SIRT3 deficiency promotes lung fibrosis by augmenting alveolar epithelial cell mitochondrial DNA damage and apoptosis

Our plain-English summary of SIRT3 deficiency promotes lung fibrosis by augmenting alveolar epithelial cell mitochondrial DNA damage and apoptosis, by R.P. Jablonski, S.J. Kim, P. Cheresh, et al (FASEB Journal, 2017).

The study

• Authors: R.P. Jablonski, S.J. Kim, P. Cheresh, D.B. Williams, L. Morales-Nebreda, Y. Cheng, A. Yeldandi, S. Bhorade, A. Pardo, M. Selman, K. Ridge, D. Gius, G.R.S. Budinger, D.W. Kamp
• Journal: FASEB Journal
• Published: 2017;31(6):2520-2532
• Cited by: 127 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Alveolar epithelial cell (AEC) mitochondrial dysfunction and apoptosis are important in idiopathic pulmonary fibrosis and asbestosis. Sirtuin 3 (SIRT3) detoxifies mitochondrial reactive oxygen species, in part, by deacetylating manganese superoxide dismutase (MnSOD) and mitochondrial 8-oxoguanine DNA glycosylase. We reasoned that SIRT3 deficiency occurs in fibrotic lungs and thereby augments AEC mtDNA damage and apoptosis. Human lungs were assessed by using immunohistochemistry for SIRT3 activity via acetylated MnSODK68 Murine AEC SIRT3 and cleaved caspase-9 (CC-9) expression were assayed by immunoblotting with or without SIRT3 enforced expression or silencing. mtDNA damage was measured by using quantitative PCR and apoptosis via ELISA. Pulmonary fibrosis after asbestos or bleomycin exposure was evaluated in 129SJ/wild-type and SIRT3-knockout mice (Sirt3-/- ) by using fibrosis scoring and lung collagen levels. Idiopathic pulmonary fibrosis lung alveolar type II cells have increased MnSODK68 acetylation compared with controls. Asbestos and H2O2 diminished AEC SIRT3 protein expression and increased mitochondrial protein acetylation, including MnSODK68 SIRT3 enforced expression reduced oxidant-induced AEC OGG1K338/341 acetylation, mtDNA damage, and apoptosis, whereas SIRT3 silencing promoted these effects. Asbestos- or bleomycin-induced lung fibrosis, AEC mtDNA damage, and apoptosis in wild-type mice were amplified in Sirt3-/- animals. These data suggest a novel role for SIRT3 deficiency in mediating AEC mtDNA damage, apoptosis, and lung fibrosis.-Jablonski, R. P., Kim, S.-J., Cheresh, P., Williams, D. B., Morales-Nebreda, L., Cheng, Y., Yeldandi, A., Bhorade, S., Pardo, A., Selman, M., Ridge, K., Gius, D., Budinger, G. R. S., Kamp, D. W. SIRT3 deficiency promotes lung fibrosis by augmenting alveolar epithelial cell mitochondrial DNA damage and apoptosis.

