Metabolism of calcium and regulation of gene expression

利用人PTH基因中钙依赖性抑制基因转录所需的负钙反应元件（nCaRE）检测细胞核因子nCaRE结合蛋白（nCaREB）。该因子与DNA元素的结合活性取决于细胞外钙的浓度。DNA结合后，nCaRE附着基因的转录受到抑制。nCaREB似乎至少由两个亚组分组成，其中一个被证明是氧化还原因子1 (ref1)，一种核蛋白。它在多种细胞和组织中的表达与nCaREB与该元素的特异性结合可以在几乎所有测试过的细胞系中检测到的事实相匹配。当细胞外钙浓度升高时，ref-1蛋白及其编码mRNA的数量增加。这一观察结果也得到了实验的支持，在实验中，向细胞中添加环己亚胺可以通过增加细胞外钙浓度来阻止nCaREB与nCaRE结合活性的增强。Ref1是一种参与DNA修复过程的内切酶，已知通过改变各种核转录因子的氧化还原状态来增强这些蛋白质的DNA结合活性。一系列实验表明(1)抗ref1抗体降低了nCaREB对nCaRE的特异性结合活性，(2)引入ref1 mRNA的反义DNA可诱导nCaRE介导的钙依赖性转录抑制消失。因此，可以推断ref1作为nCaREB的dna结合子组分起作用并诱导转录抑制。我们已经克隆出了nCaREB的另一个子成分候选基因，并一直在研究ref1和ref1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.