title: 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.
authors.affiliation: Rush University Medical Center, Department of Internal Medicine, Section of Gastroenterology and Nutrition, 1725 W. Harrison, Suite 206, Chicago, IL 60612, USA. ali_banan@rush.edu
authors.forename: A
authors.initials: A
authors.lastname: Banan
authors.forename: L J
authors.initials: LJ
authors.lastname: Zhang
authors.forename: M
authors.initials: M
authors.lastname: Shaikh
authors.forename: J Z
authors.initials: JZ
authors.lastname: Fields
authors.forename: A
authors.initials: A
authors.lastname: Farhadi
authors.forename: A
authors.initials: A
authors.lastname: Keshavarzian
mesh_terms: D015153:Blotting, Western; D018938:Caco-2 Cells; D003599:Cytoskeleton / Q000737:chemistry / Q000378:metabolism / Q000473:pathology*; D024202:Electrophoretic Mobility Shift Assay; D004789:Enzyme Activation / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007413:Intestinal Mucosa / Q000378:metabolism* / Q000473:pathology / Q000648:ultrastructure; D018613:Microscopy, Confocal; D016328:NF-kappa B / Q000187:drug effects / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress / Q000502:physiology*
chemical_list: D004791:Enzyme Inhibitors; D016328:NF-kappa B; D016877:Oxidants; D009569:Nitric Oxide; D006861:Hydrogen Peroxide; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II