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.
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.
Baregamian
Naira
N
Departments of Surgery, University of Texas Medical Branch, Galveston, Texas, USA.
Song
Jun
J
Bailey
C Eric
CE
Papaconstantinou
John
J
Evers
B Mark
BM
Chung
Dai H
DH
D000818:Animals; D001343:Autophagy; D002460:Cell Line; D020345:Enterocolitis, Necrotizing / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D004847:Epithelial Cells / Q000201:enzymology / Q000378:metabolism; D006801:Humans; D007231:Infant, Newborn; D007413:Intestinal Mucosa / Q000378:metabolism; D007422:Intestines / Q000201:enzymology; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D048848:MAP Kinase Kinase Kinase 5 / Q000235:genetics / Q000378:metabolism*; D020935:MAP Kinase Signaling System; D053078:Membrane Potential, Mitochondrial / Q000502:physiology; D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010101:Oxygen Consumption / Q000502:physiology; D010766:Phosphorylation; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048848:MAP Kinase Kinase Kinase 5