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

描述（由申请方提供）：循环磷酸盐是生长板成熟的关键决定因素。低磷酸盐血症在体内和体外损害肥大软骨细胞的凋亡，导致生长期动物佝偻病的发生。磷酸盐诱导Erk 1/2磷酸化并激活肥大但不增殖软骨细胞中的线粒体凋亡途径。Erk 1/2磷酸化的抑制在体外和体内损害磷酸盐介导的肥大软骨细胞凋亡。特定目标研究I将确定Erk 1/2磷酸化上游和下游磷酸盐介导的肥大软骨细胞凋亡的相关因素。他们将研究C-Raf在体外和体内Erk 1/2激活中的作用，并研究磷酸盐对促凋亡因子激活和亚细胞定位的影响。在特定目标II的研究将证明C-Raf在生长板成熟和肥大性软骨细胞凋亡中的关键作用，在软骨细胞特异性C-Raf消融的小鼠和从这些小鼠分离的原代软骨细胞中。与其他低磷酸盐血症小鼠模型不同，Npt 2a缺失小鼠具有高水平的1，25-二羟维生素D，并且没有报道会发生佝偻病。我们的研究揭示了这些小鼠在16日龄时的佝偻病表型，该表型在35日龄时消退。在Npt 2a缺失小鼠中损害1，25-二羟基维生素D作用导致进行性佝偻病，表明1，25-二羟基维生素D可以补偿维持正常生长板的低磷酸盐。来自我们的研究的证据表明，在佝偻病的病因学中PTH/PTHrP信号传导增强，因此我们建议从2日龄开始用慢性饱食与高剂量间歇性1，25-二羟基维生素D治疗Hyp小鼠，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反应的分子基础。将1，25-二羟维生素D对生长板和甲状旁腺功能的作用与使用封闭抗体直接抑制FGF 23作用的作用进行比较。将通过组织学、组织形态计量学和microCT分析评价这三种治疗对骨的影响。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学基础，并使用Hyp小鼠作为模型，检查治疗干预的比较疗效。 公共卫生相关性：正常的骨骼发育和成熟需要最佳的矿物质离子水平。虽然维生素D缺乏症再次成为西方国家生长和骨骼矿化受损的原因，但这种表型的大部分是循环矿物质离子水平低的直接后果。在细胞和完整动物中的研究，包括人类疾病X连锁低磷酸盐血症的小鼠模型，预计将阐明与低循环磷酸盐相关的生长板变化的分子基础。对生长板和骨的治疗干预的表征将有助于设计新的治疗方法来治疗这种疾病，以及与过度肾磷酸盐损失相关的其他疾病。

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. PUBLIC HEALTH RELEVANCE: Normal skeletal development and maturation requires optimal mineral ion levels. While vitamin D deficiency is once again emerging as a cause of impaired growth and skeletal mineralization in western countries, much of this phenotype is a direct consequence of low levels of circulating mineral ions. Studies in cells and in intact animals, including the mouse model of the human disorder X-linked hypophosphatemia, are expected to elucidate the molecular basis for the growth plate changes associated with low circulating phosphate. The characterization of the therapeutic interventions proposed, on the growth plate, as well as on bone, will help design new therapeutic approaches to the treatment of this disorder, as well as other conditions associated with excessive renal phosphate losses.