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

项目摘要/摘要： X连锁低磷血症(XLH)是最常见的遗传性低磷血症，由 磷酸调节内肽酶PHEX的功能丧失。在头面部最值得注意的是 缺陷是脊椎病牙齿，表现为弱矿化牙本质、脆性牙釉质和缺陷牙本质。 牙骨质。这些缺陷最终会导致附着丧失和自发性脓肿，从而导致 过早地脱牙。尽管在XLH患者中观察到的矿化缺陷仅是 由于血清磷水平降低，牙本质基质组织的局部破坏 成矿作用的先行和后继作用尚未得到研究。无论如何，细胞外的干扰 软骨、骨和牙骨质成熟和矿化所必需的基质(ECM)因子 在Hyp小鼠，XLH的动物模型中已有报道，表明PHEX突变和/或 低磷血症确实可能扰乱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汇聚在一个共同的，尽管定义不明确， 路径。牙本质基质蛋白1(DMP1)就是这样一种蛋白质，其功能丧失导致人类 疾病表型与XLH型相同。利用XLHPEX基因缺陷牙髓干细胞(DPSCs) 患者，我们发现细胞外基质蛋白加工受损，基质水平升高。 金属蛋白酶3(MMP3)。DMP1的组成性表达及分化介质的加入 在XLHDPSCS中，含有磷酸盐的蛋白质加工正常。以下是具体目标 目的：探讨磷酸盐和DMP1对XLH牙本质细胞外基质修复的影响 病理学。使用健康和XLH患者的DPSCs，我们将建立不同的过度表达的细胞系 DMP1的形式。通过体外条件的操作，我们将评估磷酸盐和DMP1对血管内皮细胞生长的影响。 健康DPSCs和XLHDPSCs的遗传和蛋白质组学特征，重点研究构成DPSCs 牙本质基质(目标1)。同时，我们将通过以下方式生成DMP1过表达的PEX缺陷小鼠模型 将牙齿特异的牙本质磷酸蛋白(Dspp)启动子驱动的DMP1转基因小鼠与 超级鼠标。DMP1过表达对细胞外基质蛋白时空分布的影响 将评估牙本质的矿物质质量(目标2)。拟议的研究将解决以下问题的局部影响 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。这些研究还将探讨使用DMP1介导的治疗作为 针对XLH的靶向治疗。这项研究将在伊利诺伊大学内的一所牙科学院进行 在芝加哥医学区校园，一个理想的研究环境，以实现申请人的 学术和职业目标。

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.