LYMPHOKINE PRODUCTION: EFFECT OF CYCLOSPORIN

淋巴细胞因子的产生：环孢菌素的作用

• 批准号: 3293081
• 负责人: ANGELA GRANELLI-PIPERNO
• 金额: $ 21.26万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-04-01 至 1994-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3293081
• 关键词: DNA binding protein; DNA footprinting; antibody; crosslink; cyclosporines; gel mobility shift assay; gene expression; genetic library; human genetic material tag; human tissue; interleukin 2; laboratory rabbit; messenger RNA; molecular cloning; phosphorylation; posttranslational modifications; protein biosynthesis; protein purification; protein tyrosine kinase; ultraviolet radiation

Lymphokines, the specialized products of stimulated T cells, mediate the growth and function of lymphocytes and other cell types. Lymphokine synthesis is controlled at the transcriptional level and is induced by stimulation with antigens or mitogens. The production of certain lymphokine mRNAs [IL-2, IFN-gamma, IL-4] but not the induction of other mRNAs [p55 IL-2 receptor, c-fos, a heat shock protein, IL-6, GM-CSF] is blocked selectively by the immunosuppressive drugs cyclosporin A [CSA] and FK-506. Several regulatory sequences have been identified in the promoter of the human IL-2 gene, using in vivo functional analysis and DNA-binding [electromobility shift assays EMSA]. The characterization of the factors that interact with these regulatory sites varies greatly: some have been purified and cloned while others are defined primarily by EMSA. The nuclear factor for activated T cells [NF-AT], which binds at position -289 to -269 in the 5' region of the IL-2 enhancer, remains uncharacterized. This gap is significant since NF-AT is the only known factor that is T cell restricted, requires "two signals" for induction, and is sensitive to CSA and FK-506. Other sites on the IL-2 enhancer, such as NF-KB and OCT-1, are activated by only one signal and are insensitive to CSA and FK-506. To pursue these and my preliminary findings we will: a) isolate, clone, and generate antibodies to the inducible, CSA:FK-506 sensitive factor that binds to oligonucleotide columns made with the wild-type but not mutant NF- AT site; b) similarly characterize a noninducible, CSA:FK-506 resistant modulator which was identified in the effluent of NF-AT columns and which markedly increases the activity of the NF-AT binding protein; and c) conduct a series of experiments to assess the role of these two factors in lymphokine gene expression. The latter experiments will describe the subcellular localization of the corresponding proteins and mRNA's following application of different T cell stimuli, the role of inhibitors and posttranslational modification such as tyrosine kinase dependent phosphorylation, the interaction of the two proteins with the NF-AT site as revealed by UV-crosslinking and in vitro transcription assays, the presence of comparable binding and modulator proteins in other CSA-sensitive and CSA-resistant genes, and the effects of immunosuppressive drugs on each of the above aspects of NF-AT activity.

淋巴因子，刺激T细胞的特殊产物，介导 淋巴细胞和其他类型细胞的生长和功能。淋巴因子 合成是在转录水平上控制的，并由 用抗原或有丝分裂原刺激。某些产品的生产 淋巴因子mRNAs[IL-2、干扰素-γ、IL-4]，但不诱导其他 MRNAs[p55 IL-2受体，c-fos，热休克蛋白，IL-6，GM-CSF]是 选择性地被免疫抑制药物环孢素A[CsA]和 FK-506。已在启动子中鉴定出几个调控序列 人IL-2基因的体内功能分析和DNA结合 [电迁移率移位分析EMSA]。因素的表征 与这些监管网站互动的方式千差万别：有些已经 纯化和克隆，而其他的主要由EMSA定义。这个 活化T细胞的核因子[核因子-AT]，结合于-289位 至-269，位于IL-2增强子的5‘端，但仍未被鉴定。 这一差距很大，因为核因子-AT是唯一已知的T细胞因子 限制性，需要“两个信号”进行诱导，并且对CsA敏感 和FK-506。IL-2增强子上的其他站点，如NF-KB和OCT-1，是 只被一个信号激活，对CsA和FK-506不敏感。至 根据我的初步发现，我们将：a)分离、克隆和 产生针对可诱导的CsA：FK-506敏感因子的抗体 与野生型而不是突变型的寡核苷酸柱结合- B)类似地描述了非诱导的CsA：FK-506抗性 在纳滤-AT柱出水中发现的调节剂 显著增加核因子-AT结合蛋白的活性；以及c) 进行一系列实验来评估这两个因素在 淋巴因子基因的表达。后面的实验将描述 相应蛋白质和信使核糖核酸的亚细胞定位 不同T细胞刺激的应用、抑制物的作用和 翻译后修饰，如酪氨酸激酶依赖性 磷酸化，两种蛋白质与核因子-AT位点的相互作用 UV-交联法和体外转录实验表明， 类似的结合和调节蛋白在其他CsA敏感和 CsA耐药基因及免疫抑制药物对每种基因的影响 以上几个方面说明了核因子-AT的活性。