Enamelysin Processing Mechanisms in Amelogenesis

釉质生成中的釉质加工机制

• 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
• 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

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.

该应用的目的是确定正确的牙本质 - 烯烃的关键细胞和分子步骤 交界处（DEJ）组。在矿物质形成之前，将木材细胞和牙本质细胞通过碱基分开 膜。细胞信号传导可诱导木材细胞降解地下室膜，移入 矿化牙本质的粗糙表面和分泌启动搪瓷形成的搪瓷基质蛋白。作为 当它们各自的组织增厚时 Odontoblasts在管道内维护功能性细胞过程，这些细胞过程穿透了牙本质的整个宽度。那里- 前面，牙粒细胞过程在地下膜附近留下。基质金属蛋白酶20（MMP20） 由odontoblasts和amelellasts表示和分泌。 MMP20优先（主要）和非 - 优先（次级）牙釉质基质裂解位点和裂解位点顺序对 适当的搪瓷形成。 MMP20还裂解了钙粘蛋白的外生域，从而从内部释放β-catenin 细胞膜的表面。令人惊讶的是，MMP20消融导致脆弱的搪瓷，从 基础牙本质。相反，小鼠MMP20的过表达导致明显柔软 含有细胞浸润的搪瓷，这归因于β-catenin释放和信号传导增加。然而， DEJ通常如何发展成如此韧性和弹性结构的基本机制，以及 MMP20如何调节此过程，仍然未知。缺乏知识给我们带来了关键的障碍 了解牙齿发育。当我们确定MMP20蛋白水解处理如何支持dej for- 事实，它将为未来的步骤奠定必要的基础，专注于设计高效的仿生 粘合剂材料。我们将确定MMP20消融导致DEJ的破坏的机制 （目标1）。我们假设在正常发育中，MMP20有助于地下室的降解 布雷恩和切割牙釉质基质蛋白，但在没有MMP20的情况下，该过程受到了损害 膜膜和基质蛋白保持完整，这可能会形成障碍物，以抑制牙本质 - 烯酰胺ad- 此处。我们将确定MMP20过表达对DEJ的影响（AIM 2）。我们假设这一点 正确DEJ需要地下膜水解和牙釉质基质蛋白的裂解顺序 形成，MMP20的过表达将导致通常以串联裂解的位点裂解 同时，导致DEJ受损，并且MMP20过多也会改变细胞信号传导。我们将确定 - 挖掘MMP20来源影响DEJ的形成程度（AIM 3）。虽然两种成熟的细胞 和Odontoblasts Secret MMP20，我们假设MMP20在具有细胞的Odontoblasts中的表达 DEJ附近的过程将挽救KRT14-CRE-MMP20FL/FL小鼠中的DEJ组。结果预计 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 用分子术语，搪瓷和牙本质如何与这种强度结合在一起。