Etiology and Therapy of Rickets in the Hyp mouse model of XLH

描述（由申请人提供）：循环磷酸盐是生长板成熟的关键决定因素。低磷血症在体内和体外损害肥厚软骨细胞的凋亡，导致生长动物佝偻病的发展。磷酸盐诱导Erk1/2磷酸化并激活肥大但不增生软骨细胞的线粒体凋亡途径。体外和体内抑制Erk1/2磷酸化可损害磷酸盐介导的肥大软骨细胞凋亡。在Specific Aim I中的研究将确定在Erk1/2磷酸化的上游和下游参与磷酸盐介导的肥大软骨细胞凋亡的因素。他们将在体外和体内研究C-Raf在Erk1/2激活中的作用，并研究磷酸盐对促凋亡因子的激活和亚细胞定位的影响。特异性Aim II的研究将证明C-Raf在软骨细胞特异性C-Raf消融小鼠生长板成熟和肥大软骨细胞凋亡中的关键作用，以及从这些小鼠中分离的原代软骨细胞。与其他低磷小鼠模型不同，Npt2a缺失小鼠具有高水平的1,25-二羟基维生素D，并且没有报道发生佝偻病。我们的研究揭示了这些小鼠在16日龄时的佝偻病表型，在35天时消退。在Npt2a缺失小鼠中，1,25-二羟维生素D的作用受损会导致进行性佝偻病，这表明1,25-二羟维生素D可以补偿低磷酸盐，以维持正常的生长板。我们的研究证据表明PTH/PTHrP信号在佝偻病的病因学中增强，因此我们建议从2日龄开始治疗Hyp小鼠，慢性补充与高剂量间歇性1,25-二羟基维生素D对照，以确定这些“生理”或“药理”干预是否使Hyp小鼠的生长板正常化。还将检查甲状旁腺功能和PTHrP表达，以剖析对这两种给药方案的反应的分子基础。1,25-二羟基维生素D对生长板和甲状旁腺功能的影响将与使用阻断抗体直接抑制FGF23的作用进行比较。这三种治疗方法对骨的影响将通过组织学、组织形态计量学和显微ct分析进行评估。因此，本提案中的研究旨在解决XLH中观察到的异常的病理生理基础，并以Hyp小鼠为模型，检查治疗干预的相对疗效。

DESCRIPTION (provided by applicant): Circulating phosphate is a critical determinant of growth plate maturation. Hypophosphatemia impairs apoptosis of hypertrophic chondrocytes in vivo and in vitro, leading to the development of rickets in growing animals. Phosphate induces Erk1/2 phosphorylation and activates the mitochondrial apoptotic pathway in hypertrophic but not proliferative chondrocytes. Inhibition of Erk1/2 phosphorylation in vitro and in vivo impairs phosphate-mediated hypertrophic chondrocyte apoptosis. Studies in Specific Aim I will identify the factors involved in phosphate-mediated apoptosis of hypertrophic chondrocytes upstream to and downstream of Erk1/2 phosphorylation. They will examine the role of C-Raf in Erk1/2 activation in vitro and in vivo and examine the effect of phosphate on activation of, and subcellular localization of, proapoptotic factors. Investigations in Specific Aim II will demonstrate the critical role of C-Raf in growth plate maturation and hypertrophic chondrocyte apoptosis in mice with chondrocyte-specific C-Raf ablation and in primary chondrocytes isolated from these mice. Unlike other hypophosphatemic mouse models, Npt2a null mice have high levels of 1,25-dihydroxyvitamin D and had not been reported to develop rickets. Our studies revealed a rachitic phenotype in these mice at 16 days of age, which resolves by 35 days. Impairing 1,25-dihydroxyvitamin D action in Npt2a null mice leads to progressive rickets, demonstrating that 1,25-dihydroxyvitamin D can compensate for low phosphate in maintaining a normal growth plate. Evidence from our studies implicate enhanced PTH/PTHrP signaling in the etiology of rickets, thus we propose to treat Hyp mice from 2 days of age, with chronic repletion versus high dose intermittent 1,25-dihydroxyvitamin D to determine whether these "physiological" or "pharmacological" interventions normalize the growth plates of the Hyp mice. Parathyroid function and PTHrP expression will also be examined to dissect the molecular basis for the response to these two dosing regimens. The effects of 1,25-dihydroxyvitamin D on the growth plate and on parathyroid function will be compared to that of direct inhibition of FGF23 action using blocking antibodies. The effects of these three treatments on bone will be evaluated histologically, histomorphometrically and by microCT analyses. Thus, the investigations in this proposal are directed at addressing the pathophysiologic basis for the abnormalities observed in XLH and at examining the comparative efficacy of therapeutic interventions, using the Hyp mouse as a model.