The Role of Leucine-Rich Amelogenin Peptide (LRAP) in Enamel Formation

富含亮氨酸的釉原肽 (LRAP) 在牙釉质形成中的作用

• 批准号: 7788063
• 负责人: Thuan Quoc Le
• 金额: $ 11.59万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-05 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7788063
• 关键词: Ameloblasts; Applications Grants; Atomic Force Microscopy; Attention; Automobile Driving; Biomedical Engineering; Calvaria; Cell Differentiation process; Cell Proliferation; Cells; Cellular Morphology; Complementary DNA; Cytokeratin-14 Staining Method; Data; Dental; Dental Enamel; Dentin; Development; Electron Microscopy; Enamel Formation; Enamel Organ; Epithelial; Epithelial Cells; Escherichia coli; Extracellular Matrix; Eye; Gene Expression; Genes; Genetic; Goals; Histocompatibility Testing; Histocytochemistry; Human; Immunochemistry; In Situ Hybridization; In Vitro; Incisor; Intervention; Knockout Mice; Lentivirus Vector; MMP-20; Mastication; Mesenchymal; Messenger RNA; Minerals; Modeling; Mus; Natural regeneration; Nodule; Odontoblasts; Organ; Phenotype; Play; Population; Process; Property; Protein Biosynthesis; Proteins; RNA Splicing; Raman Spectrum Analysis; Recombinants; Regulation; Reporter Genes; Resistance; Role; Scanning; Scanning Electron Microscopy; Signal Pathway; Signal Transduction; Signaling Molecule; Sorting - Cell Movement; Staging; Stellate Reticulum; Stratum Intermedium; System; Testing; Tetanus Helper Peptide; Tissues; Tooth Germ; Tooth structure; Transgenic Mice; Trichrome stain; Western Blotting; amelogenin; brain cell; enamel matrix proteins; expression vector; extracellular; in vivo; leucine-rich amelogenin peptide; mineralization; mouse model; overexpression; peptide E (adrenal medulla); promoter; public health relevance; reconstitution; vector

DESCRIPTION (provided by applicant): Amelogenins, secreted by ameloblasts, are the predominant proteins in the enamel extracellular matrix, regulating enamel formation. Leucine-rich amelogenin peptide (LRAP) is one of the most abundant alternatively spliced amelogenins. LRAP has been shown to be present in both dental epithelial and mesenchymal tissues, suggesting that it has a role in regulation of odontogenic cell proliferation and/or differentiation. Our preliminary data have shown that LRAP promotes enamel organ epithelial cells to differentiate in vitro. In addition, our results indicated that amelogenin-null mice showed disorganized enamel epithelial cells, characterized by poorly defined stratum intermedium and stellate reticulum, and lacked of secretory ameloblasts without evidence of Tomes' processes and limiting enamel matrix secretion. In contrast, LRAP overexpressor in amelogenin-null (LRAP+/null) background rescued the phenotype of stratum intermedium, stellate reticulum, and restored enamel matrix secretion. However, LRAP+/null mice had a delayed in enamel mineralization, associated with evidence of retained enamel matrix proteins, suggesting that LRAP does not promote mineralization, but only acts as a signaling molecule. In this grant proposal, we will explore the signaling role of LRAP, both in vivo and in vitro. The overall hypothesis of these studies is that LRAP promotes enamel organ epithelial cell differentiation. Our specific aims are as follows: Aim 1:To determine the effect of LRAP on differentiation of enamel organ epithelial cell in vitro. Enamel matrix gene expression, protein synthesis, and in vitro mineral formation by ex vivo human enamel organs and dental epithelial cells treated with exogenous addition of recombinant human LRAP, stably transfected with Tet-regulated promotor within epithelial cell-specific mammalian expression vector containing cDNA encoding for LRAP will be compared to the controls without LRAP intervention. The use of LRAP overexpression in dental epithelial cells will determine the effect of posttranslational modified LRAP on cell differentiation as compared to the unposttranslational modified LRAP from E. coli. The incorporation of Tet- responsive system within mammalian expression vector will allow the regulation of LRAP signaling to promote dental epithelial cell differentiation. Aim 2: To determine the effect of LRAP on enamel epithelial cell differentiation and enamel formation in vivo. Molars and incisors of wild-type, amelogenin-null mice and transgenic mice, including LRAP over- expressors in wild-type (LRAP+/WT) and amelogenin-null (LRAP+/null) backgrounds will be examined under H&E and Trichrome staining for differences in enamel organ cellular morphology and matrix at different stages of development. Alterations in expression of enamel matrix proteins related to enamel epithelial cell differentiation will be analyzed at both mRNA and protein levels. Enamel nanohardness properties of these mice will be compared using atomic force microscopy (AFM), and enamel structural differences will be examined with scanning electron microscopy (SEM) analysis. Public Health Relevance: Tooth regeneration has been gaining significant attention in the recent years from reconstitution studies of epithelial and mesenchymal cells isolated from the developing tooth buds. However, the mechanisms of dental epithelial cell differentiation into functional enamel- forming cells are not well understood. Elucidating the signaling pathways of enamel epithelial cell differentiation is critical for advancement toward bioengineering tooth enamel. In addition, these studies of LRAP signaling may also be applied as a broader model to study the potential multifunctionality of the protein in other cells and tissue types such mesenchymal cells, brain, eye, calvariae, where LRAP expression has also been detected. Our goal is to understand the mechanisms of LRAP signaling to promote dental epithelial cell differentiation. We hypothesize that under the effect of LRAP signaling, enamel organ epithelial cells differentiate into functional enamel-forming cells.

