NF-κB Inhibition Enhances Peroxynitrite-Induced Enterocyte Apoptosis

Abstract

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-κB 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-κB inhibition increases cytokine-induced enterocyte apoptosis; however, the effect of NF-κB on ONOO−-induced enterocyte apoptosis is unknown.Materials and methods. Rat intestinal epithelial cells (IEC-6) were transfected with the adenoviral vector AdIκB or AdlacZ. AdIκB contains a mutated form of IκB which functions as a superrepressor of NF-κB. Cells were then treated with 50 μM 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-κB activation was determined via electrophoretic mobility shift assay (EMSA).Results. Inhibition of NF-κB with AdIκB significantly enhanced ONOO−-induced apoptosis in IEC-6 cells. ONOO− treatment did not activate NF-κB 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 AdIκB-transfected cells. Baseline procaspase 3 activation was increased in AdIκB-transfected cells.Conclusions. NF-κB inhibition enhances ONOO−-induced enterocyte apoptosis, suggesting that NF-κB 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-κB over baseline levels of activation.