Musikacharoen T, Matsuguchi T, Kikuchi T, Yoshikai Y
Laboratory of Host Defense and Germfree Life, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Nagoya, Japan.
J Immunol. 2001 Apr 1;166(7):4516-24. doi: 10.4049/jimmunol.166.7.4516.
Toll-like receptor 2 (TLR2) is involved in the innate immunity by recognizing various bacterial components. We have previously reported that TLR2 gene expression is rapidly induced by LPS or inflammatory cytokines in macrophages, and by TCR engagement or IL-2/IL-15 stimulation in T cells. Here, to investigate the mechanisms governing TLR2 transcription, we cloned the 5' upstream region of the mouse TLR2 (mTLR2) gene and mapped its transcriptional start site. The 5' upstream region of the mTLR2 gene contains two NF-kappa B, two CCAAT/enhancer binding protein, one cAMP response element-binding protein, and one STAT consensus sequences. In mouse macrophage cell lines, deletion of both NF-kappa B sites caused the complete loss of mTLR2 promoter responsiveness to TNF-alpha. NF-kappa B sites were also important but not absolutely necessary for LPS-mediated mTLR2 promoter activation. In T cell lines, mTLR2 responsiveness to IL-15 was abrogated by the 3' NF-kappa B mutation, whereas 5' NF-kappa B showed no functional significance. The STAT binding site also seemed to contribute, as the deletion of this sequence significantly reduced the IL-15-mediated mTLR2 promoter activation. EMSAs confirmed nuclear protein binding to both NF-kappa B sites in macrophages following LPS and TNF-alpha stimulation and to the 3' NF-kappa B site in T cells after IL-15 treatment. Thus, NF-kappa B activation is important but differently involved in the regulation of mTLR2 gene expression in macrophages and T cells following LPS or cytokine stimulation.
Toll样受体2（TLR2）通过识别各种细菌成分参与固有免疫。我们之前报道过，巨噬细胞中TLR2基因表达可被脂多糖（LPS）或炎性细胞因子迅速诱导，而在T细胞中可被T细胞受体（TCR）激活或白细胞介素-2（IL-2）/白细胞介素-15（IL-15）刺激迅速诱导。在此，为了研究调控TLR2转录的机制，我们克隆了小鼠TLR2（mTLR2）基因的5'上游区域并定位了其转录起始位点。mTLR2基因的5'上游区域包含两个核因子κB（NF-κB）、两个CCAAT/增强子结合蛋白、一个环磷酸腺苷反应元件结合蛋白和一个信号转导及转录激活因子（STAT）共有序列。在小鼠巨噬细胞系中，两个NF-κB位点的缺失导致mTLR2启动子对肿瘤坏死因子-α（TNF-α）的反应性完全丧失。NF-κB位点对于LPS介导的mTLR2启动子激活也很重要，但并非绝对必需。在T细胞系中，3' NF-κB突变消除了mTLR2对IL-15的反应性，而5' NF-κB则无功能意义。STAT结合位点似乎也有作用，因为该序列的缺失显著降低了IL-15介导的mTLR2启动子激活。电泳迁移率变动分析（EMSA）证实，LPS和TNF-α刺激后巨噬细胞中核蛋白与两个NF-κB位点结合，IL-15处理后T细胞中核蛋白与3' NF-κB位点结合。因此，NF-κB激活很重要，但在LPS或细胞因子刺激后，其在巨噬细胞和T细胞中对mTLR2基因表达的调控作用有所不同。
