Molecular Genetics Of Tooth Development

牙齿发育的分子遗传学

• 批准号: 6814544
• 负责人: Ashok B. KULKARNI
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6814544
• 关键词: congenital dentition disorder; dental development; dental disorder; dentin; dentinogenesis; developmental genetics; disease /disorder model; extracellular matrix; gene expression; gene targeting; genetically modified animals; growth factor; laboratory mouse; molecular cloning; molecular genetics; normal ossification; osteogenesis imperfecta; transcription factor

Tooth development depends on reciprocal interactions between oral epithelium and mesenchyme. The mesenchymal-derived odontoblasts secrete several collagenous and non-collagenous proteins to form a unique extracellular matrix . Among the non-collagenous proteins, dentin sialoprotein (Dsp) and dentin phosphoproteins (Dpp) are highly tooth-specific and are believed to play a crucial role in converting predentin to form mineralized dentin. These two proteins are derived from the cleavage of a 940 amino acid polypeptide, dentin sialophosphoprotein (Dspp). The Dspp gene consists of 5 exons spanning 16 kb and is transcribed as 4.4 kb mRNA . Messenger RNA for Dspp is expressed predominantly by odontoblasts and transiently by pre-ameloblasts. However, recently low levels of Dspp were observed in the ear and in bone. The Dsp, the amino terminal part of Dspp, is sialic acid rich and glycosylated protein that shares similarities with the other sialoproteins including bone sialoprotein, dentin matrix protein-1, and osteopontin. The Dpp, a highly phosphorylated protein with repeats of aspartic acid and phosphoserine, is thought to play a key role in the nucleation of hydroxyapatite formation during dentin calcification. Due to its restricted expression, and its physical localization on the human chromosome 4q within the dentinogenesis imperfecta-II (DGI-II) locus, Dspp was implicated as a potential candidate gene for this disorder. The other important genes involved in biomineralization mapped to this region of chromosome 4q include dentin matrix protein-1, bone sialoprotein, DSPP and osteopontin. Dentinogenesis imperfecta (DGI), an autosomal dominant disorder of the tooth that primarily affects dentin biomineralization, is classified into three subtypes based on the clinical features in an order of degree severity with type-I being the least severe and type-III the most severe . DGI-I is associated with osteogenesis imperfecta while the more severe forms (DGI-II and DGI-III) are restricted to the dentin. Opalescent dentin with an obliterated pulp chambers are the characteristic features in DGI-II. The teeth of patients with DGI-III are referred to as shell teeth in which the dentin mineralization does not occur after mantle dentin is formed. Radiographically, the pulp cavities in these teeth appear as enlarged pulp chambers along with high incidence of pulp exposures .Recently, several mutations in the Dspp gene have been identified in families with DGI-II disorder. These mutations include a C-T transition at the end of 3rd exon (Gln45stop) resulting in a premature termination of Dspp protein, G-A transition mutation in intron 3 (splice donor site) causing exon skipping, and Pro17Thr and Val18Phe transitions. Dentin dysplasia-II (DD-II), another human disorder of dentin mineralization, that shares similarities with DGI-II, and is attributed to a Tyr6Asp protein transition mutation in the hydrophobic core sequence of dspp gene. This mutation disabled the entry of Dspp into the endoplasmic reticulum . All these mutations indicate a potential function for Dspp in tooth mineralization. In order to characterize the molecular events that control dentin mineralization during normal tooth development and disease, we have deleted the entire Dspp coding region in embryonic stem (ES) cells and generated Dspp -/- mice. These null mice displayed an enlarged pulp cavity, widened predentin zone, decreased dentin width and high incidence of pulp exposures similar to DGI-III. Additionally, these mice showed an increased accumulation of biglycan and decorin within the widened predentin and scalloped (void spaces) regions in the dentin correlating well with defective mineralization. Amelogenins form a major protein component of developing enamel and are predominantly involved in the formation of enamel. Additionally, amelogenins have been implicated in the formation and maintenance of cementum. However, their precise expression profile and molecular role in the cementogenesis are not well understood. We have identified for the first time expression of the splice variants of amelogenin mRNA in the periodental region of tooth roots of the wild-type mice but not in the amelogenin-null mice. Progressive cementum defects in the amelogenin-null mice are associated with the increased expression of RANKL, an essential molecule for osteoclastogenesis, in cementoblast/periodontal ligament cells. These findings indicate a novel role for the amelogenin proteins, derived from the splice variants, in regulating RANKL pathway affecting the maintenance of cementum through osteoclastogenisis.