描述（由申请人提供）：由成成木分泌的氨基蛋白蛋白是搪瓷细胞外基质中的主要蛋白质，调节搪瓷的形成。富含亮氨酸的氨基蛋白肽（LRAP）是最丰富的蛋白蛋白之一。 LRAP已被证明存在于牙齿上皮和间质组织中，这表明它在调节牙源性细胞增殖和/或分化中具有作用。我们的初步数据表明，LRAP促进牙釉质器官上皮细胞在体外区分。此外，我们的结果表明，阿米他素无效的小鼠表现出混乱的牙釉质上皮细胞，其特征在于定义较差的层中间和星状网状网状细胞，并且缺乏分泌的熟细胞细胞，而没有任何含量的过程，并且限制了搪瓷基质的分泌。相比之下，Amelogenin-null（LRAP+/NULL）背景中的LRAP过表达挽救了地层中间，星状网状网状和恢复的搪瓷基质分泌的表型。然而，LRAP+/NULL小鼠的搪瓷矿化延迟，与保留的搪瓷基质蛋白的证据相关，表明LRAP不会促进矿化，而只能充当信号分子。在这项赠款建议中，我们将探讨LRAP在体内和体外的信号传导作用。 这些研究的总体假设是LRAP促进牙釉质器官上皮细胞分化。我们的具体目的如下：目标1：确定LRAP在体外对搪瓷器官上皮细胞分化的影响。牙釉质基质基因表达，蛋白质合成和体外矿物质形成，由体内人牙釉质器官和牙皮上皮细胞用外源性添加的重组人LRAP处理，稳定地通过TET受调节的促销子在上皮细胞中含有特定细胞特异性乳腺表达载体，含有TET调控的促销。对于LRAP，将与没有LRAP干预的对照进行比较。与来自大肠杆菌的非静电转换改性LRAP相比，在牙齿上皮细胞中使用LRAP过表达将确定翻译后修饰的LRAP对细胞分化的影响。哺乳动物表达载体中TET响应系统的掺入将允许调节LRAP信号传导以促进牙齿上皮细胞分化。目标2：确定LRAP对体内牙釉质上皮细胞分化和搪瓷形成的影响。野生型，氨基蛋白酶无小鼠和转基因小鼠的磨牙和牙，包括野生型（LRAP+/WT）中的LRAP超表情，以及在H＆E下检查H＆E的差异，并将检查差异，并在H＆E染色下检查差异。在发育的不同阶段，在搪瓷器官细胞形态和基质中。将在mRNA和蛋白质水平上分析与搪瓷上皮细胞分化相关的牙釉质基质蛋白表达改变。将使用原子力显微镜（AFM）比较这些小鼠的搪瓷纳米性质，并通过扫描电子显微镜（SEM）分析检查搪瓷结构差异。 公共卫生相关性：近年来，由于从发育中的牙齿芽中分离出的上皮细胞和间质细胞的重建研究，牙齿再生引起了人们的关注。然而，尚不清楚牙齿上皮细胞分化为功能牙釉质细胞的机制。阐明搪瓷上皮细胞分化的信号传导途径对于促进生物工程牙釉质的发展至关重要。此外，这些对LRAP信号传导的研究也可以作为更广泛的模型，以研究其他细胞和组织类型中蛋白质的潜在多功能性，例如间充质细胞，脑，眼睛，钙钙在其中也检测到了LRAP表达。 我们的目标是了解LRAP信号传导的机制，以促进牙齿上皮细胞分化。我们假设在LRAP信号传导的作用下，搪瓷器官上皮细胞分化为功能性搪瓷形成细胞。