Nell-1, A Cbfa 1 Downstream Target, In Bone Formation

描述（由申请人提供）：项目概述/摘要本竞争性修订申请是对通知号（NOT-OD-09-058）和通知标题的回应：NIH宣布竞争性修订申请的恢复法案资金的可用性。我们之前鉴定了骨诱导分子，长Nell-1 [Nell-1；这是一种在编码表皮生长因子（EGF）样结构域的神经组织中强烈表达的蛋白质]，来自于颅缝闭闭（CS）患者的过早融合缝合线(许多这些研究是在鉴定短的Nell-1， SNell-1亚型之前进行的。为了澄清，当我们确定涉及长格式时，我们将使用“Nell-1”代替Nell-1。同时，Nell-1可以同时指代LNell-1和SNell-1)。LNell-1是一种分泌性蛋白，具有信号肽，nh2末端血栓反应蛋白-1 （TSP1-N）样模块，五个样弦状富半胱氨酸（CR）结构域和egf样结构域。这项竞争性修订和家长资助提案的长期目标是了解CS患者过度骨生长背后的机制，以便我们能够利用和引导相关分子用于临床治疗以再生骨。为此，我们不断努力，以更好地了解LNell-1的分子调控和功能，为LNell-1的治疗应用奠定坚实的科学基础。我们的进展报告和母补助金竞争性更新的初步数据表明：1)runt相关转录因子2 （Runx2）通过顺式作用元件2 （OSE2）响应元件（REs）调控LNell-1；2) LNell-1促进成骨细胞分化，可部分补偿Runx2单倍体不足；3) Nell-1缺失的动物表现为软骨细胞肥大受损，骨形成延迟，Runx2表达减少，sterix表达增加。在这个为期一年的竞争性修订中，我们建议通过集中于鉴定Nell-1受体和/或结合蛋白（R/ bp）来显著加快我们目前对Nell-1机制调控的研究节奏。为了实现这一目标，我们获得了初步的T7噬菌体展示和基于质谱（MS）的蛋白质组学数据，证明LNell-1与膜和细胞质蛋白结合。这些数据导致假设Nell-1通过作为细胞特异性和潜在的新型受体/结合蛋白的配体在骨软骨分化中发挥其功能。为了鉴定LNell-1 R/ bp，将进行三个目标：目标a)候选受体方法，以检查LNell-1与整合素家族的相互作用。在结构上，LNell-1的TSP1-N样结构域与整合素结合位点所在的TSP1-N结构域具有高度的相似性。目的B)利用T7噬菌体展示系统鉴定LNell-1 R/BP。目的C)基于质谱的功能蛋白质组学研究膜蛋白片段结合。这项竞争性修订还将刺激经济，使雇用更多的工作人员和增加目前兼职工作人员的工作时间。

DESCRIPTION (provided by applicant): Project Summery/Abstract This Competitive Revision Application is in response to Notice Number (NOT-OD-09-058) and Notice Title: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications. We previously identified osteoinductive molecule, long Nell-1 [LNell-1; a protein strongly expressed in neural tissue encoding epidermal growth factor (EGF)-like domain] from prematurely fusing sutures in craniosynostosis (CS) patients (Many of these studies were performed before identification of the short Nell-1, SNell-1 isoform. For clarification, we will use "LNell-1" in place of Nell-1 when we are certain that the long form is involved. Meanwhile, Nell-1 can refer to both LNell-1 and SNell-1). LNell-1 is a secretory protein with a signal peptide, an NH2-terminal thrombospondin-1 (TSP1-N)-like module, five chordin-like cysteine-rich (CR) domains, and EGF-like domains. The long term goal of this Competitive Revision and parent grant proposal is to understand the mechanisms behind excessive bone growth in CS patients, so that we can harness and channel the molecules responsible into clinical therapies to regenerate bone. Towards this end, we have worked continuously to better understand the molecular regulation and function of LNell-1 so as to build a strong scientific foundation for therapeutic LNell-1 application. Our progress report and preliminary data for the parent grant Competitive Renewal demonstrate that: 1) runt-related transcription factor 2 (Runx2) regulates LNell-1 through cis-acting element 2 (OSE2) response elements (REs); 2) LNell-1 increases osteoblastic differentiation and can partially compensate for Runx2 haploinsufficiency; and 3) Nell-1 deletion animals demonstrate impaired chondrocyte hypertrophy with delayed bone formation as well as decreased Runx2 and increased osterix expression. For this one-year Competitive Revision we propose to significantly accelerate the tempo of our current investigations on mechanistic regulation of Nell-1 by a concentrated focus on identification of LNell-1 receptors and/or binding proteins (R/BPs). To accomplish this objective, we have obtained preliminary T7 phage display and mass spectrometry (MS)-based proteomic data demonstrating LNell-1 binding to membranous and cytosolic proteins. These data have led to the hypothesis that Nell-1 exerts its function in osteochondral differentiation by serving as a ligand to cell specific and potentially novel receptors/binding proteins. In order to identify LNell-1 R/BPs, three aims representing a logical extension of the parent proposal will be undertaken: Aim A) Candidate receptor approach to examine interaction of LNell-1 with integrin family. Structurally, the TSP1-N-like domain of LNell-1 shares a high degree of similarity with TSP1-N domain where integrin binding sites reside. Aim B) LNell-1 R/BP identification using a T7 phage display system. Aim C) MS-based functional proteomics on the membranous protein fraction binding. This Competitive Revision will also stimulate the economy by enabling hiring of additional staff and increasing hours of current part-time staff. PUBLIC HEALTH RELEVANCE: RELEVANCE TO PUBLIC HEALTH STATEMENT Nell-1, a cause of excessive bone growth in craniosynostosis patients has shown significant promise as a potent bone forming agent in preclinical studies. A better understanding of how and why Nell-1 works to form bone can accelerate the development of clinical therapies to help patients grow better and faster bone.