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Physiological Function and Gene-Expression Regulation of Protein Crosslinking Enzyme Transglutaminase

蛋白质交联酶转谷氨酰胺酶的生理功能及基因表达调控

基本信息

批准号：
03660083
负责人：
IKURA Koji
金额：
$ 1.41万
依托单位：
Kyoto Institute of Technology (1992)Kyoto University (1991)
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

项目摘要

This research project aims to understand the physiological functions and regulatory mechanism of gene expression of the protein crosslinking enzyme transglutaminase on the molecular level. The following results were obtained.1. Treatment of human hepato blastoma HepG2 cells with interleukin-6 (IL-6) increased their transglutaminase activity in a timeand dose-dependent way. Dexamethasone strengthened the stimulation by IL-6. Half-maximum stimulation of transglutaminase activity in the cells occurred at a dose of 40 pM IL-6 regardless of the presence of dexamethasone. Based on its immunoreactivity, the transglutaminase induced was identified as tissue-type transglutaminase. Immunoblot analysis showed that the increase in transglutaminase activity was related to an increase in the amount of transglutaminase protein. Nothern blot analysis showed that exposure of HepG2 cells to IL-6 increased the mRNA level of the enzyme, and the increase was detectable 3 h after IL-6 was added. Induction of the mRNA was maximum between 10 and 14 h. The increase in the mRNA was not blocked by the presence of cycloheximide, suggesting that the increase was independent of protein synthesis. Injections of substances that induces inflammation such as turpentine and lipopolysaccharides into mice increased the liver transglutaminase activity. These results indicated that transglutaminase may be involved in some biological processes in hepatocytes regulated by IL-6 signaling.2. A 5' flanking region of the guinea pig liver transglutaminase gene was cloned and sequenced. The sequence for TATA box and potential binding sites of some regulatory factors were found in this region. The promoter activity of this region was shown by transfecting its fusion-construct with the chloramphenicol acetyltransferase gene into human hepatoblastoma HepG2 cells. The promoter activity was enhanced by interleukin-6.

本课题旨在从分子水平上了解谷氨酰胺转胺酶蛋白交联酶的生理功能和基因表达调控机制。得到了以下结果：白细胞介素-6 （IL-6）处理人肝母细胞瘤HepG2细胞，使其谷氨酰胺转氨酶活性呈时间和剂量依赖关系。地塞米松增强了IL-6的刺激作用。无论是否存在地塞米松，在40 pM IL-6剂量下，细胞中转谷氨酰胺酶活性的半最大刺激发生。根据其免疫反应性，鉴定其为组织型转谷氨酰胺酶。免疫印迹分析表明，转谷氨酰胺酶活性的增加与转谷氨酰胺酶蛋白量的增加有关。northern blot分析显示，HepG2细胞暴露于IL-6后，该酶的mRNA水平升高，且在加入IL-6 3 h后可检测到升高。mRNA的诱导在10 ~ 14 h之间达到最大值。mRNA的增加不被环己亚胺的存在所阻断，这表明mRNA的增加与蛋白质合成无关。将松节油和脂多糖等引起炎症的物质注射到小鼠体内，增加了肝脏转谷氨酰胺酶的活性。这些结果提示转谷氨酰胺酶可能参与了IL-6信号调节的肝细胞的一些生物学过程。克隆了豚鼠肝脏转谷氨酰胺酶基因的5'侧区并对其进行了测序。在该区域发现了TATA box的序列和一些调控因子的潜在结合位点。通过将其与氯霉素乙酰转移酶基因融合构建体转染人肝母细胞瘤HepG2细胞，证实了该区域的启动子活性。白细胞介素-6可增强启动子活性。