Enamelysin Processing Mechanisms in Amelogenesis

这项应用的目标是确定形成适当牙本质牙釉质的关键细胞和分子步骤。 结合部(DEJ)队形。在矿物形成之前，成釉细胞和成牙本质细胞被一个碱基分开- 膜，膜。细胞信号诱导成釉细胞降解基底膜，进入 矿化牙本质的粗糙表面，并分泌启动釉质形成的釉质基质蛋白。AS 成釉细胞和成牙本质细胞胞体在各自的组织增厚时相互远离， 成牙本质细胞在穿透整个牙本质宽度的小管内维持功能细胞过程。在那里- 因此，成牙本质细胞突起位于基底膜附近。基质金属蛋白酶20(MMP20) 由成牙本质细胞和成釉细胞表达和分泌。MMP20有优先(主要)和非优先 优先的(二次)牙釉质基质解理位置和解理位置的顺序是必不可少的 适当的釉质形成。MMP20还能裂解钙粘附素的胞外结构域，从内部释放β-连环蛋白 细胞膜的表面。引人注目的是，MMP20的消融会导致脆性牙釉质脱落。 潜在的牙本质。相反，在小鼠体内过度表达MMP20会导致明显的软化。 含有细胞渗出物的釉质，这是由于增加了β-连环蛋白的释放和信号。然而， DEJ通常如何发展成这样一个坚韧和有弹性的结构的基本机制，以及 MMP20是如何调控这一过程的，目前尚不清楚。这种知识的缺乏对我们的 对牙齿发育的理解。当我们确定MMP20蛋白降解过程如何支持DEJ时- 它将为未来专注于设计高效仿生药物的步骤奠定必要的基础 粘合材料。我们将确定MMP20消融导致DEJ中断的机制 (目标1)。我们推测，在正常发育过程中，MMP20有助于基底膜的降解- 膜和裂解釉质基质蛋白，但在缺乏MMP20的情况下，这一过程受到损害，因此碱基- 膜和基质蛋白保持完好，这可能形成一道屏障，抑制牙本质-牙釉质-牙本质 赫伦斯。我们将确定MMP20过表达对DEJ的影响(目标2)。我们假设 基底膜的水解和釉质基质蛋白的切割顺序是正确DEJ所必需的 MMP20的过度表达将导致通常被串联切割的位点被切割 一下子，导致DEJ受损，过多的MMP20也会改变细胞信号。我们会阻止- 挖掘MMP20来源对DEJ形成的影响程度(目标3)。虽然两种成釉细胞 成牙本质细胞分泌MMP20，我们推测MMP20在有细胞的成牙本质细胞中表达 DEJ附近的过程，将挽救KRT14-Cre-Mmp20fl/fl小鼠的DEJ形成。预计结果将是 有重要的积极影响，因为它们从根本上推动了牙科研究领域的发展 在分子方面，牙釉质和牙本质是如何以这样的强度结合在一起的。

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.