Oh C K, Metcalfe D D
Allergic Diseases Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892.
J Immunol. 1994 Jul 1;153(1):325-32.
TCA3 is a pro-inflammatory cytokine expressed in mast cells after activation. To examine how this gene is regulated, TCA3 mRNA levels were examined and nuclear run-on experiments and promoter analysis were performed. Northern blot analysis showed that TCA3 message appeared in mast cells within 2 h after induction, with a t1/2 of 30 min. Nuclear run-on experiments revealed that the appearance of TCA3 mRNA occurred in large part because of an increase in the level of de novo transcription. Analysis of the promoter region demonstrated that inducible gene expression was directed by a region extending between 1.324 kb and 0.082 kb upstream from the transcription start site. There was a 60- to 80-fold induction with TCA3 CAT constructs extending between 1.324 kb and 0.324 kb upstream from the transcription start site after treatment with PMA/A23187 and a 30- to 40-fold induction with Fc epsilon RI cross-linking. There was a seven- to eightfold induction with the region extending between 0.136 kb and 0.082 kb upstream from the transcription start site. TCA3 CAT constructs containing regions encompassing either the 0.042 kb or 2.0 kb sequence from the transcription start site were not able to direct CAT-protein synthesis. The TCA3 5' flanking sequence contained negative regulatory activity. Electrophoretic mobility shift assays revealed that protein in nuclear extracts of activated mast cells bound to an NF-kappa B element of the TCA3 gene. These findings demonstrate that the TCA3 gene is regulated transcriptionally in mast cells, with minimal promoter sequences contained within the 0.082 kb upstream region of the TCA3 gene; a putative enhancer NF-kappa B element is contained between 0.324 kb and 0.136 kb and a putative inhibitory element is contained between 1.324 kb and 2.0 kb upstream from the transcription start site.
TCA3是一种在激活后的肥大细胞中表达的促炎细胞因子。为了研究该基因是如何被调控的，检测了TCA3 mRNA水平，并进行了核转录实验和启动子分析。Northern印迹分析表明，TCA3信息在诱导后2小时内出现在肥大细胞中，半衰期为30分钟。核转录实验显示，TCA3 mRNA的出现很大程度上是由于从头转录水平的增加。对启动子区域的分析表明，可诱导基因表达由转录起始位点上游1.324 kb至0.082 kb之间的区域指导。用PMA/A23187处理后，转录起始位点上游1.324 kb至0.324 kb之间的TCA3 CAT构建体有60至80倍的诱导，FcεRI交联有30至40倍的诱导。转录起始位点上游0.136 kb至0.082 kb之间的区域有7至8倍的诱导。包含转录起始位点0.042 kb或2.0 kb序列区域的TCA3 CAT构建体不能指导CAT蛋白合成。TCA3 5'侧翼序列具有负调控活性。电泳迁移率变动分析显示，活化肥大细胞核提取物中的蛋白质与TCA3基因的NF-κB元件结合。这些发现表明，TCA3基因在肥大细胞中受到转录调控，TCA3基因上游0.082 kb区域内含有最小启动子序列；假定的增强子NF-κB元件位于0.324 kb至0.136 kb之间，假定的抑制元件位于转录起始位点上游1.324 kb至2.0 kb之间。
