Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Reactive oxygen species regulate Smac mimetic/TNFα-induced necroptotic signaling and cell death

Oncogene volume 34, pages 5796–5806 (2015)Cite this article

Subjects

Abstract

Necroptosis represents a key programmed cell death pathway involved in various physiological and pathophysiological conditions. However, the role of reactive oxygen species (ROS) in necroptotic signaling has remained unclear. In the present study, we identify ROS as critical regulators of BV6/tumor necrosis factor-α (TNFα)-induced necroptotic signaling and cell death. We show that BV6/TNFα-induced cell death depends on ROS production, as several ROS scavengers such as butylated hydroxyanisole, N-acetylcysteine, α-tocopherol and ethyl pyruvate significantly rescue cell death. Before cell death, BV6/TNFα-stimulated ROS generation promotes stabilization of the receptor-interacting protein kinase 1 (RIP1)/RIP3 necrosome complex via a potential positive feedback loop, as on the one hand radical scavengers attenuate RIP1/RIP3 necrosome assembly and phosphorylation of mixed lineage kinase domain like (MLKL), but on the other hand silencing of RIP1 or RIP3 reduces ROS production. Although MLKL knockdown effectively decreases BV6/TNFα-induced cell death, it does not affect RIP1/RIP3 interaction and only partly reduces ROS generation. Moreover, the deubiquitinase cylindromatosis (CYLD) promotes BV6/TNFα-induced ROS generation and necrosome assembly even in the presence of BV6, as CYLD silencing attenuates these events. Genetic silencing of phosphoglycerate mutase 5 or dynamin-related protein 1 (Drp1) fails to protect against BV6/TNFα-induced cell death. By demonstrating that ROS are involved in regulating BV6/TNFα-induced necroptotic signaling, our study provides new insights into redox regulation of necroptosis.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

Similar content being viewed by others

References

  1. Vanden Berghe T, Linkermann A, Jouan-Lanhouet S, Walczak H, Vandenabeele P . Regulated necrosis: the expanding network of non-apoptotic cell death pathways. Nat Rev Mol Cell Biol 2014; 15: 135–147.

    Article  CAS  PubMed  Google Scholar 

  2. Cai Z, Jitkaew S, Zhao J, Chiang HC, Choksi S, Liu J et al. Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis. Nat Cell Biol 2014; 16: 55–65.

    Article  CAS  PubMed  Google Scholar 

  3. Wang H, Sun L, Su L, Rizo J, Liu L, Wang LF et al. Mixed lineage kinase domain-like protein MLKL causes necrotic membrane disruption upon phosphorylation by RIP3. Mol Cell 2014; 54: 133–146.

    Article  CAS  PubMed  Google Scholar 

  4. Dondelinger Y, Declercq W, Montessuit S, Roelandt R, Goncalves A, Bruggeman I et al. MLKL compromises plasma membrane integrity by binding to phosphatidylinositol phosphates. Cell Rep 2014; 7: 971–981.

    Article  CAS  PubMed  Google Scholar 

  5. Wang Z, Jiang H, Chen S, Du F, Wang X . The mitochondrial phosphatase PGAM5 functions at the convergence point of multiple necrotic death pathways. Cell 2012; 148: 228–243.

    Article  CAS  PubMed  Google Scholar 

  6. Fulda S, Vucic D . Targeting IAP proteins for therapeutic intervention in cancer. Nat Rev Drug Discov 2012; 11: 109–124.

    Article  CAS  PubMed  Google Scholar 

  7. Bertrand MJ, Milutinovic S, Dickson KM, Ho WC, Boudreault A, Durkin J et al. cIAP1 and cIAP2 facilitate cancer cell survival by functioning as E3 ligases that promote RIP1 ubiquitination. Mol Cell 2008; 30: 689–700.

    Article  CAS  PubMed  Google Scholar 

  8. Wright A, Reiley WW, Chang M, Jin W, Lee AJ, Zhang M et al. Regulation of early wave of germ cell apoptosis and spermatogenesis by deubiquitinating enzyme CYLD. Dev Cell 2007; 13: 705–716.

