Regulation of Bone Formation by Ephrin B1 Signaling Pathways

描述（由申请人提供）：肝配蛋白配体及其受体已显示在多种组织的生长和发育中起关键作用，并且肝配蛋白配体与其受体的相互作用导致双向信号的激活，其中受体和配体都激活下游信号级联。人类ephrin B1基因突变导致颅额鼻综合征，而小鼠ephrin B1基因缺失导致围产期死亡和骨骼模式缺陷，因此表明ephrin B1是骨骼发育所必需的。基于ephrin B1基因突变的人类和小鼠表型的严重性，我们的重点是研究ephrin B1介导的反向信号和Eph受体介导的正向信号在体内调节骨形成中的作用，并确定ephrin B1反向信号调节成骨细胞（OB）分化和峰值骨量的分子机制。为此，基于我们的初步研究结果，我们提出了以下两个假设：1）OB中ephrin B1反向信号的破坏会损害OB的分化和骨形成; 2）肝配蛋白B1介导的反向信号传导的激活诱导TAZ从含有NHERF，PTPN 13，PP 2A和14-3-3用于随后转运至细胞核以结合主转录因子如Runx 2，从而调节对于骨髓基质（BMS）细胞分化成成骨细胞至关重要的基因的转录。为了检验假设1，设计遗传拯救实验以表征转基因（Tg）小鼠的骨表型，所述转基因小鼠具有能够诱导正向和反向信号传导的全长肝配蛋白B1或能够在内源性肝配蛋白B1无效背景中仅诱导正向信号传导的PDZ结构域截短的肝配蛋白B1的过表达。将通过5-CT和组织形态计量学分析评价骨骼表型。为了验证假设2，我们将进行免疫共沉淀实验，以鉴定BMS细胞中与磷酸化肝配蛋白B1和TAZ相互作用的含有PDZ结构域的蛋白质。我们将通过Western印迹和TAZ-GFP蛋白的核运输检查核TAZ，确定肝配蛋白B1反向信号的激活是否导致PP 2A对TAZ的去磷酸化，以及随后的核转位。我们还将评估使用慢病毒shRNA对候选基因进行ephrin B1反向信号转导干预对Runx 2靶基因表达和OB分化的影响。本研究的结果将有助于我们进一步了解ephrin B1反向信号调节骨形成的分子机制，为骨质疏松等骨疾病的治疗和预防提供新的治疗策略。 公共卫生相关性：发展诊断和治疗骨质疏松症的策略将需要对参与骨形成过程调控的分子途径和基因的透彻理解。通过基因拯救和分子实验成功完成拟议的研究，应促进我们对肝配蛋白B1和/或其受体如何调节成骨细胞分化和骨形成的理解。由于ephrin B1在小鼠和人类之间是保守的，因此未来确认ephrin B1信号在调节人类骨形成中的作用将最终导致更好地理解为什么有些人的峰值骨量减少以及纠正这些患者骨形成缺陷的治疗方案。

DESCRIPTION (provided by applicant): Ephrin ligands and their receptors have been shown to play key roles in the growth and development of multiple tissues, and the interaction of ephrin ligands with its receptors leads to the activation of a bidirectional signal, in which both the receptors and the ligands activate downstream signaling cascades. Mutations of ephrin B1 in humans cause craniofrontonasal syndrome while deletion of ephrinB1 gene in mice results in perinatal lethality and defects in skeletal patterning, thus suggesting that ephrin B1 is required for skeletal development. Based on the severity of the phenotypes in humans and mice with mutations in ephrin B1 gene, our focus in this grant is to examine the role of ephrin B1 mediated reverse signaling and Eph receptor mediated forward signaling in regulating bone formation in vivo, and identify the molecular mechanism by which ephrin B1 reverse signaling regulates osteoblast (OB) differentiation and peak bone mass. To this end, we propose based on our preliminary data the following 2 hypotheses in this study: 1) disruption of ephrin B1 reverse signaling in OBs will impair OB differentiation and bone formation; 2) activation of ephrin B1 mediated reverse signaling induces the release of TAZ from ephrin B1 scaffolding complex containing NHERF, PTPN13, PP2A and 14-3-3 for subsequent transport to nucleus to bind to master transcription factors such as Runx2 to modulate the transcription of genes critical for differentiation of bone marrow stromal (BMS) cells into osteoblasts. To test the hypothesis 1, genetic rescue experiments are designed to characterize the bone phenotypes of transgenic (Tg) mice with over- expression of full length ephrin B1 capable of inducing both forward and reverse signaling or PDZ domain truncated ephrin B1 capable of inducing only forward signaling in endogenous ephrin B1 null background. Skeletal phenotypes will be evaluated by 5-CT and histomorphometry analyses. To test the hypothesis 2, we will perform co-immunoprecipitation experiments to identify PDZ domain containing proteins that interact with phosphorylated ephrin B1 and TAZ in BMS cells. We will determine if activation of ephrin B1 reverse signaling leads to TAZ dephosphorylation by PP2A, and subsequent nuclear translocation by examining nuclear TAZ by Western blot and nuclear trafficking of TAZ-GFP protein. We will also evaluate the consequence of intervention of ephrin B1 reverse signaling using lentiviral shRNA to candidate genes on the expression of Runx2 target genes and OB differentiation. The results of this application will advance our understanding of the molecular mechanisms of ephrin B1 reverse signaling in regulating bone formation, and provide new therapeutic strategies for treatment and prevention of bone diseases such as osteoporosis. PUBLIC HEALTH RELEVANCE: Developing strategies to diagnose and treat osteoporosis would require a thorough understanding of the molecular pathways and the genes involved in the regulation of bone formation process. Successful completion of the proposed studies by genetic rescue and molecular experiments should advance our understanding of how ephrin B1 and/or its receptors regulate osteoblast differentiation and thereby bone formation. Because ephrin B1 is conserved between mice and humans, future confirmation of a role for ephrin B1 signaling in regulating bone formation in humans will eventually lead to a better understanding of why some people have reduced peak bone mass and of treatment options to correct bone formation deficiency in these patients.