Project Summary/Abstract Tobacco exposure is the major risk factor for Chronic Obstructive Pulmonary Disease (COPD), which is currently the third leading cause of death in the US. Although the precise molecular mechanisms involved and, importantly, the age-related aspects of COPD are poorly understood, the prevailing thinking is that oxidative stress and proteolytic imbalance in the lungs are important. The anti-aging gene Klotho has an extra-cellular domain that circulates providing important endocrine and paracrine functions impacting anti-oxidant and anti- fibrotic functions in distal organs, including the lungs. We recently showed that Klotho preserves AEC mitochondrial (mt) DNA integrity, a key intracellular target that integrates cell survival/death signaling following oxidative stress. The attenuation of Klotho expression after renal injury is mediated in part by epigenetic modification of the Klotho promoter causing transcriptional repression. Our preliminary studies show that CSEs induce AEC mtDNA damage, apoptosis, and Klotho depletion (mRNA and protein). Notably, Miz1 binds on the methylation-prone locus of the Klotho proximal promoter. Further, serum Klotho protein and mRNA levels are reduced in SPC-Cre+/Miz1 (POZ)fl/fl mice that spontaneously develop emphysema with aging. We hypothesize that the Klotho/Miz1 axis is important for limiting tobacco-induced AEC injury that promotes emphysema. Our SPECIFIC AIMS that will be examined over the next 2 years include: (1) To determine whether methylation of the Klotho promoter blocks Miz1 binding and thereby promotes AEC mtDNA damage and lung tissue remodeling. We will perform locus-specific CpG methylation analysis of the Klotho promoter using bisulfite pyrosequencing. Using methylation inhibitors, we will assess the effects of Klotho promoter methylation on Miz1 binding and subsequent AEC injury. We will use SPC-Cre+/Miz1 (POZ)fl/fl mice to determine whether loss of Miz1 in AT2 cell augments CSE-induced Klotho promoter methylation, Klotho loss and emphysema development. (2) To determine whether non-epigenetic mechanisms involving AEC mtROS account for CSE-induced reductions in Klotho and/or Miz1 expression that causes mtDNA damage and apoptosis. To address this, we will use genetic (MCAT transgenic mice) and pharmacologic approaches (Euk- 134) as well as Miz1 and Klotho silencing and over-expression studies. The relevance of our findings will be assessed in a cohort observational study of humans with COPD. (GOLD1-4). Innovation: These studies will elucidate the importance of the Klotho / Miz1 axis and mtROS in altering AEC mtDNA integrity crucial for promoting CSE-induced AEC mtDNA damage and apoptosis during COPD/emphysema development. The studies proposed will provide the scientific basis for an innovative therapeutic approach (epigenetic regulation of the Klotho/Miz1 axis) in protecting AECs for preventing common smoking related lung diseases of aging (i.e. COPD, lung cancer, emphysema...