Yamamoto Koh, Shibata Fumi, Miyasaka Nobuyuki, Miura Osamu
Department of Hematology and Oncology, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, 113-8519, Tokyo, Japan.
Biochem Biophys Res Commun. 2002 Oct 11;297(5):1245-52. doi: 10.1016/s0006-291x(02)02378-1.
IL-12 activates STAT4 by inducing tyrosine phosphorylation, homo-dimerization, and nuclear translocation in NK cells and thereby stimulates proliferation and activation of these cells. The pore-forming protein perforin is a key effector protein for NK cell- and cytotoxic T lymphocyte-mediated cytolysis. Here we demonstrate that IL-12 induces the expression of the perforin gene in human NK cell line, NKL. Electrophoretic mobility shift assays using a probe containing two putative STAT-binding sequences located at -1085 and -1059 in the human perforin gene showed that STAT4 or STAT5 activated by IL-12 or IL-2, respectively, in NKL cells binds this region. Further analyses using various probes with or without mutated STAT-binding sequences showed that, although either of the two tandem STAT-binding sequences binds STAT4 weakly, the presence of both is required for significant binding of activated STAT4 and for formation of the STAT4-DNA-binding complex with lower electrophoretic mobility. Furthermore, mutation of either of the tandem STAT-binding sequences abolished the IL-12-induced activation of the perforin gene promoter in reporter gene assays. These results indicate that the IL-12-induced expression of the perforin gene in NK cells is directly regulated by STAT4, which binds, most likely as a homo-tetramer, to the tandem STAT-binding sequences in the perforin gene promoter.
白细胞介素-12（IL-12）通过诱导酪氨酸磷酸化、同源二聚化以及在自然杀伤细胞（NK细胞）中的核转位来激活信号转导和转录激活因子4（STAT4），从而刺激这些细胞的增殖和活化。成孔蛋白穿孔素是NK细胞和细胞毒性T淋巴细胞介导的细胞溶解的关键效应蛋白。在此，我们证明IL-12可诱导人NK细胞系NKL中穿孔素基因的表达。使用包含位于人穿孔素基因-1085和-1059处的两个假定STAT结合序列的探针进行的电泳迁移率变动分析表明，在NKL细胞中分别由IL-12或IL-2激活的STAT4或STAT5结合该区域。使用具有或不具有突变STAT结合序列的各种探针进行的进一步分析表明，尽管两个串联STAT结合序列中的任何一个与STAT4的结合都较弱，但两者的存在对于活化的STAT4的有效结合以及形成具有较低电泳迁移率的STAT4-DNA结合复合物是必需的。此外，在报告基因分析中，串联STAT结合序列中的任何一个发生突变都会消除IL-12诱导的穿孔素基因启动子的激活。这些结果表明，IL-12诱导的NK细胞中穿孔素基因的表达直接受STAT4调控，STAT4很可能以同源四聚体的形式与穿孔素基因启动子中的串联STAT结合序列结合。
