Functional Studies of Kallikrein 4

激肽释放酶 4 的功能研究

• 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
• 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

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（Klk 4）是在牙釉质形成期间表达的丝氨酸蛋白酶。在人类中，KLK 4缺陷导致常染色体隐性遗传、色素沉着、发育不良的釉质发生障碍，这是一种了解不多的疾病，目前尚无治愈方法。通过基因靶向，我们已经开发了Klk 4基因敲除/β-半乳糖苷酶基因敲入小鼠，其表现出与人类状况同源的釉质表现。与野生型小鼠不同，Klk 4缺失小鼠在釉质层中保留釉质蛋白，并且釉质在牙本质-釉质连接处或附近分层。Klk 4敲除/β-半乳糖苷酶敲入小鼠提供了独特的机会来更好地定义Klk 4表达的时间和空间模式，并获得关于Klk 4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程序，用于分离基质金属蛋白酶-20和Klk 4的体外分析。我们最重要的假设是Klk 4是釉质成熟所需的，并且作为用于去除釉质蛋白的系统的一部分起作用，所述釉质蛋白在釉质发生的分泌阶段期间被分泌和部分消化。提出了五个具体目标：SA1：确定Klk 4在成釉细胞分泌、过渡和成熟阶段以及在基础成牙本质细胞中的时空表达。SA 2：表征在Klk 4表达不存在下的釉质形成。SA 3：表征Klk 4对釉原蛋白、成釉蛋白和釉蛋白的酶活性。SA 4：研究成釉细胞蛋白酶激活受体（PARs）的表达。SA 5：鉴定表达Klk 4的其他器官和组织。Klk 4在成釉细胞和成牙本质细胞中的表达通过使用β-半乳糖苷酶（lacZ）表达测定的组织化学和通过光镜和电镜水平的免疫组织化学来确定。Klk 4缺失小鼠的釉质层通过SEM、TEM、射线照相术、microCT、努氏显微硬度测试以及通过测定沿发育中的上颌骨和下颌切牙沿着规则间隔增量的蛋白质和矿物质含量来表征。提取Klk 4缺失小鼠中的残留釉质蛋白并表征，并用Klk 4消化以了解Klk 4如何降解成熟阶段釉质的有机部分。我们通过RT-PCR和原位杂交确定PARs是否在发育中的牙齿中表达，并使用合成的荧光肽确定哪些PARs可以被Klk 4切割。我们期望证明Klk 4在釉质形成的成熟阶段积极地切割釉质蛋白，并且这种功能对于釉质晶体的适当成熟至关重要。 激肽释放酶4（Klk 4）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，KLK 4基因的缺陷会导致遗传性釉质畸形，称为常染色体隐性遗传性釉质发育不良，这是一种知之甚少的疾病，目前还没有治愈方法。通过基因靶向，我们已经开发出了患有这种疾病的Klk 4敲除小鼠。我们将描述Klk 4敲除的特征，以更好地了解人类患者的病理学，并希望最终找到治愈方法。