Apoptosis Signal-regulating Kinase 1 (ASK1)-p38 Pathway-dependent Cytoplasmic Translocation of the Orphan Nuclear Receptor NR4A2 Is Required for Oxidative Stress-induced Necrosis

Abstract

p38 mitogen-activated protein kinases (MAPKs) play important roles in various cellular stress responses, including cell death, which is roughly categorized into apoptosis and necrosis. Although p38 signaling has been extensively studied, the molecular mechanisms of p38-mediated cell death are unclear. ASK1 is a stress-responsive MAP3K that acts as an upstream kinase of p38 and is activated by various stresses, such as oxidative stress. Here, we show that NR4A2, a member of the NR4A nuclear receptor family, acts as a necrosis promoter downstream of ASK1-p38 pathway during oxidative stress. Although NR4A2 is well known as a nucleus-localized transcription factor, we found that it is translocated into the cytosol after phosphorylation by p38. Because the phosphorylation site mutants of NR4A2 cannot rescue the cell death-promoting activity, ASK1-p38 pathway-dependent phosphorylation and subsequent cytoplasmic translocation of NR4A2 may be required for oxidative stress-induced cell death. In addition, NR4A2-mediated cell death does not depend on caspases and receptor-interacting protein 1 (RIP1)-RIP3 complex, suggesting that NR4A2 promotes an RIP kinase-independent necrotic type of cell death. Our findings may enable a more precise understanding of molecular mechanisms that regulate oxidative stress-induced and p38-mediated necrosis.

Keywords: Necrosis; Nuclear Receptor; Oxidative Stress; Phosphorylation; Signal Transduction; Stress Response; p38 MAPK.

FIGURE 1.

The ASK1-p38 pathway is required for 3 mm H₂O₂-induced caspase-independent cell death. A–C, after HeLa cells were treated with the indicated concentration of H₂O₂ for 8 h, cells were subjected to immunocytochemistry analysis using an anti-active caspase-3 antibody (A), and caspase-3 activity (B) or LDH release was measured (C). Error bars, S.E. (***, p < 0.001, one-way analysis of variance and Bonfferoni's multiple comparison test). D and E, after incubation with 3 mm H₂O₂ with or without 10 μm Z-VAD-fmk for the indicated time periods, caspase-3 (D) and LDH assays (E) were performed. Error bars, S.E. (**, p < 0.01, Student's t test). F and H, 48 h after transfection of the ASK1 siRNAs, HeLa cells were treated with the indicated concentration of H₂O₂. 8 h later, the caspase-3 (F) and LDH assays (H) were performed. Error bars, S.E. (*, p < 0.05; **, p < 0.01, one-way analysis of variance and Dunnett's multiple comparison test compared with the control). G and I, HeLa cells pretreated with 10 μm SP600125, 10 μm SB202190, and 10 μm SB203580, respectively, were incubated with the indicated concentration of H₂O₂. 8 h later, caspase-3 (G) and LDH assays (I) were performed. Error bars, S.E. (*, p < 0.05; **, p < 0.01, one-way analysis of variance and Dunnett's multiple comparison test compared with the control). J, HeLa cells pretreated with the indicated concentration of PH797804 were incubated with the indicated concentration of H₂O₂ for the time indicated and were subjected to immunoblotting. K, HeLa cells pretreated with 1 μm PH797804, 10 μm SB202190, and 10 μm SP600125 were incubated with the indicated concentration of H₂O₂. 8 h later, LDH assays were performed. Error bars, S.E. (**, p < 0.01, one-way analysis of variance and Dunnett's multiple comparison test compared with the control). L, 48 h after transfection of the ASK1 siRNAs, HeLa cells were treated with 3 mm H₂O₂ for the indicated time periods and subjected to immunoblotting. N.S., not significant.

FIGURE 2.

