DNA damage response pathway regulates Nrf2 in response to oxidative stress

Abstract

Nrf2 acts as a transcriptional master regulator to orchestrate antioxidant responses and maintain redox balance. However, the cellular pathway for translating oxidative stress signals into Nrf2-dependent antioxidant responses remain incompletely understood. Here, we show that reactive oxygen species (ROS) function as signaling molecules in modulating Nrf2's stability and transcriptional activity by activating the DNA damage response (DDR) signaling pathway. When activated, CHK2 phosphorylates the autophagy adaptor protein p62 at serine-349, promoting its interaction with Keap1 and disrupting the Keap1-Nrf2 interaction, thereby inhibiting Nrf2 ubiquitination-dependent degradation. In addition, CHK2 directly phosphorylates Nrf2 at serine-566/serine-577, enhancing its transcriptional activity and antioxidant capacity. Consistent with these effects, Chk2^-/- mice show impaired expression of Nrf2 and its downstream antioxidant target genes, along with more severe renal tissue damage in an ROS-dependent model of renal ischemia/reperfusion injury. Our study reveals a direct mechanism linking the DDR signaling pathway to ROS-triggered Nrf2-dependent antioxidant responses, providing critical insight into cellular protection against oxidative stress-induced damage.

Fig. 1.. ROS-ATM-CHK2 pathway stabilizes Nrf2 under metabolic stress conditions.

(A) H1299 cells were pretreated with NAC (5 mM) for 2 hours before hypoxia treatment for 18 hours. (B) H1299 cells were pretreated with NAC (5 mM) for 2 hours before glucose (Glu) starvation for 6 hours. (C) H1299 cells stably expressing either ATM shRNA (shATM) or a negative control shRNA (shNC) were subjected to hypoxic or normoxic conditions for 18 hours. (D) H1299 cells stably expressing either ATM shRNA or shNC were cultured with or without glucose starvation for 6 hours. (E) H1299 stably expressing CHK2 shRNA (shCHK2) or shNC were subjected to hypoxic or normoxic conditions for 18 hours. (F) H1299 cells stably expressing shCHK2 or shNC were cultured with or without glucose starvation for 6 hours. (G) H1299 cells stably expressing shCHK2 or shNC and CHK2-knockdown H1299 cells with reconstituted expression of the CHK2 protein were treated with or without H₂O₂ (100 μM) for 1 hour. (H) Exogenous Flag-tagged CHK2-WT or CHK2-T68A plasmids [engineered with synonymous mutations conferring resistance to single guide RNA (sgRNA) targeting endogenous CHK2] were transfected into CHK2-knockout HEK293 cells generated by sgRNA. After 36 hours of transfection, cells were treated with or without H₂O₂ (100 μM) for 1 hour, followed by analysis of Nrf2 protein accumulation. (A to H) Immunoblot analyses were performed with the indicated antibodies. Graphs were presented as means ± SD from three independent experiments, and statistical significance was assessed using one-way and two-way analysis of variance (ANOVA). *P < 0.05; **P < 0.01; ***P < 0.001. p, phospho; NT, untreated.

Fig. 2.. CHK2 phosphorylates p62 at S349.

(A and B) HCT116 cells were treated with H₂O₂ (100 μM) for the indicated periods of time. Whole-cell lysates were immunoprecipitated (IP) with anti-CHK2 (A) or anti-p62 (B). IgG, immunoglobulin G. (C) Glutathione S-transferase (GST)–CHK2 FL and truncated fusion proteins incubated with Flag-p62 protein and subjected to an in vitro GST pull-down assay. (D) A schematic illustration of the CHK2 protein sequence with FL and various domains. NLS, nuclear localization signal; SQ/TQ, Ser-Gln/Thr-Gln. (E) GST-p62 FL and truncated fusion proteins incubated with Flag-CHK2 protein and subjected to GST pull-down assay. (F) A schematic illustration of the p62 protein sequence with FL and various domains. (G) Purified GST-p62 FL and truncated fusion proteins were incubated with Flag-CHK2 protein and subjected to GST pull-down assay. (H) HCT116 cells were pretreated with CHK2 inhibitor (20 μM) for 3 hours before H₂O₂ (100 μM) stimulation for 0.5 hours. (I) Flag-CHK2 WT or Flag-CHK2 T68A were transfected into HCT116 cells with or without H₂O₂ (100 μM) for 0.5 hours. (J) An in vitro kinase assay was performed by incubating GST-p62 with CHK2 protein in the presence of adenosine 5′-O-(3-thiotriphosphate) (ATP-γ-S). (K) Purified GST-p62 was phosphorylated by CHK2 in vitro and analyzed by mass spectrometry. m/z, mass/charge ratio. (L) Alignment of protein sequences spanning p62 S349 from different species. (M) An in vitro kinase assay was performed by incubating GST-p62 WT or GST-p62 S349A with CHK2 protein in the presence of adenosine 5′-triphosphate. (N) H1299 cells stably expressing indicated shRNA were treated with or without H₂O₂ (100 μM) for 1 hour. (O) H1299 cells stably expressing indicated shRNA were treated with or without H₂O₂ (100 μM) for 1 hour. (A to C, E, G, H to J, and M to O) Immunoblot analyses were performed with the indicated antibodies. Graphs were presented as means ± SD from three independent experiments, and statistical significance was assessed using two-way ANOVA. *P < 0.05; ***P < 0.001. CBB, coomassie brilliant blue.

