Enamelysin Processing Mechanisms in Amelogenesis

釉质生成中的釉质加工机制

基本信息

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
项目类别：
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

项目摘要

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）：该补充剂的重点是确定组织蛋白酶K是否对于适当的牙齿牙釉质形成至关重要。原始应用（R01 DE016276）重点是定义namelysin（矩阵金属蛋白酶20; MMP20）如何处理搪瓷蛋白，并着重于确定这种处理如何允许正确的搪瓷开发。这种补充应用扩展并增强了原始研究，因为它的重点是识别以其在骨骼重塑中的作用而闻名的蛋白酶的功能（calter蛋白蛋白酶K），但迄今为止却不称为搪瓷产生细胞（ameleblasts）。在没有变性期间，牙釉质基质蛋白被降解并去除以生成实际的成熟搪瓷的无蛋白质矿物质。蛋白水解加工对于牙釉质形成至关重要，因为植物牙釉质蛋白酶，MMP20或Kallikrein-4（KLK4）都会导致缺陷的低压搪瓷搪瓷。我们研究的长期目标是在牙齿牙釉质达到其最终硬化形式时更好地了解蛋白质降解和去除。在此补充应用中，我们提供了初步数据，表明组织蛋白酶K由成成成成成成成糖，并且其表达随着搪瓷发育的进展而增加。我们还表明，组织蛋白酶在体外迅速降解重组蛋白质。值得注意的是，我们发现与对照组相比，组织蛋白酶k无小鼠的牙釉质硬度大大降低。因此，我们假设组织蛋白酶k积极参与通过成熟阶段的成熟片剂吸收的牙釉质基质蛋白的降解。该提案的总体目标是确定组织蛋白酶K是否是适当牙齿牙釉质发育的必不可少的蛋白酶。为此，我们将确定在发育中的牙齿中总和活跃的组织蛋白酶K的位置（AIM 1）。我们将通过使用免疫金标记将组织蛋白酶K定位在小鼠切牙中，并通过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AIM 2中，我们将确定在组织蛋白酶NULL小鼠中是否改变了搪瓷发育。牙齿形态，体积和密度将通过射线照相和MicroCT分析来检查。搪瓷晶体结构和矿物表面特征将以纳米凹痕和SEM为特征。这些研究的结果将实现我们的总体目标，即了解组织蛋白酶K是否在牙釉质发育中发挥作用。我们还将进一步了解牙釉质如何以蛋白质富含蛋白质的组织开头，但以几乎无蛋白质的矿化硬组织结束。与公共卫生的相关性：这些研究将使人们更好地了解牙齿在牙齿上的形成。这些知识是最终了解如何用遗传畸形牙釉质对待个体的必要第一步。首先，应知道并表征牙釉质形成所需的基因。这些研究也是必要的，以增强组织工程技术最终提供治疗干预措施的可能性。公共卫生相关性：关于如何从成熟的牙齿牙釉质中降解和去除的搪瓷基质蛋白知之甚少。我们表明，负责牙釉质形成的细胞（成纤维细胞）表达一种降解蛋白质的酶（组织蛋白酶K）。该提案试图证明，组织蛋白酶K通过从成熟的搪瓷中去除蛋白质而降解搪瓷基质蛋白在牙釉质形成中起着至关重要的作用。