Conditional Isolation of Fgf23 Activity

描述(由申请人提供)：适当控制血磷浓度是保持骨骼完整性的必要条件。我们先前发现成纤维细胞生长因子-23(FGF23)的错义突变是常染色体显性遗传性低磷血症(ADHR)的原因，ADHR的特征是继发于孤立的肾脏磷酸盐消耗和代谢性骨病的低磷血症。我们随后证实，失活FGF23突变会导致ADHR的镜像障碍，即家族性肿瘤钙质沉着症(TC)，表现为血磷显著升高，通常是严重的异位和血管钙化。此外，FGF23在X连锁低磷血症(XLH)患者中升高，循环中FGF23增加也与慢性肾脏疾病(CKD)患者的死亡风险增加5-6倍相关。目前，对于上述综合征，没有治愈方法，只有维持治疗。虽然在了解磷酸盐代谢的基础和临床方面已经取得了很大进展，但在正常情况下和在疾病中在骨骼水平上调节FGF23的基本机制尚不清楚。成纤维细胞生长因子23在成骨细胞和骨细胞中表达，然而，控制这种激素的产生和分泌的细胞类型(S)还不清楚。重要的是，我们最近确定成骨细胞/骨细胞的原代培养增加了FGF23对甲状旁腺素和磷酸盐的反应，这代表了当前解释FGF23调控的模型的创新转变。与新提出的体内系统相结合，这些培养物为测试FGF23的细胞特异性控制提供了一种新的方法。现有的FGF23基因敲除和转基因小鼠模型已经提供了关于FGF23生物活性的重要体内数据，包括与其辅助受体a-Klotho(KL)的相互作用，但也存在一些限制，包括FGF23全局敲除在断奶时严重受损并在8-10周死亡，以及转基因小鼠无法控制地过度表达FGF23。因此，FGF23条件零模型是探索FGF23在非致死状态下的病理生理效应以及分离其在人类疾病中的作用的必要工具。这些拟议的研究将解决以下中心假设：FGF23以细胞特异性的方式调控，在疾病中会发生改变，通过承担以下特定目标：1)培育携带FGF23等位基因的小鼠，该等位基因可以在Cre重组酶存在的情况下被特异性中断；以及2)利用条件缺失的Fgf23/Flox小鼠测试FGF23的细胞特异性调控。FGF23条件缺失动物的成功研制将为我们和其他研究人员提供独特而重要的资源。大多数在正常情况下和矿物质代谢紊乱情况下指导FGF23产生和调控的分子机制尚不清楚，因此这个模型将允许测试其他情况下不可能的假设。 公共卫生相关性：调节血磷浓度对于正常的骨骼形成和细胞功能至关重要。涉及成纤维细胞生长因子-23(FGF23)的磷酸盐稳态的病理性紊乱，如孟德尔病、常染色体显性遗传性低磷血症、X连锁低磷血症、常染色体隐性遗传性低磷血症、家族性肿瘤钙质沉着症或常见疾病，如肾功能衰竭，可导致严重的内分泌和骨骼疾病。这些疾病目前没有得到足够的治疗。我们期望FGF23条件性缺失动物的发展将揭示涉及磷酸盐稳态的新机制，并将提供新的治疗靶点。

DESCRIPTION (provided by applicant): Proper control of serum phosphate concentrations is required to maintain skeletal integrity. We previously identified missense mutations in Fibroblast growth factor-23 (FGF23) as the cause of autosomal dominant hypophosphatemic rickets (ADHR), characterized by hypophosphatemia secondary to isolated renal phosphate wasting and metabolic bone disease. We subsequently demonstrated that inactivating FGF23 mutations result in the mirror-image disorder to ADHR, familial tumoral calcinosis (TC), which is manifested by markedly elevated serum phosphate and often severe ectopic and vascular calcifications. Further, FGF23 is elevated in X-linked hypophosphatemic rickets (XLH) and increased circulating FGF23 is also associated with a 5-6 fold higher mortality risk in patients with chronic kidney disease (CKD). There are currently no cures, only maintenance treatments, for the aforementioned syndromes. Although much progress has been made towards understanding both basic and clinical aspects of phosphate metabolism, the fundamental mechanisms regulating Fgf23 at the level of the skeleton under normal conditions and in disease are unknown. FGF23 is expressed in osteoblasts and osteocytes, however the cell type(s) that 'sense' changes in serum phosphate to control production and secretion of this hormone are also unknown. Importantly, we recently determined that primary cultures of osteoblasts/osteocytes increase Fgf23 in response to PTH and to phosphate, which represents an innovative shift in the current models explaining Fgf23 regulation. In combination with the newly- proposed in vivo systems, the cultures provide a novel approach for testing cell-specific control of Fgf23. The available Fgf23 knock-out and transgenic mouse models have provided essential in vivo data regarding FGF23 bioactivity, including interactions with its co-receptor a-Klotho (KL), however there are several limitations, including that the Fgf23 global knock-out is severely compromised at weaning and dies at 8-10 weeks, and the transgenic mice uncontrollably over-express FGF23. Therefore, an Fgf23 conditional-null model is a necessary tool to explore the pathophysiologic effects of Fgf23 in a non-lethal state and for isolating its role in human disorders. These proposed studies will address the central hypothesis that: FGF23 is regulated in a cell-specific manner, which is altered in disease, through undertaking the following specific aims: 1) To develop a mouse that will carry an Fgf23 allele that can be specifically interrupted in the presence of Cre recombinase; and 2) To test for cell-specific regulation of Fgf23 utilizing the conditional-null Fgf23flox/flox mice. Successful development of an Fgf23 conditional-null animal will provide a unique and important resource for us and for other investigators. Most of the molecular mechanisms guiding Fgf23 production and regulation under normal circumstances and in disorders of mineral metabolism are unknown, therefore this model will permit testing of hypotheses that are otherwise not possible. PUBLIC HEALTH RELEVANCE: The regulation of serum phosphate concentrations is critical for normal skeletal formation and cellular function. Pathogenic disturbances in phosphate homeostasis involving Fibroblast growth factor-23 (FGF23), such as those in the Mendelian disorders autosomal dominant hypophosphatemic rickets (ADHR), X-linked hypophosphatemic rickets (XLH), autosomal recessive hypophosphatemic rickets (ARHR), and familial tumoral calcinosis (TC), or common disorders such as renal failure, lead to severe endocrine and skeletal disease. These disorders currently have inadequate treatments. We expect that development of an Fgf23 conditional-null animal will reveal new mechanisms involved in phosphate homeostasis, and will provide novel therapeutic targets.