Functional Studies of Kallikrein 4

激肽释放酶 4 的功能研究

• 批准号: 8074502
• 负责人: JAMES P SIMMER
• 金额: $ 34.39万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8074502
• 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

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）：钾化酶4 （Klk4）是牙釉质形成过程中表达的丝氨酸蛋白酶。在人类中，KLK4缺陷导致常染色体隐性、色素沉着、不成熟的无染色体发育不全，这是一种尚不清楚且无法治愈的疾病。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，其牙釉质表现与人类相似。与野生型小鼠不同，Klk4缺失小鼠在牙釉质层中保留了牙釉质蛋白，并且牙釉质在牙本质-牙釉质连接处或附近分层。Klk4敲除/ss-半乳糖苷酶敲入小鼠提供了独特的机会，以更好地定义Klk4表达的时空模式，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，我们已经开发了有效的方法分离基质金属蛋白酶-20和Klk4进行体外分析。我们最重要的假设是，Klk4是牙釉质成熟所必需的，并作为系统的一部分，在成釉发生的分泌阶段去除分泌和部分消化的牙釉质蛋白。本研究提出了五个具体目标：SA1：确定Klk4在成釉细胞分泌、转化和成熟阶段以及潜在的成牙细胞中的时空表达。SA2：在没有Klk4表达的情况下表征牙釉质的形成。SA3：表征Klk4对成釉素、成釉素和釉素的酶活性。目的：探讨蛋白酶激活受体（PARs）在成釉细胞中的表达。SA5：鉴定其他表达Klk4的器官和组织。Klk4在成釉细胞和成牙细胞中的表达是通过半乳糖苷酶（lacZ）表达法和免疫组织化学在光镜和电镜下测定的。通过扫描电镜（SEM）、透射电镜（TEM）、x线摄影（radiography）、显微ct （microCT）、Knoop显微硬度测试，以及沿发育中的上颌和下颌门牙有规律间隔地测定蛋白质和矿物质含量，对Klk4空白小鼠的牙釉质层进行了表征。对Klk4缺失小鼠的残牙釉质蛋白进行提取和表征，并用Klk4进行消化，了解Klk4如何降解成熟期牙釉质的有机部分。我们通过RT-PCR和原位杂交来确定PARs是否在发育中的牙齿中表达，并使用合成的荧光肽来确定哪些PARs可以被Klk4切割。我们希望证明Klk4在成釉发育成熟阶段积极地切割釉质蛋白，这一功能对于釉质晶体的适当成熟是必不可少的。