Fig. 3.. CHK2-mediated p62 phosphorylation activates the p62-Keap1-Nrf2 pathway.

(A) HCT116 cells stably expressing shCHK2 or shNC were treated with or without H₂O₂ (100 μM) for 1 hour. Whole-cell lysates were immunoprecipitated with anti-Keap1, and the precipitated proteins were immunoblotted with anti-p62 and anti-Keap1 antibodies. (B) HCT116 cells were pretreated with CHK2 inhibitor (20 μM) for 3 hours before H₂O₂ (100 μM) stimulation for 1 hour. Whole-cell lysates were immunoprecipitated with anti-Keap1, and the precipitated proteins were immunoblotted with anti-p62 and anti-Keap1 antibodies. (C) Flag-CHK2 WT or Flag-CHK2 T68A were transfected into HCT116 cells. After 36 hours, cells were treated with or without H₂O₂ (100 μM) for 1 hour. Whole-cell lysates were immunoprecipitated with anti-Keap1, and the precipitated proteins were immunoblotted with anti-p62 and anti-Keap1 antibodies. (D) Flag-p62 WT or Flag-p62 S349A was transfected into nontargeting control sgRNA or single guide RNA targeting CHEK2–treated HEK293 cells. After 36 hours, cells were treated with or without H₂O₂ (100 μM) for 1 hour. Whole-cell lysates were immunoprecipitated with anti-Flag, and the precipitated proteins were immunoblotted with anti-Keap1 and anti-Flag antibodies. (E) Exogenous Flag-tagged p62 WT or p62 S349A plasmids (engineered with synonymous mutations conferring resistance to shRNA targeting endogenous p62) were transfected into p62-knockdown HCT116 cells that are stably expressing shCHK2 or shNC. After 36 hours, cells were pretreated with MG132 (20 μM) for 0.5 hours before H₂O₂ (100 μM) stimulation for 1 hour. Whole-cell lysates were immunoprecipitated with anti-Nrf2, and the precipitated proteins were immunoblotted with anti-Keap1 and anti-Nrf2 antibodies. (F and G) Flag-p62 WT or Flag-p62 S349A was transfected into HCT116 cells stably expressing shCHK2 or shNC. After 36 hours, cells were pretreated with MG132 (20 μM) for 0.5 hours before H₂O₂ (100 μM) stimulation for 1 hour. Whole-cell lysates were immunoprecipitated with anti-Nrf2, and the precipitated proteins were immunoblotted with anti-ubiquitin (Ub) and anti-Nrf2 antibodies. (A to G) Immunoblot analyses were performed with the indicated antibodies.

Fig. 4.. CHK2 phosphorylates Nrf2 at S566 and S577.

