Activation of Synoviolin Promoter in Rheumatoid Synovial Cells by a Novel Transcription Complex of Interleukin Enhancer Binding Factor 3 and GA Binding Protein α
Activation of Synoviolin Promoter in Rheumatoid Synovial Cells by a Novel Transcription Complex of Interleukin Enhancer Binding Factor 3 and GA Binding Protein α
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DOI：
10.1002/art.24178
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发表时间：
2009-01-01
影响因子：
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通讯作者：
Nakajima, Toshihiro
中科院分区：
文献类型：
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作者：
Izumi, Toshihiko;Fujii, Ryoji;Nakajima, Toshihiro
目标。滑膜小提琴是一种E3泛素连接酶，其过表达与类风湿关节炎（RA）的发病机制有关。我们之前报道过滑膜小提琴启动子内的Ets结合位点1 （EBS-1）对滑膜小提琴的表达至关重要，GA结合蛋白（GABP）结合在这个位点上。本研究旨在通过ebs -1方法阐明转录调控的确切机制。我们纯化并鉴定了与eb -1结合的复杂组分，并检查了它们在类风湿滑膜细胞中对滑膜收缩的转录调节中的作用。我们对具有eb -1结合活性的蛋白进行了生化纯化，并利用液相色谱串联质谱分析对其进行了鉴定。通过电泳迁移迁移和共免疫沉淀实验，验证了所鉴定的蛋白在EBS-1上募集并形成复合物。此外，通过报告基因实验和RNA干扰实验检测了它们的转录活性。我们发现白细胞介素增强子结合因子3 （ILF-3）是该复合物中的一个新因子。ILF-3通过与类风湿滑膜细胞中GABP α的关联激活滑膜启动子。此外，还观察到ILF-2和GAB β的进一步激活，这两种物质分别是之前报道的ILF-3和GABPa的相互作用物。此外，ilf -3敲低实验显示滑膜基因表达降低。我们的研究结果表明，已知在T细胞中调节IL-2表达的ILF-3可以上调类风湿滑膜细胞中GABP α的滑膜膜表达。ILF-3可能通过其对T细胞和类风湿滑膜细胞中IL-2的作用而成为RA治疗的靶点。
Objective. Synoviolin is an E3 ubiquitin ligase, and its overexpression is implicated in the pathogenesis of rheumatoid arthritis (RA). We reported previously that Ets binding site 1 (EBS-1) within the synoviolin promoter is crucial for the expression of synoviolin, and GA binding protein (GABP) binds to this site. This study was undertaken to elucidate the precise mechanisms of transcriptional regulation via EBS-1.Methods. We performed purification and identification of complex components that bind to EBS-1 and inspected their contributions to the transcriptional regulation of synoviolin in rheumatoid synovial cells. We biochemically purified proteins that had EBS-1 binding activity and identified the proteins using liquid chromatography tandem mass spectrometry analysis. The identified proteins were verified to recruit and form the complex on EBS-1 using electrophoretic mobility shift assay and coimmunoprecipitation assay. Furthermore, their transcription activities were tested by reporter assays and RNA interference experiments.Results. We identified interleukin enhancer binding factor 3 (ILF-3) as a novel factor in the complex. ILF-3 was demonstrated to activate the synoviolin promoter via association with GABP alpha in rheumatoid synovial cells. In addition, further activation was observed with ILF-2 and GAB beta, previously reported interactants of ILF-3 and GABPa, respectively. Moreover, ILF-3-knockdown experiments showed reduced expression of the synoviolin gene.Conclusion. Our findings indicate that ILF-3, which has been known to regulate IL-2 expression in T cells, up-regulates synoviolin expression with GABP alpha in rheumatoid synovial cells. ILF-3 might be a target for RA treatment through its effect on IL-2 in T cells and synoviolin in rheumatoid synovial cells.