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In plain English

This laboratory study combined human lung tissue staining, cultured alveolar epithelial cells (AECs) and knockout mice to test whether sirtuin 3 (SIRT3) deficiency contributes to lung fibrosis. In a pilot comparison of deidentified lung tissue from 3 people with normal lungs and 6 with idiopathic pulmonary fibrosis (IPF), the authors reported a statistically significant near doubling of per-cell acetylated MnSOD-K68 (a surrogate marker of SIRT3 activity) in alveolar type II cells from IPF lungs (110 IPF versus 121 normal cells). In A549 and MLE-12 cells, amosite asbestos and hydrogen peroxide reduced SIRT3 protein. SIRT3 mRNA, measured in A549 cells, was unchanged at 24 hours. Mitochondrial protein acetylation increased after asbestos in MLE-12 cells and after asbestos or hydrogen peroxide in primary rat AT2 cells. Enforced SIRT3 expression reduced mitochondrial DNA damage and apoptosis, whereas SIRT3 silencing increased them. After intratracheal crocidolite asbestos, Sirt3-knockout mice showed more fibrosis, more cleaved caspase-3-positive cells at bronchoalveolar duct junctions (17.8% versus 9.3% in wild type) and doubled AT2 cell mtDNA damage compared with wild-type mice.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Experimental laboratory study combining human lung tissue immunohistochemistry (pilot), cell-culture experiments with gene overexpression and silencing, and asbestos- and bleomycin-induced fibrosis in wild-type and Sirt3-knockout mice
• Who was studied: Deidentified human lung tissue from 3 people with normal lungs and 6 patients with IPF; A549 and MLE-12 cell lines; primary rat and murine AT2 cells; male and female 129SJ wild-type and Sirt3-knockout mice aged 8-10 weeks (Chicago, USA)
• Exposure: UICC reference crocidolite and amosite amphibole asbestos. In cells: amosite at 5-25 micrograms per square centimetre or H2O2 at 100-250 micromolar for up to 24 h. In mice: 100 micrograms intratracheal crocidolite (TiO2 control) or bleomycin
• Main results:
  • Alveolar type II cells from IPF lungs had roughly double the per-cell acetylated MnSOD-K68 staining of normal control lungs (110 IPF cells versus 121 normal cells)
  • In A549 and MLE-12 cells, asbestos and H2O2 lowered SIRT3 protein in a time- and dose-dependent way; SIRT3 mRNA, measured in A549 cells only, was unchanged at 24 h
  • Amosite asbestos increased global mitochondrial protein lysine acetylation in MLE-12 cells over time
  • In primary rat AT2 cells, asbestos and H2O2 increased mitochondrial protein lysine acetylation and acetylated MnSOD-K68 and reduced SIRT3 protein
  • Enforced SIRT3 expression in A549 cells reduced mtDNA damage, cleaved caspase-9 and acetylated MnSOD-K68, and attenuated DNA fragmentation after amosite asbestos or H2O2
  • SIRT3 silencing in A549 cells increased mtDNA damage and raised baseline apoptosis 2.5-fold on DNA fragmentation compared with scramble siRNA controls
  • Asbestos-induced lung fibrosis (histopathology, fibrosis score, lung collagen) was greater in Sirt3-knockout mice than in wild-type mice. TiO2-exposed mice of both genotypes had near-normal lungs
  • Cleaved caspase-3-positive cells at bronchoalveolar duct junctions after asbestos: 9.3% in wild-type versus 17.8% in Sirt3-knockout mice per 220 BAD junction cells (P less than 0.05). With TiO2: 0.8% versus 1.0%, not significant
  • Apoptotic AT2 cells (CC-3 and SFTPC copositive) in asbestos-exposed mice: 2.9% in wild-type versus 5.1% in Sirt3-knockout mice; this difference did not reach statistical significance (n = 3)
  • In AT2 cells isolated 3 weeks after intratracheal crocidolite, mtDNA damage was doubled in Sirt3-knockout compared with wild-type mice (0.075 versus 0.15 lesions per 10-kB frequency per fragment; P less than 0.05)
  • Bleomycin instillation also increased fibrosis score and lung collagen more in Sirt3-knockout than in wild-type mice
• The authors' conclusion: The authors concluded that their data suggest a novel role for SIRT3 deficiency in mediating AEC mitochondrial DNA damage, apoptosis and lung fibrosis
• Limitations the authors state:
  • The authors noted the ongoing difficulty of separating direct SIRT3 effects from indirect changes, given the many reported SIRT3 targets
  • The authors stated that SIRT3 may act through other mitochondrial targets or other cell types, and that other sirtuins may also matter
  • The authors stated that the human tissue differences could reflect age, smoking status or comorbidities, and that larger prospective studies are needed
  • The authors stated that they studied only young mice to avoid the confounding effect of aging
  • The authors stated that mitochondrial SIRT3 protein could not be reliably assessed in the human tissue because suitable antibodies were lacking

Topics

Medical terms in this study, explained in our Lexicon: Fibrosis, Apoptosis, Idiopathic, Pulmonary Fibrosis, Reactive Oxygen Species (ROS), Superoxide, Immunohistochemistry, Collagen.

Related studies

• The Role of Mitochondrial DNA in Mediating Alveolar Epithelial Cell Apoptosis and Pulmonary Fibrosis — S.J. Kim, P. Cheresh, et al. (2015)
• Mitochondrial catalase overexpressed transgenic mice are protected against lung fibrosis in part via preventing alveolar epithelial cell mitochondrial DNA damage — S.J. Kim, P. Cheresh, et al. (2016)
• Mitochondria-targeted Ogg1 and aconitase-2 prevent oxidant-induced mitochondrial DNA damage in alveolar epithelial cells — S.J. Kim, P. Cheresh, et al. (2014)
• SIRT3 Overexpression Ameliorates Asbestos-Induced Pulmonary Fibrosis, mt-DNA Damage, and Lung Fibrogenic Monocyte Recruitment — P. Cheresh, S.J. Kim, et al. (2021)
• The Sphingosine Kinase 1 Inhibitor, PF543, Mitigates Pulmonary Fibrosis by Reducing Lung Epithelial Cell mtDNA Damage and Recruitment of Fibrogenic Monocytes — P. Cheresh, S.J. Kim, et al. (2020)
• Asbestos-induced pulmonary fibrosis is augmented in 8-oxoguanine DNA glycosylase knockout mice — P. Cheresh, L. Morales-Nebreda, et al. (2015)

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