The ASK1-p38 pathway is required for H₂O₂-induced caspase- and RIP1-RIP3-independent necrosis. A, HeLa cells were treated with 3 mm H₂O₂ for 8 h with or without 10 μm Nec-1 and subjected to the LDH assay. Error bars, S.E. B, after MEFs were treated with the indicated concentration of H₂O₂ with or without 10 μm Z-VAD-fmk and 10 μm Nec-1 for 6 h, cells were subjected to an LDH assay. Error bars, S.E. C, after A549 cells were treated with the indicated concentration of H₂O₂ with or without 10 μm Z-VAD-fmk and 10 μm Nec-1 for 8 h, cells were subjected to an LDH assay. Error bars, S.E. D, MEFs pretreated with the indicated inhibitor compounds were incubated with 3 mm H₂O₂. The concentration of SB202190 is 10 μm. 6 h later, LDH assays were performed. Error bars, S.E. (*, p < 0.05, one-way analysis of variance and Bonfferoni's multiple comparison test). E, A549 cells pretreated with the indicated inhibitor compounds were incubated with 3 mm H₂O₂. 8 h later, LDH assays were performed. Error bars, S.E. F, HeLa cells pretreated with 1 μm PH797804 and 10 μm SB202190 were incubated with the indicated concentration of H₂O₂. 8 h later, LDH assays were performed. Error bars, S.E. (**, p < 0.01, one-way analysis of variance and Dunnett's multiple comparison test compared with the control). G, 48 h after transfection of the ASK1 siRNAs, HeLa cells were treated with the indicated concentration of H₂O₂. 8 h later, LDH assays were performed. Error bars, S.E. (**, p < 0.01, one-way analysis of variance and Bonfferoni's multiple comparison test). N.S., not significant.

FIGURE 3.

NR4A2 is required for H₂O₂-induced caspase- and RIP1-RIP3-independent necrosis. A, the knockdown efficiency of siRNAs in HeLa cells was evaluated using quantitative RT-PCR. Error bars, S.E. B, 48 h after transfection of the NR4A2 siRNAs, HeLa cells were subjected to immunoblotting. C, 48 h after transfection of the NR4A2 siRNAs, MEFs were subjected to immunoblotting. D, NR4A2 is required for caspase- and RIP1-RIP3-independent necrosis in MEF cells. 48 h after transfection of the NR4A2 siRNAs, MEFs were treated with 3 mm H₂O₂. 6 h later, LDH assays were performed. Error bars, S.E. (*, p < 0.05; ***, p < 0.001, one-way analysis of variance and Bonfferoni's multiple comparison test). E, 48 h after transfection with the NR4A2 siRNAs, the cells were treated with 3 mm H₂O₂ for 8 h and subjected to the LDH assay. Error bars, S.E. (*, p < 0.05; **, p < 0.01, one-way analysis of variance and Dunnett's multiple comparison test compared with the control).

FIGURE 4.

H₂O₂-induced phosphorylation of NR4A2 is mediated by the ASK1-p38 pathway. A, 24 h after transfection with FLAG-NR4A2, HeLa cells were treated with 3 mm H₂O₂. 15 min later, the cell lysates were incubated with or without λ-protein phosphatase (λPPase) and subjected to immunoblotting. B, 24 h after transfection with FLAG-NR4A2, HeLa cells were treated with the indicated inhibitor compounds, and 30 min later, the cells were incubated with 3 mm H₂O₂ for 15 min and subjected to immunoblotting. C, consensus phosphorylation sites for p38 ((S/T)P site) in NR4A2. D, F, G, H, and I, 24 h after transfection with the indicated expression plasmids, HeLa cells were treated with 3 mm H₂O₂ for 15 min and subjected to immunoblotting. E, consensus phosphorylation sites for p38 ((S/T)P site) in cluster II. J and K, 24 h after transfection with FLAG-NR4A2, HeLa cells were treated with the indicated inhibitor compounds, and 30 min later, the cells were incubated with 3 mm H₂O₂ for the indicated time periods and subjected to immunoblotting.

FIGURE 5.

