Enamelysin processing mechanisms in amelogenesis

首席调查员/项目主任(最后、第一、中间)：Bartlett，John，D. 本应用的目的是表征基质金属蛋白酶-20(MMP20)和N-蛋白的作用。 钙粘附素在牙釉质发育过程中成釉细胞的运动和细胞-细胞附着。MMP20 对于牙釉质的形成是必不可少的。缺乏功能性MMP20的人和小鼠的畸形程度非常显著 牙釉质：一种稀薄、柔软且容易从下面的牙本质中磨损的牙釉质。在MMP20基因缺失小鼠中， 分泌期成釉细胞不能正常进入发育的成熟期，一旦进入成熟期， 成釉细胞相互重叠生长。这表明成釉细胞-细胞附着和 在MMP20基因缺失的小鼠中，信号会发生变化。钙粘附素是一个跨越细胞膜的蛋白质家族。 介导与相邻细胞上存在的相同钙粘附素的附着。P120-连环蛋白(P120)稳定钙粘蛋白 P120的存在显著减少了细胞表面钙粘附素的存在。此前， 我们发现，在小鼠中，p120的消融也会导致畸形的釉质磨损牙齿。 因此，MMP20和钙粘附素都是牙釉质形成所必需的。我们将确定(目标1)损失如何 MMP20的表达影响成釉细胞与细胞间的相互作用。我们假设MMP20裂解细胞外 钙粘蛋白的结构域，它从被破坏的钙粘蛋白复合体中释放细胞内信号分子， 如β-连环蛋白，它们是牙釉质形成所必需的。重要的是，我们的初步数据表明 MMP20切割E-钙粘蛋白的胞外区，我们建议通过使用一种 稳定表达的成釉细胞来源细胞系(成釉细胞系，ALC) 高水平激活的MMP20。正常牙釉质有交叉(交错)的杆状图案。每根棒子都成形了 通过一个成釉细胞和每个杆保存了成釉细胞迁移路径的完整记录， 形成了它。MMP20缺失的小鼠牙釉质要么有高度发育不良的杆状图案，要么根本没有杆状图案。我们会 确定(目的2)MMP20是否促进成釉细胞运动。我们假设MMP20切割了 钙粘附素的胞外结构域，这是成釉细胞同步成行移动到 形成复杂的交叉釉质棒图案。耐人寻味的是，正是在运动开始的时候 成釉细胞从主要表达E-钙粘蛋白转变为主要表达N-钙粘附素。N-钙粘附素 上皮细胞中的表达促进了细胞的运动。这开启了激动人心的可能性，其中MMP20可以 促进钙粘蛋白的转换，并通过钙粘蛋白的水解促进细胞运动。我们将确定(目标3)是否 成釉细胞中N-钙粘附素的消融破坏了正常的交叉釉质棒模式。我们假设 E-，到N-钙粘附素的转换对于成釉细胞的运动是必不可少的，因此，对于建立 十字交叉的釉质棒。我们的总体假设是，E-到N-钙粘附素的开关允许成釉细胞 横向成排移动以形成交叉釉质棒图案，MMP20通过以下方式促进这一过程 释放这些细胞外钙粘蛋白接触和相关的细胞内信号因子。 项目说明第6页

Principal Investigator/Program Director (Last, first, middle): Bartlett, John, D. The goal of this application is to characterize the role of matrix metalloproteinase-20 (MMP20) and N- cadherin in ameloblast movement and cell-cell attachment during dental enamel development. MMP20 is essential for dental enamel formation. People and mice lacking functional MMP20 have strikingly malformed dental enamel that is thin, soft, and easily abrades from the underlying dentin. In Mmp20 null mice, the secretory stage ameloblasts do not enter the maturation stage of development properly and, once there, the ameloblasts overlap and grow atop one another. This suggests that ameloblast cell-cell attachment and signaling is altered in Mmp20 null mice. Cadherins are a family of proteins that span the cell membrane mediating attachment to identical cadherins present on adjacent cells. p120-catenin (p120) stabilizes cadherins to the cell surface and absence of p120 significantly reduces the presence cell surface cadherins. Previously, we showed that ablation of p120 in mice also results in malformed enamel that abrades from the teeth. Therefore, both MMP20 and cadherins are required for enamel formation. We will determine (AIM 1) how loss of MMP20 affects ameloblast cell-cell interaction. We hypothesize that MMP20 cleaves the extracellular domain of cadherins, which releases intracellular signaling molecules from the disrupted cadherin complex, such as β-catenin, that are essential for enamel formation. Importantly, our preliminary data demonstrate that MMP20 cleaves the extracellular domain of E-cadherin and we propose to test our hypothesis by use of a stably transfected ameloblast derived cell line (ameloblast-lineage cells, ALC) that can be induced to express high levels of activated MMP20. Normal enamel has a decussating (interlacing) rod pattern. Each rod is formed by one ameloblast and each rod preserves a complete record of the migratory path of the ameloblast that formed it. Mmp20 null mouse enamel has either a highly dysplastic rod pattern or no rod pattern at all. We will determine (AIM 2) if MMP20 enhances ameloblast movement. We hypothesize that MMP20 cleaves the extracellular domains of cadherins and that this is required for ameloblasts to move synchronously in rows to form the complex decussating enamel rod patterns. Intriguingly, it is at precisely the initiation of movement that the ameloblasts switch from expressing predominantly E-cadherin to predominantly N-cadherin. N-cadherin expression in epithelial cells promotes cell movement. This opens exciting possibilities wherein MMP20 may facilitate the cadherin switch and facilitate cell movement via cadherin hydrolysis. We will determine (AIM 3) if N-cadherin ablation in ameloblasts disrupts the normal decussating enamel rod pattern. We hypothesize that the E-, to N-cadherin switch is essential for ameloblast movement and, therefore, for establishing the decussating enamel rods. Our overall hypothesis is that the E- to N-cadherin switch allows ameloblasts to move laterally in rows to form the decussating enamel rod pattern and that MMP20 facilitates this process by releasing these extracellular cadherin contacts and associated intracellular signaling factors. Project Description Page 6