    Article  CAS  PubMed  Google Scholar 

  9. Hitomi J, Christofferson DE, Ng A, Yao J, Degterev A, Xavier RJ et al. Identification of a molecular signaling network that regulates a cellular necrotic cell death pathway. Cell 2008; 135: 1311–1323.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Han D, Ybanez MD, Ahmadi S, Yeh K, Kaplowitz N . Redox regulation of tumor necrosis factor signaling. Antioxid Redox Signal 2009; 11: 2245–2263.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Morgan MJ, Liu ZG . Reactive oxygen species in TNFalpha-induced signaling and cell death. Molecules Cells 2010; 30: 1–12.

    Article  CAS  PubMed  Google Scholar 

  12. Vanden Berghe T, Vanlangenakker N, Parthoens E, Deckers W, Devos M, Festjens N et al. Necroptosis, necrosis and secondary necrosis converge on similar cellular disintegration features. Cell Death Differ 2010; 17: 922–930.

    Article  CAS  PubMed  Google Scholar 

  13. Shulga N, Pastorino JG . GRIM-19-mediated translocation of STAT3 to mitochondria is necessary for TNF-induced necroptosis. J Cell Sci 2012; 125: 2995–3003.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Shindo R, Kakehashi H, Okumura K, Kumagai Y, Nakano H . Critical contribution of oxidative stress to TNFalpha-induced necroptosis downstream of RIPK1 activation. Biochem Biophys Res Commun 2015; 436: 212–216.

    Article  Google Scholar 

  15. Kim YS, Morgan MJ, Choksi S, Liu ZG . TNF-induced activation of the Nox1 NADPH oxidase and its role in the induction of necrotic cell death. Mol Cell 2007; 26: 675–687.

    Article  CAS  PubMed  Google Scholar 

  16. Vanlangenakker N, Vanden Berghe T, Bogaert P, Laukens B, Zobel K, Deshayes K et al. cIAP1 and TAK1 protect cells from TNF-induced necrosis by preventing RIP1/RIP3-dependent reactive oxygen species production. Cell Death Differ 2011; 18: 656–665.

    Article  CAS  PubMed  Google Scholar 

  17. Zhang DW, Shao J, Lin J, Zhang N, Lu BJ, Lin SC et al. RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis. Science 2009; 325: 332–336.

    Article  CAS  PubMed  Google Scholar 

  18. Tait SW, Oberst A, Quarato G, Milasta S, Haller M, Wang R et al. Widespread mitochondrial depletion via mitophagy does not compromise necroptosis. Cell Rep 2013; 5: 878–885.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Laukens B, Jennewein C, Schenk B, Vanlangenakker N, Schier A, Cristofanon S et al. Smac mimetic bypasses apoptosis resistance in FADD- or caspase-8-deficient cells by priming for tumor necrosis factor alpha-induced necroptosis. Neoplasia 2011; 13: 971–979.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Sun L, Wang H, Wang Z, He S, Chen S, Liao D et al. Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell 2012; 148: 213–227.

    Article  CAS  PubMed  Google Scholar 

  21. Cassidy-Stone A, Chipuk JE, Ingerman E, Song C, Yoo C, Kuwana T et al. Chemical inhibition of the mitochondrial division dynamin reveals its role in Bax/Bak-dependent mitochondrial outer membrane permeabilization. Dev Cell 2008; 14: 193–204.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Dixon SJ, Stockwell BR . The role of iron and reactive oxygen species in cell death. Nat Chem Biol 2014; 10: 9–17.

    Article  CAS  PubMed  Google Scholar 

  23. Circu ML, Aw TY . Reactive oxygen species, cellular redox systems, and apoptosis. Free Radic Biol Med 2010; 48: 749–762.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Vernon PJ, Tang D . Eat-me: autophagy, phagocytosis, and reactive oxygen species signaling. Antioxid Redox Signal 2013; 18: 677–691.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Lin Y, Choksi S, Shen HM, Yang QF, Hur GM, Kim YS et al. Tumor necrosis factor-induced nonapoptotic cell death requires receptor-interacting protein-mediated cellular reactive oxygen species accumulation. J Biol Chem 2004; 279: 10822–10828.

