ZBP1 orchestrates dynamic transitions between cell death pathways in response to arsenic and hyperosmotic stress

Abstract

Z-DNA binding protein 1 (ZBP1), a sensor of Z-form nucleic acids, plays a crucial role in cell death and inflammation. While its functions in infection and development are well-established, its involvement in environmental stress responses remains largely unexplored. In this study, we uncovered novel mechanisms by which ZBP1 mediates cell death under arsenic and hyperosmotic stress. We demonstrate that ZBP1 initiates necroptosis during the early stages of these stresses. As the stress persists, the cell death mode evolves, shifting towards apoptosis and pyroptosis in later stages. This transition is particularly pronounced when the necroptotic pathway is compromised. Interestingly, despite previous implications of stress granules (SGs) in arsenic-dependent cell death, we found that neither SGs formation inhibitors nor the ablation of SGs components significantly impacts cell death under arsenic or hyperosmotic stress. This suggests that environmental stress-induced necroptosis operates largely independent of SGs formation. Employing genome-wide CRISPR/Cas9 library screening, we identified that ZBP1-dependent cell death in response to arsenic is primarily driven by reactive oxygen species and the KEAP1-NRF2 signaling pathway. Notably, ZBP1 KO mice demonstrated resistance to arsenic-induced cell death and tissue injury, further substantiating our findings. Our research provides important new insights into ZBP1's role in environmental stress-induced cell death, expanding our understanding beyond its established functions in infection and development. These findings offer potential implications for comprehending stress-related tissue injury mechanisms.

Keywords: ZBP1; apoptosis; necroptosis; pyroptosis; stress granules.

Figure 1

Arsenic and osmotic stress induce ZBP1-dependent cell death.A, WT L929 were treated with different concentrations of sodium arsenite (0, 50, 100, 150, 200 μM) or sodium chloride (0, 100, 150, 200, 250 mM) for 4 h with or without 10 ng ml⁻¹ IFNγ pretreatment. Cell viability was determined by neutral red staining (mean values ± SD; Student's t test; ∗p < 0.05, ∗∗∗∗p < 0.0001, n = 2 independent experiments, six replicates per experiment). B, microscopic analysis of WT L929 for cell death induced by sodium arsenite or NaCl stress in different concentrations after IFNγ pretreatment. Arrows indicate dead cells. Bar, 50 μm. C, WT and ZBP1-deficient L929 cells were treated with 100 μM sodium arsenite or 200 mM NaCl for 4 h with or without 10 ng ml⁻¹ IFNγ pretreatment. Cell viability was determined by neutral red staining (mean values ± SD; Student's t test; ∗∗∗∗p < 0.0001, n = 3 independent experiments, five or six replicates per experiment). D, microscopic analysis of WT or ZBP1-deficient L929 for cell death induced by stress of 100 μM sodium arsenite or 200 mM NaCl for 4 h with or without 10 ng ml⁻¹ IFNγ pretreatment. Arrows indicate dead cells. Bar, 50 μm. E, WT or RIPK3-deficient L929 were treated with 100 μM sodium arsenite or 200 mM NaCl for 4 h with or without 10 ng ml⁻¹ IFNγ pretreatment. Cell viability was determined by neutral red staining (mean values ± SD; Student's t test; ∗∗∗∗p < 0.001, n = 2 independent experiments, six replicates per experiment). F, WT, ZBP1-deficient, RIPK3-deficient and mixed lineage kinase domain-like protein (MLKL)-deficient L929 were treated with 100 μM sodium arsenite or 200 mM NaCl for 4 h with or without 10 ng ml⁻¹ IFNγ pretreatment. Cell viability was determined by neutral red staining (mean values ± SD; Student's t test; ∗∗∗∗p < 0.0001, n = 2 independent experiments, five replicates per experiment). G, WT and ZBP1-deficient mouse embryonic fibroblast were treated with 100 μM sodium arsenite or 200 mM NaCl for 4 h with or without 10 ng ml⁻¹ IFNγ pretreatment. Cell viability was determined by neutral red staining (mean values ± SD; Student's t test; ∗∗∗∗p < 0.0001, n = 2 independent experiments, six replicates per experiment). MLKL, mixed lineage kinase domain-like protein.

