Enamelysin Processing Mechanisms in Amelogenesis

描述（由申请人提供）：本申请的目的是表征基质金属蛋白酶-20 （MMP20）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MMP20的人和老鼠的牙釉质明显畸形，又薄又软，很容易从底层牙本质上磨损。在Mmp20缺失的小鼠中，分泌期成釉细胞不能正常进入发育的成熟阶段，一旦进入成熟阶段，成釉细胞就会重叠并生长在彼此的上面。这表明在Mmp20缺失的小鼠中，成釉细胞-细胞附着和信号传导发生了改变。钙粘蛋白是一种跨越细胞膜的蛋白家族，介导与相邻细胞上相同的钙粘蛋白的附着。p120-catenin （p120）使钙粘蛋白稳定在细胞表面，p120的缺失显著降低了细胞表面钙粘蛋白的存在。先前，我们发现p120在小鼠中消融也会导致牙釉质畸形，从牙齿上磨损。因此，牙釉质的形成需要MMP20和钙粘蛋白。我们将确定（AIM 1） MMP20的缺失如何影响成釉细胞-细胞相互作用。我们假设MMP20切割了钙粘蛋白的细胞外结构域，钙粘蛋白从被破坏的钙粘蛋白复合体中释放细胞内信号分子，如¿-catenin，这对牙釉质的形成至关重要。重要的是，我们的初步数据表明MMP20切割E-cadherin的细胞外结构域，我们建议通过使用稳定转染的成釉细胞衍生细胞系（成釉细胞谱系细胞，ALC）来验证我们的假设，该细胞系可以诱导表达高水平的活化MMP20。正常的牙釉质呈交错状。每个杆由一个成釉细胞形成，每个杆保存了形成它的成釉细胞迁移路径的完整记录。Mmp20缺失小鼠牙釉质要么具有高度发育不良的棒状图案，要么根本没有棒状图案。我们将确定MMP20是否能增强成釉细胞的运动。我们假设MMP20切割了钙粘蛋白的细胞外结构域，这是成釉细胞同步成行移动以形成复杂的牙釉质棒模式所必需的。有趣的是，正是在运动开始时，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。上皮细胞中N-cadherin的表达促进细胞运动。这开启了令人兴奋的可能性，其中MMP20可能促进钙粘蛋白开关，并通过钙粘蛋白水解促进细胞运动。我们将确定（AIM 3） n -钙粘蛋白消融在成釉细胞中是否会破坏正常的牙釉质棒模式。我们假设E-到n -钙粘蛋白的开关对于成釉细胞的运动是必不可少的，因此，对于建立相互作用的搪瓷棒是必不可少的。我们的总体假设是，E-到n -钙粘蛋白开关允许成釉细胞横向成行移动，形成牙釉质棒模式，MMP20通过释放这些细胞外钙粘蛋白接触和相关的细胞内信号因子促进了这一过程。

DESCRIPTION (provided by applicant): 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 ¿-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.