Novel effect of NF-κB activation:: carbonylation and nitration injury to cytoskeleton and disruption of monolayer barrier in intestinal epithelium
Novel effect of NF-κB activation:: carbonylation and nitration injury to cytoskeleton and disruption of monolayer barrier in intestinal epithelium
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DOI：
10.1152/ajpcell.00146.2004
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发表时间：
2004-10-01
影响因子：
5.5
通讯作者：
Keshavarzian, A
中科院分区：
文献类型：
--
作者：
Banan, A;Zhang, LJ;Keshavarzian, A
使用单层肠细胞，我们报道了诱导型一氧化氮合酶（iNOS）的上调是氧化损伤所必需的，NF-κ B的激活是细胞骨架不稳定的关键。在本研究中，我们假设NF-kappaB激活对于氧化剂诱导的iNOS上调及其有害后果至关重要：细胞骨架氧化和硝化以及单层功能障碍。用NF-κ B抑制剂预处理野生型（WT）细胞，暴露或不暴露于氧化剂（H2 O2）。其他细胞用IkappaB α突变体（NF-κ B的抑制剂）转染。相对于暴露于溶媒的WT细胞，氧化剂暴露导致IkappaB α不稳定性、NF-κ B亚基活化、iNOS相关活性（NO、氧化应激、微管蛋白硝化）、微管解体和不稳定性（单体微管蛋白增加和聚合微管蛋白减少）以及单层破坏增加。用NF-κ B抑制剂（MG-132，lactacystin）预处理的单层被保护免于氧化，显示NF-κ B--> iNOS --> NO途径的所有测量值降低。IkappaB α的显性突变体稳定化为JNF-kappaB抑制了iNOS/NO上调的所有措施，同时保护单层细胞免受氧化剂损伤。在这些突变体中，我们发现微管蛋白硝化和氧化的预防和细胞骨架和单层稳定性的增强。我们得出结论：1）NF-κ B是氧化剂诱导的iNOS上调以及随后的细胞骨架硝化和氧化所必需的; 2）NF-κ B激活导致NO驱动过程上调后的细胞骨架损伤; 3）NF-κ B不稳定作用的分子事件似乎是细胞骨架亚基组分羰基化和硝基酪氨酸化的增加。促进NO过量产生和细胞骨架硝化/氧化的能力是一种新的机制，以前没有归因于细胞中的NF-κ B。
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 (H2O2). 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.