Figure 2

RIPK3 and MLKL is essential for early necroptotic cell death induced by SA and NaCl in the presence or absence of IFNγ.A, immunoblot analysis with indicated antibodies in WT L929 cells stressed with 100 μM sodium arsenite or 200 mM NaCl for 4 h with or without 10 ng ml⁻¹ IFNγ pretreatment (n = 2 independent experiments). B, immunoblot analysis with indicated antibodies in WT and ZBP1-deficient L929 cells stressed with 100 μM sodium arsenite or 200 mM NaCl for 4 h with or without 10 ng ml⁻¹ IFNγ pretreatment (n = 2 independent experiments). C and D, Immunoblot analysis with indicated antibodies in WT, ZBP1-, RIPK3- and MLKL-deficient L929 cells stressed with 100 μM sodium arsenite or 200 mM NaCl with or without IFNγ pretreatment. Arrows indicate cleaved CASP3 (p17) and cleaved CASP8 (p43) (n = 2 independent experiments). MLKL, mixed lineage kinase domain-like protein.

Figure 3

ZBP1 mediates a temporal switch in cell death pathways in response to arsenic or hyperosmotic Stress.A and B, WT, ZBP1-, RIPK3- and MLKL-deficient L929 were treated with 100 μM sodium arsenite for 6 and 12 h with or without 10 ng ml⁻¹ IFNγ pretreatment. Cell viability was determined by neutral red staining. Representative pics of cell death were shown (mean values ± SD; Student's t test; ∗∗∗∗p < 0.0001, n = 2 independent experiments, three replicates per experiment). C and D, WT, ZBP1-, RIPK3- and MLKL-deficient L929 were treated with 200 mM NaCl for 6 and 12 h with or without 10 ng ml⁻¹ IFNγ pretreatment. Cell viability was determined by neutral red staining. Representative pics of cell death were shown (mean values ± SD; Student's t test; ∗∗∗∗p < 0.0001, n = 2 independent experiments, three replicates per experiment). E and F, immunoblot analysis with indicated antibodies in WT and MLKL-deficient L929 stressed with 100 μM sodium arsenite or 200 mM NaCl with or without IFNγ pretreatment. Arrows indicate cleaved CASP3 (p17), cleaved CASP8 (p43), cleaved CASP9 (p37) and cleaved gasdermin E (GSDME) (n = 2 independent experiments). G, immunoblot analysis of with indicated antibodies in MLKL-deficient L929 that were treated with 100 μM sodium arsenite with or without IFNγ pretreatment. In addition, Z-VAD-FMK was used to inhibit activation of Caspases (n = 2 independent experiments). H, MLKL KO L929 was treated with 100 μM sodium arsenite for 12 h with IFNγ pretreatment. Cell viability was determined by neutral red staining (mean values ± SD; Student's t test; ∗∗∗∗p < 0.0001, n = 2 independent experiments, five replicates per experiment). In addition, Z-VAD-FMK was used to inhibit activation of Caspases. GSDME, gasdermin E; MLKL, mixed lineage kinase domain-like protein.

Figure 4

ZBP1 mediated apoptosis and pyroptosis in the absence of necroptosis.A–D, MLKL KO and MLKL/ZBP1 DKO L929 were treated with 100 μM sodium arsenite or 200 mM NaCl for 12 h with or without 10 ng ml⁻¹ IFNγ pretreatment. Cell viability was determined by neutral red staining (mean values ± SD; Student's t test; ∗∗∗∗p < 0.0001, n = 2 independent experiments, six replicates per experiment). E and F, immunoblot analysis with indicated antibodies in MLKL KO and MLKL/ZBP1 DKO L929 that were treated with 100 μM sodium arsenite or 200 mM NaCl with or without IFNγ pretreatment. Arrows indicate cleaved CASP3 (p17), cleaved CASP8 (p43), cleaved CASP9 (p37) and cleaved GSDME (n = 2 independent experiments). GSDME, gasdermin E; MLKL, mixed lineage kinase domain-like protein.

