Interleukin-8 Gene Repression by Clarithromycin is mediated by AP-1 Binding Site in Human Bronchial Epithelial Cells

已知大环内酯类抗生素可有效治疗慢性炎症性呼吸道疾病，包括弥漫性泛细支气管炎、慢性支气管炎和哮喘。除抗微生物活性外，大环内酯类化合物还具有“抗炎”作用，如抑制细胞因子的产生。在本研究中，我们利用人支气管上皮细胞系BET-1A细胞，研究了克拉霉素对白介素8(IL-8)基因表达及其蛋白水平的影响。Northern分析表明，CAM以剂量和孵育时间依赖的方式抑制肿瘤坏死因子α(TNF)诱导的IL-8基因表达。IL-8基因5‘侧翼序列与荧光素酶报告基因融合基因在BET-1A细胞中的感染研究显示，在174bp的片段(相对于转录起始点的-130~+44bp)有很强的启动子活性。该片段包括AP-1和NF-1；lambda；B-like位点，对TNF的反应最强。在该片段中，肿瘤坏死因子诱导的启动子活性受到CAM的显著抑制。然而，156个碱基(-112~+44个碱基)片段不包括AP-1位点，但包含一个NF-1；lambda&gt；与启动子分析一致的是，凝胶迁移率改变分析表明，在经肿瘤坏死因子处理的BET-1A细胞中，CAM抑制AR-1的结合，而在BET-1A细胞中，肿瘤坏死因子既能诱导AP-1结合，又能诱导核因子B结合活性。提示CAM等大分子物质主要通过AP-1结合部位抑制人支气管上皮细胞IL-8基因转录。我们的发现为大环内酯类化合物的抗炎作用提供了一个新的机制，为开发治疗慢性呼吸道炎症的新药提供了一个靶点。

Macrolide antibiotics are known to be effective for the treatment of chronic inflammatory airway diseases including diffuse panbronchiolitis, chronic bronchitis and bronchial asthma. Other than anti-microbial activities, macrolides have "anti-inflammatory" effects, such as the inhibition of cytokine production. In the present study, we investigated the effects of clarithromycin (CAM) on the interleukin-8 (IL-8) gene expression and its protein levels using a human bronchial epithelial cell line, BET-1A cells. Northern analyses demonstrated that CAM inhibited the tumor necrosis factor alpha (TNF)-induced IL-8 gene expression in dose and incubation time-dependent fashions. The half-life of IL-8 mRNA transcripts in TN F-treated BET-1A cells did not change with CAM.Transfection studies with BET-1A cells, using fusion genes composed of the 5'-flanking sequences of the IL-8 gene and a luciferase reporter gene, demonstrated a potent promoter activity in a 174.bp segment (-130 to +44 bp relative to the transcription start site). This segment includes AP-1 and NF-_<lambda>B-like sites, and exhibited a strongest response to TNF.TNF-induced promoter activity in this segment showed a significant repression by CAM.However, a 156-bp segment (-112 to +44 bp), which does not include an AP-1 site but includes a NF-_<lambda>B-like site, did not show a significant repression of TNF-induced promoter activity by CAM.Consistent with promoter analyses, an electrophoretic mobility shift assay demonstrated that CAM repressed the AR-1 binding in TNF-treated BET-1A cells, however, TNF induced both AP-1 and NF-_<lambda>B binding activities in BET-1A cells. These data suggest that macrolidessuch as CAM repress the IL-8 gene transcription mainly via the AP-1 binding site in human bronchial epithelial cells. Our findings provide a novel mechanism of the anti-inflammatory function of macrolides, implicating a target for the development of a new drug for the treatment of chronic airway inflammation.