REGULATION OF NF-KAPPA B BY NITRIC OXIDE IN THE LUNG

核因子-kappaB是一种转录因子，调节多种炎症反应基因。在这种炎症状态下，细胞产生的一氧化氮(NO)会增加。根据细胞类型、刺激、浓度和递送形式的不同，已发现NO对NF-Kappa B具有刺激或抑制作用。本研究旨在测试NO或NO相关分子对呼吸道上皮细胞中NF-Kappa B活性的影响。我们的初步研究表明，NO对呼吸道上皮细胞中的核因子-kappaB具有抑制作用。NO发挥这种作用的机制(S)尚不清楚。我们假设，NO通过在p50-p65异源二聚体或激活途径中的另一种蛋白上形成亚硝硫醇来调节核因子-kappaB。为了研究这一假说，我们计划：1.确定外源性和内源性NO或NO相关分子对呼吸道上皮细胞NF-kappaB活性的影响，并确定这些影响是否对细胞内硫醇水平敏感。2.体外分析重组的NF-Kappa B蛋白，确定它们是否可以亚硝化，以及亚硝化发生在分子上的哪个位点(S)。3.确定这些经S硝化处理的核因子-kappaB蛋白是否存在于细胞内。4.确定这些蛋白质的S亚硝化改变其细胞功能的机制。申请者的长期目标是成为一名独立的研究人员，研究肺中基因转录的调控，特别是与氧化和亚硝化应激有关的基因转录调控。有了这个奖项，申请者将获得实现这一目标所需的分子生物技术方面的培训和经验。

NF-Kappa B is a transcription factor which regulates numerous inflammatory response genes. Cellular production of nitric oxide (NO) is increased in such inflammatory states. Depending upon the cell type, stimulus, concentration, and form of delivery, NO has been found to be either stimulatory or inhibitory to NF-Kappa B. This proposed research is designed to test the effects of NO or NO-related molecules on NF-Kappa B activity in the respiratory epithelium. Our preliminary studies indicate that NO has an inhibitory effect on NF-Kappa B in respiratory epithelial cells. The mechanism(s) by which NO exerts this effect is not understood. We hypothesize that NO regulates NF-Kappa B via the formation of a nitrosothiol on the p50-p65 heterodimer or another protein in the activation pathway. To investigate this hypothesis we plan to: 1. Determine the effects of exogenous and endogenous NO or NO-related molecules on NF-Kappa B activity in respiratory epithelial cells and determine whether these effects are sensitive to intracellular thiol levels. 2. Analyze recombinant NF-Kappa B proteins in vitro to determine whether they can be nitrosylated and at which site(s) this occurs on the molecule. 3. Determine whether these S-nitrosylated NF-Kappa B proteins are present intracellularly. 4. Determine the mechanism by which S-nitrosylation of these proteins alters their cellular function. The applicants long term goals are to become an independent investigator studying the regulation of gene transcription in the lung, in particular, as it relates to oxidative and nitrosative stress. With this award, the applicant will obtain the necessary training and experience in molecular biological techniques to achieve this goal.