Hormonal and Molecular Etiology of Skeletal Abnormalities in XLH

XLH 骨骼异常的激素和分子病因学

• 批准号: 9757666
• 负责人: Marie Demay
• 金额: $ 36.75万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-21 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9757666
• 关键词: Ablation; Acute; Address; Adjuvant; Affect; Animals; Apoptosis; Apoptotic; Biological Availability; Biomechanics; Brush Border; Cells; Child; Chondrocytes; Chronic Kidney Failure; Data; Dihydroxycholecalciferols; Disease; Endopeptidases; Epiphysial cartilage; Etiology; Excretory function; Exhibits; Familial hypophosphatemic bone disease; Fibroblast Growth Factor Receptors; Gene Expression; Genes; Genetic; Growth; Hormonal; Hormones; Hour; Human; Hypercalcemia; Hypophosphatemia; Impairment; In Vitro; Incidence; Injections; Intestines; Investigation; KDR gene; Kidney; Knockout Mice; MAP2K1 gene; MAPK3 gene; Mediating; Mitochondria; Molecular; Mus; Mutation; Nephrocalcinosis; Osteomalacia; Pathway interactions; Patients; Phenotype; Phosphorylation; Process; Property; Protein Isoforms; Receptor Signaling; Resistance; Rickets; Role; Secondary Hyperparathyroidism; Serum; Signal Pathway; Signal Transduction; Signaling Molecule; Supplementation; Tubular formation; Urine; VEGFA gene; Vascular Endothelial Growth Factors; Vascular calcification; Vegf inhibition; Vitamin D; absorption; base; bone; brush border membrane; extracellular; gastrointestinal; gene induction; improved; in vivo; inhibitor/antagonist; inorganic phosphate; mouse model; novel; prevent; response; screening; side effect; skeletal; skeletal abnormality; small molecule; small molecule inhibitor; therapeutic effectiveness; urinary

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.

本提案中的研究旨在确定低磷酸盐血症 导致佝偻病，以及1，25-二羟维生素D（1，25 D）治疗佝偻病有效性的分子基础。 X连锁低磷血症（XLH）。虽然磷酸盐和1，25 D联合治疗XLH已被广泛接受， 在过去的40年中，1，25 D有益作用的分子基础尚未完全了解。我们 在Hyp小鼠XLH模型中，用1，25 D进行单一疗法可以改善生长，预防佝偻病， 并改善了骨骼的微结构和生物力学特性， 增加循环FGF 23水平。尽管FGF 23，1，25 D治疗显著增加， 降低Hyp小鼠的尿磷酸盐。因此，1，25 D对骨和肾磷酸盐具有有益作用 在XLH中的处理，尽管进一步增加FGF 23。我们研究了1，25 D对FGF 23的影响， 信号传导以鉴定1，25 D拮抗FGF 23的磷酸尿效应的机制。 初步数据表明，1，25 D导致Npt 2a/NHERF 1在肾刷状缘膜上滞留， Hyp小鼠通过拮抗FGF 23/FGF受体相互作用下游的FGF 23信号传导。研究将 在WT和Hyp小鼠的肾脏、肾小管细胞和刷状缘膜中进行。 低磷酸盐血症损害肥大软骨细胞凋亡，导致生长期动物佝偻病， 人类，包括XLH患者。我们已经证明磷酸盐诱导ERK 1/2磷酸化， 在体外和体内肥大软骨细胞中激活线粒体凋亡途径所需的。 我们还发现，在软骨细胞中A-、B-和C-Raf的消融可消除磷酸盐诱导的ERK 1/2 磷酸化损害肥大软骨细胞凋亡并导致佝偻病。Raf; MEK 1/2; ERK 1/2可以是 通过几种途径激活。因此，我们进行了小分子抑制剂筛选，以确定 磷酸盐诱导ERK 1/2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ERK 1/2磷酸化需要信号传导。 这些研究将解决磷酸盐激活VEGFR 2信号传导的假设，特别是在 肥大的软骨细胞，并将确定是否需要增加VEGFA释放/分泌， 这些影响。他们还将讨论软骨细胞特异性消融VEGFR 2损伤 磷酸盐介导的体外肥大软骨细胞凋亡和体内正常生长板成熟。 将检查1，25 D对该信号通路的影响，以确定1，25 D如何预防佝偻病。 低磷酸盐血症。因此，提出的研究将确定诱导的分子基础， 肥大软骨细胞凋亡的磷酸盐，并将确定如何1，25 D调节这一进程， 损害肾脏中FGF 23的磷酸尿作用。我们的研究将有重要的影响， XLH患者的治疗，并将确定磷酸盐激活信号通路的机制。