Although 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, that acts in part by inducing oxidative stress and proteolytic imbalance in the lungs, the precise molecular mechanisms involved and, importantly, the age-related aspects of COPD are poorly understood. The anti-aging gene Klotho, which is primarily made in the kidney, 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. In the kidney, attenuation of Klotho expression after renal injury is mediated in part by epigenetic modification of the Klotho gene locus resulting in transcriptional repression. Our preliminary studies show that CSEs induce AEC mtDNA damage, apoptosis, Klotho depletion (mRNA and protein), and diminished Miz1 expression. Notably, serum Klotho protein and mRNA levels are reduced in Miz1fl/fl-spc-Cre+ mice that spontaneously develop emphysema with aging. We reason that Klotho/Miz1 axis has an important role in limiting emphysema by protecting the AECs from smoking. 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 (mtDNA damage/apoptosis). We will use SPC-Cre+/Miz1 (POZ)fl/fl mice to determine whether AT2 cell specific loss of Miz1 augments CS-induced AEC hyper-methylation of the Klotho promoter, Klotho loss and development of CSE-induced emphysema. (2) To determine whether CSEs reduce Klotho expression by decreasing Miz1 levels resulting in AEC mtDNA damage and apoptosis. Using Miz1 silencing / over-expression studies, we will assess CSE-induced AEC mtDNA damage and apoptosis. We will determine whether reKlotho mitigates CSE-exposed AEC mtDNA damage and apoptosis in Miz1 silencing. We will also measure Klotho level from the blood of patients with COPD (GOLD1-4) and a healthy smokers, as a control group. Innovation: These studies will elucidate the importance of the Klotho / Miz1 axis in maintaining AEC mtDNA integrity crucial for preventing 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, CPFE etc.).
|Effective start/end date||9/1/19 → 8/31/20|
- National Institute on Aging (5R21AG060211-02)
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