Tumor Necrosis Factor-Alpha and Apoptosis Signal-Regulating Kinase 1 Control Reactive Oxygen Species Release, Mitochondrial Autophagy, and c-Jun N-terminal kinase/p38 Phosphorylation During Necrotizing Enterocolitis

Oxid Med Cell Longev. Nov-Dec 2009;2(5):297-306. doi: 10.4161/oxim.2.5.9541.


Background: Oxidative stress and inflammation may contribute to the disruption of the protective gut barrier through various mechanisms; mitochondrial dysfunction resulting from inflammatory and oxidative injury may potentially be a significant source of apoptosis during necrotizing enterocolitis (NEC). Tumor necrosis factor (TNF)-alpha is thought to generate reactive oxygen species (ROS) and activate the apoptosis signal-regulating kinase 1 (ASK1)-c-Jun N-terminal kinase (JNK)/p38 pathway. Hence, the focus of our study was to examine the effects of TNF-alpha/ROS on mitochondrial function, ASK1-JNK/p38 cascade activation in intestinal epithelial cells during NEC.

Results: We found (a) abundant tissue TNF-alpha and ASK1 expression throughout all layers of the intestine in neonates with NEC, suggesting that TNF-alpha/ASK1 may be a potential source (indicators) of intestinal injury in neonates with NEC; (b) TNF-alpha-induced rapid and transient activation of JNK/p38 apoptotic signaling in all cell lines suggests that this may be an important molecular characteristic of NEC; (c) TNF-alpha-induced rapid and transient ROS production in RIE-1 cells indicates that mitochondria are the predominant source of ROS, demonstrated by significantly attenuated response in mitochondrial DNA-depleted (RIE-1-rho) intestinal epithelial cells; (d) further studies with mitochondria-targeted antioxidant PBN supported our hypothesis that effective mitochondrial ROS trapping is protective against TNF-alpha/ROS-induced intestinal epithelial cell injury; (e) TNF-alpha induces significant mitochondrial dysfunction in intestinal epithelial cells, resulting in increased production of mtROS, drop in mitochondrial membrane potential (MMP) and decreased oxygen consumption; (f) although the significance of mitochondrial autophagy in NEC has not been unequivocally shown, our studies provide a strong preliminary indication that TNF-alpha/ROS-induced mitochondrial autophagy may play a role in NEC, and this process is a late phenomenon.

Methods: Paraffin-embedded intestinal sections from neonates with NEC and non-inflammatory condition of the gastrointestinal tract undergoing bowel resections were analyzed for TNF-alpha and ASK1 expression. Rat (RIE-1) and mitochondrial DNA-depleted (RIE-1-rho) intestinal epithelial cells were used to determine the effects of TNF-alpha on mitochondrial function.

Conclusions: Our findings suggest that TNF-alpha induces significant mitochondrial dysfunction and activation of mitochondrial apoptotic responses, leading to intestinal epithelial cell apoptosis during NEC. Therapies directed against mitochondria/ROS may provide important therapeutic options, as well as ameliorate intestinal epithelial cell apoptosis during NEC.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Autophagy*
  • Cell Line
  • Enterocolitis, Necrotizing / enzymology
  • Enterocolitis, Necrotizing / metabolism*
  • Enterocolitis, Necrotizing / pathology
  • Epithelial Cells / enzymology
  • Epithelial Cells / metabolism
  • Humans
  • Infant, Newborn
  • Intestinal Mucosa / metabolism
  • Intestines / enzymology
  • JNK Mitogen-Activated Protein Kinases / metabolism
  • MAP Kinase Kinase Kinase 5 / genetics
  • MAP Kinase Kinase Kinase 5 / metabolism*
  • MAP Kinase Signaling System
  • Membrane Potential, Mitochondrial / physiology
  • Mice
  • Mitochondria / metabolism*
  • Mitogen-Activated Protein Kinases / metabolism*
  • Oxygen Consumption / physiology
  • Phosphorylation
  • RNA Interference
  • RNA, Small Interfering / metabolism
  • Rats
  • Reactive Oxygen Species / metabolism*
  • Tumor Necrosis Factor-alpha / metabolism*
  • p38 Mitogen-Activated Protein Kinases / metabolism


  • RNA, Small Interfering
  • Reactive Oxygen Species
  • Tumor Necrosis Factor-alpha
  • JNK Mitogen-Activated Protein Kinases
  • Mitogen-Activated Protein Kinases
  • p38 Mitogen-Activated Protein Kinases
  • MAP Kinase Kinase Kinase 5