Characterization of DMP1 A Dentin Phosphoprotein

描述（由申请人提供）：在骨和牙本质生物矿化中，沉积的羟基磷灰石晶体的性质受到胶原模板和非胶原酸性蛋白的精确控制。这些酸性蛋白被认为在新生矿物成核、调节羟基磷灰石晶体大小及其形态中起关键作用。酸性基质蛋白以有序的方式结合到羟基磷灰石的晶格中，从而使牙本质成为具有高抗拉强度的复合材料。胶原基质为矿物晶体的形成提供了空间和环境。因此，牙本质形成是一个涉及细胞和细胞外事件级联的动态过程。细胞-细胞、细胞-基质和基质-基质等特定的相互作用事件负责成牙本质分化和矿化牙本质基质的组装。矿化过程中的问题在许多牙齿病理中都很明显。牙本质再生和修复的成功依赖于将成牙本质细胞前体招募到原位的能力，随后是这些细胞的成熟过程、基质沉积和有机基质的最终矿化。牙本质基质蛋白1 （DMP1）是我们首次从矿化牙本质基质中克隆到的细胞外基质蛋白。DMP1是典型的多阴离子，具有高含量的天冬氨酸、谷氨酸和磷酸丝氨酸残基，使其成为一种非常适合钙结合的蛋白质。我们对这种矿化基质的起始和调控机制知之甚少。本研究的目的是从DMP1的鉴定和定位到研究该蛋白的表达调控，以及它在矿化过程中的潜在参与。我们现在提出以下具体目标：(1)表征DMP1启动子并确定3kb上游序列中参与细胞和组织特异性表达的特定位点；(2)通过构建性过表达DMP1，探讨胚胎间充质细胞向成牙细胞样细胞分化的机制；(3)证明DMP1作为羟基磷灰石形成的潜在成核剂的作用；(4)研究DMP1蛋白在牙本质基质中的分布，从而确定DMP1在矿化过程中的功能作用。了解矿化过程对生物医学和牙科领域很重要，因为正常生物矿化的破坏可能导致病理性矿化或脱矿过程。长期目标是了解DMP1在牙本质矿化中的调控机制。

DESCRIPTION (provided by applicant): In bone and dentin biomineralization, the nature of the hydroxyapatite crystals deposited are under the precise control of the collagen template as well as the noncollagenous acidic proteins. These acidic proteins have been postulated to play a critical role in de novo mineral nucleation, regulating hydroxyapatite crystal size as well as its morphology. Acidic matrix proteins are incorporated into the crystal lattice of hydroxyapatite in a well ordered manner, thus making dentin a composite with high tensile strength. The collagen matrix provides the space and milieu in which the mineral crystals are initiated. Therefore, dentinogenesis is a dynamic process that involves a cascade of cellular and extracellular events. Specific interactive events like cell-cell, cell-matrix and matrix-matrix are responsible for odontoblast differentiation and the assembly of the mineralized dentin matrix. Problems in the mineralization process are evident in a number of dental pathologies. Successful dentin regeneration and repair in dentistry relies on the ability to recruit odontoblast precursors to the site followed by maturation process of these cells, matrix deposition and ultimately mineralization of the organic matrix. Dentin matrix protein 1 (DMP1) is an extracellular matrix protein that was first cloned by us from the mineralized dentin matrix. DMP1 is typically polyanionic having a high content of aspartic, glutamic and phosphoserine residues making it a protein highly suitable for calcium binding. Very little is known about the mechanism responsible for initiation and regulation of this mineralized matrix. The objective of this proposed investigation is to move forward from the identification and localization of the DMP1 to the study of the regulation of the expression of the protein, and to its potential involvement in the mineralization process. We now propose the following specific aims: (1) To characterize the DMP1 promoter and to identify specific sites within the 3 kb upstream sequence that are involved in cell and tissue specific expression; (2) To identify the mechanisms involved during the differentiation of embryonic mesenchymal cells to odontoblast-like cells by constitutive overexpression of DMP1; (3) To demonstrate the role of DMP1 as a potential nucleator for hydroxyapatite formation; (4) To study the distribution of DMP1 protein in the dentin matrix and thus determine the functional role of DMP1 during the process of mineralization. Understanding the mineralization process is important to the biomedical and dentistry fields because the disruption of normal biomineralization can lead to pathological mineralization or demineralization process. The long-term goal is to understand the regulatory mechanism of DMP1 in dentin mineralization.