D'Ambrosio D, Trotta R, Vacca A, Frati L, Santoni A, Gulino A, Testi R
Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Italy.
Eur J Immunol. 1993 Nov;23(11):2993-7. doi: 10.1002/eji.1830231140.
The 5' flanking region of the human interleukin (IL)-2 gene was investigated for enhancer activity in response to CD69-generated signals, using a chloramphenicol acetyltransferase (CAT)-driven transient expression system in Jurkat cells. The region extending from -317 to +47 relative to the initiation site of IL-2 gene transcription was shown to contain sequences able to respond to CD69 cross-linking, by enhancing by about 100% a phorbol 12-myristate 13-acetate (PMA)-plus-ionomycin stimulation of CAT activity. A similar increase in CAT activity produced by PMA-plus-anti-CD3 mAb was induced by CD69 cross-linking, while a 200% increase over that obtained by PMA-plus-anti-CD28 mAb stimulation was seen. Analysis of enhancer deletion mutants revealed that proximal AP-1, OCT-1/octamer-associated protein and nuclear factor of activated T cells (NFAT) binding regions were all necessary to allow CD69-mediated enhancement of CAT activity. By gel mobility shift analysis, cyclosporin A-sensitive NFAT-binding induction and enhancement of AP-1 binding activity could be detected in nuclear extracts of both Jurkat and peripheral blood T cells after simultaneous CD69 and protein kinase C stimulation. Finally, CD69-mediated signals could increase NFAT and AP-1 binding activity following PMA and ionomycin stimulation in peripheral blood T cells. Collectively, these data suggest that CD69-generated signals participate in the control of the IL-2 gene expression at the transcriptional level, likely acting through NFAT and AP-1 transcription factor complexes.
利用氯霉素乙酰转移酶（CAT）驱动的瞬时表达系统，在Jurkat细胞中研究了人类白细胞介素（IL）-2基因5'侧翼区域对CD69产生的信号的增强子活性。相对于IL-2基因转录起始位点，从-317至+47延伸的区域显示含有能够响应CD69交联的序列，通过将佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）加离子霉素刺激的CAT活性提高约100%来实现。CD69交联诱导了由PMA加抗CD3单克隆抗体产生的类似CAT活性增加，同时观察到比PMA加抗CD28单克隆抗体刺激所获得的活性增加200%。增强子缺失突变体分析表明，近端活化蛋白-1（AP-1）、八聚体转录因子-1（OCT-1）/八聚体相关蛋白和活化T细胞核因子（NFAT）结合区域对于CD69介导的CAT活性增强都是必需的。通过凝胶迁移率变动分析，在Jurkat细胞和外周血T细胞的核提取物中，同时进行CD69和蛋白激酶C刺激后，可检测到环孢素A敏感的NFAT结合诱导和AP-1结合活性增强。最后，在外周血T细胞中，PMA和离子霉素刺激后，CD69介导的信号可增加NFAT和AP-1结合活性。总体而言，这些数据表明，CD69产生的信号在转录水平参与IL-2基因表达的调控，可能通过NFAT和AP-1转录因子复合物发挥作用。
