Regulation of Bone Formation by Ephrin B1 Signaling Pathways

说明书(申请人提供)：已证明肾上腺素配体及其受体在多种组织的生长和发育中起关键作用，而肾上腺素配体与其受体的相互作用导致双向信号的激活，在该双向信号中，受体和配体都激活下游的信号级联。在人类中，ewitin B1的突变会导致颅前鼻窦综合征，而在小鼠中，eferinB1基因的缺失会导致围产期的死亡和骨骼模式的缺陷，从而表明ePhrin B1是骨骼发育所必需的。基于人类和小鼠的表型的严重程度，我们在这项研究中的重点是研究ePhin 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ePhin B1反向信号调节成骨细胞分化和峰值骨量的分子机制。为此，我们在本研究的初步数据基础上提出了以下两个假说：1)在OB中eaffin B1反向信号的干扰将损害OB的分化和成骨；2)eaffin B1介导的反向信号的激活诱导含有NHERF、PTPN13、PP2A和14-3-3的ePhin B1支架复合体释放TAZ，随后运输到细胞核，与主要转录因子如Runx2结合，调节骨髓基质细胞向成骨细胞分化的关键基因的转录。为了验证假设1，设计了基因拯救实验来表征转基因(TG)小鼠的骨表型，这些转基因小鼠的全长ePhin B1能够诱导正向和反向信号，或者PDZ结构域截短的ePhin B1能够在内源性ePhin B1缺失背景中仅诱导正向信号。骨骼表型将通过5-CT和组织形态计量学分析进行评估。为了验证假设2，我们将进行免疫共沉淀实验，以确定在BMS细胞中包含PDZ结构域的蛋白质与磷酸化的ePhin B1和TAZ相互作用。我们将通过Western印迹检测核TAZ和TAZ-GFP蛋白的核转运来确定ePhin B1反向信号的激活是否导致PP2A对TAZ去磷酸化，以及随后的核转位。我们还将评估利用慢病毒shRNA对候选基因进行ePhin B1反向信号干预对Runx2靶基因表达和OB分化的影响。这一应用的结果将加深我们对ePhin B1反向信号调控骨形成的分子机制的理解，并为骨质疏松等骨病的治疗和预防提供新的治疗策略。 公共卫生相关性：制定诊断和治疗骨质疏松症的策略需要彻底了解参与骨形成过程调控的分子途径和基因。通过基因挽救和分子实验的成功完成拟议的研究，应该会促进我们对eaffin B1和/或其受体如何调控成骨细胞分化从而骨形成的理解。由于在小鼠和人类之间ePhrin B1是保守的，未来对ephin B1信号在调节人类骨形成中的作用的确认最终将有助于更好地理解为什么一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选择。

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.