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，旨在研究釉原蛋白前mRNA剪接产物在牙齿形态发生过程中参与上皮-间充质信号的潜力。体内实验表明，特定的成釉蛋白基因剪接产物具有骨形态发生蛋白样活性，可诱导异位软骨/骨的形成，并可诱导培养的间充质细胞表达软骨/骨表型分子。我们已经证明了两个特定的剪接产物，长度分别为73和59个氨基酸，通过调节转录因子的表达而发挥作用。它们在培养过程中对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影响。虽然釉原蛋白基因被认为只在牙齿上皮细胞中表达，但釉原蛋白基因产物已被证明存在于外套质牙本质中，我们通过原位杂交获得的证据表明，它们可能直接在早期成牙本质细胞中产生。其他研究表明，釉原蛋白可能参与了牙骨质的诱导。在这些数据的基础上，我们假设釉原蛋白基因的小剪接产物多肽是转录调节因子，在上皮-间充质信号中发挥作用，而上皮-间充质信号是牙齿发育的重要部分。本研究提出三个具体目标：1)利用原位杂交技术证实成牙本质细胞中是否存在成釉蛋白mRNAs，并用免疫组织化学方法确定成牙本质细胞在不同发育阶段表达成釉蛋白基因信息，并显示多肽产物在成牙本质细胞中的表达情况；2)利用基因芯片和其他技术详细阐明多肽在牙齿发育过程中指导成牙本质细胞和成牙本质细胞表型表达的作用；以及3)更完整地确定多肽与间充质细胞相互作用改变其表型表达的机制。这些研究的合乎逻辑的扩展将是，在目标2下，检查多肽在更一般的异型上皮-间充质重组实验中的使用，以及，在目标3)下，分析两种多肽对骨形成蛋白样活性的联合影响，以及在骨诱导和牙齿发育中间充质细胞表型表达的影响。在这次修订中，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.