Functional Studies of Kallikrein 4

激肽释放酶 4 的功能研究

基本信息

批准号：
7693629
负责人：
JAMES P SIMMER
金额：
$ 36.93万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
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：
2009-09-01 至 2014-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7693629
关键词：
Affect Alleles Ameloblasts Amelogenesis Amelogenesis Imperfecta Amino Acid Sequence Antibodies Biological Assay Cleaved cell Complementary DNA Defect Dental Dental Enamel Dentin Dentinoenamel junction Development Diagnostic radiologic examination Digestion Disease Electron Microscopy Employment Enamel Formation Enzyme Precursors Exhibits Exons Family Family suidae Figs - dietary Galactosidase Gene Targeting Generations Genes Hardness Histocytochemistry Human Hydrolysis Immunohistochemistry In Situ Hybridization In Vitro Incisor Inherited Knock-in Mouse Knock-out Knockout Mice LacZ Genes Learning Light MMP-20 Mandible Maturation-Stage Ameloblast Maxilla Messenger RNA Minerals Mus Mutation Odontoblasts Organ Pathology Patients Pattern Peptide Hydrolases Peptides Phase Pigments Procedures Proteinase-Activated Receptors Proteins Protocols documentation Recombinants Research Residual state Reverse Transcriptase Polymerase Chain Reaction Role Serine Protease Signal Transduction Site Staging Structure System Terminator Codon Testing Thick Tissues Tooth structure Translations Wild Type Mouse amelogenin base cDNA Library enamelin in vivo kallikrein 4 malformation member premature public health relevance stable cell line

项目摘要

DESCRIPTION (provided by applicant): Kallikrein 4 (Klk4) is a serine protease expressed during dental enamel formation. In humans, KLK4 defects cause autosomal recessive, pigmented, hypomaturation amelogenesis imperfecta, a poorly understood condition for which there is no cure. Through gene targeting we have developed a Klk4 knock-out/ss-galactosidase knock-in mouse exhibiting enamel manifestations homologous to the human condition. Unlike wild-type mice, the Klk4 null mice retain enamel proteins in the enamel layer, and the enamel delaminates at or near the dentino-enamel junction. The Klk4 knock-out/ss-galactosidase knock- in mouse offers unique opportunities to better define the temporal and spatial patterns of Klk4 expression and to gain valuable information concerning Klk4's role in dental enamel formation in vivo. Furthermore we have developed efficient procedures for the isolation of matrix metalloproteinase-20 and Klk4 for in vitro analyses. Our overriding hypothesis is that Klk4 is required for enamel maturation and functions as a part of the system for removing enamel proteins that were secreted and partially digested during the secretory stage of amelogenesis. Five specific aims are posed: SA1: To determine the temporal and spatial expression of Klk4 in ameloblasts during the secretory, transition, and maturation stages and in the underlying odontoblasts. SA2: To characterize enamel formation in the absence of Klk4 expression. SA3: To characterize the enzymatic activity of Klk4 on amelogenin, ameloblastin and enamelin. SA4: To investigate the expression of protease activated receptors (PARs) by ameloblasts. SA5: To identify other organs and tissues that express Klk4. The expression of Klk4 in ameloblasts and odontoblasts is determined by histochemistry using the ss- galactosidase (lacZ) expression assay and by immunohistochemistry at the light and electron microscopy levels. The enamel layer of the Klk4 null mouse is characterized by SEM, TEM, radiography, microCT, Knoop microhardness testing, and by determining protein and mineral contents in regularly-spaced increments along the developing maxillary and mandibular incisors. The residual enamel protein in the Klk4 null mouse is extracted and characterized, and digested with Klk4 to learn how Klk4 degrades the organic portion of maturation stage enamel. We determine if PARs are expressed in developing teeth by RT-PCR and in situ hybridization, and determine which PARs can be cleaved by Klk4 using synthetic fluorescent peptides. We expect to prove that Klk4 aggressively cleaves enamel proteins during the maturation stage of amelogenesis and that this function is essential for the proper maturation of enamel crystals. PUBLIC HEALTH RELEVANCE: Kallikrein 4 (Klk4) is a serine protease expressed during dental enamel formation. In humans, defects in the KLK4 gene cause inherited enamel malformations known as autosomal recessive hypomaturation amelogenesis imperfecta, a poorly understood condition for which there is no cure. Through gene targeting we have developed a Klk4 knock-out mouse that has this disease. We will characterize the Klk4 knock-out to better understand the pathology in human patients and hope, eventually, to discover a cure.