The ASK1-p38 pathway mediates H₂O₂-induced cytoplasmic translocation of NR4A2. A, HeLa cells were treated with 3 mm H₂O₂ for 8 h with or without 100 nm actinomycin D and subjected to the LDH assay. Error bars, S.E. B and E, 24 h after transfection with the ASK1 siRNA, FLAG-NR4A2 was transiently transfected into HeLa cells. Twenty-four hours later, the cells were treated with 3 mm H₂O₂ for 90 min and subjected to immunocytochemistry analysis with anti-FLAG antibody. The images were monitored using confocal microscopy (B), and the cytoplasmic intensity per total intensity of FLAG-NR4A2 was quantitated using a cell image analyzer (E). Error bars, S.E. (*, p < 0.05; **, p < 0.01, one-way analysis of variance and Dunnett's multiple comparison test compared with the control) (E). C and F, 48 h after transfection with FLAG-NR4A2, HeLa cells that were pretreated with 10 μm SB202190 for 30 min were incubated with 3 mm H₂O₂. After the indicated periods, the cells were subjected to immunocytochemistry analysis with the anti-FLAG antibody. The images were monitored using confocal microscopy (C), and H₂O₂-induced translocation of each NR4A2 was quantified using a cell image analyzer (F). Error bars, S.E. (**, p < 0.01, Student's t test). D, the intensity of nuclear and cytoplasmic NR4A2, which is enclosed by green and red lines, respectively, was detected from 200 cells using a cell image analyzer. G, 48 h after transfection with FLAG-NR4A2, HeLa cells that were pretreated with 1 μm PH797804 and 5 μm K811 for 30 min were incubated with 3 mm H₂O₂. After the indicated periods, the cells were lysed and separated into nucleic and cytoplasmic fractions and then subjected to immunoblotting. H, 48 h after transfection with FLAG-NR4A2, HeLa cells that were pretreated for the indicated times with the indicated concentrations of leptomycin B (LMB) were incubated with 3 mm H₂O₂. After the indicated periods, the cells were subjected to immunocytochemistry analysis with the anti-FLAG antibody. I, there are two NESs in NR4A2. J, 48 h after transfection with FLAG-NR4A2, HeLa cells were incubated with 3 mm H₂O₂. After the indicated periods, the cells were lysed and separated into nucleic and cytoplasmic fractions and then were subjected to immunoblotting. K, 48 h after transfection with FLAG-NR4A2, HeLa cells were incubated with 3 mm H₂O₂. After the indicated periods, the cells were subjected to immunocytochemistry analysis. N.S., not significant.

FIGURE 6.

Alanine substitution of Ser¹²⁶, Thr¹²⁹, and Thr¹³² reduces H₂O₂-induced cytoplasmic translocation of NR4A2 and necrosis. A, 48 h after transfection with the indicated expression plasmids, HeLa cells were treated with 3 mm H₂O₂ for 90 min. After the indicated periods, the cells were subjected to immunocytochemistry analysis with the anti-FLAG antibody and phospho-NR4A2 antibody. The images were monitored using confocal microscopy. B–D, 48 h after transfection with the indicated expression plasmids, HeLa cells were treated with 3 mm H₂O₂ for 90 min. After the indicated periods, the cells were subjected to immunocytochemistry analysis with the anti-FLAG antibody. The images were monitored using confocal microscopy (B). H₂O₂-induced translocation of each NR4A2 was quantified using a cell image analyzer (**, p < 0.01, one-way analysis of variance and Dunnett's multiple comparison test compared with the control) (C and D). E, siRNA-resistant constructs of NR4A2 were successfully expressed. 24 h after transfection of the NR4A2 siRNA, the indicated NR4A2-expressing plasmids were transiently transfected, and 24 h later, HeLa cells were lysed and subjected to immunoblotting. F and G, 24 h after transfection of the NR4A2 siRNA and the indicated siRNA-resistant NR4A2-expressing constructs, the cells were treated with 3 mm H₂O₂ for 8 h and subjected to the LDH assay. Error bars, S.E. (**, p < 0.01, one-way analysis of variance and Bonfferoni's multiple comparison test). H, schematic model of this study.