Gonsky Rivkah, Deem Richard L, Bream Jay, Young Howard A, Targan Stephan R
Inflammatory Bowel Disease Research Center, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, CA 90048, USA.
J Immunol. 2004 Nov 15;173(10):6241-7. doi: 10.4049/jimmunol.173.10.6241.
IFN-gamma is an important immunoregulatory protein with tightly controlled expression in activated T and NK cells. Three potential STAT binding regions have been recognized within the IFN-gamma promoter: 1) an IL-12-mediated STAT4 binding site at -236 bp; 2) a newly identified IL-2-induced STAT5 binding element at -3.6 kb; and 3) CD2-mediated STAT1 and STAT4 binding to an intronic element in mucosal T cells. However, functional activation of these sites remains unclear. In this study we demonstrate CD2-mediated activation of the newly characterized -3.6-kb IFN-gamma STAT5 binding region. CD2 signaling of human PBMC results in activation of the -3.6-kb IFN-gamma promoter, whereas mutation of the -3.6-kb STAT5 site attenuates promoter activity. Functional activation is accompanied by STAT5A but little STAT5B nucleoprotein binding to the IFN-gamma STAT5 site, as determined by competition and supershift assays. STAT5 activation via CD2 occurs independent of IL-2. Western and FACS analysis shows increased phospho-STAT5 following CD2 signaling. AG490, a tyrosine kinase inhibitor affecting Jak proteins, inhibits CD2-mediated IFN-gamma mRNA expression, secretion, and nucleoprotein binding to the IFN-gamma STAT5 site in a dose-dependent fashion. This report is the first to describe CD2-mediated activation of STAT5 and supports STAT5 involvement in regulation of IFN-gamma expression.
干扰素-γ是一种重要的免疫调节蛋白，在活化的T细胞和NK细胞中表达受到严格控制。在干扰素-γ启动子内已识别出三个潜在的信号转导和转录激活因子（STAT）结合区域：1）位于-236 bp处的白细胞介素-12介导的STAT4结合位点；2）新发现的位于-3.6 kb处的白细胞介素-2诱导的STAT5结合元件；3）CD2介导的STAT1和STAT4与黏膜T细胞内含子元件的结合。然而，这些位点的功能激活仍不清楚。在本研究中，我们证明了CD2介导的新鉴定的-3.6 kb干扰素-γ STAT5结合区域的激活。人外周血单核细胞（PBMC）的CD2信号传导导致-3.6 kb干扰素-γ启动子的激活，而-3.6 kb STAT5位点的突变会减弱启动子活性。通过竞争和超迁移分析确定，功能激活伴随着STAT5A而非STAT5B核蛋白与干扰素-γ STAT5位点的结合。通过CD2激活STAT5独立于白细胞介素-2发生。蛋白质免疫印迹法（Western）和荧光激活细胞分选术（FACS）分析显示，CD2信号传导后磷酸化STAT5增加。AG490是一种影响Jak蛋白的酪氨酸激酶抑制剂，它以剂量依赖的方式抑制CD2介导的干扰素-γ mRNA表达、分泌以及核蛋白与干扰素-γ STAT5位点的结合。本报告首次描述了CD2介导的STAT5激活，并支持STAT5参与干扰素-γ表达调控。
