Hormonal and Molecular Etiology of Skeletal Abnormalities in XLH

这项建议中的研究旨在确定低磷血症的机制 导致软骨病，以及1，25-二羟基维生素D(1，25D)治疗的分子基础。 X连锁低磷血症(XLH)。而磷酸盐和1，25D联合治疗黄体生成素一直是 在过去的40年里，1，25D的有益作用的分子基础还没有完全被理解。我们 已经表明，在Hyp小鼠模型中使用1，25D可以促进生长，预防软骨病 并改善了骨骼的微结构和生物力学性能，尽管已经翻了一番 循环中FGF23水平升高。尽管FGF23显著增加，但1，25D治疗显著 降低Hyp小鼠的尿磷排泄量。因此，1，25D对骨骼和肾脏磷酸盐有有益的影响 在XLH处理，尽管进一步增加了FGF23。我们建议研究1，25D对FGF23的影响 信号转导以确定1，25D拮抗FGF23的磷酸尿酸效应的机制。 初步数据表明，1，25D导致Npt2a/NHERF1在肾刷状缘膜上滞留 通过拮抗FGF23信号下游与FGF23/FGF23受体的相互作用而抑制Hyp小鼠。研究将会是 在WT和Hyp小鼠的肾脏、肾小管上皮细胞和刷状缘膜上进行。 低磷血症损害肥大的软骨细胞凋亡，导致生长动物和 人类，包括那些患有XLH的人。我们已经证明，磷酸对ERK1/2磷酸化的诱导是 在体外和体内肥大的软骨细胞中激活线粒体凋亡途径所需的。 我们还表明，软骨细胞中A-、B-和C-Raf的消融可以消除磷酸诱导的ERK1/2 磷酸化，损害肥大的软骨细胞凋亡，并导致软骨病。RAF；MEK1/2；ERK1/2可以是 被几条通路激活。因此，我们进行了小分子抑制物筛选，以确定 磷酸诱导ERK1/2磷酸化的途径。这些研究表明，VEGFR 在原代肥大软骨细胞中，磷酸盐诱导的ERK1/2磷酸化需要信号转导。 提出的研究将解决这一假设，即磷酸盐激活VEGFR2信号，特别是在 并将确定是否需要增加VEGFA的释放/分泌 这些影响。他们还将解决这样的假设，即软骨细胞特异性地去除VEGFR2会损害 磷酸盐介导的肥大软骨细胞体外凋亡和体内正常生长板成熟。 将研究1，25D对这一信号通路的影响，以确定1，25D如何预防软骨病。 低磷血症的设定。因此，所提出的研究将确定诱导 并将定义1，25D如何调节这一过程和 削弱FGF23在肾脏中的磷酸作用。我们的研究将对 治疗XLH患者，并将确定磷酸盐激活信号通路的机制。

The investigations in this proposal are directed at identifying the mechanism by which hypophosphatemia leads to rickets, and the molecular basis for the therapeutic effectiveness of 1,25-dihydroxyvitamin D (1,25D) in X-linked hypophosphatemia (XLH). While the combination of phosphate and 1,25D therapy for XLH has been used for the past 4 decades, the molecular basis for the beneficial effects of 1,25D is not fully understood. We have shown that monotherapy with 1,25D in the Hyp mouse model of XLH improves growth, prevents rickets and improves the microarchitectural and biomechanical properties of bone despite doubling the already increased circulating FGF23 levels. Despite this major increase in FGF23, 1,25D treatment significantly decreases urinary phosphate in Hyp mice. Thus, 1,25D has beneficial effects on bone and renal phosphate handling in XLH, in spite of further increasing FGF23. We propose to examine the effects of 1,25D on FGF23 signaling to identify the mechanism by which 1,25D antagonizes the phosphaturic effects of FGF23. Preliminary data suggest that 1,25D causes retention of Npt2a/NHERF1 at renal brush border membranes of Hyp mice by antagonizing FGF23 signaling downstream of FGF23/FGF receptor interactions. Studies will be performed in kidneys, renal tubular cells and brush border membranes of WT and Hyp mice. Hypophosphatemia impairs hypertrophic chondrocyte apoptosis leading to rickets in growing animals and humans, including those with XLH. We have shown that phosphate induction of ERK1/2 phosphorylation is required for activation of the mitochondrial apoptotic pathway in hypertrophic chondrocytes in vitro and in vivo. We have also shown that ablation of A-, B- and C-Raf in chondrocytes abolishes phosphate-induced ERK1/2 phosphorylation, impairs hypertrophic chondrocyte apoptosis and leads to rickets. Raf;MEK1/2;ERK1/2 can be activated by several pathways. We, therefore, undertook a small molecule inhibitor screen to identify the pathway by which phosphate induces ERK1/2 phosphorylation. These studies demonstrated that VEGFR signaling is required for phosphate induced ERK1/2 phosphorylation in primary hypertrophic chondrocytes. The studies proposed will address the hypothesis that phosphate activates VEGFR2 signaling specifically in hypertrophic chondrocytes and will determine whether increased VEGFA release/secretion is required for these effects. They will also address the hypothesis that chondrocyte-specific ablation of VEGFR2 impairs phosphate-mediated hypertrophic chondrocyte apoptosis in vitro and normal growth plate maturation in vivo. The effects of 1,25D on this signaling pathway will be examined to determine how 1,25D prevents rickets in the setting of hypophosphatemia. Thus, the studies proposed will identify the molecular basis for induction of hypertrophic chondrocyte apoptosis by phosphate and will define how 1,25D modulates this process and impairs the phosphaturic effects of FGF23 in the kidney. Our studies will have important implications for the treatment of XLH patients and will identify the mechanism by which phosphate activates signaling pathways.