Quan Rong, Fu Yi, He Weiyong, Feng Wen-Hai
State key laboratory of Agrobiotechnology, Department of Microbiology and Immunology, College of Biological Science, China Agricultural University, Beijing 100193, China.
Vet Immunol Immunopathol. 2012 May 15;146(3-4):277-82. doi: 10.1016/j.vetimm.2012.02.010. Epub 2012 Mar 14.
Interleukin 10 (IL-10) is an anti-inflammatory and immunosuppressive cytokine that plays an important role in regulating the immune response. Therefore, understanding how IL-10 is regulated is important. The regulatory elements have been well studied in human and mouse promoters and several transcription factors have been showed to be involved in IL-10 transcription. In our study, a 1.5 kb fragment of the 5' flanking region of IL-10 gene was cloned and functionally characterized. Several putative regulatory elements including IRF, AP-1, Sp1, C/EBP, and STAT binding sites were found in the porcine IL-10 (pIL-10) promoter. The pIL-10 promoter deletion mutants were analyzed for their ability to direct luciferase expression in a porcine macrophage cell line (CRL 2843), human gastric carcinoma cell lines with or without Epstein-Barr virus (EBV), AGS-EBV and AGS cell lines. Our data showed that the minimal active pIL-10 promoter region was from -605 to +19, with the inducible activity requiring only one key DNA element, the Sp1 binding site (-398 to -393) upstream of the IL-10 gene starting point in both LPS-stimulated CRL 2843 and AGS-EBV cells. Moreover, our results suggested that the two IRF binding sites (-950 to -942 and -662 to -640) may have a positive role in the activation of the pIL-10 promoter in AGS-EBV cells, but not in LPS-stimulated CRL 2843 cells. These data implicate that the cloned porcine IL-10 promoter could be used to explore the molecular mechanisms underlying the regulation of IL10 production in pigs.
白细胞介素10（IL-10）是一种抗炎和免疫抑制细胞因子，在调节免疫反应中起重要作用。因此，了解IL-10是如何被调控的很重要。其调控元件在人类和小鼠启动子中已得到充分研究，并且已表明几种转录因子参与IL-10转录。在我们的研究中，克隆了IL-10基因5'侧翼区域的1.5 kb片段并对其进行了功能表征。在猪IL-10（pIL-10）启动子中发现了几个推定的调控元件，包括IRF、AP-1、Sp1、C/EBP和STAT结合位点。分析了pIL-10启动子缺失突变体在猪巨噬细胞系（CRL 2843）、有或没有爱泼斯坦-巴尔病毒（EBV）的人胃癌细胞系、AGS-EBV和AGS细胞系中指导荧光素酶表达的能力。我们的数据表明，最小活性pIL-10启动子区域为-605至+19，在脂多糖刺激的CRL 2843和AGS-EBV细胞中，诱导活性仅需要一个关键DNA元件，即IL-10基因起始点上游的Sp1结合位点（-398至-393）。此外，我们的结果表明，两个IRF结合位点（-950至-942和-662至-640）可能对AGS-EBV细胞中pIL-10启动子的激活有积极作用，但对脂多糖刺激的CRL 2843细胞没有作用。这些数据表明，克隆的猪IL-10启动子可用于探索猪中IL10产生调控的分子机制。
