Blocking of Interferon Regulatory Factor 1 Reduces Tumor Necrosis Factor α-Induced Interleukin-18 Bioactivity in Rheumatoid Arthritis Synovial Fibroblasts by Induction of Interleukin-18 Binding Protein a Role of the Nuclear Interferon Regulatory Factor 1-NF-κB-c-Jun Complex
Blocking of Interferon Regulatory Factor 1 Reduces Tumor Necrosis Factor α-Induced Interleukin-18 Bioactivity in Rheumatoid Arthritis Synovial Fibroblasts by Induction of Interleukin-18 Binding Protein a Role of the Nuclear Interferon Regulatory Factor 1-NF-κB-c-Jun Complex
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DOI：
10.1002/art.30583
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发表时间：
2011-11-01
影响因子：
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通讯作者：
Koch, Alisa E.
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文献类型：
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作者：
Marotte, Hubert;Tsou, Pei-Suen;Koch, Alisa E.
Objective.研究干扰素调节因子1（IRF-1）在肿瘤坏死因子α（TNF α）诱导的类风湿关节炎滑膜成纤维细胞（RASFs）白细胞介素18结合蛋白a（IL-18 BPa）表达中的作用。通过实时定量聚合酶链反应和蛋白质印迹法评估TNF α诱导的IRF-1表达。使用细胞核和细胞质提取物、蛋白质印迹和免疫荧光评估TNF α对IRF-1的影响。使用NF-κ B或MAP激酶的化学抑制剂分析TNF α诱导的IRF-1表达和IRF-1核转位的信号通路。使用对照和IRF-1小干扰RNA（siRNA）来分析IRF-1下调对TNF α诱导的IL-18 BP表达的影响。采用酶联免疫吸附试验检测IL-18 BPa的表达，采用KG-1细胞检测IL-18的转录水平和生物活性。TNF α诱导RASF IRF-1在信使RNA和蛋白水平的表达，在2小时时具有最大作用（P < 0.05; n >= 3）。TNF α诱导IRF-1发生核转位，在2 h时达到最大值（与6倍诱导相似）（P < 0.05; n = 4）。阻断NF-κ B或JNK-2通路分别使TNF α诱导的IRF-1核转位减少35%和50%（P < 0.05; n >= 4）。使用siRNA敲低IRF-1，我们观察到IL-18 BPa表达降低。此外，当使用siRNA敲低IRF-1表达时，IL-18的生物活性更高。这些结果表明，IRF-1是RASF中IL-18 BPa表达和IL-18生物活性的关键调节因子。调节IRF-1的表达将成为RA治疗的新靶点。
Objective. To examine the role of interferon regulatory factor 1 (IRF-1) in tumor necrosis factor alpha (TNF alpha)-induced interleukin-18 binding protein a (IL-18BPa) expression in rheumatoid arthritis synovial fibroblasts (RASFs).Methods. TNF alpha-induced IRF-1 expression was assessed by real-time quantitative polymerase chain reaction and Western blotting. The effect of TNF alpha on IRF-1 was assessed using nuclear and cytoplasmic extracts, Western blots, and immunofluorescence. Chemical inhibitors of NF-kappa B or MAP kinases were used to analyze the signaling pathways of TNF alpha induced IRF-1 expression and IRF-1 nuclear translocation. Control and IRF-1 small interfering RNA (siRNA) were used to analyze the effect of IRF-1 down-regulation on TNF alpha-induced IL-18BP expression. IL-18BPa expression was assessed by enzyme-linked immunosorbent assay, and IL-18 was assessed at the transcription and bioactivity levels using KG-1 cells.Results. TNF alpha induced RASF IRF-1 expression at the messenger RNA and protein levels, with a maximal effect at 2 hours (P < 0.05; n >= 3). Furthermore, TNF alpha induced nuclear translocation of IRF-1, with maximal translocation at 2 hours (similar to 6 fold-induction) (P < 0.05; n = 4). Blocking of the NF-kappa B or JNK-2 pathways reduced TNF alpha-induced IRF-1 nuclear translocation by 35% and 50%, respectively (P < 0.05; n >= 4). Using siRNA to knock down IRF-1, we observed reduced IL-18BPa expression. Additionally, IL-18 bioactivity was higher when siRNA was used to knock down IRF-1 expression.Conclusion. These results show that IRF-1 is a key regulator of IL-18BPa expression and IL-18 bioactivity in RASFs. Regulation of IRF-1 will be a new therapeutic target in RA.