Functional Studies of Kallikrein 4

激肽释放酶 4 的功能研究

基本信息

批准号：
8272467
负责人：
JAMES P SIMMER
金额：
$ 35.1万
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/8272467
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 Immunohistochemistry In Situ Hybridization In Vitro Incisor Inherited Knock-in Mouse Knock-out Knockout Mice LacZ Genes Learning 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 light microscopy 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）：Kallikrein 4（KLK4）是牙齿搪瓷形成期间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，色素沉着，不饱和的ameleogenes imperfecta，这是一种无法治愈的疾病。通过基因靶向，我们开发了一种KLK4敲除/SS-半乳糖苷酶敲除小鼠，其表现出与人类状况同源的搪瓷表现。与野生型小鼠不同，KLK4无效的小鼠保留在牙釉质层中的搪瓷蛋白，并且牙釉质在丹蒂诺 - 内胺交界处或附近分离。小鼠中的KLK4敲除/SS-半乳糖苷酶敲除可以更好地定义KLK4表达的时间和空间模式，并获得有关KLK4在体内牙齿搪瓷形成中的作用的宝贵信息。此外，我们为分离基质金属蛋白酶-20和KLK4开发了有效的程序，用于体外分析。我们的压倒性假设是，KLK4是牙釉质成熟所必需的，并且是去除在没有分泌阶段的分泌阶段分泌并部分消化的系统系统中的一部分。提出了五个特定的目标：SA1：确定分泌，过渡和成熟阶段以及基础的牙本质细胞中KLK4的时间和空间表达。 SA2：在没有KLK4表达的情况下表征搪瓷的形成。 SA3：表征KLK4对氨基蛋白蛋白，杏仁蛋白和搪瓷蛋白的酶活性。 SA4：通过成成布研究蛋白酶活化受体（PAR）的表达。 SA5：识别表达KLK4的其他器官和组织。 KLK4在成成布和odontoblasts中的表达通过使用SS-半乳糖苷酶（LACZ）表达测定法和通过光和电子显微镜水平的免疫组织化学来确定。 KLK4无效小鼠的搪瓷层的特征是SEM，TEM，X射线照相，微观，小刀片微硬度测试，并通过沿着发育中的上皮和下颌切牙的正常间隔来确定蛋白质和矿物质含量。提取和表征KLK4无效小鼠中的残留牙釉质蛋白，并用KLK4消化，以了解KLK4如何降解成熟阶段搪瓷的有机部分。我们确定在通过RT-PCR和原位杂交发育中表达PARS是否表达了PAR，并确定使用合成荧光肽可以通过KLK4裂解哪些PAR。我们预计，KLK4会在增生的成熟阶段积极切割牙釉质蛋白，并且该功能对于搪瓷晶体的适当成熟至关重要。公共卫生相关性：Kallikrein 4（KLK4）是在牙齿搪瓷形成期间表达的丝氨酸蛋白酶。在人类中，KLK4基因中的缺陷导致遗传的搪瓷畸形被称为常染色体隐性膜性不饱和ameleogeness imperfecta，这是一种无法治愈的疾病。通过基因靶向，我们开发了一种具有这种疾病的KLK4敲除小鼠。我们将表征KLK4淘汰赛，以更好地了解人类患者的病理，并希望最终发现治疗方法。