牙齿的发育依赖于口腔上皮和间充质之间的相互作用。间充质源性成牙本质细胞分泌多种胶原和非胶原蛋白，形成独特的细胞外基质。在非胶原蛋白中，牙本质涎蛋白（Dsp）和牙本质磷蛋白（Dpp）是高度牙齿特异性的，并且被认为在前牙本质转化为矿化牙本质中起关键作用。这两种蛋白质来源于940个氨基酸的多肽牙本质唾液磷蛋白（Dspp）的切割。Dspp基因由5个外显子组成，跨越16 kb，转录为4.4 kb的mRNA。Dspp的信使RNA主要由成牙本质细胞表达，并由前成釉细胞短暂表达。然而，最近在耳和骨中观察到低水平的Dspp。Dsp是Dspp的氨基末端部分，是富含唾液酸的糖基化蛋白，与其他唾液蛋白包括骨唾液蛋白、牙本质基质蛋白-1和骨桥蛋白具有相似性。Dpp是一种高度磷酸化的蛋白质，具有天冬氨酸和磷酸丝氨酸的重复序列，被认为在牙本质钙化期间羟基磷灰石形成的成核中起关键作用。由于其有限的表达，和它的物理定位在人类染色体4 q上的牙本质发生的dentinogenesis approximta-II（DGI-II）基因座内，Dspp被认为是这种疾病的潜在候选基因。其他参与生物矿化的重要基因定位在染色体4 q的这个区域，包括牙本质基质蛋白-1，骨唾液酸蛋白，DSPP和骨桥蛋白。牙本质生成障碍（DGI）是一种主要影响牙本质生物矿化的常染色体显性遗传疾病，根据临床特征按严重程度分为三种亚型，I型最轻，III型最重。DGI-I与骨生成相关，而更严重的形式（DGI-II和DGI-III）仅限于牙本质。乳光牙本质和闭塞的髓室是DGI-II的特征性特征。DGI-III患者的牙齿被称为壳牙，其中在套牙本质形成后不发生牙本质矿化。放射学上，这些牙齿的髓腔表现为沿着增大的髓室，具有较高的牙髓暴露率。这些突变包括第3外显子末端的C-T转换（Gln 45 stop）导致Dspp蛋白提前终止，内含子3（剪接供体位点）中的G-A转换突变导致外显子跳跃，以及Pro 17 Thr和Val 18 Phe转换。Dentin dysplasia-II（DD-II）是另一种人类牙本质矿化疾病，与DGI-II有相似之处，并且归因于dspp基因疏水核心序列中Tyr 6Asp蛋白转换突变。该突变使Dspp不能进入内质网。所有这些突变表明Dspp在牙齿矿化中的潜在功能。为了表征正常牙齿发育和疾病过程中控制牙本质矿化的分子事件，我们删除了胚胎干细胞（ES）中的整个Dspp编码区，并生成Dspp -/-小鼠。这些无效小鼠表现出髓腔扩大，前牙本质区增宽，牙本质宽度减少，牙髓暴露的发生率高，与DGI-III相似。此外，这些小鼠显示出在牙本质中加宽的前牙本质和扇形（空隙空间）区域内双糖链蛋白聚糖和核心蛋白聚糖的积累增加，这与矿化缺陷很好地相关。釉原蛋白形成发育中的釉质的主要蛋白质组分，并且主要参与釉质的形成。此外，釉原蛋白与牙骨质的形成和维持有关。然而，其确切的表达谱和在牙骨质形成中的分子作用尚不清楚。我们已经确定了第一次表达的釉原蛋白mRNA的剪接变体在牙周区的牙根的野生型小鼠，但不是在釉原蛋白基因敲除小鼠。釉原蛋白缺失小鼠中进行性牙骨质缺损与成牙骨质细胞/牙周韧带细胞中破骨细胞生成的必需分子RANKL表达增加相关。这些发现表明，一种新的作用，为釉原蛋白蛋白，来自剪接变异体，在调节RANKL途径影响通过破骨细胞生成的牙骨质的维护。