Enamelysin Processing Mechanisms in Amelogenesis

本申请的目的是确定正确的牙本质-釉质的关键细胞和分子步骤 结（DEJ）形成。在矿物质形成之前，成釉细胞和成牙本质细胞被一个碱基分开， 隔膜细胞信号转导诱导成釉细胞降解基底膜，进入细胞内， 牙釉质基质蛋白是矿化牙本质的粗糙表面，并分泌启动牙釉质形成的牙釉质基质蛋白。作为 成釉细胞和成牙本质细胞的细胞体在它们各自的组织破裂时彼此远离， 成牙本质细胞在穿透牙本质的整个宽度的小管内维持功能性细胞突起。在那里- 成牙本质细胞突起位于基底膜附近。基质金属蛋白酶20（MMP 20） 由成牙本质细胞和成釉细胞表达和分泌。MMP 20具有优先（主要）和非- 优先的（次级）釉质基质切割位点和切割位点的顺序对于 正常的釉质形成MMP 20还切割钙粘蛋白胞外结构域，其从内表面释放β-连环蛋白。 细胞膜的表面。引人注目的是，MMP 20的消融导致脆性牙釉质，其福尔斯以片状脱落， 下面的牙本质相反，Mmp 20在小鼠中的过度表达导致比正常情况下明显更软。 牙釉质含有细胞浸润，这归因于β-连环蛋白释放和信号传导增加。然而，在这方面， DEJ通常如何发展成如此坚韧和有弹性的结构的潜在机制，以及 MMP 20如何调节这一过程仍然未知。这种知识的缺乏对我们的研究构成了严重的障碍。 了解牙齿发育。当我们确定MMP 20蛋白水解加工如何支持DEJ时- 信息，它将奠定必要的基础，为未来的步骤集中在设计高效仿生 粘合剂材料。我们将确定Mmp 20消融导致DEJ中断的机制， (Aim 1）。我们假设在正常发育中，MMP 20有助于基底膜的降解， 膜和切割釉基质蛋白，但在MMP 20的情况下，这一过程受到损害，所以基础- 牙本质膜和基质蛋白保持完整，这可能会形成一个屏障，抑制牙本质-牙釉质和牙本质-牙釉质之间的相互作用。 赫伦斯。我们将确定MMP 20过表达对DEJ的影响（目的2）。我们假设 适当的DEJ需要基底膜水解和釉质基质蛋白的切割顺序 MMP 20的过度表达将导致通常连续切割的位点被切割 同时，导致DEJ受损，过多的MMP 20也会改变细胞信号传导。我们会阻止- 挖掘Mmp 20的来源对DEJ形成的影响程度（目标3）。虽然成釉细胞 而成牙本质细胞分泌MMP 20，我们推测Mmp 20在成牙本质细胞中的表达， 在DEJ附近的过程，将拯救KRT 14-Cre-Mmp 20 fl/fl小鼠中的DEJ形成。结果预计 具有重要的积极影响，因为它们从根本上推进了牙科研究领域， 从分子的角度来解释牙釉质和牙本质是如何结合在一起的。

The goal of this application is to identify the key cellular and molecular steps for proper dentin–enamel junction (DEJ) formation. Prior to mineral formation, ameloblasts and odontoblasts are separated by a base- ment membrane. Cell signaling induces the ameloblasts to degrade the basement membrane, move into the rough surface of the mineralizing dentin and secrete enamel matrix proteins that initiate enamel formation. As ameloblasts and odontoblast cell bodies move away from each other when their respective tissues thicken, the odontoblasts maintain functional cell processes within tubules that penetrate the entire width of the dentin. There- fore, the odontoblastic processes are left near the basement membrane. Matrix metalloproteinase-20 (MMP20) is expressed and secreted by both odontoblasts and ameloblasts. MMP20 has preferential (primary) and non- preferential (secondary) enamel matrix cleavage sites and the cleavage site sequential order is essential for proper enamel formation. MMP20 also cleaves cadherin ectodomains, which releases β-catenin from the inner surface of the cell membrane. Strikingly, MMP20 ablation results in brittle enamel that falls away in sheets from the underlying dentin. Conversely, over-expression of Mmp20 in mice results in significantly softer than normal enamel that contains a cell infiltrate, which is attributed to increased β-catenin release and signaling. However, the underlying mechanisms for how the DEJ normally develops into such a tough and resilient structure, and how MMP20 regulates this process, remain unknown. This lack of knowledge presents a critical obstacle to our understanding of tooth development. When we establish how MMP20 proteolytic processing supports DEJ for- mation, it will lay the necessary foundation for future steps focused on designing highly effective biomimetic adhesive materials. We will determine the mechanism by which Mmp20 ablation causes disruption of the DEJ (Aim 1). We hypothesize that in normal development, MMP20 contributes to degradation of the basement mem- brane and cleaves enamel matrix proteins, but in the absence of MMP20, this process is compromised so base- ment membrane and matrix proteins remain intact and this may form a barrier that inhibits dentin–enamel ad- herence. We will determine the impact of MMP20 over-expression on the DEJ (Aim 2). We hypothesize that basement membrane hydrolysis and the cleavage order of enamel matrix proteins are required for proper DEJ formation, and that MMP20 over-expression will cause the sites that are normally cleaved in series to be cleaved all at once, resulting in a compromised DEJ, and that too much MMP20 also alters cell signaling. We will deter- mine the extent to which the source of Mmp20 impacts formation of the DEJ (Aim 3). Although both ameloblasts and odontoblasts secrete MMP20, we hypothesize that Mmp20 expression in odontoblasts, which have cell processes near the DEJ, will rescue DEJ formation in KRT14-Cre-Mmp20fl/fl mice. The results are expected to have an important positive impact because they fundamentally advance the field of dental research by defining in molecular terms how enamel and dentin bind together with such strength.