Figure 5

RIPK1 and RIPK3 promote ZBP1-mediated apoptosis and pyroptosis in the absence of necroptosis.A and B, MLKL KO and MLKL/RIPK3 DKO L929 were stressed with 100 μM sodium arsenite or 100 μM sodium arsenite for 12 h with 10 ng ml⁻¹ IFNγ pretreatment. Cell viability was determined by neutral red staining (mean values ± SD; Student's t test; ∗∗∗∗p < 0.0001, n = 2 independent experiments, six replicates per experiment). C, MLKL KO and MLKL/RIPK1 DKO L929 were treated with 100 μM sodium arsenite or 100 μM sodium arsenite for 12 h with or without 10 ng ml⁻¹ IFNγ pretreatment. Cell viability was determined by neutral red staining (mean values ± SD; Student's t test; ∗∗∗∗p < 0.0001, n = 2 independent experiments, six replicates per experiment). D, immunoblot analysis with indicated antibodies in MLKL KO and MLKL/RIPK3 DKO L929 stressed with 100 μM sodium arsenite or 200 mM NaCl for 12 h with or without IFNγ pretreatment. Arrows indicate cleaved ZBP1, cleaved CASP3 (p17), cleaved CASP8 (p43), cleaved CASP9 (p37) and cleaved GSDME. E, immunoblot analysis with indicated antibodies in MLKL KO and MLKL/RIPK1 DKO L929 that were treated with 100 μM sodium arsenite or 200 mM NaCl for 12 h with or without IFNγ pretreatment. Arrows indicate cleaved ZBP1, cleaved CASP3 (p17), cleaved CASP8 (p43), cleaved CASP9 (p37) and cleaved GSDME (n = 2 independent experiments). GSDME, gasdermin E; MLKL, mixed lineage kinase domain-like protein.

Figure 6

Stress granulesformation is dispensable for arsenic- and hyperosmotic stress-induced cell death.A and B, representative images of cells stained with DAPI or immunostained for G3BP1 and graphs depicting quantification of the proportion of G3BP1⁺ cells in IFNγ primed WT L929 cells stressed with 100 μM sodium arsenite or 200 mM NaCl for 2 h with or without 50 μg ml⁻¹ cycloheximide pretreatment. Arrows indicate cells that form G3BP1 aggregation, which is a characterization of stress granules formation. Bar, 50 μm (n = 2 independent experiments). C and D, IFNγ primed L929 were stressed with 100 μM sodium arsenite or 200 mM NaCl for 4 h (WT) or 8 h (MLKL KO) with or without 50 μg ml⁻¹ cycloheximide pretreatment. Cell viability was determined by neutral red staining (mean values ± SD; Student's t test; ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, n = 2 independent experiments, six replicates per experiment). E and F, Representative images of cells stained with DAPI or immunostained for TIA1 and graphs depicting quantification of the proportion of TIA1⁺ cells in IFNγ primed WT or G3BP1/2 DKO L929 cells stressed with 100 μM sodium arsenite or 200 mM NaCl for 2 h. Arrows indicate cells that form TIA1 aggregation, which is a characterization of stress granules formation. Bar, 50 μm (n = 2 independent experiments). G and H, IFNγ primed WT or G3BP1/2 DKO L929 were stressed with sodium arsenite or NaCl of different concentrations for 4 h Cell viability was determined by neutral red staining (mean values ± SD; Student's t test; ns p > 0.5, n = 2 independent experiments, six replicates per experiment). MLKL, mixed lineage kinase domain-like protein.

