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

基因序列与核蛋白相互作用对血栓调节蛋白的转录调节

基本信息

批准号：
06672202
负责人：
HORIE Shuichi
金额：
$ 1.41万
依托单位：
Faculty of Pharmaceutical Sciences, Teikyo University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

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.

我们报道，用全反式视黄酸（RA）处理内皮细胞，通过转录加速，血栓调节蛋白（TM）（激活蛋白C的抗凝辅助因子）上调。在本研究中，通过人胰腺BxPC-3细胞中的瞬时表达测定研究了TM基因5'-非翻译区的转录调控序列。含有TM启动子和氯霉素乙酰转移酶（CAT）报告基因的质粒构建体的缺失突变体表现出近端（-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。这两个序列都通过 RA 增加了启动子活性，并且引入每个序列的突变证实了 RA 治疗的功能反应。反式激活研究表明，含有两种序列的质粒与 RA 受体的表达质粒（例如 RAR-α、-β、-γ 和 RXR-α）共转染，不会增强 RA 依赖性启动子活性，尽管用 RA 处理的 BxPC 细胞中 RAR-β mRNA 增加。凝胶阻滞实验表明与 Spl 序列相互作用的是 Spl 蛋白，而不是 AP2 蛋白其中不包含 Spl 删除序列。通过用RA处理细胞，观察到Spl、SPR-1和SPR-2的mRNA水平呈时间依赖性增加。我们得出结论，基因组 DNA 区域的两个位点参与了 RA 依赖性的 TM 基因表达增强。