Development of detection method for novel calcium- and phosphate- regulating hormone (phosphatonin)

无机磷酸盐（磷酸盐）是糖酵解、糖异生、能量代谢和骨骼矿化过程中必需的营养素。肾刷状边界膜（BBM）上表达的II型钠依赖性磷酸盐转运蛋白（NPT2）在生理和病理生理上调节磷酸盐稳态。在遗传性x连锁低磷血症（XLH）中，NPT2蛋白含量和相关mRNA含量降低，也显示BBM磷酸盐运输活性降低。导致XLH的基因被鉴定为PHEX（与x染色体上的内多肽酶同源的磷酸盐调节基因），一种抑制磷酸盐运输的体液因子（磷酸素）可能是XLH患者肾脏磷酸盐丢失的原因。为了直接验证这一假设，我们制备了表达含有人类NPT2基因启动子的荧光素酶基因的OK-B2400细胞。结果表明，低磷因子是肾脏中磷酸盐再吸收的正常调节因子，因此通常存在于血清中，但在XLH中变得异常。斯坦钙素（STC）是硬骨鱼类斯坦钙小体产生的一种钙和磷酸盐调节激素。最近报道了STC的哺乳动物同源物（STC 1），它刺激肾脏的磷酸盐摄取。从人骨肉瘤cDNA文库中克隆了第二个哺乳动物STC2。采用STC2转染CHO细胞的培养基，首次检测了STC2对肾NPT2启动子活性的影响。这一发现表明STC2会在转录水平上抑制肾NPT2的表达。在制备抗STC2抗体后，建立了STC2的检测方法。STC2在许多器官中广泛表达。STC2从中国仓鼠卵巢（CHO-K1）细胞组成性分泌，但不从对鼠肾细胞系（OK-B）分泌。然而，钙内流介导的1,25 (OH) 2D3显著刺激了OK-B细胞的STC2排泄，表明肾小管细胞对SCT2的排泄具有调节性。因此，我们认为STC2将提供骨和矿物质代谢调节的另一个维度，并且是假定的磷酸蛋白的良好候选者。少

Inorganic phosphate (phosphate) is an essential nutrient in the processes of glycolysis, gluconeogenesis, energy metabolism and skeletal mineralization. Type II sodium-dependent phosphate transporter (NPT2) expressed on renal brush border membrane (BBM) serves to physiologically and pathophysiologically regulate phosphate homeostasis. In hereditary X-linked hypophosphatemia (XLH), the NPT2 protein content and the related mRNA content is reduced showing also reduced BBM phosphate transport activity. The gene causing XLH was identified as PHEX (phosphate regulating gene with homologies to endopeptidase on the X-chromosome) and a humoral factor (phosphatonin) inhibiting phosphate transport may be responsible for the renal phosphate loss observed in XLH.To test this hypothesis directly, we prepared OK-B2400 cells expressing the luciferase gene containing human NPT2 gene promoter. The findings suggest that the hypophosphatemic factor is a normal regulator of phosphate reabsorption in the ki … More dney, and hence normally present in serum, but which becomes aberrant in XLH.Stanniocalcin (STC) is a calcium- and phosphate-regulating hormone produced by the corpuscles of Stannius in bony fishes. The mammalian homologue of STC has recently been reported (STC 1), which stimulates the phosphate uptake of the kidney. The cloning of a second mammalian stanniocalcin (STC2) from the human osteosarcoma cDNA library was identified in our laboratory. The effect of STC2 on the promoter activity of renal NPT2 was first examined, using the culture medium of STC2-transfected CHO cells. The finding suggested that STC2 would inhibit the expression of a renal NPT2 at the transcriptional level. Furthermore, we established the assay method of STC2 after preparing anti-STC2 antibody. STC2 expression is widely distributed an many organs. STC2 is constitutively excreted from chinese hamster ovary (CHO-K1) cells, but not from opposum kidney cell line (OK-B).However excretion of STC2 from OK-B cell was dramatically stimulated by 1,25 (OH) 2D3 mediated by calcium influx, indicating regulatory excretion of SCT2 from renal tubular cells. Therefore, it is concluded the STC2 will provide another dimension of the regulation of bone and mineral metabolism and a good candidate for the putative phosphatonin. Less