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Regulation of FGF23 by DMP1 in Health and in Chronic Kidney Disease (CKD)

DMP1 在健康和慢性肾脏病 (CKD) 中对 FGF23 的调节

基本信息

批准号：
9750663
负责人：
Aline C Martin
金额：
$ 39.76万
依托单位：
NORTHWESTERN UNIVERSITY AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-07-01 至 2021-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9750663
关键词：
Acids Amino Acids Antibodies Area Binding Biological Biological Assay Blood Circulation Bone Marrow Bone Matrix C-terminal Calcinosis Calcitriol Calcium Cardiac Cardiovascular Diseases Cardiovascular system Chronic Kidney Failure Cleaved cell Co-Immunoprecipitations Coupled Data Development Plans Diet Disease Disease Progression Endopeptidases Enzyme-Linked Immunosorbent Assay Enzymes Equilibrium Event Familial hypophosphatemic bone disease Fluorescein-5-isothiocyanate GALNT3 gene Genes Genetic Transcription Goals Health Histology Homeostasis Hormones Hypophosphatemia Impairment In Vitro Individual Inherited Interferometry Kidney Knowledge Luciferases Measures Mediating Messenger RNA Metalloendopeptidases Modeling Molecular Morphology Mus Mutation Osteoblasts Osteocytes Peptides Post-Translational Protein Processing Production Proteins Regulation Renal function Reporter Research Research Personnel Rickets Role Scanning Electron Microscopy Site Stromal Cells Syndrome System Testing Therapeutic Toxic effect Transfection Transgenic Mice Transgenic Model Transgenic Organisms Western Blotting Work X Chromosome bone cardiovascular disorder risk career development dentin matrix protein 1 experimental study expression vector extracellular fibroblast growth factor 23 glycosylation human model improved improved outcome in vivo innovation inorganic phosphate mortality mouse model new therapeutic target novel therapeutic intervention overexpression promoter public health relevance therapeutic target tumor wasting

项目摘要

DESCRIPTION (provided by applicant): Chronic kidney disease (CKD) is characterized by an early and progressive increase in osteocyte production of fibroblast growth factor (FGF)-23. Elevated FGF23 levels independently predict cardiovascular events, CKD progression, and mortality, suggesting a need to develop novel therapeutic approaches to reduce FGF23 levels in CKD. Our preliminary data indicate that both increased transcription and reduced cleavage of FGF23 contribute to increased circulating levels of biologically active hormone in CKD, but the mechanisms of these alterations are unknown. To date, most knowledge of FGF23 regulation comes from study of hereditary hypophosphatemic rickets caused by inactivating mutations of the osteocyte proteins, dentin matrix protein (DMP)-1 and phosphate-regulating gene with homology to endopeptidase on the X chromosome (PHEX), that cause elevated FGF23 levels. Like CKD, increased transcription and reduced cleavage of FGF23 drive increased circulating levels in states of DMP1 and PHEX inactivation. Our preliminary data suggest that direct binding of DMP1 to PHEX occurs through the ASARM motif present in the C-terminal fragment of DMP1 (cDMP1) peptide, and that this binding is an essential regulatory mechanism of FGF23 transcription and FGF23 cleavage. Although cDMP1 and PHEX are proven local regulators of FGF23 in bone, no studies have tested the hypothesis that altered PHEX and cDMP1 interactions mediate dysregulated FGF23 transcription and cleavage in CKD. In this proposal, we will apply our expertise in FGF23 regulation in hereditary hypophosphatemia to understand local bone mechanisms regulating FGF23 in CKD. In Aim 1, we will perform interferometry and co-immunoprecipitation experiments to determine if cDMP1 is the active fragment of DMP1 that binds PHEX and inhibits FGF23 transcription. In Aim 2, we will test whether cDMP1 inhibits GALNT3, the enzyme responsible for protecting FGF23 from degradation by glycosylating its cleavage site. In both aims, we will use calcitriol, calcium or phosphate loading as different approaches to induce FGF23 transcription in wild-type (WT) and cDMP1 transgenic (cDMP1Tg) mice and their bone marrow stromal cells (BMSC). We will also use MC3T3-E1 osteoblasts transfected with a luciferase reporter FGF23 promoter or an inducible FGF23 expression vector. In Aim 3, we will use the validated Col4a3ko mouse model of progressive CKD to test our hypothesis that decreased cDMP1 concentrations contribute to increased FGF23 levels in CKD. We will investigate osteocyte organization and morphology using acid etched scanning electron microscopy and FITC-Imaris quantitative modelisation, we will quantify cDMP1 peptides and test whether overexpression of cDMP1 blunts the expected FGF23 increase in Col4a3ko mice, during the course of CKD. These innovative Aims form the basis of the career development plan of a new investigator working in a highly productive FGF23 research team. The results will define an essential role of the PHEX/cDMP1 axis in regulating FGF23, and identify novel therapeutic targets to treat disorders of FGF23 excess, including CKD.

