Mitochondrial 8-oxoguanine DNA glycosylase mitigates alveolar epithelial cell PINK1 deficiency, mitochondrial DNA damage, apoptosis, and lung fibrosis

Abstract

Alveolar epithelial cell (AEC) apoptosis, arising from mitochondrial dysfunction and mitophagy defects, is important in mediating idiopathic pulmonary fibrosis (IPF). Our group established a role for the mitochondrial (mt) DNA base excision repair enzyme, 8-oxoguanine-DNA glycosylase 1 (mtOGG1), in preventing oxidant-induced AEC mtDNA damage and apoptosis and showed that OGG1-deficient mice have increased lung fibrosis. Herein, we determined whether mice overexpressing the mtOGG1 transgene ( mtOgg1tg) are protected against lung fibrosis and whether AEC mtOGG1 preservation of mtDNA integrity mitigates phosphatase and tensin homolog-induced putative kinase 1 (PINK1) deficiency and apoptosis. Compared with wild type (WT), mtOgg1tg mice have diminished asbestos- and bleomycin-induced pulmonary fibrosis that was accompanied by reduced lung and AEC mtDNA damage and apoptosis. Asbestos and H2O2 promote the MLE-12 cell PINK1 deficiency, as assessed by reductions in the expression of PINK1 mRNA and mitochondrial protein expression. Compared with WT, Pink1-knockout ( Pink1-KO) mice are more susceptible to asbestos-induced lung fibrosis and have increased lung and alveolar type II (AT2) cell mtDNA damage and apoptosis. AT2 cells from Pink1-KO mice and PINK1-silenced (siRNA) MLE-12 cells have increased mtDNA damage that is augmented by oxidative stress. Interestingly, mtOGG1 overexpression attenuates oxidant-induced MLE-12 cell mtDNA damage and apoptosis despite PINK1 silencing. mtDNA damage is increased in the lungs of patients with IPF as compared with controls. Collectively, these findings suggest that mtOGG1 maintenance of AEC mtDNA is crucial for preventing PINK1 deficiency that promotes apoptosis and lung fibrosis. Given the key role of AEC apoptosis in pulmonary fibrosis, strategies aimed at preserving AT2 cell mtDNA integrity may be an innovative target.

The Sphingosine Kinase 1 Inhibitor, PF543, Mitigates Pulmonary Fibrosis by Reducing Lung Epithelial Cell mtDNA Damage and Recruitment of Fibrogenic Monocytes

“…Mice were exposed to a single IT-instillation of crocidolite asbestos (100 μg /50 μL PBS) or nonfibrogenic control particulate (TiO 2 100 μg /50 μL PBS) for either 21 d (short-term) or 60 d (long-term) and following exposure were treated with either PF543 (1mg/kg) or vehicle IP injection every other day from day 14−21 or day 30−60 ( Figure 1 ) [ 21 , 35 ]. Consistent with our prior studies [ 6 , 29 , 31 ], compared to TiO 2 , asbestos fibers increased pulmonary fibrosis at 21 d in B6 mice as evidenced by histology (H&E and trichrome staining– Figure 4 A), lung fibrosis score assessed by a pathologist blinded to the experimental protocol ( Figure 4 B), and lung collagen levels ( Figure 4 C). PF543 post-treatment begun 7 d following asbestos IT-instillation significantly reduced asbestos-induced lung fibrosis at 21 d as compared with untreated mice ( Figure 4 ).…”

“…The mechanism by which PF543 attenuates mtDNA damage is not established from our studies but several lines of evidence suggest it results from reduced mitochondrial ROS levels. Work by our group and others have established that mitochondrial ROS are crucial for promoting mtDNA damage evident in humans with IPF and animal models of pulmonary fibrosis [ 6 , 7 , 8 , 9 , 10 , 11 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 33 , 49 ]. Further, we recently showed that inhibition of SPHK1 with PF543 reduced bleomycin- and TGF-β-mediated mtROS levels in HLF and that MitoTEMPO, which scavenges mitochondrial ROS, ameliorated TGF-β-dependent expression of FN and α-SMA, suggesting a direct link between SPHK1-driven mitochondrial ROS levels and fibroblast differentiation.…”

“…Although not examined in this study, PF543 may also mitigate mtDNA damage by augmenting mtDNA repair pathways, such as mtOGG1 and/or other BER enzymes. MtOGG1 may be of particular interest given that mtOGG1 prevents oxidant-induced AEC apoptosis by mitigating mtDNA damage [ 7 , 8 , 49 ] and that asbestos- and bleomycin-induced lung fibrosis is increased in Ogg1 -/- mice but reduced in mice globally overexpressing the human mitochondria-targeted OGG1 subunit 1-alpha transgene ( mtOgg1 tg ) with concordant changes in AT2 cell mtDNA damage and apoptosis [ 29 , 31 ]. MtDNA release, which was not investigated here, can occur from TGFβ-exposed HLF and apoptotic AECs, the latter of which can promote TLR9 pro-fibrotic signaling [ 32 , 33 ].…”

“…IT-instillation of asbestos or TiO 2 to induce lung fibrosis was performed as described [ 6 , 29 , 31 ]. Crocidolite asbestos was suspended in Phosphate Buffered Saline (PBS) and 15 mM HEPES at 2 mg/mL and sonicated for 8 min at 40% power (Sonicator: Branson, Danbury, CT, USA) to disrupt fiber clumps.…”

“…Mitochondrial dysfunction and mtDNA damage can activate intrinsic AEC apoptosis in patients with IPF and appear crucial in the pathobiology of murine models of lung fibrosis, such as bleomycin (resolving model of lung fibrosis) and asbestos fibers (nonresolving model of lung fibrosis (asbestosis) [ 1 , 6 , 7 , 8 , 9 , 10 , 22 , 23 , 24 , 25 , 26 , 27 , 28 ]. We showed that mitochondrial 8-oxoguanine DNA glycosylase 1 (mtOGG1), a key mtDNA base excision repair (BER) enzyme, prevents oxidant-induced AEC apoptosis and lung fibrosis by mitigating mtDNA damage [ 8 , 29 , 30 , 31 ]. Furthermore, mitochondrial catalase enforced-expression ( MCAT ) transgenic mice have reduced asbestos- and bleomycin-induced lung fibrosis due in part to reductions in AT2 cell mitochondrial ROS production, mtDNA damage and apoptosis [ 6 ].…”