Figure 7

KEAP1-NRF2 Axis protects against arsenic-induced cell death.A, the visualization analysis of CRISPR screening results. The red dots indicate significance gene (−log10 > 1.3), while the blue dots indicate insignificance gene (−log10 < 1.3). Sig: significance, Notsig: not significance, pos|lfc: positive|log2 fold change. B, WT or KEAP1 KO L929 was stressed with 100 μM sodium arsenite or 200 mM NaCl for 6 h with or without IFNγ pretreatment. Cell viability was determined by neutral red staining (mean values ± SD; Student's t test; ns p > 0.1, ∗∗∗∗p < 0.0001, n = 2 independent experiments, six replicates per experiment). C, immunoblotting analysis with indicated antibodies in WT, KEAP1-deficient, MLKL-deficient and MLKL/KEAP1 DKO L929. D, KEAP1-deficient L929 cells reconstituted with or without KEAP1 were stressed with 100 μM sodium arsenite for 6 h with or without IFNγ pretreatment (mean values ± SD; Student's t test; ∗∗∗∗p < 0.0001 n = 2 independent experiments, five replicates per experiment). E, DCFH-DA staining of WT or KEAP1-deficient L929 those were stress with 100 μM sodium arsenite for 6 h to evaluate intracellular ROS levels. F, immunoblot analysis with indicated antibodies in WT or KEAP-deficient L929 stressed with 100 μM sodium arsenite for 6 h with or without IFNγ pretreatment (n = 2 independent experiments). G, unprimed or IFNγ-primed WT L929 were stressed with 100 μM sodium arsenite or 200 mM NaCl for 6 h with or without pretreatment of 10 mM NAC, which is a ROS scavenger, cell viability was assayed using neutral red staining. Representative pics of cells were shown, arrows indicate dead cells (mean values ± SD; Student's t test; ns p > 0.1, ∗∗∗∗p < 0.0001, n = 2 independent experiments, five replicates per experiment). H, immunoblot analysis with indicated antibodies in unprimed or IFNγ-primed WT L929 cells stressed with 100 μM sodium arsenite or 200 mM NaCl for 6 h with or without NAC pretreatment (n = 2 independent experiments). MLKL, mixed lineage kinase domain-like protein.

Figure 8

ZBP1 drives arsenic-induced tissue injury.A, immunoblotting analysis of the expression level of RIPK3 and ZBP1 in the liver, spleen, lung, brain, large intestine, and small intestine from WT and Zbp1^−/− mice. B, representative H&E-stained sections of spleen, lung, liver and kidney from WT and Zbp1^−/− mice supplied with drinking water with or without sodium arsenite (n = 8 mice for each group). C, spleen weight index and representative pics of spleens of WT or Zbp1^−/− mice supplied with drinking water with or without sodium arsenite (mean values ± SD; Student's t test; ns p > 0.5, ∗∗∗∗p < 0.0001, n = 8 mice for each group). D, representative images of sections stained for cleaved caspase-3, p-MLKL, DAPI or TUNEL of liver and lung from WT or Zbp1^−/− mice supplied with drinking water with or without sodium arsenite (n = 3 mice for each group). E, the MDA and glutathione levels were assessed to determine the lipid oxidation levels and antioxidant capacity in the spleen of WT and Zbp1^−/− mice supplied with drinking water with or without sodium arsenite (mean values ± SD; Student's t test; ns p > 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n = 6 mice for each group). F, The MDA and glutathione levels were assessed to determine the lipid oxidation levels and antioxidant capacity in the lung of WT or Zbp1^−/− mice supplied with drinking water with or without sodium arsenite (mean values ± SD; Student's t test; ns p > 0.05, ∗p < 0.05, ∗∗∗p < 0.001, n = 6 mice for each group). MDA, malondialdehyde; MLKL, mixed lineage kinase domain-like protein.