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。间充质来源的成牙本质细胞分泌多种胶原蛋白和非胶原蛋白，形成独特的细胞外基质。在非胶原蛋白中，牙本质唾液蛋白(DSP)和牙本质磷蛋白(DPP)具有高度的牙齿特异性，被认为在转化前牙本质形成矿化牙本质过程中起着至关重要的作用。这两种蛋白质是由940个氨基酸的多肽--牙本质涎磷蛋白(Dspp)裂解而来的。Dspp基因由5个外显子组成，全长16kb，转录为4.4kb的mRNA。Dspp的信使RNA主要由成牙本质细胞表达，并由成釉细胞前瞬时表达。然而，最近在耳朵和骨骼中观察到低水平的DSPP。DSPP的氨基末端是一种富含唾液酸的糖基化蛋白，与其他唾液酸化蛋白如骨唾液蛋白、牙本质基质蛋白-1和骨桥蛋白有相似之处。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过程中羟基磷灰石的成核过程中发挥关键作用。由于Dspp基因的表达受到限制，并且定位于人类染色体4Q上的牙本质发育不全基因(DGI-II)，因此Dspp基因可能是该疾病的候选基因。与生物矿化有关的其他重要基因包括牙本质基质蛋白-1、骨涎蛋白、DSPP和骨桥蛋白。牙本质发育不全是一种常染色体显性遗传性牙病，主要影响牙本质的生物矿化。根据临床特征，牙本质发育不全可分为三种亚型，I型最轻，III型最重。DGI-I与成骨不全有关，而更严重的类型(DGI-II和DGI-III)仅限于牙本质。乳白色牙本质与牙髓腔闭塞是DGI-II的特征。DGI-III患者的牙齿称为壳牙，其中牙本质形成后不会发生牙本质矿化。放射学上，这些牙齿的牙髓腔表现为扩大的牙髓腔，并伴有高发生率的牙髓暴露。最近，在DGI-II疾病家系中发现了Dspp基因的几个突变。这些突变包括导致Dspp蛋白提前终止的第三外显子末端的C-T转换(Gln45stopp)，内含子3(剪接供体位点)的G-A转换突变导致外显子跳过，以及Pro17Thr和Val18Phe转换。牙本质发育不良-II(DD-II)是另一种人类牙本质矿化障碍，与DGI-II有相似之处，被归因于dspp基因疏水核心序列中的Tyr6Asp蛋白转换突变。这种突变使Dspp无法进入内质网。所有这些突变都表明Dspp在牙齿矿化中具有潜在的功能。为了确定在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中的整个Dspp编码区，并产生了Dspp-/-小鼠。这些空白小鼠表现出牙髓腔扩大，前牙本质区增宽，牙本质宽度减少，牙髓暴露的发生率与DGI-III相似。此外，这些小鼠在牙本质中扩大的前牙本质区域和扇形(空隙)区域显示出增加的Biglycan和Decorin的积累，这与缺陷矿化有很好的相关性。釉原蛋白是釉质发育过程中的主要蛋白质成分，主要参与釉质的形成。此外，釉原蛋白还与牙骨质的形成和维持有关。然而，它们在牙骨质形成中的确切表达谱和分子作用还不是很清楚。我们首次在野生型小鼠的牙根牙周区发现了釉原蛋白mRNA的剪接变异体的表达，但在釉原蛋白缺失的小鼠中没有表达。釉原蛋白缺失小鼠进行性牙骨质缺陷与成牙骨质细胞/牙周膜细胞中RANKL的表达增加有关，RANKL是破骨细胞发生的关键分子。这些发现表明，来自剪接变异体的釉原蛋白在调节RANKL途径中发挥了新的作用，RANKL途径通过破骨形成影响牙骨质的维持。