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.