Pathophysiologic Regulation of Fgf-23 in Phosphate Homeostasis: Role of Vitamin D

说明(申请人提供)：成纤维细胞生长因子-23(成纤维细胞生长因子-23)是最近发现的一种分子，与多种人类疾病的发病机制有关，包括X-连锁低磷血症(XLH)、肿瘤性骨软化症(OOM)、常染色体显性遗传性低磷血症(ADHR)、家族性肿瘤钙化(FTC)和慢性肾脏疾病。成纤维细胞生长因子-23是维持磷酸盐稳态和骨骼矿化的最重要和决定性因素之一。这项拨款提案的长期目标是确定在生理和病理生理条件下成纤维细胞生长因子-23的体内功能和调节。作为实现这些目标的初步步骤，我们最近培育了一只小鼠，在小鼠中，通过同源重组成功地去除了成纤维细胞生长因子-23基因。这些fgf-23基因缺失的小鼠表现出高磷血症、维生素D活性增加、骨骼过度矿化和软组织异常钙化。在这项拨款申请中，我们建议分析三种基本成分，磷酸盐，成纤维细胞生长因子-23和维生素D的效果和相互关系，使用成纤维细胞生长因子-23基因缺失的小鼠。为了确定成纤维细胞生长因子-23在体内的作用和调节，我们建议通过产生成纤维细胞生长因子-23-/钠-磷酸共转运体2a-/-双突变小鼠来确定钠-磷酸共转运体(Napi)在成纤维细胞生长因子-23缺失小鼠异常的磷酸盐稳态中的作用，如(特定目标1A)所述。还建议进行进一步的研究，以确定烟酰胺降低成纤维细胞生长因子-23缺失动物的血磷的效果(特异性目标1B)。我们还将研究循环中的成纤维细胞生长因子-23是否足以挽救成纤维细胞生长因子-23缺失动物的异常系统表型。因此，我们建议建立一种完全去除内源性成纤维细胞生长因子-23的小鼠模型，但在成骨细胞中表达成纤维细胞生长因子-23，然后释放到循环中(特定目标2)。此外，我们建议通过建立和分子鉴定成纤维细胞生长因子-23/1a羟基酶和成纤维细胞生长因子-23/维生素D受体双突变小鼠(成纤维细胞生长因子-23-/-/1a(OH)酶-/-；成纤维细胞生长因子-23-/DR-/-)(特异性目标3A、3B)来研究维生素D在成纤维细胞生长因子-23介导的功能中的作用。此外，我们将在不依赖于维生素D信号的正常钙/常磷微环境中检测成纤维细胞生长因子-23的体内生物活性(具体目标3B)。最后，我们建议使用颅骨成骨细胞和外植体在体外分析成纤维细胞生长因子-23的自分泌功能，并将探讨其作为矿化抑制因子的作用(特定目标4)。成功完成这项拟议的赠款申请将产生数据，这些数据将成为设计策略的基础，以操纵患有一系列疾病的患者的异常磷酸盐稳态和骨骼矿化缺陷，这些疾病包括软骨病、XLH、ADHR、OOM、FTC和慢性肾功能衰竭，使用FGF-23或其相互作用的分子作为潜在的治疗工具。

DESCRIPTION (provided by applicant): Fibroblast growth factor-23 (FGF-23) is a recently identified molecule, and implicated in the pathogenesis of various human diseases, including in X-linked hypophosphatemia (XLH), oncogenic osteomalacia (OOM), autosomal dominant hypophosphatemic rickets (ADHR), familial tumor calcinosis (FTC) and chronic renal diseases. FGF-23 is one of the most important and determinant factors in maintaining phosphate homeostasis, and skeletal mineralization. The long-term objective of this grant proposal is to determine in vivo function, and regulation of FGF-23 in physiological and pathophysiological conditions. As a preliminary step of obtaining such objectives, we have recently generated mice, in which the Fgf-23 gene has been successfully ablated by homologous recombination. These Fgf-23 null mice exhibit hyperphosphatemia, increased vitamin-D activities, excessive mineralization in bone, and abnormal calcifications in the soft tissues. In this grant application, we propose to analyze the effects and interrelationship of three essential components, phosphate, Fgf-23 and vitamin-D, using Fgf-23 null mice. To determine the in vivo roles and regulations of Fgf-23, we propose to define the role of sodium-phosphate co-transporters (NaPi) in abnormal phosphate homeostasis in Fgf-23 null mice by generating Fgf-23-/- / NaPi 2a-/- double mutant mice, as outlined in (Specific Aim 1A). Further studies are also proposed to determine the effects of lowering serum phosphate by nicotinamide in Fgf-23 null animals (Specific Aim 1B). We will also investigate whether circulating FGF-23, exclusively derived from ?1(l) collagen (2.3 kb promoter) expressing osteoblasts, is sufficient to rescue the abnormal systemic phenotype of Fgf-23 null animals. We, therefore, propose to generate a mouse model that is completely ablated for endogenous Fgf-23, but expresses FGF-23 in osteoblasts which is then released into circulation (Specific Aim 2). Furthermore, we proposed to study the role of vitamin D in Fgf-23 mediated functions, by generating and molecular characterization of Fgf-23/1a hydroxylase and Fgf-23/vitamin D receptor double mutant mice (Fgf-23-/-/1a(OH)ase-/-; Fgf-23-/- / \/DR-/-) (Specific Aims 3A, 3B). In addition, we will examine the In vivo bioactivities of Fgf-23 in a normocalcemic/normophosphatemic microenvironment that are independent of vitamin D signaling (Specific Aim 3B). Finally, we propose to analyze the autocrine function of Fgf-23 in vitro using calvarial osteoblasts and explants and will investigate its role as an inhibitor of mineralization (Specific Aim 4). Successful completion of this proposed grant application would generate data that will form the basis to design strategies to manipulate abnormal phosphate homeostasis and defective skeletal mineralization in patients suffering from a wide range of diseases including rickets, XLH, ADHR, OOM, FTC, and chronic renal failure, using FGF-23 or its interacting molecules as a potential therapeutic tool.