Signal transduction of T cell activation and regulation of IL-3 and GM-CSF gene expression

T 细胞激活的信号转导以及 IL-3 和 GM-CSF 基因表达的调节

• 批准号: 03454544
• 负责人: YOKOTA Takashi
• 金额: $ 3.97万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-03454544/
• 关键词: interleukin 3; interleukin 5; colony stimulating factor; cis-regulatory element; gene expression; T cell activation signal; DNA binding protein; cAMP; シグナル伝達; DNA結合タンパク質; 転写因子; エンハンサー配列; トランスアクチベーター; インタ-ロイキン3; コロニ-刺激因子; エンハンサ-配列; トランスアクチベ

The CLE2/GC-box (at positions -95 - -73, which is homologous to the NF-kB binding site) and CLE0 (at positions -54 - -40) of the mouse GM-CSF promoter are essential for transcriptional activation in response to phorbol 12-myristate 13 acetate (PMA) and calcium ionophore (A23187). GM-kB defines a binding site for a protein induced by PMA which is related to NF-kB.CLE0 binding proteins, which are responsive to PMA/calcium signal and also sensitive to cyclosporin A, is composed of AP1 and NFAT, a T-cell-specific trnascription factor. CLE0-like elements are found in the promoters of IL-3, IL-4, IL-5, as well as in the IL-2 and GM-CSF promoters. NFAT or related factors may account for the coordinate induction of these cytokine genes during T-cell activation.The CT/GC-rich sequence (at positions -76 to -57) of the human IL-3 gene is one of the upstream elements that mediate the response to HTLV-I Tax. We isolated cDNAs encoding DB1, a novel zinc-finger DNA binding protein, as well as previou … More sly described EGR1 and EGR2, all of which bind to this CT/GC-rich sequence. DB1 has six Cys_2/His_2-type zinc finger motifs and a glutamine-stretch which is often found in other transcription factors. Electrophoretic mobility shift assay (EMSA), using specific antibodies, showed that DB1 constitutively binds to this region whereas EGR1 binds in a T cell activation-dependent manner. Forced expression of EGR1, EDR2, or DB1 proteins in Jurkat cells increased the transcription activity of the IL-3 promoter in the presence of upstream elements and T cell activation signals. These results suggest that these zinc finger proteins support the constitutive and inducible promoter activity of the IL-3 gene through the CT/GC-rich sequence.Mouse thymoma cell line EL-4 produces IL-5 as well as IL-2, IL-3, IL-4, IL-10 and GM-CSF in response to PMA.Although the production of IL-5 by PMA alone is very weak, co-stimulation with PMA and cAMP significantly increased the level of the IL-5 mRNA and the protein. Transient transfection assay employing EL-4 cells revealed that the IL-5 promoter was synergistically activated by PMA and cAMP.Co-transfection of the catalytic subunit of protein kinase A (PKA) mimicked cAMP response. These results indicated that IL-5 production upon T cell activation in vivo is mediated by a signal(s) that modulate cAMP-PKA pathway. Activation of the IL-5 promoter in response to cAMP and PMA was dependent on two distinct regions located at nucleotide positions -970 to -930 (region I) and -141 to -80 (region II). In contrast, cAMP almost completely inhibited the PMA-dependent activation of endogenous IL-2 and GM-CSF genes as well as that of the transfected IL-2 promoter. These results indicate that the IL-5 gene is positively regulated by cAMP in a manner opposite to IL-2 and GM-CSF genes in EL-4 cells. Less

小鼠GM-CSF启动子的CLE 2/GC盒（在位置-95 --73，其与NF-κ B结合位点同源）和CLE 0（在位置-54 - -40）对于响应佛波醇12-肉豆蔻酸酯13乙酸酯（PMA）和钙离子载体（A23187）的转录激活是必需的。CLE 0结合蛋白由AP 1和T细胞特异性转录因子NFAT组成，对PMA/钙信号有反应，对环孢菌素A也敏感。CLE 0样元件存在于IL-3、IL-4、IL-5的启动子以及IL-2和GM-CSF启动子中。人IL-3基因的富含CT/GC的序列（在位置-76至-57）是介导对HTLV-1 Tax的应答的上游元件之一。我们分离了编码DB 1的cDNA，DB 1是一种新的锌指DNA结合蛋白， ...更多信息 Sly描述了EGR 1和EGR 2，它们都与这个富含CT/GC的序列结合。DB 1具有6个Cys_2/His_2型锌指基序和一个谷氨酰胺延伸区，这在其他转录因子中也很常见。电泳迁移率变动分析（EMSA），使用特异性抗体，表明DB 1组成型结合到这个区域，而EGR 1结合在T细胞活化依赖性的方式。在存在上游元件和T细胞活化信号的情况下，Jurkat细胞中EGR 1、EDR 2或DB 1蛋白的强制表达增加了IL-3启动子的转录活性。小鼠胸腺瘤细胞系EL-4对PMA反应产生IL-5、IL-2、IL-3、IL-4、IL-10和GM-CSF，虽然单独的PMA产生IL-5的能力很弱，PMA和cAMP共刺激可显著提高IL-5 mRNA和蛋白水平。瞬时转染EL-4细胞，发现PMA和cAMP协同激活IL-5启动子，共转染蛋白激酶A催化亚基（PKA）模拟cAMP反应。这些结果表明，体内T细胞活化时的IL-5产生是由调节cAMP-PKA途径的信号介导的。响应cAMP和PMA的IL-5启动子的激活依赖于位于核苷酸位置-970至-930（区域I）和-141至-80（区域II）的两个不同区域。相反，cAMP几乎完全抑制PMA依赖的内源性IL-2和GM-CSF基因以及转染的IL-2启动子的激活。这些结果表明，IL-5基因在EL-4细胞中以与IL-2和GM-CSF基因相反的方式被cAMP正调控。少

项目成果

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