Identification and Function of nuDMP1 in Odontoblast Differentiation

nuDMP1在成牙本质细胞分化中的鉴定及其功能

• 批准号: 8637971
• 负责人: Yongbo Lu
• 金额: $ 32.85万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8637971
• 关键词: Ablation; Binding; Biochemistry; Biology; Blood; Bone Matrix; Bypass; Cell Cycle; Cell Differentiation process; Cell Nucleus; Cells; Cephalic; Clinical Treatment; Co-Immunoprecipitations; Defect; Dental; Dentin; Deposition; Differentiation Antigens; Disease; Endoplasmic Reticulum; Failure; Familial hypophosphatemic bone disease; Genetic; Goals; Human; Hypophosphatemia; Image; In Vitro; Initiator Codon; Knockout Mice; Length; Mass Spectrum Analysis; Minerals; Molecular; Mus; Mutagenesis; N-terminal; Nuclear; Nucleic Acid Regulatory Sequences; Odontoblasts; Osteoblasts; Osteocytes; Peptide Signal Sequences; Phenotype; Principal Investigator; Process; Production; Protein Isoforms; Proteins; Research; Research Personnel; Secondary to; Serum; Staging; Testing; Time; Tooth structure; Transgenes; Transgenic Mice; Transgenic Organisms; Translating; Wild Type Mouse; Work; base; bone; dentin matrix protein 1; face bone structure; fibroblast growth factor 23; in vivo; inorganic phosphate; insight; mineralization; novel; osteoblast differentiation; overexpression; promoter; public health relevance; skeletal; therapy development; transcription factor

DESCRIPTION (provided by applicant): The loss of dentin matrix protein 1 (DMP1) function in humans and mice results in autosomal recessive hypophosphatemic rickets (ARHR), characterized by hypomineralization in dentin and bone, hypophosphatemia and the elevation of circulating fibroblast growth factor 23 (FGF23) levels. The dental and skeletal defects of Dmp1-deficient subjects have been attributed to the combined effects of the intrinsic differentiation defects of Dmp1-null odontoblasts and osteoblasts/osteocytes (local effect) and hypophosphatemia (systemic effect). However, the way in which the loss of DMP1 function causes the differentiation defects of odontoblasts and osteoblasts/osteocytes remains largely unknown. The goal of this application is to identify a nuclear isoform(s) of DMP1 (referred to as "nuDMP1") and to study its function in odontoblast differentiation. We hypothesize that nuDMP1 is translated from alternative downstream in-frame AUG start codon(s) and controls odontoblast differentiation by regulating Dspp expression. This hypothesis is based on our recent in vitro and in vivo findings, which include: 1) nuclear DMP1 is observed in cells transfected with a construct expressing full-length DMP1 with the endoplasmic reticulum (ER)-entry signal peptide sequence; 2) a construct expressing full-length DMP1 without the ER-entry signal peptide sequence produces two proteins, a full-length DMP1 and a putative nuDMP1; 3) a construct producing the putative nuDMP1 is more potent at stimulating Dspp promoter activity than those producing full-length DMP1 with or without the ER-entry signal peptide; and 4) the transgenic overexpression of DSPP driven by a 3.6 kb Col1a1 promoter rescues the dental defects but not the other manifestations of Dmp1-null mice. To test our central hypothesis, three specific aims are proposed. Aim 1 will determine the mechanisms by which the nuDMP1 is generated and subsequently translocated into the nuclei. Aim 2 will investigate the function of the nuDMP1 in odontoblast differentiation in vivo by generating and characterizing the 3.6 kb Col1a1-nuDMP1 transgenic mice. Aim 3 will determine how Dspp expression is activated through nuDMP1 and if the failure of this process is solely responsible for the tooth phenotype in Dmp1-null mice. Successful completion of this novel proposal will provide unique insights into how DMP1 regulates cell differentiation via the nuDMP1, which is independent of its function in regulating matrix mineralization. The identification of this novel nuDMP1 and its function will not only help elucidate the molecular mechanisms through which DMP1 controls the formation of tooth and bone but will also provide a scientific basis for developing therapies for treatment of hypophosphatemic rickets.

描述（由申请人提供）：人类和小鼠中牙本质基质蛋白1（DMP1）功能的丧失导致常染色体隐性低磷酸盐症（ARHR），其特征是牙本质和骨骼低磷酸盐，低磷酸盐症和循环纤维纤维生长因子的升高（FGFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFRENCA22323（FGFFFFFFFFFFFFFFFFFFRENCA2 23） DMP1缺陷受试者的牙齿和骨骼缺陷已归因于DMP1-NULL ODONTOBLASTS和成骨细胞/成骨细胞（局部效应）和低磷酸酯（全身效应）的内在分化缺陷的综合作用。但是，DMP1函数丢失的方式会导致牙糖细胞和成骨细胞/骨细胞的分化缺陷在很大程度上尚不清楚。该应用的目的是识别DMP1的核同工型（称为“ nudMP1”），并研究其在Odontoblast分化中的功能。我们假设NUDMP1从替代的下游框架内启动密码子转化，并通过调节DSPP表达来控制Odontoblast分化。该假设基于我们最近的体外和体内发现，其中包括：1）核DMP1在用表达全长DMP1的细胞中观察到核DMP1，该细胞具有内质网（ER） - 居住信号肽序列的全长DMP1； 2）表达不带ER入门信号肽序列的全长DMP1的构建体产生两种蛋白质，一个全长DMP1和一个推定的NUDMP1； 3）产生推定的NUDMP1的构建体在刺激DSPP启动子活动方面比产生或不具有ER-ER-ENTRY信号肽的全长DMP1的构造更有效。 4）由3.6 kb col1a1启动子驱动的DSPP的转基因过表达挽救了牙齿缺陷，但没有挽救DMP1无效小鼠的其他表现。为了检验我们的中心假设，提出了三个具体目标。 AIM 1将确定生成NUDMP1并随后转移到核中的机制。 AIM 2将通过产生和表征3.6 Kb Col1a1-NudMP1转基因小鼠来研究NUDMP1在体内odontoblast分化中的功能。 AIM 3将确定如何通过NUDMP1激活DSPP表达，并且该过程的失败是否完全负责DMP1-NULL小鼠中的牙齿表型。这项新建议的成功完成将为DMP1如何通过NUDMP1调节细胞分化提供独特的见解，NUDMP1与其在调节基质矿化方面的功能无关。这种新型裸子及其功能的鉴定不仅将有助于阐明DMP1控制牙齿和骨骼形成的分子机制，而且还将为开发治疗低磷酸rick鼠的疗法提供科学基础。