Mechanisms of Asbestos-Induced Alveolar Epithelial Cell Injury

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 DESCRIPTION (provided by applicant): Asbestos causes asbestosis (pulmonary fibrosis due to asbestos) and malignancies (lung cancer and mesothelioma) by mechanisms that are not fully elucidated. The extent of alveolar epithelial cell (AEC) injury and repair are critical determinants of the fibrogenic potential of toxins, such as asbestos. Sirtuin 3 (SIRT3), the major mitochondrial deactylase, regulates mitochondrial metabolism that detoxifies mitochondrial reactive oxygen species (ROS). We have shown that mitochondrial ROS mediate asbestos-induced AEC DNA damage and apoptosis by a p53- and mitochondria-regulated death pathway as well as a novel mechanism by which mitochondrial human 8-oxoguanine-DNA glycosylase 1 (mt-hOGG1) prevents ROS-induced AEC apoptosis by preserving mitochondrial aconitase (ACO-2), which in turn prevents mitochondrial DNA (mtDNA) damage. Compared to wild type (WT) mice, we showed that Ogg1-/- mice have increased asbestos-induced lung fibrosis due in part to alveolar type II (AT2) cell apoptosis from reduced ACO-2 levels and increased mtDNA damage. SIRT3, which controls the function of mitochondrial proteins including OGG1, ACO-2, and others, can augment mtDNA repair in the setting of oxidative stress in nerve cells. Our preliminary studies show that asbestos reduces AEC SIRT3 expression; that SIRT3 deficiency promotes asbestos-induced AEC mtDNA damage, apoptosis, and pulmonary fibrosis; and that SIRT3 enforced expression (EE) attenuates oxidant-induced AEC ACO-2 depletion, mtDNA damage, and apoptosis. We reason that AEC mtDNA is a key target that integrates cell survival / death signals following exposure to asbestos by a SIRT3-regulated mechanism. HYPOTHESIS: SIRT3 attenuates asbestos-induced AEC mtDNA damage by preserving mt-OGG1/ ACO-2 function and reducing mitochondrial dysfunction important for limiting apoptosis and lung fibrosis. Our SPECIFIC AIMS that will be examined over the next 4 years include: (1) To determine if SIRT3 deficiency promotes AEC mtDNA damage and intrinsic apoptosis due to alterations in OGG1 and/or ACO-2 acetylation that directs protein function. We will assess the effects of SIRT3 deficiency on acetylation of OGG1 and ACO-2, mtDNA damage, and apoptosis. Sirt3-/- and lung epithelial cell specific Sirt3-/- mice will be used to assess whether SIRT3 deficiency augments asbestos- and bleomycin-induced AEC MnSOD, OGG1 and ACO-2 acetrylation, mtDNA damage, apoptosis, and pulmonary fibrosis. (2) To determine whether SIRT3 enforced expression (EE) prevents asbestos-induced AEC mitochondrial protein acetylation (total, OGG1, ACO-2, MnSOD), mtDNA damage, and apoptosis resulting from altered OGG1 / ACO-2. We will use a combination of pharmacologic (resveratrol / viniferin) and genetic approaches in vitro. We will use Sirt3-EE mice available to us and lung epithelial cell specific Sirt3 mice that we will develop to assess whether SIRT3 attenuates asbestos- and bleomycin-induced lung fibrosis by preventing AEC mitochondrial protein acetylation (OGG1 and ACO-2), ACO-2 depletion, mtDNA damage, and apoptosis. (3) To determine if mitochondrial hOGG1-EE attenuates the deleterious effects of SIRT3 deficiency as occurs following asbestos exposure. In vitro studies using MLE-12 cells that overexpress mt-Ogg1 and are SIRT3 depleted will be used to assess asbestos-induced mitochondrial protein acetylation (total, OGG1, ACO-2, and MnSOD), ACO-2 levels, mtDNA damage, and intrinsic apoptosis. We will also use Mitochondrial hOgg1-EE mice in the absence and presence of SIRT3 silencing to ascertain if mt-hOGG1-EE attenuates lung fibrosis (asbestos / bleomycin) by preserving AEC ACO-2 levels and mtDNA while diminishing apoptosis. Innovation: These studies will elucidate the importance of SIRT3 in maintaining AT2 cell mtDNA integrity crucial for preventing AT2 cell apoptosis and pulmonary fibrosis. The studies proposed will advance our understanding of lung fibrosis that may have broader implications for more common lung disorders (i.e. IPF and lung cancer) present in the veteran population for which effective treatments are needed.