Roles of the Phosphate-regulating Proteins, PHEX and DMP1, in the Dentin Matrix of XLH Patients

磷酸盐调节蛋白 PHEX 和 DMP1 在 XLH 患者牙本质基质中的作用

• 批准号: 10201569
• 负责人: Elizabeth Guirado
• 金额: $ 4.75万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2022-05-05
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10201569
• 关键词: Abscess; Address; Age-Months; Animal Model; Animals; Biochemical; Biological Assay; C-terminal; Cartilage; Cell Culture Techniques; Cell Differentiation process; Chicago; Clinical; Clinical Pathology; Collagen Fiber; DSPP gene; Data; Defect; Dental; Dental Cementum; Dental Enamel; Dental Pulp; Dentin; Dentin Formation; Deposition; Disease; Dyes; Endopeptidases; Environment; Extracellular Domain; Extracellular Matrix; Extracellular Matrix Proteins; Familial hypophosphatemic bone disease; Gene Expression; Genes; Genetic; Goals; Hour; Hypophosphatemia; Illinois; Impairment; In Vitro; Inherited; Label; Lead; Length; Link; Mandible; Mediating; Medical; Metabolic Diseases; Metalloproteases; Minerals; Molecular; Monitor; Mus; Mutation; N-terminal; Odontoblasts; Pathologic; Pathology; Pathway interactions; Patients; Pattern; Peptide Hydrolases; Phenotype; Proteins; Proteomics; Pulp Chambers; Reporting; Research; Role; Scanning Electron Microscopy; Serum; Signal Transduction; Skeleton; Small Interfering RNA; Thinness; Time; Tooth structure; Transgenes; Transgenic Mice; Universities; Western Blotting; Width; X Chromosome; base; bone; college; craniofacial; craniofacial complex; density; dentin matrix protein 1; design; disease phenotype; edentulism; established cell line; genetic regulatory protein; human disease; improved; inorganic phosphate; loss of function; microCT; mineralization; mouse model; novel; novel therapeutic intervention; overexpression; premature; promoter; protein expression; repaired; response; screening; small molecule; sodium phosphate; spatiotemporal; stem cells; targeted treatment

Project Summary/Abstract: X-linked hypophosphatemia (XLH) is the most common hereditary hypophosphatemic disorder resulting from loss-of-function of the phosphate-regulating endopeptidase, PHEX. In the craniofacial region the most notable defect is the rachitic tooth, which presents with weak hypomineralized dentin, brittle enamel, and defective cementum. These defects ultimately lead to attachment loss and spontaneous abscesses that result in premature edentulism. Although the mineralization defects observed in XLH patients have been solely attributed to decreased serum phosphate levels, the local disruption in the organization of the dentin matrix that precedes and sustains mineralization has not been studied. Neverthesless, disruption of extracellular matrix (ECM) factors necessary for the maturation and mineralization of cartilage, bone and cementum have been reported in Hyp mice, the animal model for XLH, suggesting that PHEX mutations and/or hypophosphatemia may indeed disrupt ECM deposition. An increasing amount of evidence suggests that PHEX and the non-collagenous proteins of bone and teeth converge on a common, albeit poorly defined, pathway. Dentin matrix protein 1 (DMP1) is one such protein whose loss-of-function results in a human disease phenotypically identical to XLH. Using PHEX-deficient dental pulp stem cells (DPSCs) from XLH patients, we have demonstrated impaired processing of ECM proteins and elevated levels of matrix metalloprotease 3 (MMP3). Constitutive expression of DMP1 or the addition of differentiation medium containing phosphate revealed normal protein processing in XLH DPSCs. The following specific aims are designed to investigate the influence of phosphate and DMP1 in restoring the impaired dentin ECM in XLH pathology. Using DPSCs from healthy and XLH patients, we will establish cell lines overexpressing different forms of DMP1. By manipulating in vitro conditions, we will evaluate the effect of phosphates and DMP1 on the genetic and proteomic profile of healthy and XLH DPSCs, focusing on the ECM proteins that constitute the dentin matrix (Aim 1). In parallel, we will generate a DMP1-overexpressing Phex-deficient mouse model by crossing our tooth-specific dentinsialophosphoprotein (Dspp) promoter-driven DMP1 transgenic mouse with the Hyp mouse. The effect of DMP1-overexpression on the spatiotemporal distribution of ECM proteins and the mineral quality of tooth dentin will be evaluated (Aim 2). The proposed studies will address the local effects of PHEX mutations on dentin matrix deposition and will further clarify the predicted relationship between PHEX, DMP1, and phosphate. The studies will also address the possibility of using DMP1-mediated treatments as targeted therapies for XLH. This research will be conducted at a dental college within the University of Illinois at Chicago’s medical district campus, an ideal research environment for the fulfillment of the applicant’s academic and professional goals.

项目摘要/摘要： X连锁低磷酸血症（XLH）是由 磷酸盐调节肽酶的功能丧失，PHEX。在颅面地区最著名的 缺陷是rachitic牙齿，其呈弱降低牙本质，脆性搪瓷和有缺陷的牙齿 骨质。这些缺陷最终导致依恋损失和发起脓肿，导致 虽然XLH患者观察到的矿化缺陷过早 归因于血清磷酸盐水平降低，这是牙本质基质组织的局部破坏 在此之前，自杀矿化尚未研究。无关，细胞外的破坏 基质（ECM）软骨，骨骼和骨质成熟和矿化所需的因素 在XLH的动物模型催眠小鼠中报道了PHEX突变和/或 下降磷酸血症确实可能破坏ECM沉积。越来越多的证据表明 PHEX和骨骼和牙齿的非胶原蛋白在一个共同的（尽管定义较差）上汇聚 路径。牙本质基质蛋白1（DMP1）是一种这样的蛋白质，其功能丧失导致人类 疾病与XLH表面上相同。使用XLH的pHEx缺陷牙髓干细胞（DPSC） 患者，我们已经证明了ECM蛋白的加工受损和基质水平升高 金属蛋白酶3（MMP3）。 DMP1的组成型表达或添加分化培养基 含有磷酸盐揭示了XLH DPSC中的正常蛋白质加工。以下具体目的是 旨在研究磷酸盐和DMP1在恢复XLH中受损的牙本质ECM的影响 病理。使用来自健康和XLH患者的DPSC，我们将建立过表达不同的细胞系 DMP1的形式。通过操纵体外条件，我们将评估磷酸盐和DMP1对 健康和XLH DPSC的遗传和蛋白质组学特征，重点是构成ECM蛋白 牙本质矩阵（AIM 1）。同时，我们将通过通过 跨越我们的牙齿特异性牙本质磷蛋白（DSPP）启动子驱动的DMP1转基因小鼠 催眠老鼠。 DMP1过表达对ECM蛋白和时空分布的影响 将评估牙齿牙本质的矿物质质量（AIM 2）。拟议的研究将解决 牙本质基质沉积的PHEX突变，并将进一步阐明PHEX之间的预测关系 DMP1和磷酸盐。这些研究还将解决使用DMP1介导的治疗作为 XLH的靶向疗法。这项研究将在伊利诺伊大学的一所牙科学院进行 在芝加哥医疗区校园，这是申请人的理想研究环境 学术和专业目标。