Project Summary/Abstract The pathogenesis of age-related idiopathic pulmonary fibrosis (IPF) and asbestosis is complex and incompletely understood, though accumulating evidence firmly implicates mitochondrial DNA (mtDNA) damage that lead to alveolar epithelial cell (AEC) apoptosis as a key event in disease development. We previously showed that the extent of AEC mtDNA damage, mitochondrial aconitase (ACO-2) depletion and apoptosis are critical determinants of the fibrogenic potential of asbestos. Sirtuin 3 (SIRT3) is the major mitochondrial deacetylase and considered the ?guardian of the mitochondrial genome? through its role in regulating mitochondrial proteins that detoxify oxidative stress and preserve mtDNA via modulation of mitochondrial 8- oxoguanine DNA glycosylase (OGG1) and ACO-2 activities. Our preliminary studies in preparation for this proposal show that oxidative stress (asbestos or H2O2) decreases AEC SIRT3 protein expression; that SIRT3 silencing augments while SIRT3 enforced expression (EE) attenuates oxidant-induced AEC ACO-2 depletion, mtDNA damage, and apoptosis; that SIRT3 deficiency enhances asbestos- and bleomycin-induced pulmonary fibrosis in association with increased AEC mtDNA damage; and that lungs from patients with idiopathic pulmonary fibrosis (IPF) have increased acetylation of OGG1 and MnSOD. Taken together, these data suggest a novel role for SIRT3 in the maintenance of a healthy alveolar epithelium and prevention of fibrotic lung diseases. We hypothesize that honokiol, a small molecule SIRT3 inducer, attenuates oxidant-induced AEC mtDNA damage, mitochondrial ROS production, and apoptosis as well as pulmonary fibrosis in part by maintaining expression and activity of OGG1, ACO-2, and MnSOD. We have formulated two related aims to test this hypothesis. In Aim 1, we will determine whether honokiol prevents oxidant-induced AEC mtDNA damage and intrinsic apoptosis in vitro via a SIRT3-dependent mechanism involving preservation of AEC mitochondrial protein (OGG1, ACO-2, MnSOD) function and reduction in protein acetylation and mitochondrial ROS. In Aim 2, we will use wild-type and Sirt3-/- mice to determine whether honokiol and resveratrol (another small molecule sirtuin inducer) mitigate pulmonary fibrosis (asbestos or bleomycin) in vivo by a SIRT3- dependent mechanism and whether protection is associated with reductions in alveolar epithelial type 2 cell (AT2) OGG1 and MnSOD acetylation, mtDNA damage, and apoptosis. These studies will elucidate the importance of honokiol-induced SIRT3-EE in preserving AT2 cell mtDNA integrity and preventing lung fibrosis which may have broad implications for the treatment of IPF and other fibrotic lung diseases.