THE MECHANISM OF GENE REGULATION BY EXTRA CELLULAR CALCIUM THROUGH CALCIUM-SENSING RECEPTOR

负性钙反应元件(NCaRE)通过与其结合蛋白(NCaREB)与胞外钙(Ca^2+_e)的特异性结合，使携带nCaRE的基因，如人甲状旁腺激素(PTH)基因，受到Ca^2+_e的负性调控，Ku抗原通过与氧化还原因子蛋白ref1相互作用，介导Ca^2+_e对基因的负性调控。虽然Ku抗原的序列非特异性DNA结合活性已经被很好地表征，但其序列特异性DNA结合的机制仍然不清楚，在这里，我们报道Ku抗原与另一种蛋白质refl的特异性结合导致具有新的序列特异性的DNA-蛋白质复合体的形成，从而调节基因的表达。接下来，我们检查了最近发现的膜钙敏感受体是否参与了这种类型的基因调控。尽管转染钙敏感受体表达载体的培养细胞表现出抑制的nCaRE报告活性，但这些发现与细胞外钙浓度无关。另一方面，我们发现其中一种nCaRE寡聚B在血管活性基因中非常保守，如加压素和心钠素多肽基因。此外，前一个基因中的寡核苷酸B在整个进化过程中都是保守的。我们证明了寡核苷酸B与其结合的核蛋白包括氧化还原因子1(ReFl)之间的结合被氯化钠(NaC L)产生的高渗透压降低，但不被尿素所降低。这种减弱的结合可以通过马铃薯酸性磷酸酶去磷酸化一些核蛋白来逆转，这表明氯化钠处理诱导了这些核蛋白的磷酸化，从而削弱了它们与寡聚B的结合活性。此外，这些核事件允许高渗透压介导的转录刺激在培养细胞中携带这种DNA元件的基因。

Through the specific binding of a negative calcium responsive element (nCaRE) to its binding protein (nCaREB) in response to extracellular Ca (Ca^2+_e), nCaRE-bearing genes, such as the human parathyroid hormone (PTH) gene, are negatively regulated by Ca^2+_e. The Ku antigen mediated negative gene regulation by Ca^2+_e by interacting with a redox factor protein, refl. Though sequence-nonspecific DNA binding activity of the ku antigen has been well characterized, the mechanism of its sequence-specific DNA binding remained obscure, Here, we report that the specific binding of the Ku antigen to another protein, refl, leads to DNA-protein complex formation with a novel sequence-specificity and thereby regulates gene expression. We next examined whether the recently-identified membrane calcium-sensing receptor was involved in this type of gene regulation. Though cultured cells transfected with the calcium-sensing receptor expression vector potentiated the binding between nCaRE and nCaREB as exhibited repressed nCaRE-bearing reporter activity, these findings were observed irrespective of the extracellular calcium concentrations.On the other hand, we have found that one of the nCaREs, oligo B,is very well conserved among such vasoactive genes as the vasopressin and atrial natriuretic polypeptide genes. Further, the oligo B in the former gene is conserved throughout evolution. We demonstrate that the binding between oligo B and its binding nuclear proteins including a redox factor 1 (refl) was reduced by hyperosmolarity generated by sodium chloride (NaCl) but not by urea. Such attenuated binding was reversed by dephosphorylating some of the nuclear proteins by a potato acid phosphatase, suggesting that NaCl treatment elicited phosphorylation of these nuclear proteins to weaken their binding activity to oligo B.Furthermore, these nuclear events let to hyperosmolarity-mediated transcriptional stimulation of the genes bearing this DNA element in the cultured cells.