Mouri Fumihiko, Tsukada Junichi, Mizobe Takamitsu, Higashi Takehiro, Yoshida Yasuhiro, Minami Yasuhiro, Izumi Hiroto, Kominato Yoshihiko, Kohno Kimitoshi, Tanaka Yoshiya
The first Department of Internal Medicine, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan.
Eur J Haematol. 2008 Jan;80(1):10-9. doi: 10.1111/j.1600-0609.2007.00981.x.
High mobility group box 1 protein (HMGB1), originally described as a non-histone, DNA binding protein, was recently identified as a late mediator of inflammation via its extracellular release from activated macrophages/monocytes. In the present study, we report that intracellular HMGB1 synergizes with a macrophage/monocyte-specific E26 transformation-specific sequence (Ets) transcription factor PU.1 to transactivate the promoter of the IL1B gene coding a 31-kDa proIL-1beta protein. The -131 to +12 IL1B promoter, which possesses a PU.1 binding motif essential for its transactivation, was induced when HMGB1 expression vector was transfected into murine RAW264.7 macrophage cells. Our glutathione S-transferase-pulldown and coimmunoprecipitation assays demonstrated direct physical interaction of HMGB1 with PU.1. Deletion of the PU.1 winged helix-turn-helix DNA-binding domain inhibited the association of the two proteins. In electrophoretic mobility shift assay using recombinant PU.1 protein, a ternary complex of PU.1, HMGB1 and PU.1-binding element within the IL1B promoter was generated. The importance of PU.1 was further supported by our observation that induction of the IL1B promoter was obtained only after PU.1 expression in PU.1-deficient murine EL4 thymoma cells. Thus, our data raise the possibility of a novel mechanism which sustains and amplifies inflammatory reactions through physical interaction of PU.1 with intracellular HMGB1 in macrophages/monocytes.
高迁移率族蛋白B1（HMGB1）最初被描述为一种非组蛋白DNA结合蛋白，最近被确定为炎症的晚期介质，它通过从活化的巨噬细胞/单核细胞中释放到细胞外发挥作用。在本研究中，我们报告细胞内HMGB1与巨噬细胞/单核细胞特异性的E26转化特异性序列（Ets）转录因子PU.1协同作用，以反式激活编码31 kDa前白细胞介素-1β蛋白的IL1B基因的启动子。当将HMGB1表达载体转染到小鼠RAW264.7巨噬细胞中时，具有反式激活所必需的PU.1结合基序的-131至+12 IL1B启动子被诱导。我们的谷胱甘肽S-转移酶下拉和免疫共沉淀试验证明了HMGB1与PU.1之间存在直接的物理相互作用。PU.1翼状螺旋-转角-螺旋DNA结合结构域的缺失抑制了这两种蛋白质的结合。在使用重组PU.1蛋白的电泳迁移率变动分析中，在IL1B启动子内产生了PU.1、HMGB1和PU.1结合元件的三元复合物。我们观察到在PU.1缺陷的小鼠EL4胸腺瘤细胞中只有在PU.1表达后才诱导IL1B启动子，这进一步支持了PU.1的重要性。因此，我们的数据提出了一种新机制的可能性，即通过巨噬细胞/单核细胞中PU.1与细胞内HMGB1的物理相互作用来维持和放大炎症反应。
