Asbestosis is regulated by Rac1-mediated mitochondrial H2O2 levels

描述(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，人们对此知之甚少。肺泡巨噬细胞产生的ROS，尤其是H_2O_2，在肺纤维化的发展中起着不可或缺的作用，因为应用过氧化氢酶可以减轻纤维化。然而，调节石棉诱导的肺泡巨噬细胞产生过氧化氢的来源和分子机制(S)尚不清楚。Rac1是Rho GTP结合蛋白家族的成员，它调节多种细胞功能，包括过氧化氢的产生。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过氧化氢生成的机制(S)尚未被描述。我们的数据表明，在石棉肺患者肺泡巨噬细胞的线粒体中存在着rac1基因，而且rac1基因的导入需要C端的半胱氨酸残基(Cys-189)，而C端的半胱氨酸残基被香叶酸修饰。香叶素转移酶抑制剂(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对过氧化氢的产生至关重要，因为突变体(C178S)对过氧化氢水平的降低程度与C189S相似。在线粒体膜间间隙(IMS)中，rac1与细胞色素c相互作用，细胞色素c受其氧化还原状态的电子传递调节。具有胶原性结构的巨噬细胞受体(MARCO)是已知的对吸入颗粒物提供天然免疫防御的分子。与正常人相比，石棉肺患者肺泡巨噬细胞表达明显更多的MARCO，但MARCO的表达与线粒体氧化应激无关。Rac1信号与PI3K/Akt信号通路的相互作用可以是正向的，也可以是负向的。目前还没有研究PI3K/Akt对rac1介导的线粒体过氧化氢生成的调节作用。我们发现，结构性活性Akt的过度表达增强了rac1进入线粒体的能力。这些观察表明，一种新的连接Marco、PI3K/Akt和rac1进入线粒体的机械途径，在那里它与细胞色素c相互作用产生ROS，这与肺纤维化有关。假设：rac1进入肺泡巨噬细胞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Geranylgeranyation的调节。在目标1中，我们将研究在线粒体的输入过程中是否需要rac1的香叶酰化，以及是否由rac1介导的线粒体中的H_2O_2是从细胞色素c到Cys-178的电子传递的次要环节。在目标2中，我们将研究石棉是否与Marco结合并调节rac1的输入，以及PI3K/Akt是否调节rac1的geranylgerananyation。在目标3中，我们将研究巨噬细胞rac1条件C178S或C189S突变的转基因小鼠是否可以预防肺纤维化，以及用GGTI抑制rac1老年性突变是否可以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.