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抵抗软骨病(XLH)是一种较为常见的遗传性维生素D抵抗软骨病，由显著的肾脏磷酸盐泄漏引起。经连锁分析，其致病基因为PHEX，与中性内肽酶有同源性，突变导致其功能丧失。其致病机制假设如下。体液磷酸因子(S)在正常情况下被苯丙氨酸立即降解，可能通过苯丙氨酸功能的丧失而避开这一降解机制，从而对肾脏磷吸收产生影响。然而，磷酸尿酸因素仍然是假设的。在这项研究中，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小鼠接受正常的bo…更多的Ne骨髓显示出所有疾病参数的改善，如低磷血症、维生素D代谢异常和低矿化。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。此外，血磷水平与植入的正常细胞质量呈正相关。从这一结果来看，PHEX的天然底物可能在表达PHEX的细胞附近表达，或者表达PHEX的细胞本身。2)在NEP抑制剂中，硫吗芬可以引起野生型小鼠的一过性低磷血症。然而，FGF23被认为是致癌性骨软化症和常染色体显性遗传性低磷血症性软骨病的致病因素。这一信息促使我们探索其在XLH发病机制中的作用。为此，我们研制了抗FGF23的多克隆抗体，并检测了其蛋白表达。此外，我们还通过RT-PCR检测了Hyp小鼠的mRNA表达。在成年Hyp小鼠中，即使用RT-PCR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FGF23以外的其他因子作为磷酸因子。为了进一步的实验，我们建立了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。较少

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