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: 8483222
• 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/8483222
• 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（由申请方提供）：人和小鼠牙本质基质蛋白1（DMP 1）功能丧失导致常染色体隐性低磷血症性佝偻病（ARHR），其特征为牙本质和骨矿化不足、低磷血症和循环成纤维细胞生长因子23（FGF 23）水平升高。Dmp 1缺陷受试者的牙齿和骨骼缺陷归因于Dmp 1缺失的成牙本质细胞和成骨细胞/骨细胞的内在分化缺陷（局部效应）和低磷酸盐血症（全身效应）的综合效应。然而，DMP 1功能的丧失导致成牙本质细胞和成骨细胞/骨细胞分化缺陷的方式在很大程度上仍然未知。本申请的目的是鉴定DMP 1的核同种型（称为“nuDMP 1”）并研究其在成牙本质细胞分化中的功能。我们假设nuDMP 1是从另一个下游符合读码框的AUG起始密码子翻译而来，并通过调节Dspp表达来控制成牙本质细胞的分化。这一假设是基于我们最近的体外和体内研究结果，包括：1）在用表达具有内质网（ER）进入信号肽序列的全长DMP 1的构建体转染的细胞中观察到核DMP 1; 2）表达不具有ER进入信号肽序列的全长DMP 1的构建体产生两种蛋白质，全长DMP 1和推定的nuDMP 1; 3）产生推定的nuDMP 1的构建体比产生具有或不具有ER进入信号肽的全长DMP 1的构建体更有效地刺激Dspp启动子活性;和4）由3.6 kb Col 1a 1启动子驱动的DSPP转基因过表达可以挽救牙齿缺陷，但不能挽救Dmp 1缺失小鼠的其他表现。为了检验我们的中心假设，提出了三个具体目标。目的1将确定nuDMP 1产生并随后易位到细胞核中的机制。目的2通过建立3.6kb Col 1a 1-nuDMP 1转基因小鼠模型，研究nuDMP 1在成牙本质细胞分化中的作用。目的3将确定Dspp表达是如何通过nuDMP 1激活的，以及该过程的失败是否是Dmp 1缺失小鼠牙齿表型的唯一原因。成功完成这一新的提案将提供独特的见解DMP 1如何通过nuDMP 1调节细胞分化，这是独立于其功能，在调节基质矿化。这种新型nuDMP 1的鉴定及其功能不仅有助于阐明DMP 1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血症性佝偻病的疗法提供科学依据。