    Article  CAS  PubMed  Google Scholar 

  26. Cho YS, Challa S, Moquin D, Genga R, Ray TD, Guildford M et al. Phosphorylation-driven assembly of the RIP1–RIP3 complex regulates programmed necrosis and virus-induced inflammation. Cell 2009; 137: 1112–1123.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Shulga N, Pastorino JG . Mitoneet mediates TNFalpha-induced necroptosis promoted by exposure to fructose and ethanol. J Cell Sci 2014; 127: 896–907.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Irrinki KM, Mallilankaraman K, Thapa RJ, Chandramoorthy HC, Smith FJ, Jog NR et al. Requirement of FADD, NEMO, and BAX/BAK for aberrant mitochondrial function in tumor necrosis factor alpha-induced necrosis. Mol Cell Biol 2011; 31: 3745–3758.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Roca FJ, Ramakrishnan L . TNF dually mediates resistance and susceptibility to mycobacteria via mitochondrial reactive oxygen species. Cell 2013; 153: 521–534.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Ye YC, Wang HJ, Yu L, Tashiro SI, Onodera S, Ikejima T . RIP1-mediated mitochondrial dysfunction and ROS production contributed to tumor necrosis factor alpha-induced L929 cell necroptosis and autophagy. Int Immunopharmacol 2012; 14: 674–682.

    Article  CAS  PubMed  Google Scholar 

  31. Davis CW, Hawkins BJ, Ramasamy S, Irrinki KM, Cameron BA, Islam K et al. Nitration of the mitochondrial complex I subunit NDUFB8 elicits RIP1- and RIP3-mediated necrosis. Free Radic Biol Med 2010; 48: 306–317.

    Article  CAS  PubMed  Google Scholar 

  32. Huang CY, Kuo WT, Huang YC, Lee TC, Yu LC . Resistance to hypoxia-induced necroptosis is conferred by glycolytic pyruvate scavenging of mitochondrial superoxide in colorectal cancer cells. Cell Death Dis 2013; 4: e622.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Coupienne I, Fettweis G, Rubio N, Agostinis P, Piette J . 5-ALA-PDT induces RIP3-dependent necrosis in glioblastoma. Photochem Photobiol Sci 2011; 10: 1868–1878.

    Article  CAS  PubMed  Google Scholar 

  34. Song KJ, Jang YS, Lee YA, Kim KA, Lee SK, Shin MH . Reactive oxygen species-dependent necroptosis in Jurkat T cells induced by pathogenic free-living Naegleria fowleri. Parasite Immunol 2011; 33: 390–400.

    Article  CAS  PubMed  Google Scholar 

  35. Chen TY, Chi KH, Wang JS, Chien CL, Lin WW . Reactive oxygen species are involved in FasL-induced caspase-independent cell death and inflammatory responses. Free Radic Biol Med 2009; 46: 643–655.

    Article  CAS  PubMed  Google Scholar 

  36. He S, Wang L, Miao L, Wang T, Du F, Zhao L et al. Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha. Cell 2009; 137: 1100–1111.

    Article  CAS  PubMed  Google Scholar 

  37. Gorrini C, Harris IS, Mak TW . Modulation of oxidative stress as an anticancer strategy. Nat Rev Drug Discov 2013; 12: 931–947.

    Article  CAS  PubMed  Google Scholar 

  38. Held JM, Gibson BW . Regulatory control or oxidative damage? Proteomic approaches to interrogate the role of cysteine oxidation status in biological processes. Mol Cell Proteom 2012; 11: 013037.

    Article  Google Scholar 

  39. Vanlangenakker N, Bertrand MJ, Bogaert P, Vandenabeele P, Vanden Berghe T . TNF-induced necroptosis in L929 cells is tightly regulated by multiple TNFR1 complex I and II members. Cell Death Dis 2011; 2: e230.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Kulathu Y, Garcia FJ, Mevissen TE, Busch M, Arnaudo N, Carroll KS et al. Regulation of A20 and other OTU deubiquitinases by reversible oxidation. Nat Commun 2013; 4: 1569.

    Article  PubMed  Google Scholar 

  41. Arslan SC, Scheidereit C . The prevalence of TNFalpha-induced necrosis over apoptosis is determined by TAK1-RIP1 interplay. PLoS One 2011; 6: e26069.

