Metabolism of calcium and regulation of gene expression

利用人甲状旁腺素基因中存在的钙依赖性抑制基因转录所需的负性钙响应元件（nCaRE），我们可以检测细胞核因子nCaRE结合蛋白（nCaREB）。该因子与DNA元件的结合活性取决于细胞外钙浓度。DNA结合后，抑制了与nCaRE连接的基因的转录。nCaREB似乎由至少两个亚组分组成，其中之一是氧化还原因子1（ref 1），一种核蛋白。其在多种细胞和组织中的表达与几乎所有测试过的细胞系中都可以检测到nCaREB与该元件的特异性结合的事实很好地匹配。当细胞外钙离子浓度升高时，ref-1蛋白和编码ref-1蛋白的mRNA的量增加。这一观察结果也得到了实验的支持，在该实验中，向细胞中加入放线菌酮通过增加细胞外钙浓度来阻断nCaREB与nCaRE的结合活性的增强。Ref 1是一种参与DNA修复过程的核酸内切酶，已知通过改变这些蛋白质的氧化还原状态来增强各种核转录因子的DNA结合活性。一系列实验表明：（1）抗ref 1抗体可降低nCaREB与nCaRE的特异性结合活性;（2）ref 1 mRNA的反义DNA的导入可使nCaRE介导的钙依赖性转录抑制消失，从而推断ref 1作为nCaREB的DNA结合亚组分发挥作用并诱导转录抑制。我们已经克隆了nCaREB的另一个子组分的候选物，并一直在研究ref 1之间的关系。

By using the negative calcium-responsive element (nCaRE) which is found in the human PTH gene and required for calcium-dependent suppression of the gene's transcription, we could detect the cellular nuclear factor, nCaRE-binding protein (nCaREB). The binding activity of this factor to the DNA element is dependent on the extracellular concentration of calcium. The DNA binding is followed by suppression of transcription of the gene attached to nCaRE.nCaREB seems to consist of at least two subcomponents, one of which turned out to be redox factor 1 (ref1), a nuclear protein. Its expression over the wide variety of cells and tissues matches well the fact that specific binding of nCaREB to the element can be detected in almost all the cell lines ever tested. The amounts of ref-1 protein and mRNA coding for it increase when extracellular calcium concentration rises. This observation is also supported by the experiment in which addition of cycloheximide to the cells blocks enhancement of the binding activity of nCaREB to nCaRE by an increase in the extracellular calcium concentration. Ref1 is an endonuclease involved in the DNA repair process and known to enhance DNA-binding activity of various nuclear transcription factors by altering the redox state of those proteins. A series of experiments has shown that (1) specific binding activity of nCaREB to nCaRE is decreased by anti-ref1 antibody, (2) introduction of antisense DNA of ref1 mRNA induces disappearance of calcium-dependent suppression of transcription mediated by nCaRE.It is, thus, deduced that ref1 functions as a DNA-binding subcomponent of nCaREB and induces transcriptional repression. We have already cloned the candidate for another subcomponent of nCaREB and have been studying the relationship between ref1.