ROLE OF NITRIC OXIDE IN THE PATHOGENESIS OF LUNG DISEASE

人诱导型一氧化氮合酶（hNOS）是负责一氧化氮合成的一种酶。 一氧化氮（NO）的合成反应的炎症介质。 调控 hiNOS主要发生在转录水平。的 hiNOS启动子近端8296 bp区域含有许多潜在的 包括AP-1和NF-kB的药物应答元件。AP-1和 NF-κ B是一种普遍存在的转录因子和多效性调节因子 许多编码蛋白质的基因参与调节 炎症和宿主防御过程。的 AP-1和NF-kB转录因子参与 苦参碱对人诱导型一氧化氮合酶的诱导作用 （hiNOS）启动子活性。荧光素酶报告质粒， 在AP-1和NF-kB位点含有突变， 在A549细胞（人肺上皮腺癌细胞）中表达 line）和用CM（a）处理后测定启动子活性 含有IL-1 β、IFN-γ和TNF-α的细胞因子混合物）。 转录起始点上游约5301 bp的AP-1七肽突变 位点使基因激活降低了90%。AP-1中断（-5115）， 和NF-κ B（在-115和-8283）位点导致启动子的减少 活性分别提高了45%、67%和52%。对CM的反应是 在两个NF-kB位点突变的构建体中减少了85%。凝胶 延迟分析显示CM增加AP-1-和NF-κ B- 约束力用NF-kB序列，仅观察到蛋白质/DNA复合物 CM治疗后。诱导最高时，第一次测试在1 h，随刺激时间延长而降低。相比之下， 在未处理的细胞和细胞因子刺激中发现AP-1结合 以双相方式增加结合;最大结合为3， 24小时Supershift分析确定Jun D和Fra-2为 AP-1复合物。有趣的是，每个kB位点结合不同的互补物， NF-kB/Rel家族成员（近端位点，Rel A/p50;远端位点，Rel A/Rel A）。在蛋白质水平上，Jun D和p50是组成性的 在CM处理的细胞中表达并诱导Fra 2。同意 目前的NF-kB调节模型，Rel A最大，而 CM处理1小时后，IkB-α在细胞核中表达最低， 对应于NF-κ B结合活性的峰值。 总之， 这些结果表明AP-1和NF-kB是重要的顺式元件 细胞因子对A549细胞hiNOS基因转录的诱导作用 细胞

Human inducible nitric oxide synthase (hiNOS) is responsible for nitric oxide (NO) synthesis in response to inflammatory mediators. Regulation of (hiNOS) occurs predominantly at the transcriptional level. The proximal 8296-bp region of the hiNOS promoter contains many potential cytokine-response elements including those for AP-1 and NF-kB. AP-1 and NF-kB are ubiquitous transcription factors and pleiotropic regulators of many genes encoding proteins involved in the modulation of inflammatory and host defense processes in eukaryotic cells. The involvement of AP-1 and NF-kB transcription factors in cytokine-mediated induction of human inducible nitric oxide synthase (hiNOS) promoter activity was examined. Luciferase reporter plasmids, containing mutations in AP-1 and NF-kB sites, were transiently expressed in A549 cells (a human lung epithelial adenocarcinoma cell line) and promoter activity was determined after treatment with CM (a cytokine mixture containing IL-1beta, IFN-gamma, and TNF-alpha). Mutation of the AP-1 heptad -5301 bp upstream of the transcription start site decreased gene activation by 90%. Disruption of AP-1 (at -5115), and NF-kB (at -115 and -8283) sites resulted in reduction of promoter activity by 45%, 67%, and 52%, respectively. Responsiveness to CM was reduced by 85% in constructs mutated in both NF-kB sites. Gel retardation analyses revealed that CM increased AP-1- and NF-kB- binding. With NF-kB sequences, protein/DNA complexes were observed only after treatment with CM. Induction was highest when first tested at 1 h and decreased with longer stimulation. In contrast, constitutive AP-1-binding was found in untreated cells and cytokine stimulation increased binding in a biphasic manner; with maximal binding at 3 and 24 h. Supershift analysis identified Jun D and Fra-2 as components of AP-1 complexes. Interestingly, each kB site bound different complements of NF-kB/Rel family members (proximal site, Rel A/p50; distal site, Rel A/Rel A). At the protein level, Jun D and p50 were constitutively expressed and Fra 2 was induced in CM-treated cells. In agreement with the current model of NF-kB regulation, Rel A was maximally, while IkB-alpha was minimally, expressed in nuclei after 1 h of CM treatment, corresponding with the peak in NF-kB-binding activity. Altogether, these findings suggest that AP-1 and NF-kB are important cis-elements in the transcriptional induction of hiNOS gene by cytokines in A549 cells.