Enamelysin processing mechanisms in amelogenesis

主要研究者/项目负责人（最后，第一，中间）：Bartlett，John，D. 本申请的目的是表征基质金属蛋白酶-20（MMP 20）和N- 钙粘蛋白在牙釉质发育过程中成釉细胞运动和细胞-细胞附着中的作用。MMP20 对牙釉质的形成至关重要。缺乏功能性MMP 20的人和小鼠具有惊人的畸形 薄、软且容易从下面的牙本质磨损的牙釉质。在Mmp 20敲除小鼠中， 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， 成釉细胞重叠并在彼此之上生长。这表明成釉细胞-细胞附着和 在Mmp 20缺失小鼠中信号传导改变。钙粘蛋白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蛋白的附着。p120-连环蛋白（p120）稳定钙粘蛋白 p120的缺乏显著减少了细胞表面钙粘蛋白的存在。在此之前， 我们表明，小鼠中p120的消融也导致从牙齿磨损的畸形牙釉质。 因此，MMP 20和钙粘蛋白都是釉质形成所必需的。我们将确定（目标1）如何损失 MMP 20的表达影响成釉细胞间的相互作用。我们假设MMP 20切割细胞外的 钙粘蛋白的结构域，其从被破坏的钙粘蛋白复合物释放细胞内信号分子， 例如β-连环蛋白，其对于釉质形成是必需。重要的是，我们的初步数据表明， MMP 20切割E-钙粘蛋白的细胞外结构域，我们建议通过使用一个 稳定转染的成釉细胞来源的细胞系（成釉细胞系细胞，ALC），其可以被诱导表达 高水平的活化MMP 20。正常的牙釉质有交叉（交错）杆模式。每根杆都是由 每个棒保存了成釉细胞迁移路径的完整记录， Mmp 20无效小鼠牙釉质具有高度发育异常的杆图案或根本没有杆图案。我们将 确定（AIM 2）MMP 20是否增强成釉细胞运动。我们假设MMP 20切割了 细胞外结构域的钙粘蛋白，这是所需的成釉细胞同步移动行， 形成复杂的交叉釉质棒图案。有趣的是，正是在运动的开始， 成釉细胞从主要表达E-钙粘蛋白转变为主要表达N-钙粘蛋白。N-cadherin 在上皮细胞中的表达促进细胞运动。这开启了令人兴奋的可能性，其中MMP 20可以 促进钙粘蛋白转换并通过钙粘蛋白水解促进细胞运动。我们将确定（AIM 3），如果 成釉细胞中的N-钙粘蛋白消融破坏了正常的交叉釉质棒模式。我们假设 E-钙粘蛋白到N-钙粘蛋白的转换对于成釉细胞的运动是必不可少的，因此， 交叉的釉质棒我们的总体假设是，E-到N-钙粘蛋白开关允许成釉细胞， 以行的方式横向移动以形成交叉釉质棒图案，MMP 20通过以下方式促进该过程： 释放这些细胞外钙粘蛋白接触和相关的细胞内信号传导因子。 项目描述第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