Enamelysin processing mechanisms in amelogenesis

釉质形成中的釉质溶解加工机制

• 批准号: 9225454
• 负责人: JOHN D BARTLETT
• 金额: $ 2.52万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-02-16 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9225454
• 关键词: Enamelysin; processing; mechanisms; amelogenesis

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

首席研究员/项目总监（最后、第一、中间）：Bartlett, John, D. 本应用的目标是表征基质金属蛋白酶 20 (MMP20) 和 N-的作用 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。基质金属蛋白酶20 对于牙釉质的形成至关重要。缺乏功能性 MMP20 的人和小鼠具有惊人的畸形 牙釉质薄、软且容易从下面的牙本质磨损。在 Mmp20 缺失小鼠中， 分泌阶段的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， 成釉细胞相互重叠并生长。这表明成釉细胞-细胞附着和 Mmp20 缺失小鼠中的信号传导发生改变。钙粘蛋白是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 的缺失会显着降低细胞表面钙粘蛋白的存在。之前， 我们发现，小鼠体内 p120 的消融也会导致牙釉质畸形，从而导致牙齿磨损。 因此，MMP20 和钙粘蛋白都是牙釉质形成所必需的。我们将确定（AIM 1）损失如何 MMP20 影响成釉细胞与细胞之间的相互作用。我们假设 MMP20 裂解细胞外 钙粘蛋白结构域，从破坏的钙粘蛋白复合物中释放细胞内信号分子， 例如β-连环蛋白，它对于牙釉质的形成至关重要。重要的是，我们的初步数据表明 MMP20 裂解 E-钙粘蛋白的细胞外结构域，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），可被诱导表达 高水平的活化 MMP20。正常牙釉质具有交叉（交错）棒状图案。每根杆都形成 由一个成釉细胞组成，每根杆都保留了成釉细胞迁移路径的完整记录 形成了它。 Mmp20 缺失小鼠牙釉质要么具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 是否增强成釉细胞运动。我们假设 MMP20 裂解 钙粘蛋白的细胞外结构域，这是成釉细胞同步移动到行中所必需的 形成复杂的交叉牙釉质棒图案。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-钙粘蛋白 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) 如果 成釉细胞中的 N-钙粘蛋白消融破坏了正常的交叉牙釉质棒模式。我们假设 E-钙粘蛋白到N-钙粘蛋白的转换对于成釉细胞运动至关重要，因此，对于建立 交错的牙釉质棒。我们的总体假设是 E-钙粘蛋白到 N-钙粘蛋白的转换允许成釉细胞 成排横向移动以形成交叉牙釉质棒图案，并且 MMP20 通过以下方式促进此过程 释放这些细胞外钙粘蛋白接触和相关的细胞内信号因子。 项目描述第6页

项目成果

Kallikrein-related peptidase-4 (KLK4): role in enamel formation and revelations from ablated mice.

与Kallikrein相关的肽酶4（KLK4）：在消融小鼠的搪瓷形成和启示中的作用。

• DOI: 10.3389/fphys.2014.00240
• 发表时间: 2014
• 期刊: Frontiers in physiology
• 影响因子: 4
• 作者: Bartlett JD;Simmer JP
• 通讯作者: Simmer JP

Why Does Enamel in Klk4-Null Mice Break above the Dentino-Enamel Junction?

• DOI: 10.1159/000324260
• 发表时间: 2011-01-01
• 期刊: CELLS TISSUES ORGANS
• 影响因子: 2.7
• 作者: Simmer, James P.;Hu, Yuanyuan;Hu, Jan C. -C.
• 通讯作者: Hu, Jan C. -C.

Transcription factor FoxO1 is essential for enamel biomineralization.

• DOI: 10.1371/journal.pone.0030357
• 发表时间: 2012
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Poché RA;Sharma R;Garcia MD;Wada AM;Nolte MJ;Udan RS;Paik JH;DePinho RA;Bartlett JD;Dickinson ME
• 通讯作者: Dickinson ME

Cleavage site specificity of MMP-20 for secretory-stage ameloblastin.

• DOI: 10.1177/0022034510366903
• 发表时间: 2010-08
• 期刊: Journal of dental research
• 影响因子: 7.6
• 作者: Chun YH;Yamakoshi Y;Yamakoshi F;Fukae M;Hu JC;Bartlett JD;Simmer JP
• 通讯作者: Simmer JP

MMP20 and KLK4 activation and inactivation interactions in vitro.

• DOI: 10.1016/j.archoralbio.2013.08.005
• 发表时间: 2013-11
• 期刊: ARCHIVES OF ORAL BIOLOGY
• 影响因子: 3
• 作者: Yamakoshi, Yasuo;Simmer, James P.;Bartlett, John D.;Karakida, Takeo;Oida, Shinichiro
• 通讯作者: Oida, Shinichiro