Enamelysin Processing Mechanisms in Amelogenesis

描述（由申请人提供）：本补充品的重点是确定组织蛋白酶K对牙釉质形成是否必要。最初的申请（R01 DE016276）侧重于确定釉质蛋白酶（matrix metalloproteinase-20; MMP20）如何处理釉质蛋白，并侧重于确定这种处理如何允许适当的釉质发育。这一补充应用扩展和增强了原始研究，因为它的重点是确定蛋白酶（cathepsin K）的功能，蛋白酶（cathepsin K）以其在骨重塑中的作用而闻名，但迄今为止还不知道它是否在牙釉质生成细胞（成釉细胞）中表达。在成釉过程中，牙釉质基质蛋白被降解和去除，生成成熟牙釉质中几乎不含蛋白质的矿物质。蛋白水解过程对牙釉质的形成至关重要，因为牙釉质蛋白酶MMP20或钾化钾素-4 （KLK4）的纯合突变会导致牙釉质低矿化缺陷。我们研究的长期目标是更好地理解牙釉质最终硬化时蛋白质的降解和去除。在这个补充申请中，我们提供了初步数据，证明组织蛋白酶K在成釉细胞中表达，并且随着釉质发育的进展其表达增加。我们还表明组织蛋白酶K在体外快速降解重组淀粉原蛋白。值得注意的是，我们发现与对照组相比，组织蛋白酶K缺失小鼠的牙釉质硬度显著降低。因此，我们假设组织蛋白酶K积极参与成熟成釉细胞吸收的釉质基质蛋白的降解。本提案的总体目标是确定组织蛋白酶K是否是牙釉质发育所必需的蛋白酶。为此，我们将确定总组织蛋白酶K和活性组织蛋白酶K在发育中的牙齿中的位置（目的1）。我们将使用免疫金标记法在小鼠门牙中定位组织蛋白酶K，并将使用催化组织化学测定法确定活性组织蛋白酶K的位置。在第二阶段，我们将确定组织蛋白酶K缺失小鼠的牙釉质发育是否发生改变。牙齿形态、体积和密度将通过x线摄影和微ct分析进行检查。利用纳米压痕和扫描电镜对釉质晶体结构和矿物表面特征进行表征。这些研究的结果将实现我们了解组织蛋白酶K是否和/或在多大程度上在牙釉质发育中起作用的总体目标。我们还将进一步了解牙釉质是如何从富含蛋白质的组织开始，但最终成为几乎不含蛋白质的矿化硬组织的。与公众健康相关：这些研究将有助于更好地了解牙釉质是如何在牙齿上形成的。这些知识是最终了解如何治疗遗传牙釉质畸形的必要的第一步。首先，牙釉质形成所需的基因应该被了解和充分表征。这些研究对于提高组织工程技术最终提供治疗干预的可能性也是必要的。

DESCRIPTION (provided by applicant): The focus of this supplement is to determine if cathepsin K is essential for proper dental enamel formation. The original application (R01 DE016276) focused on defining how enamelysin (matrix metalloproteinase-20; MMP20) processes enamel proteins and focused on determining how such processing allows for proper enamel development. This supplemental application extends and enhances the original studies because it is focused on identifying the function of a protease (cathepsin K) that is well known for its role in bone remodeling, but was heretofore not known as expressed by enamel producing cells (ameloblasts). During amelogenesis, enamel matrix proteins are degraded and removed to generate the virtually protein-free mineral of mature enamel. Proteolytic processing is critical for enamel formation as homozygous mutation of either of the resident enamel proteases, MMP20 or kallikrein-4 (KLK4), results in defective hypomineralized enamel. The long-term goal of our research is to achieve a better understanding of protein degradation and removal as dental enamel attains its final hardened form. In this supplemental application we provide preliminary data demonstrating that cathepsin K is expressed by ameloblasts and that its expression increases as enamel development progresses. We also show that cathepsin K rapidly degrades recombinant amelogenin in vitro. Notably, we discovered that cathepsin K null mice have significantly reduced enamel hardness when compared to controls. Thus, we hypothesize that cathepsin K actively participates in the degradation of enamel matrix proteins that are resorbed by maturation stage ameloblasts. The overall objective of this proposal is to determine if cathepsin K is an essential protease for proper dental enamel development. To this end, we will identify the location of total and active cathepsin K within the developing tooth (Aim 1). We will localize cathepsin K in mouse incisors by use of immunogold labeling and will identify the location of active cathepsin K by use of a catalytic histochemical assay. In Aim 2 we will determine if enamel development is altered in the cathepsin K null mouse. Tooth morphology, volume, and density will be examined by radiography and microCT analysis. Enamel crystal structure and mineral surface characteristics will be characterized by nanoindentation and SEM. Results from these studies will accomplish our overall objective of understanding if, and/or to what extent, cathepsin K plays a role in enamel development. We will also further our knowledge of how enamel starts as a protein rich tissue, but ends as a virtually protein-free mineralized hard tissue. Relevance to public health: These studies will lead to a better understanding of how dental enamel forms on teeth. This knowledge is a necessary first step to eventually understand how to treat individuals with genetically malformed dental enamel. At the outset, the genes required for enamel formation should be known and well characterized. These studies are also necessary to enhance the possibility that tissue engineering techniques will eventually provide therapeutic interventions. PUBLIC HEALTH RELEVANCE: Little is known about how enamel matrix proteins are degraded and removed from maturing dental enamel. We show that the cells (ameloblasts) responsible for enamel formation, express an enzyme (cathepsin K) that degrades proteins. This proposal seeks to demonstrate that cathepsin K plays an essential role in enamel formation by degrading enamel matrix proteins as the proteins are removed from the maturing enamel.