Novel effect of NF-kappaB activation: carbonylation and nitration injury to cytoskeleton and disruption of monolayer barrier in intestinal epithelium.

Abstract

Using monolayers of intestinal cells, we reported that upregulation of inducible nitric oxide synthase (iNOS) is required for oxidative injury and that activation of NF-kappaB is key to cytoskeletal instability. In the present study, we hypothesized that NF-kappaB activation is crucial to oxidant-induced iNOS upregulation and its injurious consequences: cytoskeletal oxidation and nitration and monolayer dysfunction. Wild-type (WT) cells were pretreated with inhibitors of NF-kappaB, with or without exposure to oxidant (H(2)O(2)). Other cells were transfected with an IkappaBalpha mutant (an inhibitor of NF-kappaB). Relative to WT cells exposed to vehicle, oxidant exposure caused increases in IkappaBalpha instability, NF-kappaB subunit activation, iNOS-related activity (NO, oxidative stress, tubulin nitration), microtubule disassembly and instability (increased monomeric and decreased polymeric tubulin), and monolayer disruption. Monolayers pretreated with NF-kappaB inhibitors (MG-132, lactacystin) were protected against oxidation, showing decreases in all measures of the NF-kappaB --> iNOS --> NO pathway. Dominant mutant stabilization of IkappaBalpha to inactivate NF-kappaB suppressed all measures of the iNOS/NO upregulation while protecting monolayers against oxidant insult. In these mutants, we found prevention of tubulin nitration and oxidation and enhancement of cytoskeletal and monolayer stability. We concluded that 1) NF-kappaB is required for oxidant-induced iNOS upregulation and for the consequent nitration and oxidation of cytoskeleton; 2) NF-kappaB activation causes cytoskeletal injury following upregulation of NO-driven processes; and 3) the molecular event underlying the destabilizing effects of NF-kappaB 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-kappaB in cells.