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：
8964715
负责人：
Aline C Martin
金额：
$ 40.1万
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 2020-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8964715
关键词：
Accounting Acids Amino Acids Antibodies Area Binding 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 Genes Genetic Transcription Goals Health Histology Homeostasis Hormones Human Hypophosphatemia In Vitro Individual Inherited Interferometry Kidney Knowledge Luciferases Measures Mediating Messenger RNA Modeling Molecular Morphology Mus Mutation Osteoblasts Osteocytes Outcome Peptides Polypeptide N-acetylgalactosaminyltransferase Post-Translational Protein Processing Production Proteins Regulation Renal function Reporter Research Research Personnel Rickets Role Scanning Electron Microscopy Site Staging Stromal Cells Syndrome System Testing Therapeutic Toxic effect Transfection Transgenic Mice Transgenic Model Transgenic Organisms Western Blotting Work X Chromosome base bone cardiovascular disorder risk career development dentin matrix protein 1 expression vector extracellular fibroblast growth factor 23 glycosylation improved in vivo innovation inorganic phosphate mortality mouse model new therapeutic target novel therapeutic intervention overexpression promoter public health relevance research study therapeutic target 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（由适用提供）：慢性肾脏疾病（CKD）的特征是成纤维细胞生长因子（FGF）-23的骨细胞产生的早期和逐渐增加。 FGF23升高水平独立预测心血管事件，CKD进展和死亡率，这表明需要开发新型的治疗方法，以降低CKD中的FGF23水平。我们的初步数据表明，FGF23的转录和裂解降低均导致CKD中生物活性同种的循环水平增加，但是这些改变的机制尚不清楚。迄今为止，大多数对FGF23调节的了解来自对遗传性下降磷酸盐风险的研究，这是由于骨细胞蛋白的失活突变，牙本质基质蛋白（DMP）-1和磷酸磷酸盐调节基因与同源性基因对X染色体上的内肽酶（PHEX）（pHEx）高度升高FGFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFGFS上（phep2级高。像CKD一样，在DMP1和PHEX失活状态下，FGF23驱动的转录增加和FGF23驱动的裂解降低。我们的初步数据表明，DMP1与PHEX的直接结合是通过DMP1（CDMP1）肽的C末端片段中存在的ASARM基序进行的，并且该结合是FGF23转录和FGF23裂解的基本调节机制。尽管CDMP1和PHEX已被证明是骨骼中FGF23的局部调节剂，但尚无研究检验的假设，即改变PHEX和CDMP1相互作用介导了CKD中FGF23转录和裂解的失调。在此提案中，我们将在雌性低磷酸血症的FGF23调节中应用我们的专业知识，以了解CKD中FGF23的局部骨骼机制。确定CDMP1是否是结合PHEX并抑制FGF23转录的DMP1的活动片段。在AIM 2中，我们将测试CDMP1是否抑制GALNT3（负责通过糖基化裂解位点来保护FGF23的酶免受降解。在这两个目的中，我们都将使用钙或磷酸盐负载作为不同的方法来诱导野生型（WT）和CDMP1转基因（CDMP1TG）小鼠及其骨髓间基细胞（BMSC）中的FGF23转录。我们还将使用用荧光素酶报告基因FGF23启动子或诱导型FGF23表达载体翻译的MC3T3-E1成骨细胞。在AIM 3中，我们将使用经过验证的COL4A3KO小鼠模型的进行性CKD测试我们的假设，即提高的CDMP1浓度有助于CKD中FGF23水平的提高。我们将使用酸蚀刻的扫描电子显微镜和FITC-MIMARIS定量调节化研究骨细胞组织和形态，我们将量化CDMP1肽，并测试CDMP1的过表达在CCKD过程中，COL4A3KO小鼠的预期FGF23是否会增加预期的FGF23。这些创新的目标构成了在高产FGF23研究团队中工作的新调查员职业发展计划的基础。结果将定义PHEX/CDMP1轴在确定FGF23中的重要作用，并确定包括CKD在内的FGF23超过的新型治疗靶标，以治疗FGF23的疾病。