Gene Expression of Regulatory Protein Regucalcin of Ca^<2+>-Signaling and Its Regulatory Mechanism.

本研究旨在阐明regucalcin的基因表达及其调控机制，regucalcin可调节激素刺激引起的肝细胞Ca^<2+>信号转导。结果表明：1. Regucalcin能抑制正常大鼠肝和再生大鼠肝细胞核RNA的合成; 2. Regucalcin mRNA在转化的HepG 2细胞中表达，并能被胰岛素刺激表达; 3.从小鼠肝cDNA文库中克隆了Regucalcin基因，并进行了序列测定。Regucalcin mRNA在小鼠肝脏中唯一表达，且其表达受钙离子刺激; 4.发现大鼠regucalcin基因5 '侧翼区存在核因子，这些核因子参与regucalcin基因表达的组织特异性调控; 5.将从大鼠不同组织中获得的核蛋白用于凝胶迁移率变化分析，在肝脏和肾脏中发现了蛋白质-DNA复合物的组织特异性形成。同时给予钙调素拮抗剂可完全抑制Ca^<2+>诱导的AP-1因子与regucalcin基因的结合。将大鼠regucalcin基因的5 '侧翼区与荧光素酶报告基因连接，在H4-II-E肝癌细胞中具有启动子活性。

This investigation was undertaken to clarify the gene expression and its regulatory mechanism of regucalcin which can regulate Ca^<2+> signalin in liver cells due to hormonal stimulation. The following results were obtained.1.Regucalcin could inhibit RNA synthesis in the nuclei of normal rat liver and regenerating rat liver.2.Regucalcin mRNA was expressed in the transformed HepG2 cells and its expression was stimulated by insulin.3.The cDNA clone encoding a regucalcin was isolated from a mouse liver cDNA library and sequenced. Regucalcin mRNA in mice was uniquely expressed in the liver, and that its expression was stimulated by calcium administration.4.The existence of nuclear factors which bind to the 5'-flanking region of regucalcin gene in rats was found. These identified components was involved in the tissue-specific regulation of regucalcin gene expression.5.When nuclear proteins obtained from various rat tissues were used in gel mobility-shift assays, tissue-specific formation of a protein-DNA complex was found in the liver and kidney. Ca^<2+>-induced binding of the AP-1 factor to the regucalcin gene was completely inhibited by simultaneous administration of an antagonist of calmodulin. The 5'-flanking region of the rat regucalcin gene ligated to a luciferase reporter gene possessed the promotor activity in H4-II-E hepatoma cells.