Therapeutic strategy for inflammatory diseases by a novel negative regulatory transcriptional factor of inflammatory cytokines

类风湿关节炎(RA)的一个主要病理特征是组织中单核/巨噬细胞长时间产生包括IL-1b在内的炎性细胞因子。关于IL-1b基因的转录调控，我们揭示了转录起始点(CAP位点)上游启动子序列的重要性，并确定了-3134至-2729之间的元件位置是最大限度诱导内毒素和PMA所必需的。该增强子区域还参与了人单核细胞系THP-1、小鼠巨噬细胞系RAW-267.4细胞以及RA滑膜细胞、滑膜细胞系E11中IL-1b基因转录的上调。使用包含这些丝裂原反应元件的系列CAT质粒，我们还发现负调节区位于-2833至-2782之间。该元件的缺失导致IL-1b基因在THP-1和RAW 267.4细胞中转录增强，但在E11中没有。我们用~(32)P标记的负性调控元件的寡核苷酸进行了凝胶迁移率改变分析。单一条带可被内毒素、PMA刺激增强，且不与Jun、Fos家族和NFkB等常规转录因子抗体竞争。

Prolonged production of inflammatory cytokines including IL-lb from monocytes/ macrophages in the tissue is a major pathological feature of Rheumatoid Arthritis (RA). Regarding the transcriptional regulation of IL-lb gene, we revealed the importance of promoter sequences immediately upstream of the transcription start site (cap site) and identified the element located position between-3134 and -2729 was required for maximal LPS and PMA induction. The enhancer region also involved in the increase transcription of IL-lb gene in human monocytic cell line THP-1, murine macrophage cell line RAW 267.4 cells as well as-RA synovial cells, synovial cell line Ell. Using a serial CAT plasmid including these mitogen responsive elements, we also found negative regulatory region located between-2833 and-2782. The deletion of the element resulted in the augmentation of transcription of IL-lb gene in both THP-1 and RAW 267.4 cells but not in Ell. We conducted electrophoretic mobility shift assay using 32P-labeled oligonucleotides of the negative regulatory element. A single band was enhanced by LPS, PMA stimulation and not competed by antibodies for conventional transcriptional factors including jun and fos families and NFkB.