Enamelysin Processing Mechanisms in Amelogenesis

釉质生成中的釉质加工机制

• 批准号: 7818106
• 负责人: JOHN D BARTLETT
• 金额: $ 31.63万
• 依托单位: ADA FORSYTH INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-21 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7818106
• 关键词: Address; Ameloblasts; Amelogenesis; Amelogenesis Imperfecta; Biological Assay; Bone remodeling; Cells; Characteristics; Data; Dental Enamel; Dental Enamel Hypoplasia; Dentin; Development; Diagnostic radiologic examination; Dimensions; Enamel Formation; Enamel Organ; Enzymes; Excision; Exhibits; Extracellular Matrix; Figs - dietary; Funding; Genes; Goals; Hardness; Housing; Human; In Vitro; Incisor; Individual; Knockout Mice; Knowledge; Label; Lead; Letters; Location; MMP-20; Maturation-Stage Ameloblast; Minerals; Morphology; Mus; Mutation; Organelles; Outcome; Patients; Peptide Hydrolases; Pigments; Play; Positioning Attribute; Process; Proteins; Proteolysis; Proteolytic Processing; Public Health; Publishing; Recombinants; Recovery; Research; Research Personnel; Role; Structure; Surface; Techniques; Testing; Therapeutic Intervention; Time; Tissue Engineering; Tissues; Tooth structure; United States National Institutes of Health; Work; amelogenin; base; cathepsin K; density; enamel matrix proteins; enamelysin; experience; extracellular; kallikrein 4; nanoindentation; protein degradation; public health relevance; treatment strategy

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.

描述（由申请人提供）：本补充的重点是确定组织蛋白酶K是否是正确的牙釉质形成所必需的。最初的申请（R 01 DE 016276）集中于定义釉质溶解素（基质金属蛋白酶-20; MMP 20）如何加工釉质蛋白，并集中于确定这种加工如何允许适当的釉质发育。该补充申请扩展并增强了原始研究，因为其集中于鉴定蛋白酶（组织蛋白酶K）的功能，所述蛋白酶（组织蛋白酶K）因其在骨重塑中的作用而众所周知，但迄今为止不知道由釉质产生细胞（成釉细胞）表达。在釉质形成过程中，釉质基质蛋白被降解和去除，以产生成熟釉质的几乎不含蛋白质的矿物质。蛋白水解加工对于釉质形成是至关重要的，因为固有釉质蛋白酶MMP 20或激肽释放酶-4（KLK 4）中的任一种的纯合突变导致有缺陷的低矿化釉质。我们研究的长期目标是更好地了解牙釉质达到其最终硬化形式时蛋白质的降解和去除。在本补充申请中，我们提供了初步数据，证明组织蛋白酶K由成釉细胞表达，并且其表达随着釉质发育的进展而增加。我们还表明，组织蛋白酶K在体外快速降解重组釉原蛋白。值得注意的是，我们发现组织蛋白酶K缺失小鼠与对照组相比，牙釉质硬度显著降低。因此，我们假设组织蛋白酶K积极参与降解的釉基质蛋白被吸收的成熟阶段成釉细胞。本建议的总体目标是确定组织蛋白酶K是否是正常牙釉质发育所必需的蛋白酶。为此，我们将确定总的和活性组织蛋白酶K在发育中的牙齿中的位置（目标1）。我们将使用免疫金标记定位组织蛋白酶K在小鼠切牙，并将确定活性组织蛋白酶K的位置，通过使用催化组织化学测定。在目标2中，我们将确定在组织蛋白酶K缺失小鼠中釉质发育是否改变。将通过X线摄影和microCT分析检查牙齿形态、体积和密度。牙釉质的晶体结构和矿物表面特征将通过纳米压痕和SEM表征。这些研究的结果将实现我们的总体目标，了解组织蛋白酶K是否和/或在多大程度上在釉质发育中发挥作用。我们还将进一步了解牙釉质是如何开始作为一个富含蛋白质的组织，但最终作为一个几乎不含蛋白质的矿化硬组织。与公共卫生的相关性：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畸形牙釉质的必要的第一步。在开始时，釉质形成所需的基因应该是已知的，并得到很好的表征。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。 公共卫生相关性：关于釉质基质蛋白如何从成熟的牙釉质中降解和去除的知之甚少。我们表明，细胞（成釉细胞）负责釉质形成，表达一种酶（组织蛋白酶K），降解蛋白质。该提议试图证明组织蛋白酶K通过降解釉质基质蛋白在釉质形成中起重要作用，因为蛋白质从成熟的釉质中去除。