    Article  CAS  PubMed  Google Scholar 

  42. Kikuchi M, Kuroki S, Kayama M, Sakaguchi S, Lee KK, Yonehara S . Protease activity of procaspase-8 is essential for cell survival by inhibiting both apoptotic and nonapoptotic cell death dependent on receptor-interacting protein kinase 1 (RIP1) and RIP3. J Biol Chem 2012; 287: 41165–41173.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. He S, Liang Y, Shao F, Wang X . Toll-like receptors activate programmed necrosis in macrophages through a receptor-interacting kinase-3-mediated pathway. Proc Natl Acad Sci USA 2011; 108: 20054–20059.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Luedde M, Lutz M, Carter N, Sosna J, Jacoby C, Vucur M et al. RIP3, a kinase promoting necroptotic cell death, mediates adverse remodelling after myocardial infarction. Cardiovasc Res 2014; 103: 206–216.

    Article  CAS  PubMed  Google Scholar 

  45. Moquin DM, McQuade T, Chan FK . CYLD deubiquitinates RIP1 in the TNFalpha-induced necrosome to facilitate kinase activation and programmed necrosis. PLoS One 2013; 8: e76841.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Zhao J, Jitkaew S, Cai Z, Choksi S, Li Q, Luo J et al. Mixed lineage kinase domain-like is a key receptor interacting protein 3 downstream component of TNF-induced necrosis. Proc Natl Acad Sci USA 2012; 109: 5322–5327.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Gomes A, Fernandes E, Lima JL . Fluorescence probes used for detection of reactive oxygen species. J Biochem Biophys Methods 2005; 65: 45–80.

    Article  CAS  PubMed  Google Scholar 

  48. Remijsen Q, Goossens V, Grootjans S, Van den Haute C, Vanlangenakker N, Dondelinger Y et al. Depletion of RIPK3 or MLKL blocks TNF-driven necroptosis and switches towards a delayed RIPK1 kinase-dependent apoptosis. Cell Death Dis 2014; 5: e1004.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Murphy JM, Czabotar PE, Hildebrand JM, Lucet IS, Zhang JG, Alvarez-Diaz S et al. The pseudokinase MLKL mediates necroptosis via a molecular switch mechanism. Immunity 2013; 39: 443–453.

    Article  CAS  PubMed  Google Scholar 

  50. Juo P, Woo MS, Kuo CJ, Signorelli P, Biemann HP, Hannun YA et al. FADD is required for multiple signaling events downstream of the receptor Fas. Cell Growth Differ 1999; 10: 797–804.

    CAS  PubMed  Google Scholar 

  51. Varfolomeev E, Blankenship JW, Wayson SM, Fedorova AV, Kayagaki N, Garg P et al. IAP antagonists induce autoubiquitination of c-IAPs, NF-kappaB activation, and TNFalpha-dependent apoptosis. Cell 2007; 131: 669–681.

    Article  CAS  PubMed  Google Scholar 

  52. Gonzalez P, Mader I, Tchoghandjian A, Enzenmuller S, Cristofanon S, Basit F et al. Impairment of lysosomal integrity by B10, a glycosylated derivative of betulinic acid, leads to lysosomal cell death and converts autophagy into a detrimental process. Cell Death Differ 2012; 19: 1337–1346.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Fulda S, Strauss G, Meyer E, Debatin KM . Functional CD95 ligand and CD95 death-inducing signaling complex in activation-induced cell death and doxorubicin-induced apoptosis in leukemic T cells. Blood 2000; 95: 301–308.

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank D Vucic (Genentech Inc., South San Francisco, CA, USA) for providing Smac mimetic, X Wang (Beijing, China) for providing anti-RIP3 and anti-PGAM5 antibodies and C Hugenberg for expert secretarial assistance. This work has been partially supported by the Deutsche Forschungsgemeinschaft (SFB815) and the BMBF.

Author information

Authors and Affiliations

  1. Institute for Experimental Cancer Research in Pediatrics, Goethe-University, Frankfurt, Germany

    B Schenk & S Fulda

  2. German Cancer Consortium (DKTK), Heidelberg, Germany

    S Fulda

  3. German Cancer Research Center (DKFZ), Heidelberg, Germany

    S Fulda

Authors
  1. B Schenk
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. S Fulda
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to S Fulda.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies this paper on the Oncogene website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Schenk, B., Fulda, S. Reactive oxygen species regulate Smac mimetic/TNFα-induced necroptotic signaling and cell death. Oncogene 34, 5796–5806 (2015). https://doi.org/10.1038/onc.2015.35

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2015.35

This article is cited by

Search

Advanced search

Quick links