(A and B) HCT116 cells were treated with H₂O₂ (100 μM) for the indicated periods of time. Whole-cell lysates were immunoprecipitated with anti-CHK2 (A) or anti-Nrf2 (B). h, hours. (C) GST-Nrf2 FL and truncated fusion proteins incubated with Flag-CHK2 protein and subjected to GST pull-down assay. (D) A schematic illustration of Nrf2 protein sequence with FL and various domains. (E) GST-CHK2 FL and truncated fusion proteins incubated with Flag-Nrf2 protein and subjected to in vitro GST pull-down assay. (F) A schematic illustration of CHK2 protein sequence with FL and various domains. (G) HCT116 cells were cultured in medium containing CHK2 inhibitor (20 μM) for 3 hours. Cells were pretreated with MG132 (20 μM) for 0.5 hours before H₂O₂ (100 μM) stimulation for 1 hour. Whole-cell lysates were immunoprecipitated with anti-Nrf2, and the precipitated proteins were immunoblotted with anti-CHK2 and anti-Nrf2 antibodies. (H) Flag-CHK2 WT or Flag-CHK2 T68A was transfected into HCT116 cells stably expressing shCHK2. After 36 hours, cells were pretreated with MG132 (20 μM) for 0.5 hours before H₂O₂ (100 μM) stimulation for 1 hour. Whole-cell lysates were immunoprecipitated with anti-Flag, and the precipitated proteins were immunoblotted with anti-Nrf2 and anti-Flag antibodies. (I) An in vitro kinase assay was performed by incubating GST-Nrf2 with CHK2 protein in the presence of ATP-γ-S. The samples were then alkylated with p-Nitrobenzyl mesylate. (J) Purified GST-Nrf2 was phosphorylated by CHK2 in vitro and analyzed by mass spectrometry. (K) Alignment of protein sequences spanning Nrf2 S566 and Nrf2 S577 from different species. (L) An in vitro kinase assay was performed by incubating GST-Nrf2 WT, GST-Nrf2 S566A, or GST-Nrf2 S577A with CHK2 protein in the presence of ATP. The samples were then analyzed by immunoblotting. (A to C, E, G to I, and L) Immunoblot analyses were performed with the indicated antibodies.

Fig. 5.. CHK2-mediated phosphorylation of Nrf2 enhances its antioxidant capacity.

(A to D) RT-PCR analyses of mRNA levels of HO-1, NQO1, SOD2, and GCLM in H1299 cells stably expressing ATM shRNA or shNC treated with or without H₂O₂ (200 μM) for 6 hours. (E to H) RT-PCR analyses of mRNA levels of HO-1, NQO1, SOD2, and GCLM in H1299 cells stably expressing shCHK2 or shNC treated with or without H₂O₂ (200 μM) for 6 hours. (I to L) RT-PCR analyses of mRNA levels of HO-1, NQO1, SOD2, and GCLM in Nrf2-knockdown H1299 cells stably expressing Nrf2 WT or Nrf2 S2A protein treated with or without H₂O₂ (200 μM) for 6 hours. (M and N) H1299 cells stably expressing shCHK2 or shNC were treated with or without H₂O₂ (500 μM) for 6 hours. Flow cytometry [2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA)] was used to detect ROS levels. Total antioxidant capacity was also measured. (O and P) Nrf2-knockdown H1299 cells stably expressing vector, Nrf2 WT, or Nrf2 S2A protein were treated with or without H₂O₂ (500 μM) for 6 hours. Flow cytometry (DCFH-DA) was used to detect ROS levels. Total antioxidant capacity was also measured. Graphs were presented as means ± SD from three independent experiments, and statistical significance was assessed using two-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 6.. CHK2-mediated Nrf2 activation attenuates renal IRI and promotes tumor survival.

(A to I) Chk2^+/+ or Chk2^−/− mice (n = 6) were subjected to a renal ischemia/reperfusion operation, consisting of 45 min of ischemia, followed by 24 hours of reperfusion. (A and B) Scr and BUN were detected in blood supernatant. (C and D) Representative H&E and TUNEL staining of Chk2^+/+ or Chk2^−/− mice renal tissues were performed by optical microscope. (E to I) The expression levels of Nrf2, p-p62 S349, p62, HO-1, and NQO1 in IRI and SHAM renal tissue. Tubulin was used as a control for loading. (J to L) Therapeutic evaluation of cisplatin and CHK2 inhibitor BML-277 in A549 xenograft models. (J) Representative ex vivo tumor specimens. Tumor growth dynamics (K) and excised tumor mass (L) were systematically quantified (n = 6). (M to O) Pharmacological responses in A549 cells expressing Nrf2 variants. (M) Resected tumor macroscopic characteristics. Tumor volume progression (N) and corresponding neoplastic tissue weight (O) (n = 6). Graphs were presented as means ± SD, and statistical significance was assessed using one-way and two-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 7.. A schematic model showing how the ATM-CHK2 axis drives Nrf2-dependent antioxidant responses under oxidative stress conditions.