Hormonal and Molecular Etiology of Skeletal Abnormalities in XLH

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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.