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CSF-1 in Dental Biology

CSF-1 在牙科生物学中的应用

基本信息

批准号：
8294405
负责人：
SHERRY L ABBOUD-WERNER
金额：
$ 27.58万
依托单位：
UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-03-05 至 2014-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8294405
关键词：
Address Ameloblasts Amelogenesis Amelogenesis Imperfecta Animal Model Biology Cell Lineage Cell surface Cells Complementary DNA Data Defect Dental Dental Enamel Dentin Development Electrophoretic Mobility Shift Assay Epithelial Cells Gene Delivery Gene Expression Genes Genetic Genetic Crosses Genetic Transcription Goals Human In Situ Hybridization In Vitro Incisor Knock-out LacZ Genes Lentivirus Vector Link Macrophage Colony-Stimulating Factor Mandible Mediating Molecular Morphology Mus Newborn Infant Odontoblasts Oral health Osteocalcin Osteoclasts Phenotype Plant Roots Printing Protein Isoforms Regulatory Element Reporter Genes Resolution Scanning Series Testing Therapeutic Time Tooth Diseases Tooth eruption Tooth structure Transfection Transgenic Mice Transgenic Organisms Work base cis acting element enamel matrix proteins enamelin foot gene therapy improved in vivo kallikrein 4 lentiviral-mediated mRNA Expression mineralization mouse model novel novel therapeutics postnatal prevent promoter protein expression public health relevance research study selective expression transgene expression vector

项目摘要

DESCRIPTION (provided by applicant): Macrophage colony stimulating factor (CSF-1) is essential for tooth matrix formation and eruption. Ameloblasts and odontoblasts express soluble (s) and cell-surface (cs) forms of CSF-1; however, the precise biologic effects of these isoforms on amelogenesis and the regulatory elements in the CSF-1 gene that control their expression during tooth development have not been explored. The long-term goal of this proposal is to characterize the molecular mechanisms that control CSF- 1 expression in ameloblast lineage cells and determine the biologic effect of CSF-1 isoforms on enamel matrix formation using animal models. Our central working hypothesis is that CSF-1 is critical for amelogenesis during tooth development. Preliminary data show, for the first time, that a -774/+183 bp fragment of the CSF-1 promoter in transgenic mice confers high lacZ expression in the inner enamel epithelial (IEE) cells that differentiate into ameloblasts. Our first hypothesis is that cell-specific cis-acting elements in the -774 bp CSF-1 promoter direct gene expression in ameloblast lineage cells during tooth development. To address this issue, a series of -774 bp ]5'CSF-1 promoter deletion constructs will be tested for transcriptional activity in cultured ameloblast and non-ameloblast cells and relevant sequences will be analyzed in vivo by generating transgenic mice harboring these sequences linked to the lacZ reporter gene. In recent studies using op/op mice that lack both CSF-1 isoforms, we showed that absence of CSF-1 alters tooth matrix protein expression that, in turn, leads to enamel and dentin defects. Transgenic op/op mice expressing either csCSF-1 (op/opCS) or sCSF-1 (op/opS) in odontoblasts under the control of the osteocalcin (OC) promoter were generated and showed distinct tooth phenotypes. sCSF-1 corrected dentin and led to partial correction of enamel defects with op/opS mice showing unique features characterized by chalky white teeth and impaired root formation. These findings are novel and indicate that absence of CSF-1 in ameloblasts of op/opS teeth alters enamel matrix and root development. This is supported by our preliminary data in op/opS mice showing decreased enamelin and kallikrein-4 (KLK4, known as EMSP1) as well as shortened roots. Our second hypothesis is that CSF-1 isoforms differentially regulate enamel matrix and root formation and result in distinct phenotypes. For these experiments, the -774/+183 bp CSF-1 promoter will be used to selectively express sCSF-1 or csCSF-1 in ameloblasts. Double transgenic op/op mice carrying sCSF-1 under the OC promoter and harboring either sCSF-1 or csCSF-1 under the -774/+183 bp promoter will be established. Mice will be examined for resolution of enamel defects and teeth will be analyzed for morphology, enamel matrix protein expression, enamel integrity and mineralization. We will also test the hypothesis that lentiviral-mediated gene delivery of sCSF-1 to ameloblasts will rescue enamel/root defects in op/opS mice. Results from these studies should increase our understanding of the molecular mechanisms that regulate CSF-1 and identify distinct functional effects of sCSF-1 and csCSF-1 that may have therapeutic application for preventing enamel defects in acquired and genetic dental disorders such as amelogenesis imperfecta. PUBLIC HEALTH RELEVANCE: Macrophage colony stimulating factor (CSF-1) is a key regulatory molecule for tooth matrix formation and eruption. Work in this proposal plans to determine the biologic effect of soluble and cell surface forms of CSF-1 on enamel matrix formation and the molecular mechanisms that control CSF-1 expression during tooth development using animal models and gene therapy approaches. Results from these studies may suggest novel therapeutic strategies for enhancing enamel integrity and improving oral health in acquired and genetic dental disorders.