描述（由申请人提供）：慢性肾脏疾病（CKD）的特征是成纤维细胞生长因子（FGF）-23的骨细胞产生早期和进行性增加。FGF 23水平升高可独立预测心血管事件、CKD进展和死亡率，这表明需要开发新的治疗方法来降低CKD患者的FGF 23水平。我们的初步数据表明，增加转录和减少切割的FGF 23有助于增加循环水平的生物活性激素在CKD，但这些改变的机制是未知的。迄今为止，大多数关于FGF 23调节的知识来自于对遗传性低磷酸盐血症性佝偻病的研究，所述遗传性低磷酸盐血症性佝偻病是由骨细胞蛋白、牙本质基质蛋白（dentin matrix protein，DNT）-1和与X染色体上的内肽酶（endopeptidase on the X chromosome，PHEX）具有同源性的磷酸盐调节基因的失活突变引起的，所述失活突变引起FGF 23水平升高。与CKD一样，在DMP 1和PHEX失活状态下，FGF 23的转录增加和切割减少驱动循环水平增加。我们的初步数据表明，DMP 1与PHEX的直接结合是通过DMP 1（cDMP 1）肽的C-末端片段中存在的ASARM基序发生的，并且这种结合是FGF 23转录和FGF 23切割的重要调节机制。虽然cDMP 1和PHEX被证明是骨骼中FGF 23的局部调节因子，但没有研究测试改变的PHEX和cDMP 1相互作用介导CKD中FGF 23转录和切割失调的假设。在这项提案中，我们将运用我们在遗传性低磷酸盐血症中FGF 23调节方面的专业知识，了解CKD中FGF 23调节的局部骨机制。在目标1中，我们将进行干涉测量和免疫共沉淀实验，以确定cDMP 1是否是结合PHEX并抑制FGF 23转录的DMP 1活性片段。在目标2中，我们将测试cDMP 1是否抑制GALNT 3，GALNT 3是负责通过糖基化其切割位点来保护FGF 23免于降解的酶。在这两个目标中，我们将使用骨化三醇，钙或磷酸盐负载作为不同的方法来诱导野生型（WT）和cDMP 1转基因（cDMP 1 Tg）小鼠及其骨髓基质细胞（BMSC）中的FGF 23转录。我们还将使用用荧光素酶报告基因FGF 23启动子或诱导型FGF 23表达载体转染的MC 3 T3-E1成骨细胞。在目标3中，我们将使用经验证的进行性CKD的Col 4a 3 ko小鼠模型来检验我们的假设，即降低的cDMP 1浓度有助于增加CKD中的FGF 23水平。我们将使用酸蚀刻扫描电子显微镜和FITC-Imaris定量建模研究骨细胞组织和形态，我们将定量cDMP 1肽，并测试在CKD过程中cDMP 1的过表达是否会减弱Col 4a 3 ko小鼠中预期的FGF 23增加。这些创新的目标形成了一个新的研究人员在一个高效的FGF 23研究团队工作的职业发展计划的基础。这些结果将确定PHEX/cDMP 1轴在调节FGF 23中的重要作用，并确定新的治疗靶点来治疗FGF 23过量的疾病，包括CKD。