Studies on the function of responsible gene, PHEX, for X-linked hypophosphatemic vitamin D resistant rickets - Search for novel phosphate regulating systems

x连锁低磷血症性维生素D抗性佝偻病（XLH）是一种较为常见的维生素D抗性遗传性佝偻病，其病因是肾磷泄漏。通过连锁分析确定其负责基因为PHEX，与中性内肽酶具有同源性，突变导致其功能丧失。发病机制假设如下。正常情况下立即被PHEX降解的体液性磷酸化因子，可能由于PHEX功能的丧失而逃避这种降解机制，并可能对肾脏磷酸盐吸收发挥作用。然而，磷因子仍然是假设的。因此，我们利用Hyp小鼠进行了鉴定磷酸化因子的实验，Hyp小鼠具有与PHEX基因相同的小鼠对应物突变。在本研究项目中，我们通过以下两种方法来解决这个问题：1)小鼠骨髓移植；2)寻找PHEX抑制剂。1) Hyp小鼠接受正常骨髓治疗后，低磷血症、维生素D代谢异常、低矿化等各项疾病指标均有改善。在5%的骨髓细胞为正常供体细胞的条件下，这些影响是明显的。血清磷与移植正常细胞质量呈正相关。由此可见，PHEX的天然底物可能在表达PHEX的细胞附近表达，也可能在表达PHEX的细胞本身表达。2)在NEP抑制剂中，硫罕有可能引起野生型小鼠短暂性低磷血症。然而，FGF23被确定为致癌性骨软化和常染色体显性低磷血症佝偻病的致病因素。这些信息引导我们探索其在XLH发病机制中的作用。为此，我们制备了针对FGF23的多克隆抗体，并检测了蛋白表达。通过RT-PCR检测Hyp小鼠的mRNA表达。即使采用RT-PCR，在成年Hyp小鼠中也很难检测到FGF23信号。结合我们的骨髓移植研究，我们不能排除除FGF23以外的其他因子作为磷酸化因子。为了进一步的实验，我们开发了PHEX表达系统和更灵敏的方法来检测与重组PHEX蛋白相互作用的分子。少

X-linked hypophosphatemic vitamin D resistant rickets (XLH) is a relatively common vitamin D resistant hereditary rickets caused by remarkable renal phosphate leak. Its responsible gene was identified as PHEX, which has homology to neutral endopeptidase, from linkage analysis, and the mutations causes loss of its function. The pathogenetic mechanism has been hypothesized as follows. Humoral phosphaturic factor(s), which is degraded immediately by PHEX in normal condition, may escape this degradation mechanism by the loss of function of PHEX and may exert its effects on renal phosphate resorption. However, the phosphaturic factor is still hypothetical. Therefore we have been performing experiments to identify the phosphaturic factor by using Hyp mice, which have the same mutation in murine counterpart of PHEX gene.In this study project, we address this issue during two following approaches 1) murine bone marrow transplantation 2) search for PHEX inhibitors.1) Hyp mice received normal bo … More ne marrow showed improvements all disease parameters, such as hypophosphatemia, abnormal vitamin D metabolism and hypomineralization. These effects were evident under the conditions, in which 5% of bone marrow cells were normal donor cells. Moreover, there was positive correlation between serum phosphorus and implanted normal cell mass. From this result, natural substrate of PHEX may be expressed near the cells expressing PHEX or PHEX expressing cells themselves.2) Among NEP inhibitors, thiorphan could cause transient hypophosphatemia in wild type mice. However, FGF23 was identified as the causative factor for oncogenic osteomalacia and autosomal dominant hypophosphatemic rickets. And this information led us to explore its role in the pathogenesis of XLH. To this aid, we developed polyclonal antibody against FGF23, and examined protein expression. Moreover by RT-PCR we examined mRNA expression in Hyp mice. Even by RT-PCR, it was hard to detect FGF23 signal in adult Hyp mice. Combined with our bone marrow transplantation study, we could not exclude the other factor than FGF23 as a phosphaturic factor. For further experiment, we developed PHEX expression system and more sensitive methodology to detect the molecules which interact with recombinant PHEX protein. Less