DNA Repair Interacts with Autophagy To Regulate Inflammatory Responses to Pulmonary Hyperoxia

Abstract

Oxygen is supplied as a supportive treatment for patients suffering from acute respiratory distress syndrome. Unfortunately, high oxygen concentration increases reactive oxygen species generation, which causes DNA damage and ultimately cell death in the lung. Although 8-oxoguanine-DNA glycosylase (OGG-1) is involved in repairing hyperoxia-mediated DNA damage, the underlying molecular mechanism remains elusive. In this study, we report that ogg-1-deficient mice exhibited a significant increase of proinflammatory cytokines (TNF-α, IL-6, and IFN-γ) in the lung after being exposed to 95% oxygen. In addition, we found that ogg-1 deficiency downregulated (macro)autophagy when exposed to hyperoxia both in vitro and in vivo, which was evident by decreased conversion of LC3-I to LC3-II, reduced LC3 punctate staining, and lower Atg7 expression compared with controls. Using a chromatin immunoprecipitation assay, we found that OGG-1 associated with the promoter of Atg7, suggesting a role for OGG1 in regulation of Atg7 activity. Knocking down OGG-1 decreased the luciferase reporter activity of Atg7. Further, inflammatory cytokine levels in murine lung epithelial cell line cells were downregulated following autophagy induction by starvation and rapamycin treatment, and upregulated when autophagy was blocked using 3-methyladenine and chloroquine. atg7 knockout mice and Atg7 small interfering RNA-treated cells exhibited elevated levels of phospho-NF-κB and intensified inflammatory cytokines, suggesting that Atg7 impacts inflammatory responses to hyperoxia. These findings demonstrate that OGG-1 negatively regulates inflammatory cytokine release by coordinating molecular interaction with the autophagic pathway in hyperoxia-induced lung injury.

FIGURE 1. OGG-1 responds to hyperoxic DNA damage and inflammation in lung cells

MLE-12 cells were incubated in room air or 95% O₂. (A) DNA strand breaks were detected by a comet assay through measuring tail length (indicated by rulers) with confocal laser scanning fluorescence microscopy (CLSM). (B) Tail lengths were markedly increased in lung cells by hyperoxia compared to the control (P<0.001). (C) OGG-1 activity under 24 h hyperoxia was determined by incision enzymatic assay. (D) Increased inflammatory responses in MLE-12 cells after 6 h or 24 h exposure to hyperoxia by immunoblotting analysis. (E) Inflammatory responses in mice increased with exposure time (24, 48 and 72 h) by immunoblotting analysis. Data were representative of three experiments with similar results (student t-test, *p< 0.05, **p< 0.01).

FIGURE 2. Increased PMN, oxidation injury and inflammatory responses in lungs of ogg-1 KO mice

(A) Hyperoxia increased lung injury and inflammation as assessed by H&E staining. (B) and (C) Increased PMN infiltration and an acute inflammatory response were observed in the lung (B) and blood (C) of ogg-1 KO mice compared to WT mice (n=6) following hyperoxia for 48 h. (D)–(F), Increased inflammatory cytokines in BAL fluid of ogg-1 KO mice compared to those of WT mice by ELISA. (G) Increased expression of inflammatory cytokines in lungs of ogg-1 KO mice compared to WT mice by immunoblotting analysis. ogg-1 KO mice and WT mice were exposed to hyperoxia (95%) for 48 h. Gel data were quantified using ImageJ densitometry. Data were representative of three experiments with similar results (student t-test, *p< 0.05, **p< 0.01).

FIGURE 3. ogg-1 KO mice exhibit impaired autophagy under hyperoxia

(A) Immunoblotting analysis of Atg7, p-NF-κB and LC3 in lungs of ogg-1 KO and WT mice (n=6) exposed to hyperoxia for 48 h. (B) Decreased expression of Atg7 in ogg-1 KO mice by immunohistochemistry. (C) Decreased Atg7 in MLE-12 cells after 48 h exposure to hyperoxia by immunoblotting analysis. Gel data were quantified using densitometry with Image J. (D) Tandem GFP-RFP-LC3 plasmids and OGG-1 siRNA were transfected to MH-S cells and then cells were exposed to hyperoxia for 6 h. Arrows indicate LC3 puncta. Data were representative of three experiments with similar results (student t-test, *p< 0.05, **p< 0.01). (E) Immunoblot analysis of LC3 with RFP-GFP-LC3/siOGG-1 transfected to MLE-12 cells that were exposed to hyperoxia for 6 h (lane 1: Ctrl siRNA 0 h; lane 2:OGG-1 siRNA 0 h; lane 3: Ctrl siRNA 6 h; lane 4: OGG-1 siRNA 6 h).

FIGURE 4. OGG-1 interacts with Atg7

(A) Co-localization of OGG-1 and Atg7 was observed by microscopy (see arrows). (B) Interaction between OGG-1 and Atg7 detected using co-immunoprecipitation (CoIP) assay. IB, immunoblotting. (C) ChIP and real-time PCR analysis of OGG-1 for DNA binding of Atg7 or GAPDH in MLE-12 cells ed under hyperoxia for 24 h. (D) Normalized luciferase activity of a reporter containing the promoter constructs of Atg7 in MLE-12 cells. Cells were transfected with siRNA of OGG-1. 24 h later, the cells were transfected with Atg7-reporter plasmid and then followed by hyperoxia exposure for 24 h. Data were representative of three experiments with similar results (student t-test, *p< 0.05, **p< 0.01).

FIGURE 5. Atg7 deficiency contributes to intensified inflammatory responses under hyperoxia in vitro and in vivo

(A) Increased expression of cytokines (IL-6, TNF-α) and p-NF-κB after knocking down Atg7 with siRNA in MLE-12 cells. (B) and (C) PMN infiltration and inflammatory response were increased in the lung (B) and blood (C) of atg7 KO mice compared to WT mice. (D) Increased lung injury and inflammation as assessed by H&E staining. (E)-(G) Increased inflammatory cytokines in BAL fluid of atg7 KO mice compared to WT mice by ELISA. (H) Increased expression of inflammatory cytokines in the lungs of atg7 KO mice (n=6) compared to WT mice after 48 h hyperoxic exposure by immunoblotting analysis. Data were representative of three experiments with similar results (student t-test, *p< 0.05).

FIGURE 6. OGG-1 plays a role in regulating the translocation of NF-κB

(A) Increased expression of NF-κB in ogg-1 KO mice after 48 h hyperoxia by immunoblotting analysis. Six mice were in each goup. (B) Knocking down OGG-1 with siRNA in MLE-12 cells increased NF-κB expression under hyperoxia using immunoblotting analysis. (C) Translocation of NF-κB was observed by microscopy (arrows show the nuclear translocation). (D) Hyperoxia-induced cytokine production was inhibited by NF-κB inhibitor (SN50). Cells were pretreated with SN50 (1.8 µM) for 1 h before hyperoxia exposure. Data were representative of three experiments with similar results. (E) Schematic illustration of the signaling pathways for cell viability regulated by OGG-1 under hyperoxia.