Epigenetic regulation of the Klotho / Miz 1 axis in cigarette‐smoke extract (CSE)‐induced alveolar epithelial cell (AEC) mtDNA damage and apoptosis
Authors: Kim Seok-Jo , Cheresh Paul , Liu Jing , Singer Benjamin , Kalhan Ravi , Ridge Karen , Varga John , Kamp David 
Published: April 2020

We demonstrated that Klotho, an age‐related gene, preserves AEC mitochondrial (mt) DNA integrity, a critical target, which integrates cell survival/ death following oxidative stress (Kim et al, AJP‐Lung 2017), but the pathophysiology of this pathway in age‐related emphysema/COPD is unclear. Our group has shown that compared to controls, mice with lung AEC‐specific deletion of Miz1 develop age‐related lung changes resembling emphysema and bronchitis by middle age (12 months). To determine whether Klotho mitigates CSE‐induced AEC mtDNA damage and apoptosis and whether methylation of the Klotho promoter blocks Miz‐1 binding necessary for preventing this. AEC (A549 or MLE‐12 cells) were exposed to CSE (0–200 μg/ml) for 24h and assessed for mtDNA damage (Q‐PCR), Klotho mRNA expression (real‐time PCR), apoptosis (DNA fragmentation ELISA), and Klotho protein (ELISA). Human and mouse Klotho promoters were analyzed by ManInspect, EMBOSS Cpgplot (CpG islands) in the rRNA gene and intragenic spacer. We used 5‐Aza‐2′‐deoxycitidine (DAC) for inhibition of CpG methylation in CSE (100 μg/ml for 72h v. DMSO controls)‐exposed MLE‐12 cells. Chromatin immunoprecipitation (ChIP) assay of AECs exposed to CSE (100 μg/ml, 72h) or DMSO was performed using a Miz1 antibody or IgG. Serum Klotho protein and AT2 cell Klotho mRNA and mtDNA damage levels were assessed from young (<6 months) and aged (>1 year) SPC‐Cre+/Miz1(POZ)fl/fl or Miz1(POZ)fl/fl (control) mice. We found that CSEs reduce AEC Klotho mRNA/protein expression and augment mtDNA damage/apoptosis in MLE‐12 cells. Miz1 binding sites that are rich in CpG islands are present in both the human and murine Klotho promoter. Inhibition of CpG methylation by DAC sustains Klotho protein level in CSE‐exposed MLE‐12 cells. Using a ChIP assay, we confirmed that Miz1 binds the Klotho promoter in AECs and, interestingly, that CSE abolishes Miz1 binding. Compared to Miz1(POZ)fl/fl, AT2 cell Klotho mRNA and serum Klotho protein levels are decreased in aged‐SPC‐Cre+/Miz1(POZ)fl/fl mice and AT2 cell mtDNA damage is increased and mtDNA damage is further increased as compared to young mice. Our findings suggest that Miz1 is a novel regulator of the Klotho gene that is susceptible to CSE‐induced epigenetic methylation that promotes AEC mtDNA damage and apoptosis. We reason that epigenetic regulation of the Klotho/Miz1 axis is crucial for modulating smoking‐related AEC injury and may be an innovative therapeutic target for preventing smoking‐related lung diseases of aging (i.e. COPD, lung fibrosis, lung cancer, etc.).Support or Funding InformationNIH‐R21 (1R21AG060211‐01A1, SK)(A) Compared to Miz1(POZ)f/fl control, Klotho mRNA (AT2 cells, left) and protein (serum, right) levels are decreased in SPC‐Cre+/Miz1 (POZ)fl/fl mice. (B) mtDNA damage is increased in young SPC‐Cre+/Miz1 (POZ)fl/fl mice, and increased further in aged SPC‐Cre+/Miz1 (POZ)fl/fl mice.Figure 1