Critical contribution of oxidative stress to TNFα-induced necroptosis downstream of RIPK1 activation

Biochem Biophys Res Commun. 2013 Jun 28;436(2):212-6. doi: 10.1016/j.bbrc.2013.05.075. Epub 2013 May 29.


While apoptosis has been considered to be identical to programmed cell death, necroptosis, which is morphologically related to necrosis, has emerged as a novel type of programmed cell death. Necroptosis depends on two structurally related kinases, receptor-interacting serine-threonine kinase (RIPK)1 and RIPK3. RIPK1 is activated through oligomerization of upstream adaptor molecules such as Fas-associated protein with death domain (FADD) and TNF receptor-associated death domain (TRADD) that are triggered by TNFα or Fas ligand. Activated RIPK1 subsequently interacts with and activates RIPK3, resulting in necroptosis. However, contribution of oxidative stress to execution of necroptosis is still controversial. We found that a selective inhibitor for RIPK1, necrostatin-1 (Nec-1) significantly blocked TNFα-induced cell death and ROS accumulation in NF-κB activation-deficient cells. This suggests that these cells mostly died by necroptosis upon TNFα stimulation. Intriguingly, an antioxidant, butylated hydroxyanisole (BHA) blocked TNFα-induced necroptosis and ROS accumulation in NF-κB activation-deficient cells. However, Nec-1, but not BHA, inhibited TNFα-induced phosphorylation of RIPK1 in these cells, suggesting that ROS play a crucial role in execution of necroptosis downstream of RIPK1 activation. Structural and functional analyses using BHA related compounds revealed that both tert-butyl and hydroxy groups of BHA are crucial for its anti-necroptotic function. Together, these results suggest that TNFα-induced necroptosis is tightly associated with oxidative stress, and oxidative stress is induced downstream of RIPK1 activation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antioxidants / pharmacology
  • Apoptosis / drug effects*
  • Blotting, Western
  • Butylated Hydroxyanisole / pharmacology
  • Cell Survival / drug effects
  • Cells, Cultured
  • Dose-Response Relationship, Drug
  • Embryo, Mammalian / cytology
  • Enzyme Activation / drug effects
  • Fibroblasts / cytology
  • Fibroblasts / drug effects
  • Fibroblasts / metabolism
  • Imidazoles / pharmacology
  • Indoles / pharmacology
  • Inhibitor of Apoptosis Proteins / deficiency
  • Inhibitor of Apoptosis Proteins / genetics
  • Mice
  • Mice, Knockout
  • Models, Biological
  • Necrosis
  • Oxidative Stress*
  • Phosphorylation / drug effects
  • Reactive Oxygen Species / metabolism
  • Receptor-Interacting Protein Serine-Threonine Kinases / antagonists & inhibitors
  • Receptor-Interacting Protein Serine-Threonine Kinases / metabolism*
  • Transcription Factor RelA / deficiency
  • Transcription Factor RelA / genetics
  • Tumor Necrosis Factor-alpha / pharmacology*


  • Antioxidants
  • Imidazoles
  • Indoles
  • Inhibitor of Apoptosis Proteins
  • Reactive Oxygen Species
  • Rela protein, mouse
  • Transcription Factor RelA
  • Tumor Necrosis Factor-alpha
  • necrostatin-1
  • Butylated Hydroxyanisole
  • Receptor-Interacting Protein Serine-Threonine Kinases
  • Ripk1 protein, mouse