NF-kappaB inhibition enhances peroxynitrite-induced enterocyte apoptosis.
BACKGROUND
Sustained overproduction of nitric oxide and peroxynitrite (ONOO(-)) in conditions such as necrotizing enterocolitis and inflammatory bowel disease may promote gut barrier failure by inducing enterocyte apoptosis. NF-kappaB is upregulated in the gut during inflammation and, in addition to its proinflammatory effects, may upregulate protective or antiapoptotic factors such as inhibitor of apoptosis proteins (IAPs). We have previously demonstrated that NF-kappaB inhibition increases cytokine-induced enterocyte apoptosis; however, the effect of NF-kappaB on ONOO(-)-induced enterocyte apoptosis is unknown.


METHODS
Rat intestinal epithelial cells (IEC-6) were transfected with the adenoviral vector AdIkappaB or AdlacZ. AdIkappaB contains a mutated form of IkappaB which functions as a superrepressor of NF-kappaB. Cells were then treated with 50 microM ONOO(-) or decomposed ONOO(-). Apoptosis was then determined by flow cytometry with annexin V-FITC and propidium iodide staining. Caspase activation and IAP, Bcl-2, Bad, and Bax expression were examined using Western blot analysis, and NF-kappaB activation was determined via electrophoretic mobility shift assay (EMSA).


RESULTS
Inhibition of NF-kappaB with AdIkappaB significantly enhanced ONOO(-)-induced apoptosis in IEC-6 cells. ONOO(-) treatment did not activate NF-kappaB in IEC-6 cells as determined by EMSA. There was no difference in IAP, Bcl-2, Bad, and Bax expression between nontransfected, AdlacZ-transfected, and AdIkappaB-transfected cells. Baseline procaspase 3 activation was increased in AdIkappaB-transfected cells.


CONCLUSIONS
NF-kappaB inhibition enhances ONOO(-)-induced enterocyte apoptosis, suggesting that NF-kappaB upregulates a protective factor. This protective factor does not appear to be an IAP or Bcl-2 family member and may be expressed constitutively, since ONOO(-) did not activate NF-kappaB over baseline levels of activation.
Department of Surgery, Children's Hospital of Pittsburgh, and University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.
Douglas A
DA
Potoka
Jeffrey S
JS
Upperman
Evan P
EP
Nadler
Catarina T
CT
Wong
Xin
X
Zhou
Xiao Ru
XR
Zhang
Henri R
HR
Ford
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D020895:Enterocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005822:Genetic Vectors; D020672:I-kappa B Proteins / Q000235:genetics; D007413:Intestinal Mucosa / Q000166:cytology; D007763:Lac Operon; D016328:NF-kappa B / Q000378:metabolism*; D030421:Peroxynitrous Acid / Q000494:pharmacology*; D051381:Rats; D014162:Transfection
D020672:I-kappa B Proteins; D016328:NF-kappa B; D030421:Peroxynitrous Acid; D020169:Caspases