Signaling activities of amelogenin gene splice products.

描述（由申请人提供）：这是一份修订后的申请，旨在研究牙釉原蛋白前mRNA剪接产物参与牙齿形态发生过程中上皮-间充质信号传导的潜力。已显示特定的釉原蛋白mRNA剪接产物具有BMP样活性，当植入体内异位部位时导致软骨/骨诱导，并诱导培养的间充质细胞中软骨/骨表型分子的表达。我们已经表明，两个特定的剪接产物，73和59个氨基酸的长度，分别通过调节转录因子的表达。它们对体外培养的牙胚发育有不同的诱导作用。它们对牙本质缺损的修复和牙髓矿化也产生不同的作用。虽然釉原蛋白基因被认为只在牙上皮中表达，但釉原蛋白基因产物已被证明存在于套牙本质中，并且我们有原位杂交证据表明它们可能直接在早期前成牙本质细胞中产生。其他人已经表明，釉原蛋白可能参与诱导牙骨质。基于这些数据，我们假设釉原蛋白基因小剪接产物肽是转录调节因子，在上皮-间充质信号传导中发挥作用，而上皮-间充质信号传导是牙齿发育的重要组成部分。本研究的主要目的是：1）通过原位杂交和免疫组化技术检测成牙本质细胞中amelogenin mRNA的表达，以及成牙本质细胞中amelogenin mRNA表达的肽类产物的出现; 2）使用基因阵列和其他技术详细阐明在培养物中，肽在牙齿发育期间指导成牙本质细胞和成牙骨质细胞表型表达的作用;和3）更完全地确定肽与间充质细胞相互作用以改变其表型表达的机制。这些研究的逻辑延伸将是在目的2下检查肽在更一般的异型上皮-间充质重组实验中的用途，并且在目的3）下分析两种肽对骨诱导和牙齿发育中间充质细胞中的表型表达的BMP样活性的组合作用。在本修订版中，提供了更多的实验细节。

DESCRIPTION (provided by applicant): This is a revised application to study the potential of amelogenin pre-mRNA splice products to participate in epithelial-mesenchymal signaling during tooth morphoigenesis. Specific amelogenin mRNA splice products have been shown to have BMP-like activities leading to cartilage/bone induction when implanted into ectopic sites in vivo, and to induce expression of cartilage/bone phenotypic molecules in mesenchymal cells in culture. We have shown that two specific splice products, 73 and 59 amino acids in length, respectively, act by regulating the expression of transcription factors. They have different inductive effects on the development of tooth germs in culture. They also yield different effects on the repair of dentin defects and pulp mineralization. Although the amelogenin genes have been thought to be expressed only in the dental epithelium, amelogenin gene products have been shown to be present in the mantle dentin and we have evidence from in situ hybridization that they may be produced directly in early pre-odontoblasts. Others have shown that amelogenins may be involved in induction of tooth cementum. On the basis of these data we have hypothesized that the amelogenin gene small splice product peptides are transcriptional regulating factors that have a role in the epithelial-mesenchymal signaling that is such a prominent part of tooth development. Three specific aims are proposed for this study: 1) To use in situ hybridization to confirm the presence of amelogenin mRNAs in odontoblasts, and immunohistochemistry to determine the tooth developmental stages at which odontoblasts express the amelogenin gene message, and show the appearance of the peptide products in the odontoblasts; 2)To clarify in detail, using gene arrays and other techniques the effects of the peptides on directing the expression of odontoblast and cementoblast phenotypes during tooth development, in culture; and 3) To more completely determine the mechanisms by which the peptides interact with mesenchymal cells in general to alter their phenotypic expression. Logical extensions of these studies would be to examine, under Aim 2, the use of the peptides in more general heterotypic epithelial-mesenchymal recombination experiments, and, under Aim 3) to analyze the combined effects of the two peptides on the BMP-like activity on the phenotypic expression in mesenchymal cells in bone induction and tooth development. In this revision, more experimental details have been provided.