Abstract：
While IκB-kinase-ε (IKKε) induces immunomodulatory genes following viral stimuli, its up-regulation by inflammatory cytokines remains under-explored. Since airway epithelial cells respond to airborne insults and potentiate inflammation, IKKε expression was characterized in pulmonary epithelial cell lines (A549, BEAS-2B) and primary human bronchial epithelial cells grown as submersion or differentiated air-liquid interface cultures. IKKε expression was up-regulated by the pro-inflammatory cytokines, interleukin-1β (IL-1β) and tumour necrosis factor-α (TNFα). Thus, mechanistic interrogations in A549 cells were used to demonstrate the NF-κB dependence of cytokine-induced IKKε. Furthermore, chromatin immunoprecipitation in A549 and BEAS-2B cells revealed robust recruitment of the NF-κB subunit, p65, to one 5\' and two intronic regions within the IKKε locus (IKBKE). In addition, IL-1β and TNFα induced strong RNA polymerase 2 recruitment to the 5\' region, the first intron, and the transcription start site. Stable transfection of the p65-binding regions into A549 cells revealed IL-1β- and TNFα-inducible reporter activity that required NF-κB, but was not repressed by glucocorticoid. While critical NF-κB motifs were identified in the 5\' and downstream intronic regions, the first intronic region did not contain functional NF-κB motifs. Thus, IL-1β- and TNFα-induced IKKε expression involves three NF-κB-binding regions, containing multiple functional NF-κB motifs, and potentially other mechanisms of p65 binding through non-classical NF-κB binding motifs. By enhancing IKKε expression, IL-1β may prime, or potentiate, responses to alternative stimuli, as modelled by IKKε phosphorylation induced by phorbol 12-myristate 13-acetate. However, since IKKε expression was only partially repressed by glucocorticoid, IKKε-dependent responses could contribute to glucocorticoid-resistant disease.
摘要：
而IkB激酶-ε(IKKε)在病毒刺激后诱导免疫调节基因,其通过炎性细胞因子的上调仍未被探索。由于气道上皮细胞对空气传播的损伤有反应并增强炎症，IKKε表达在肺上皮细胞系（A549，BEAS-2B）和以浸没或分化的气液界面（ALI）培养物生长的原代人支气管上皮细胞（pHBECs）中表征。IKKε表达被促炎细胞因子上调，IL-1β和TNFα。因此，A549细胞中的机制询问用于证明细胞因子诱导的IKKε的NF-κB依赖性。此外，A549和BEAS-2B细胞中的染色质免疫沉淀显示NF-κB亚基的稳健募集，p65，到IKKε基因座（IKBKE）内的一个5'和两个内含子区域。此外,IL-1β和TNFα诱导强RNA聚合酶2募集至5'区，第一个内含子，和转录起始位点。将p65结合区稳定转染到A549细胞中，揭示了IL-1β和TNFα可诱导的报告活性，这需要NF-κB，但未被糖皮质激素抑制。虽然在5'和下游内含子区域中鉴定出关键的NF-κB基序，第一个内含子区不含功能性NF-κB基序。因此，IL-1β和TNFα诱导的IKKε表达涉及三个NF-κB结合区，含有多功能NF-κB基序，以及p65通过非经典NF-κB结合基序结合的潜在其他机制。通过增强IKKε表达，IL-1β可能引发，或者增强，对替代刺激的反应，如通过佛波醇12-肉豆蔻酸酯13-乙酸酯诱导的IKKε磷酸化建模。然而,由于IKKε表达仅被糖皮质激素部分抑制，IKKε依赖性反应可能导致糖皮质激素抗性疾病。
