Using monolayers of intestinal cells, we reported that upregulation of iNOS is required for oxidative injury and that activation of NF-B is key to cytoskeletal instability. In the current study we hypothesized that NF-B activation is crucial to oxidant-induced iNOS upregulation and its injurious consequences, cytoskeletal oxidation & nitration and monolayer dysfunction. Wild type (WT) cells were pretreated with inhibitors of NF-B ± oxidant (H₂O₂). Other cells were transfected with an I-B mutant (an inhibitor of NF-B). RESULTS: {A} Relative to WT cells exposed to vehicle, oxidant exposure caused increases in I-B instability; NF-B subunits activation; iNOS-related activity [NO, oxidative stress, tubulin nitration]; microtubule disassembly & instability [increased monomeric & decreased polymeric tubulin]; and monolayer disruption. {B} Monolayers pretreated with NF-B inhibitors (MG-132, lactacystin) were protected against oxidation, showing decreases in all measures of the NF-BiNOSNO pathway. {C} Dominant mutant stabilization of I-B to inactivate NF-B suppressed all measures of the iNOS/NO upregulation while protecting monolayers against oxidant insult. In these mutants, we found prevention of tubulin nitration & oxidation and enhancement of cytoskeletal and monolayer stability. CONCLUSIONS: [1] NF-B is required for oxidant-induced iNOS upregulation and the consequent nitration and oxidation of cytoskeleton. [2] NF-B activation causes cytoskeletal injury following upregulation of NO driven processes. [3] The molecular event underlying the destabilizing effects of NF-B appears to be increases in carbonylation and nitrotyrosination of the subunit components of cytoskeleton. The ability to promote NO overproduction and cytoskeletal nitration/oxidation is a novel mechanism not previously attributed to NF-B in cells.