描述（由申请人提供）：巨噬细胞集落刺激因子（CSF-1）对于牙基质的形成和萌出至关重要。成釉细胞和成牙质细胞表达可溶性 (s) 和细胞表面 (cs) 形式的 CSF-1；然而，这些异构体对牙釉质生成的精确生物学效应以及在牙齿发育过程中控制其表达的 CSF-1 基因中的调控元件尚未得到探索。该提案的长期目标是表征控制成釉细胞谱系细胞中 CSF-1 表达的分子机制，并使用动物模型确定 CSF-1 亚型对牙釉质基质形成的生物效应。我们的中心工作假设是 CSF-1 对于牙齿发育过程中的釉质形成至关重要。初步数据首次表明，转基因小鼠中 CSF-1 启动子的 -774/+183 bp 片段在分化为成釉细胞的内釉质上皮 (IEE) 细胞中赋予高 lacZ 表达。我们的第一个假设是，-774 bp CSF-1 启动子中的细胞特异性顺式作用元件在牙齿发育过程中指导成釉细胞谱系细胞中的基因表达。为了解决这个问题，将测试一系列-774 bp]5'CSF-1启动子缺失构建体在培养的成釉细胞和非成釉细胞中的转录活性，并通过产生含有与lacZ报告基因连接的这些序列的转基因小鼠来体内分析相关序列。在最近使用缺乏两种 CSF-1 同工型的 op/op 小鼠的研究中，我们发现 CSF-1 的缺乏会改变牙基质蛋白的表达，进而导致牙釉质和牙本质缺陷。产生了在骨钙素 (OC) 启动子控制下在成牙本质细胞中表达 csCSF-1 (op/opCS) 或 sCSF-1 (op/opS) 的转基因 op/op 小鼠，并显示出不同的牙齿表型。 sCSF-1 纠正了牙本质，并部分纠正了 op/opS 小鼠的牙釉质缺陷，这些小鼠表现出白垩白牙齿和牙根形成受损的独特特征。这些发现是新颖的，表明 op/opS 牙齿的成釉细胞中缺乏 CSF-1 会改变牙釉质基质和牙根发育。我们在 op/opS 小鼠中的初步数据支持了这一点，这些数据显示牙釉质和激肽释放酶 4（KLK4，称为 EMSP1）减少以及根缩短。我们的第二个假设是 CSF-1 亚型差异调节牙釉质基质和牙根形成并导致不同的表型。对于这些实验，-774/+183 bp CSF-1 启动子将用于在成釉细胞中选择性表达 sCSF-1 或 csCSF-1。将建立在OC启动子下携带sCSF-1并在-774/+183bp启动子下携带sCSF-1或csCSF-1的双转基因op/op小鼠。将检查小鼠牙釉质缺陷的解决情况，并分析牙齿的形态、牙釉质基质蛋白表达、牙釉质完整性和矿化情况。我们还将测试以下假设：慢病毒介导的 sCSF-1 基因递送至成釉细胞将挽救 op/opS 小鼠的牙釉质/牙根缺陷。这些研究的结果应增加我们对调节 CSF-1 的分子机制的理解，并确定 sCSF-1 和 csCSF-1 的不同功能作用，这些作用可能具有预防获得性和遗传性牙科疾病（如釉质生成不全）中牙釉质缺陷的治疗应用。公共卫生相关性：巨噬细胞集落刺激因子 (CSF-1) 是牙基质形成和萌出的关键调节分子。该提案中的工作计划利用动物模型和基因治疗方法确定可溶性和细胞表面形式的 CSF-1 对牙釉质基质形成的生物效应，以及在牙齿发育过程中控制 CSF-1 表达的分子机制。这些研究的结果可能会提出新的治疗策略，以增强牙釉质完整性并改善获得性和遗传性牙科疾病的口腔健康。