Enamelysin Processing Mechanisms in Amelogenesis

描述(申请人提供)：本申请的目标是表征基质金属蛋白酶-20(MMP20)和N-钙粘附素在成釉细胞移动和细胞-细胞附着过程中的作用。MMP20是牙釉质形成所必需的。缺乏功能性MMP20的人和小鼠有明显的畸形牙釉质，它很薄，很软，很容易从下面的牙本质中磨损。在MMP20基因缺失的小鼠中，分泌期的成釉细胞没有正确地进入发育的成熟阶段，一旦进入成釉细胞成熟阶段，成釉细胞就会重叠并相互重叠生长。这表明，在MMP20基因缺失的小鼠中，成釉细胞细胞-细胞附着和信号传递发生了变化。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-catenin(P120)使钙粘附素稳定在细胞表面，缺乏p120显著减少了细胞表面钙粘附素的存在。此前，我们在小鼠身上发现，p120的消融也会导致畸形的釉质从牙齿上磨损。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)MMP20的缺失如何影响成釉细胞细胞-细胞间的相互作用。我们假设MMP20裂解钙粘蛋白的胞外区域，从被破坏的钙粘蛋白复合体中释放出细胞内的信号分子，如β-连环蛋白，这是釉质形成所必需的。重要的是，我们的初步数据表明，MMP20可以裂解E-钙粘蛋白的胞外区，我们建议使用稳定转染的成釉细胞衍生细胞系(成釉细胞系细胞，ALC)来验证我们的假设，该细胞系可以被诱导表达高水平的激活的MMP20。正常牙釉质有交叉(交错)的杆状图案。每个成釉细胞由一个成釉细胞组成，每个成釉细胞保存了形成成釉细胞的迁移路径的完整记录。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。我们将确定(目标2)MMP20是否促进成釉细胞运动。我们假设MMP20切割钙粘附素的胞外区，这是成釉细胞同步成行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。N-钙粘附素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MMP20可以促进钙粘蛋白的开关，并通过钙粘蛋白水解酶促进细胞运动。我们将确定(目标3)成釉细胞中N-钙粘附素的消融是否会扰乱正常的交叉釉质棒模式。我们推测，E-到N-钙粘素的转换对于成釉细胞的运动是必不可少的，因此，对于建立交叉的釉质棒是必要的。我们的总体假设是，E-to-N-钙粘蛋白开关允许成釉细胞横向成排移动，形成交叉的釉质棒模式，MMP20通过释放这些细胞外钙粘素接触和相关的细胞内信号因子来促进这一过程。

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.