介导的线粒体H2 O2产生的调节。我们发现，组成性活性Akt的过度表达增强了Rac 1向线粒体的输入。这些观察结果表明，MARCO，PI 3 K/Akt和Rac 1输入到线粒体中，在线粒体中与细胞色素c相互作用产生ROS，这与肺纤维化有关。假设：将Rac 1输入到肺泡巨噬细胞的线粒体中在产生氧化应激和介导肺纤维化中是关键的，并且受MARCO、PI 3 K/Akt和香叶基香叶基化调节。在目的1中，我们将调查，如果香叶基香叶基化的Rac 1是必要的线粒体进口，如果Rac 1介导的H2 O2在线粒体是次要的电子转移从细胞色素c的Cys-178在Rac 1。在目标2中，我们将研究石棉是否与MARCO结合并调节Rac 1的输入，以及PI 3 K/Akt是否调节Rac 1的geranylgeranylation。在目标3中，我们将研究在巨噬细胞Rac 1中具有条件性C178 S或C189 S突变的转基因小鼠是否受到肺纤维化的保护，以及GGTI抑制Rac 1香叶基香叶基化是否减弱肺纤维化的发展。 公共卫生相关性：石棉肺是石棉引起的肺部疾病中最严重的一种，是肺纤维化或肺瘢痕形成的一种重要类型。肺泡巨噬细胞（肺中的防御战斗细胞）产生的过氧化氢与瘢痕形成有关。本申请中的研究将定义调节过氧化氢产生的分子机制，以开发预防肺部瘢痕形成的治疗选择。

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. PUBLIC HEALTH RELEVANCE: Asbestosis, which is the most debilitating type of asbestos-induced lung disease, is an important type of pulmonary fibrosis, or scarring in the lung. The production of hydrogen peroxide by alveolar macrophages, a defense-fighting cell in the lung, is associated with the development of scarring. The studies in this application will define the molecular mechanisms that regulate hydrogen peroxide production in order to develop a therapeutic option to prevent the scarring in the lung.