Enamelysin Processing Mechanisms in Amelogenesis

描述（由申请方提供）：本申请的目的是表征牙釉质发育过程中基质金属蛋白酶-20（MMP 20）和N-钙粘蛋白在成釉细胞运动和细胞-细胞附着中的作用。MMP 20是牙釉质形成所必需的。缺乏功能性MMP 20的人和小鼠具有明显畸形的牙釉质，其薄、软且容易从下面的牙本质磨损。在Mmp 20缺失小鼠中，分泌期成釉细胞没有正常进入发育的成熟阶段，一旦进入成熟阶段，成釉细胞重叠并在彼此之上生长。这表明成釉细胞-细胞附着和信号传导在Mmp 20缺失小鼠中改变。钙粘蛋白是跨细胞膜的蛋白质家族，其介导与存在于相邻细胞上的相同钙粘蛋白的附着。p120-连环蛋白（p120）使钙粘蛋白稳定在细胞表面，而p120的缺失显著减少了细胞表面钙粘蛋白的存在。以前，我们发现，在小鼠中消融p120也会导致牙釉质畸形，从牙齿上磨损。因此，MMP 20和钙粘蛋白都是釉质形成所必需的。我们将确定（AIM 1）MMP 20的缺失如何影响成釉细胞-细胞相互作用。我们假设MMP 20切割钙粘蛋白的细胞外结构域，从而从被破坏的钙粘蛋白复合物中释放出细胞内信号分子，如对釉质形成至关重要的连环蛋白。重要的是，我们的初步数据表明，MMP 20切割E-钙粘蛋白的胞外结构域，我们建议通过使用稳定转染的成釉细胞衍生的细胞系（成釉细胞系细胞，ALC），可以诱导表达高水平的活化MMP 20来测试我们的假设。正常的牙釉质有交叉（交错）杆模式。每个棒由一个成釉细胞形成，并且每个棒保存形成它的成釉细胞的迁移路径的完整记录。Mmp 20无效小鼠釉质具有高度发育异常的棒图案或根本没有棒图案。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切割钙粘蛋白的胞外结构域，这是成釉细胞在行中同步移动以形成复杂的交叉釉质棒图案所必需的。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。上皮细胞中的N-钙粘蛋白表达促进细胞运动。这开启了令人兴奋的可能性，其中MMP 20可以促进钙粘蛋白开关并通过钙粘蛋白水解促进细胞运动。我们将确定（AIM 3）成釉细胞中的N-钙粘蛋白消融是否破坏了正常的交叉釉质棒模式。我们推测，E-，N-钙粘蛋白开关是必不可少的成釉细胞的运动，因此，建立交叉釉质棒。我们的总体假设是，E-到N-钙粘蛋白开关允许成釉细胞横向移动行，形成交叉釉质棒模式，MMP 20促进这一过程中释放这些细胞外钙粘蛋白的联系和相关的细胞内信号传导因子。

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.