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

本研究旨在阐明在激素刺激下肝细胞中调节Ca^<2+>信号素的regucalcin的基因表达及其调控机制。得到了以下结果：Regucalcin能抑制正常大鼠肝脏和再生大鼠肝脏细胞核内RNA的合成。Regucalcin mRNA在转化后的HepG2细胞中表达，胰岛素可刺激其表达。从小鼠肝脏cDNA文库中分离到编码正则化蛋白的cDNA克隆，并对其进行测序。Regucalcin mRNA在小鼠肝脏中特异表达，且其表达受钙处理的刺激。在大鼠体内发现了与regucalcin基因5′侧区结合的核因子。这些鉴定的成分参与了组织特异性调节的regulalcin基因表达。当从各种大鼠组织中获得的核蛋白用于凝胶迁移试验时，在肝脏和肾脏中发现了组织特异性的蛋白质- dna复合物的形成。Ca^<2+>-诱导的AP-1因子与regucalcin基因的结合被同时给予钙调素拮抗剂完全抑制。在H4-II-E型肝癌细胞中，与荧光素酶报告基因连接的大鼠调钙素基因的5'-侧翼区域具有启动子活性。

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.