描述（由申请人提供）：激肽释放酶4（Klk4）是一种在牙釉质形成过程中表达的丝氨酸蛋白酶。在人类中，KLK4 缺陷会导致常染色体隐性遗传、色素沉着、发育不全的釉质生成不全，这是一种人们知之甚少且无法治愈的疾病。通过基因靶向，我们开发了 Klk4 敲除/ss-半乳糖苷酶敲入小鼠，其牙釉质表现与人类状况同源。与野生型小鼠不同，Klk4缺失小鼠在牙釉质层中保留牙釉质蛋白，并且牙釉质在牙本质-牙釉质交界处或附近分层。 Klk4 敲除/ss-半乳糖苷酶敲入小鼠提供了独特的机会，可以更好地定义 Klk4 表达的时间和空间模式，并获得有关 Klk4 在体内牙釉质形成中的作用的有价值的信息。此外，我们还开发了用于体外分析的基质金属蛋白酶-20 和 Klk4 分离的有效程序。我们最重要的假设是，Klk4 是釉质成熟所必需的，并且作为去除釉质蛋白系统的一部分，这些蛋白在釉质形成的分泌阶段被分泌和部分消化。提出了五个具体目标： SA1：确定 Klk4 在分泌、过渡和成熟阶段以及潜在成牙本质细胞中成釉细胞中的时空表达。 SA2：表征缺乏 Klk4 表达的情况下牙釉质的形成。 SA3：表征 Klk4 对牙釉蛋白、成釉蛋白和牙釉质的酶活性。 SA4：研究成釉细胞表达蛋白酶激活受体（PAR）的情况。 SA5：鉴定表达 Klk4 的其他器官和组织。 Klk4 在成釉细胞和成牙本质细胞中的表达是通过使用 ss-半乳糖苷酶 (lacZ) 表达测定的组织化学以及通过光学和电子显微镜水平的免疫组织化学来确定的。 Klk4 null 小鼠的牙釉质层通过 SEM、TEM、放射线照相、microCT、努普显微硬度测试进行表征，并通过沿发育中的上颌和下颌切牙以规则间隔的增量测定蛋白质和矿物质含量。提取 Klk4 缺失小鼠中残留的牙釉质蛋白并进行表征，并用 Klk4 消化，以了解 Klk4 如何降解成熟阶段牙釉质的有机部分。我们通过 RT-PCR 和原位杂交确定 PAR 是否在发育中的牙齿中表达，并使用合成荧光肽确定哪些 PAR 可以被 Klk4 切割。我们希望证明 Klk4 在釉质形成的成熟阶段积极裂解牙釉质蛋白，并且这种功能对于牙釉质晶体的正常成熟至关重要。公共健康相关性：激肽释放酶 4 (Klk4) 是一种在牙釉质形成过程中表达的丝氨酸蛋白酶。在人类中，KLK4 基因缺陷会导致遗传性牙釉质畸形，称为常染色体隐性遗传性低成熟釉质生成不全症，这是一种人们知之甚少且无法治愈的疾病。通过基因打靶，我们培育出了患有这种疾病的 Klk4 敲除小鼠。我们将描述 Klk4 敲除的特征，以更好地了解人类患者的病理学，并希望最终找到治疗方法。