Jedrzkiewicz S, Nakamura H, Silverman E S, Luster A D, Mansharamani N, In K H, Tamura G, Lilly C M
Combined Program in Pulmonary and Critical Care Medicine, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston 02115, Massachusetts, USA.
Am J Physiol Lung Cell Mol Physiol. 2000 Dec;279(6):L1058-65. doi: 10.1152/ajplung.2000.279.6.L1058.
Eotaxin is an asthma-related C-C chemokine that is produced in response to interleukin-1beta (IL-1beta). We detected an increase in newly transcribed eotaxin mRNA in IL-1beta-stimulated airway epithelial cells. Transient transfection assays using promoter-reporter constructs identified a region as essential for IL-1beta-induced increases in eotaxin transcription. Using site-directed mutagenesis, we found that a nuclear factor-kappaB (NF-kappaB) site located 46 bp upstream from the transcriptional start site was both necessary and sufficient for IL-1beta induction of reporter construct activity. Electrophoretic mobility shift assay demonstrated that IL-1beta-stimulated airway epithelial cells produced p50 and p65 protein that bound this site in a sequence-specific manner. The functional importance of the NF-kappaB site was demonstrated by coexpression experiments in which increasing doses of p65 expression vector were directly associated with reporter activity exclusively in constructs with an intact NF-kappaB site (r(2) = 0.97, P = 0.002). Moreover, IL-1beta-induced increases in eotaxin mRNA expression are inhibited by inhibitors of NF-kappaB. Our findings implicate NF-kappaB and its binding sequence in IL-1beta-induced transcriptional activation of the eotaxin gene.
嗜酸性粒细胞趋化因子是一种与哮喘相关的C-C趋化因子，它是在白细胞介素-1β（IL-1β）的刺激下产生的。我们检测到在IL-1β刺激的气道上皮细胞中新转录的嗜酸性粒细胞趋化因子mRNA增加。使用启动子-报告基因构建体的瞬时转染试验确定了一个区域，该区域对于IL-1β诱导的嗜酸性粒细胞趋化因子转录增加至关重要。通过定点诱变，我们发现位于转录起始位点上游46 bp处的一个核因子-κB（NF-κB）位点对于IL-1β诱导报告基因构建体活性既是必需的也是充分的。电泳迁移率变动分析表明，IL-1β刺激的气道上皮细胞产生以序列特异性方式结合该位点的p50和p65蛋白。NF-κB位点的功能重要性通过共表达实验得以证明，在该实验中，增加剂量的p65表达载体仅与具有完整NF-κB位点的构建体中的报告基因活性直接相关（r² = 0.97，P = 0.002）。此外，NF-κB抑制剂可抑制IL-1β诱导的嗜酸性粒细胞趋化因子mRNA表达增加。我们的研究结果表明NF-κB及其结合序列参与了IL-1β诱导的嗜酸性粒细胞趋化因子基因转录激活。
