DETERMINATION AND EXPRESSION OF AMELOGENIN GENE PRODUCTS

描述(由申请人提供)：该项目是NOT-OD-09-058的一部分，通知标题为“NIH宣布恢复法资金可用于竞争性修订申请”，并与父母拨款DE06988的目标一致，DE06988旨在确定釉原蛋白是否通过促进改变分化的信号来促进细胞行为。在这个项目中，我们扩展了父母赠款的特定目标3，以扩大对釉原蛋白作用的研究范围，包括它们在干细胞中诱导成骨途径的能力。这一竞争性修订项目的范围将加快发现的速度，并将为博士后科学家提供额外的就业机会。富含亮氨酸的釉原蛋白多肽(LRAP)是釉原蛋白剪接异构体之一，已被证明可在多种细胞类型中诱导成骨，包括大鼠肌肉成纤维细胞、小鼠成牙骨质细胞、小鼠口腔粘膜细胞和小鼠胚胎干细胞。我们的初步研究表明，LRAP促进成骨细胞的生成，抑制骨髓间充质干细胞(BMMSC)的脂肪生成。经典的Wnt/2-catenin信号通路在LRAP处理后被激活；而特异性Wnt抑制剂SFRP-1完全阻断LRAP介导的BMMSCs的谱系选择。我们推测LRAP能够通过激活典型的Wnt/2-catenin通路来调节BMMSCs成骨和成脂细胞系之间的相互关系。我们提出了以下具体目标来检验这一假设。目的：确定LRAP激活规范的Wnt/2-catenin通路是否在体外刺激BMMSCs成骨和抑制成脂作用的充分必要条件。目的：研究LRAP激活典型的Wnt/2-连环蛋白途径的机制，并确定LRAP的功能结构域(S)。目的：观察LRAP对骨髓表达LRAP转基因小鼠骨和脂肪组织的影响及对釉原蛋白缺失小鼠的影响。 与公共健康相关：釉原蛋白的一个以前未被认识的功能是刺激干细胞向骨形成的分化，同时抑制它们向脂肪形成细胞的分化。这一开关是由釉原蛋白刺激Wnt信号级联激活的。利用釉原蛋白可以促进出生缺陷、创伤和肿瘤引起的骨丢失的手术矫正，因为它提供了一种刺激骨形成的方法，用真实的骨取代异物。

DESCRIPTION (provided by applicant): This project is part of NOT-OD-09-058, with Notice Title, "NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications" and is consistent with the aims of parent grant DE06988 which seeks to determine if amelogenin proteins contribute to cell behavior by promoting signals that change differentiation. In this project we expand parent grant specific aim 3, to expand the scope of investigation into the role of amelogenin to include their capacity to induce an osteogenic pathway in stem cells. The scope of this Competitive Revision project will enhance the pace of discovery and it will provide additional employment for post-doctoral scientists. One of the amelogenin splicing isoforms, Leucine-rich Amelogenin Peptide (LRAP) has been shown to induce osteogenesis in various cell types, including rat muscle fibroblasts, mouse cementoblasts, mouse oral mucosal cells, and mouse embryonic stem cells. Our preliminary studies have shown that LRAP stimulates osteoblastogenesis and inhibits adipogenesis of bone marrow mesenchymal stem cells (BMMSC). Canonical Wnt/2-catenin signaling pathway is activated upon LRAP treatment; while a specific Wnt inhibitor sFRP-1, completely blocks LRAP-mediated lineage selection of BMMSCs. We hypothesize that LRAP is capable of modulating the reciprocal relationship between osteogenic and adipogenic cell lineages in BMMSCs through activating the canonical Wnt/2-catenin pathway. We propose the following specific aims to test this hypothesis. Aim I: To determine whether activation of the canonical Wnt/ 2 -catenin pathway by LRAP is necessary and sufficient to stimulate osteoblastogenesis and to inhibit adipogesis of BMMSCs in vitro. Aim II: To investigate the mechanism by which LRAP activates the canonical Wnt/2-catenin pathway and to characterize the functional domain(s) in LRAP. Aim III: To examine the in vivo effect of LRAP on bone and adipose tissue in transgenic mice expressing the LRAP transgene in bone marrow and in amelogenin null mice. PUBLIC HEALTH RELEVANCE: A previously unrecognized function of amelogenin proteins is to stimulate the differentiation of stem cells towards bone formation while suppressing their differentiation into fat forming cells. This switch is activated by amelogenin stimulating the Wnt signaling cascade. Utilizing amelogenin proteins may improve surgical correction of birth defects, trauma and tumor induced bone loss by providing a means to stimulate bone formation replacing foreign materials with real bone.