Enamelysin Processing Mechanisms in Amelogenesis

釉质生成中的釉质加工机制

基本信息

批准号：
10316206
负责人：
JOHN D BARTLETT
金额：
$ 53.62万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-01-01 至 2023-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10316206
关键词：
Ablation Adherence Adhesives Ameloblasts Amelogenesis Basement membrane Binding Biomechanics Biomimetics Cadherins Cell Nucleus Cell membrane Cell physiology Cell surface Cells Data Dental Enamel Dental Research Dentin Dentin Formation Development Electron Microscopy Facility Elements Enamel Formation Engineering Failure Foundations Fracture Future Genes Goals Human Human Resources Hydrolysis Image Ions Knowledge Left MMP-20 Membrane Membrane Proteins Minerals Molecular Mus Mutate Odontoblasts Ohio Outcome Play Prevention Process Proteins Proteolytic Processing Research Resistance Resolution Role Series Signal Transduction Site Source Structure Surface Testing Therapeutic Three-Dimensional Image Tissues Tooth structure Universities Vent Width Work base beta catenin calcification cell motility design enamel matrix proteins enamelysin experience falls mineralization overexpression restorative dentistry

项目摘要

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.

该应用的目标是确定正确牙本质-牙釉质的关键细胞和分子步骤在矿物质形成之前，成釉细胞和成牙本质细胞被碱基分开。细胞信号传导诱导成釉细胞降解基底膜，进入细胞膜。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。当成釉细胞和成牙本质细胞细胞体各自的组织增厚时，它们的细胞体会相互远离，成牙本质细胞在小管内维持功能性细胞过程，穿透牙本质的整个宽度。因此，成牙本质细胞过程留在基底膜附近。 MMP20 由成牙本质细胞和成釉细胞表达和分泌，具有优先性（初级）和非优先性（初级）。优先（次要）牙釉质基质裂解位点和裂解位点的顺序对于 MMP20 也会裂解钙粘蛋白胞外域，从内部释放 β-连环蛋白。引人注目的是，MMP20 的消融会导致牙釉质变脆，呈片状脱落。小鼠中 Mmp20 的过度表达导致牙本质明显比正常情况软。牙釉质中含有细胞浸润，这是由于 β-连环蛋白释放和信号传导增加所致。 DEJ 通常如何发展成如此坚韧和有弹性的结构的基本机制，以及 MMP20 如何调节这一过程仍然未知，这种知识的缺乏给我们带来了一个关键障碍。当我们了解 MMP20 蛋白水解加工如何支持 DEJ 时，化，它将为未来专注于设计高效仿生材料的步骤奠定必要的基础我们将确定 Mmp20 消融导致 DEJ 破坏的机制。（目标 1）我们努力发现，在正常发育过程中，MMP20 会导致基底内存退化。膜并裂解牙釉质基质蛋白，但在缺乏 MMP20 的情况下，该过程会受到损害，因此牙本质膜和基质蛋白保持完整，这可能形成抑制牙本质-牙釉质粘附的屏障因此，我们将确定 MMP20 过度表达对 DEJ 的影响（目标 2）。正确的 DEJ 需要基底膜水解和牙釉质基质蛋白的裂解顺序 MMP20 的过度表达会导致通常串联切割的位点被切割一次，导致 DEJ 受损，而且过多的 MMP20 也会改变细胞信号传导。探究 Mmp20 来源对 DEJ 形成的影响程度（目标 3）。成牙本质细胞分泌 MMP20，我们捕获了 Mmp20 在成牙本质细胞中的表达，其具有细胞 DEJ 附近的过程将挽救 KRT14-Cre-Mmp20fl/fl 小鼠中的 DEJ 形成。具有重要的积极影响，因为它们通过定义从根本上推进了牙科研究领域从分子角度来说，牙釉质和牙本质如何以如此强度结合在一起。