The DNA glycosylase Ogg1 defends against oxidant-induced mtDNA damage and apoptosis in pulmonary artery endothelial cells

Abstract

Emerging evidence suggests that mitochondrial (mt) DNA damage may be a trigger for apoptosis in oxidant-challenged pulmonary artery endothelial cells (PAECs). Understanding the rate-limiting determinants of mtDNA repair may point to new targets for intervention in acute lung injury. The base excision repair (BER) pathway is the only pathway for oxidative damage repair in mtDNA. One of the key BER enzymes is Ogg1, which excises the base oxidation product 8-oxoguanine. Previously we demonstrated that overexpression of mitochondrially targeted Ogg1 in PAECs attenuated apoptosis induced by xanthine oxidase (XO) treatment. To test the idea that Ogg1 is a potentially rate-limiting BER determinant protecting cells from oxidant-mediated death, PAECs transfected with siRNA to Ogg1 were challenged with XO and the extent of mitochondrial and nuclear DNA damage was determined along with indices of apoptosis. Transfected cells demonstrated significantly reduced Ogg1 activity, which was accompanied by delayed repair of XO-induced mtDNA damage and linked to increased XO-mediated apoptosis. The nuclear genome was undamaged by XO in either control PAECs or cells depleted of Ogg1. These observations suggest that Ogg1 plays a critical and possibly rate-limiting role in defending PAECs from oxidant-induced apoptosis by limiting the persistence of oxidative damage in the mitochondrial genome.

Copyright © 2010 Elsevier Inc. All rights reserved.

Figures

FIGURE 1

Abundance of Ogg1 mRNA and Ogg1-like activity in pulmonary artery endothelial cells transfected with Ogg1-specific siRNA. (A) Decrease in Ogg1 mRNA accumulation normalized to the abundance of 28S RNA in PAECs transfected with Ogg1-specific siRNA compared with sham-transfected cells 24 and 48 hours after transfection. (Mean ± SE, N = 6, *p < 0.05). (B) Oligonucleotide cleavage assay for Ogg1-like activity in PAECs transfected with Ogg1-specific siRNA (Ogg1) and in cells transfected with a scrambled siRNA (Scr) 48 hours after transfection. LEFT: Representative autoradiograph of gel separation of 32P-labeled substrate (S) and product (P) of the Ogg1 enzymatic reaction. RIGHT: Results of oligonucleotide cleavage assays calculated as the ratio of product to substrate band intensities ratio and displayed as mean ± SE (N = 5, *p < 0.05).

FIGURE 2

Lack of nuclear DNA fragmentation as detected by the Fpg-FLARE Comet assay in pulmonary artery endothelial cells transfected with either scrambled (A) or Ogg1-specific (B) siRNA and harvested immediately after 1 hour treatment with xanthine oxidase (5 mU/ml). Note lack of Comet “tails”. Negative results also were obtained in cells treated with 2 and 10 mU/ml of XO using either the conventional Comet assay or the Fpg-FLARE. (C) Pulmonary artery endothelial cells without any treatment were used as a negative control. (D) PAECs treated with 1 mM hydrogen peroxide for 15 min at 4°C were used as a positive control for the assay.

FIGURE 3

Quantitative Southern blot analysis of Fpg-sensitive mtDNA damage in pulmonary artery endothelial cells. (A) Representative autoradiograph of Southern blot of mtDNA from PAECs transfected with scrambled (Scr) and Ogg1-specific (Ogg1) siRNA immediately after 1 h treatment with xanthine oxidase (XO, 2 or 5 mU/ml; 0 h) or without treatment (XO, 0 mU/ml; 0 h) and 1, 2 or 4 hours later. (B) Calculated changes in equilibrium lesion density in mtDNA normalized to 10 kb in PAECs immediately after 60 min treatment with XO, 2 or 5 mU/ml and at 1, 2 or 4 hours after removal of XO. Bars reflect means ± SE of 4 determinations. Note that lesions in mtDNA from Ogg1 siRNA transfected cells are significantly higher (*p < 0.05) at 2 h post XO treatment in comparison to sham-transfected cells.

FIGURE 4

Immunocytochemical analysis of apoptotic markers in pulmonary artery endothelial cells transfected with scrambled and Ogg1-specific siRNA 4 h after cell treatment with xanthine oxidase. (A – D) Photomicrographs of PAECs transfected with scrambled ( A and C) or Ogg1-specific ( B and D) siRNA without treatment (A and B, Control) or after 1 h exposure to xanthine oxidase (C and D, XO 5 mU/ml) stained with Hoechst 33258 (blue) and antibodies against activated caspase 3 (red). (E) Pooled data displaying means ± SE of the percentage of apoptotic cells in cultures of siRNA transfected PAECs 4 h after exposure to xanthine oxidase (XO) in the indicated concentrations. (N = 4, *p < 0.05).

FIGURE 5

Western blot analysis of pulmonary artery endothelial cells transfected with scrambled and Ogg1-specific siRNA for active caspase 3 after cell exposure to xanthine oxidase. (A) Representative Western blot analysis for active caspase 3 in PAECs transfected with scrambled (Scr) or Ogg1-specific (Ogg1) siRNA (Control) or harvested 4 hours after treatment with xanthine oxidase (2 or 5 mU/ml). (B) Pooled data for relative band intensities of activated caspase 3 hybridization displayed as mean ± SE (N = 3, *p < 0.05).