Yu C R, Ortaldo J R, Curiel R E, Young H A, Anderson S K, Gosselin P
Division of Basic Sciences, Laboratory of Experimental Immunology, National Cancer Institute, Frederick, MD, USA.
J Immunol. 1999 Mar 1;162(5):2785-90.
The pore-forming protein perforin is preferentially expressed in NK and cytotoxic T cells. To investigate the molecular regulation of human perforin gene transcription, the activity of the human perforin promoter was analyzed in human NK and T cell lines using various promoter fragments linked to a luciferase reporter gene. A core promoter was identified within 55 bp upstream of the transcription start site. This promoter region contains a guanine/cytosine box and has basal activity in YT, Kit225-k6, and Jurkat cells. A strong enhancer activity was identified between positions -1136 and -1076, a region that includes a STAT-like element. This enhancer region was active in YT cells, which have constitutive perforin expression and activated STAT3 protein, but not in Kit225-k6 or Jurkat cells, which do not have constitutive perforin expression. Mutation of the STAT binding site resulted in a dramatic down-regulation of promoter activity. Electrophoretic mobility shift assays, using a probe containing the STAT element of the perforin promoter, indicated that this element can bind STAT3 from YT cells. Moreover, the STAT element was shown to bind STAT5a/b induced by IL-2 as well as STAT1alpha induced by IL-6 in human NK cells. Together, these results suggest that STAT proteins play a key role in perforin gene transcription and provide a model by which cytokines can regulate perforin gene expression.
穿孔素是一种形成孔道的蛋白质，在自然杀伤细胞（NK细胞）和细胞毒性T细胞中优先表达。为了研究人类穿孔素基因转录的分子调控机制，我们使用与荧光素酶报告基因相连的各种启动子片段，在人类NK细胞系和T细胞系中分析了人类穿孔素启动子的活性。在转录起始位点上游55 bp范围内鉴定出一个核心启动子。该启动子区域包含一个鸟嘌呤/胞嘧啶盒，在YT细胞、Kit225-k6细胞和Jurkat细胞中具有基础活性。在-1136至-1076位之间鉴定出一种强大的增强子活性，该区域包含一个类似信号转导和转录激活因子（STAT）的元件。该增强子区域在具有组成型穿孔素表达和激活的STAT3蛋白的YT细胞中具有活性，但在不具有组成型穿孔素表达的Kit225-k6细胞或Jurkat细胞中无活性。STAT结合位点的突变导致启动子活性显著下调。使用包含穿孔素启动子STAT元件的探针进行的电泳迁移率变动分析表明，该元件可以结合YT细胞中的STAT3。此外，在人类NK细胞中，该STAT元件还显示出能结合由白细胞介素-2（IL-2）诱导的STAT5a/b以及由IL-6诱导的STAT1α。总之，这些结果表明STAT蛋白在穿孔素基因转录中起关键作用，并提供了一种细胞因子可调节穿孔素基因表达的模型。
