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

项目概要/摘要： X连锁低磷酸盐血症（XLH）是最常见的遗传性低磷酸盐血症， 磷酸调节内肽酶（PHEX）功能丧失。在颅面区域， 缺陷是佝偻病牙齿，表现为弱的低矿化牙本质、脆的釉质和有缺陷的 牙骨质这些缺陷最终会导致依恋的丧失和自发的情感缺失， 过早缺牙尽管在XLH患者中观察到的矿化缺陷仅是 由于血清磷酸盐水平降低，牙本质基质组织的局部破坏， 在矿化之前和维持矿化的过程中没有被研究过。无神经细胞，细胞外破坏 软骨、骨和牙骨质的成熟和矿化所必需的基质（ECM）因子 在Hyp小鼠（XLH的动物模型）中报告，表明PHEX突变和/或 低磷酸盐血症确实可以破坏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聚集在一个共同的，尽管定义不清， 通路牙本质基质蛋白1（DMP1）是一种这样的蛋白质，其功能丧失导致人牙本质缺损。 与XLH表型相同的疾病。使用来自XLH的PHEX缺陷型牙髓干细胞（DPSC） 我们已经证明了ECM蛋白的加工受损，基质水平升高， 金属蛋白酶3（MMP 3）。DMP1的组成型表达或分化培养基的添加 含有磷酸盐的XLH DPSC显示了正常的蛋白质加工。具体目标如下： 探讨磷酸盐和DMP1对XLH损伤牙本质ECM修复的影响 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对细胞增殖的影响。 健康和XLH DPSC的遗传和蛋白质组学特征，重点是构成 牙本质基质（Aim 1）。同时，我们将通过以下方法产生DMP1过表达Phex缺陷小鼠模型： 将我们的牙齿特异性牙本质磷蛋白（Dspp）启动子驱动的DMP1转基因小鼠与 Hyp老鼠DMP1过表达对ECM蛋白时空分布的影响及对细胞增殖的影响 评价牙本质的矿物质质量（目标2）。拟议的研究将探讨下列因素对当地的影响： PHEX突变对牙本质基质沉积的影响，并将进一步阐明PHEX， DMP1和磷酸盐。这些研究还将解决使用DMP1介导的治疗作为治疗的可能性。 XLH的靶向治疗。这项研究将在伊利诺斯大学的一所牙科学院进行 在芝加哥的医疗区校园，一个理想的研究环境，为申请人的履行 学术和专业目标。

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.