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.
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.
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