Transcriptional Regulation of Thrombomodulin by Interactions of the Gene Sequences with Nuclear Proteins

我们报道了凝血调节素（TM），激活蛋白C的抗凝辅助因子，在全反式维甲酸（RA）处理内皮细胞后通过转录加速而上调。本研究在人胰腺BxPC-3细胞中，采用瞬时表达法研究了TM基因5′-非翻译区转录调控序列。含有TM启动子和氯霉素乙酰转移酶（CAT）报告基因的质粒缺失突变体显示出近端（-202至207）和远端（-1515至-1531）ra响应区域的存在。远端包含RA反应元件，近端包含DNA序列中的Spl位点。这两个序列是RA对启动子活性增加的加性作用，每个序列中引入的突变证实了RA处理后的功能反应。转激活研究表明，含有RA受体表达质粒（rar - α、- β、- γ和rxr - α）序列的质粒共转染RA依赖性启动子活性不增强，但RA处理的BxPC细胞中rar - β mRNA升高。凝胶阻滞实验表明，Spl蛋白与Spl序列相互作用，而非AP2蛋白与Spl缺失序列相互作用。通过RA细胞处理，观察到Spl、spr - 1和SPR-2 mRNA水平的时间依赖性升高。我们得出结论，基因组DNA区域的两个位点参与了ra依赖性TM基因表达的增强。

We reported that thrombomodulin (TM) , the anticoagulant cofactor for activation of protein C,was up-regulated by treatment of endothelial cells with all-trans retinoic acid (RA) throgh the transcriptional acceleration. In the present investigation, the transcriptional regulatory sequence of the 5'-untranslated region of TM gene studied by transient expression assay in human pancreas BxPC-3 cells. Deletion mutants of plasmid constructs containing the TM promoter and chloramphenicol acetyltransferase (CAT) reporter gene exhibited that presence of proximal (-202 to 207) and distal (-1515 to -1531) RA-responsive regions. The distal one contains RA response element and the proximal one contains Spl site in the DNA sequence. The both sequences were additive in the increase in promoter activity by RA and mutations introduced into each sequence confirmed the functional response with RA-treatment. Transactivation study showed that cotransfection of the plasmid containing both the sequences with expression plasmid for RA receptor, such as RAR-alpha, -beta, -gamma and RXR-alpha, did not enhance RA-dependent promoter activity, though RAR-beta mRNA increased in BxPC cells treated with RA.Gel retardation experiment indicated the Spl protein not AP2 protein interacted with the Spl sequence where not with the Spl-deleted sequence. By treatment of the cells with RA time-dependent increases in mRNA levels of Spl, SPR-l and SPR-2 were observed. We concluded that two sites of genomic DNA regions particpated in the RA-dependent augmentation of TM gene expression.