CSF-1 in Dental Biology

CSF-1 在牙科生物学中的应用

• 批准号: 7882575
• 负责人: SHERRY L ABBOUD-WERNER
• 金额: $ 27.86万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-03-05 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7882575
• 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; 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; Messenger RNA; 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; mineralization; mouse model; novel; novel therapeutics; postnatal; prevent; promoter; protein expression; public health relevance; research study; selective expression; transgene expression; vector

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）是牙齿基质形成和萌牙所必需的。成釉细胞和成牙细胞表达可溶性和细胞表面形式的CSF-1；然而，这些异构体在牙齿发育过程中对成釉发育的确切生物学作用以及CSF-1基因中控制其表达的调控元件尚未被探索。本研究的长期目标是通过动物模型研究控制成釉细胞谱系中CSF-1表达的分子机制，并确定CSF-1同种异构体对釉质基质形成的生物学作用。我们的中心工作假设是，在牙齿发育过程中，CSF-1对成釉发育至关重要。初步数据显示，在转基因小鼠中，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）细胞中具有高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直接成釉细胞系细胞的基因表达。为了解决这一问题，我们将在培养的成釉细胞和非成釉细胞中测试一系列-774 bp]5'CSF-1启动子缺失构建子的转录活性，并通过产生含有这些与lacZ报告基因相关序列的转基因小鼠，在体内分析相关序列。在最近的研究中，我们发现缺乏CSF-1亚型的op/op小鼠会改变牙基质蛋白的表达，从而导致牙釉质和牙本质缺陷。在骨钙素（OC）启动子的控制下，生成了在成牙细胞中表达csCSF-1 （op/opCS）或sCSF-1 （op/opS）的转基因op/op小鼠，并表现出不同的牙齿表型。sCSF-1对牙本质进行矫正，使op/opS小鼠的牙釉质缺损得到部分矫正，表现出牙齿呈白垩白，牙根形成受损的独特特征。这些发现是新颖的，表明在op/opS牙的成釉细胞中缺乏CSF-1会改变牙釉质基质和牙根的发育。我们在op/opS小鼠中的初步数据支持了这一点，显示出釉素和缩钙素-4 （KLK4，称为EMSP1）减少以及根缩短。我们的第二个假设是CSF-1同种异构体对牙釉质基质和根的形成有不同的调节作用，并导致不同的表型。在这些实验中，将使用-774/+183 bp的CSF-1启动子在成釉细胞中选择性表达sCSF-1或csCSF-1。建立OC启动子下携带sCSF-1、-774/+183 bp启动子下携带sCSF-1或csCSF-1的双转基因op/op小鼠。研究人员将检测小鼠牙釉质缺损的消退情况，并分析牙齿形态学、牙釉质基质蛋白表达、牙釉质完整性和矿化情况。我们还将验证慢病毒介导的sCSF-1基因传递到成釉细胞是否能修复op/opS小鼠的牙釉质/牙根缺陷。这些研究的结果将增加我们对CSF-1调控的分子机制的理解，并确定sCSF-1和csCSF-1的不同功能作用，可能在预防获得性和遗传性牙齿疾病（如无釉发育不全）的牙釉质缺陷方面具有治疗应用。