Regulation of Bone Formation by Ephrin B1 Signaling Pathways

Ephrin B1 信号通路对骨形成的调节

基本信息

批准号：
7896362
负责人：
Weirong Xing
金额：
$ 20.49万
依托单位：
LOMA LINDA VETERANS ASSN RESEARCH & EDUC
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-04-01 至 2012-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7896362
关键词：
Adaptor Signaling Protein Binding Binding Proteins Biological Assay Biological Models Bone Diseases Bone Marrow Breeding C-terminal Calvaria Candidate Disease Gene Cell Differentiation process Cell Lineage Cell Nucleus Cell physiology Cells Chromatin Co-Immunoprecipitations Collagen Type I Complex Cytoplasm Cytoplasmic Tail DEXA Data Defect Diagnosis Docking Embryo Embryonic Development Enhancers Eph Family Receptors Ephrin B Receptor Ephrin-B1 Extracellular Domain Family member Fas-associated phosphatase-1 Future GTP-Binding Protein Regulators Gene Expression Gene Targeting Genes Genetic Genetic Transcription Grant Growth Growth Factor Growth Factor Receptors Growth and Development function Human Immunoprecipitation Intervention JUN gene Knock-out Lead Length Ligands Measures Mediating Membrane Mesenchymal Stem Cells Metabolism Modeling Molecular Molecular Target Mus Muscle Cells Mutation Neural Crest Cell Nuclear Nuclear Translocation Osteoblasts Osteoclasts Osteogenesis Osteoporosis PDZ protein Pathway interactions Patients Pattern Perinatal Phenotype Phosphorylation Phosphotransferases Play Point Mutation Precipitation Prevention Process Protein Dephosphorylation Protein Phosphatase 2A Regulatory Subunit PR53 Protein Tyrosine Phosphatase Protein phosphatase Proteins Publishing Recruitment Activity Regulation Reporter Research Role Serum Severities Signal Pathway Signal Transduction Site Skeletal Development Stromal Cells Subfamily lentivirinae TFAP2A gene Tail Testing Tissues Transactivation Transcriptional Activation Transgenic Organisms Tyrosine Western Blotting Wild Type Mouse Zebrafish base beta catenin bone bone cell bone mass cell type craniofrontonasal syndrome cranium design glutamate receptor interacting protein in vivo innovation inositol-1,4,5-trisphosphate 5-phosphatase novel novel therapeutics nucleocytoplasmic transport osteoblast differentiation overexpression postnatal postsynaptic density protein promoter public health relevance receptor receptor binding research study scaffold skeletal small hairpin RNA sodium-hydrogen exchanger regulatory factor src Homology Region 2 Domain stem cell differentiation trafficking transcription factor transgene expression treatment strategy

项目摘要

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.

描述（申请人提供）：肝配蛋白配体及其受体已被证明在多种组织的生长和发育中发挥关键作用，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，其中两者受体和配体激活下游信号级联。人类肝配蛋白 B1 突变会引起颅额鼻综合征，而小鼠肝配蛋白 B1 基因缺失会导致围产期死亡和骨骼模式缺陷，因此表明肝配蛋白 B1 是骨骼发育所必需的。基于肝配蛋白 B1 基因突变的人和小鼠表型的严重程度，我们本次资助的重点是检查肝配蛋白 B1 介导的反向信号传导和 Eph 受体介导的正向信号传导在调节体内骨形成中的作用，并确定肝配蛋白 B1 反向信号传导调节成骨细胞 (OB) 分化和峰值骨量的分子机制。为此，我们根据初步数据在本研究中提出以下2个假设：1）OB中肝配蛋白B1反向信号传导的破坏将损害OB分化和骨形成； 2) 激活肝配蛋白 B1 介导的反向信号传导，诱导 TAZ 从含有 NHERF、PTPN13、PP2A 和 14-3-3 的肝配蛋白 B1 支架复合物中释放，随后转运至细胞核，与 Runx2 等主转录因子结合，从而调节对于骨髓基质（BMS）细胞分化为成骨细胞至关重要的基因。为了检验假设 1，设计了基因拯救实验来表征转基因 (Tg) 小鼠的骨表型，这些小鼠过度表达能够诱导正向和反向信号传导的全长肝配蛋白 B1 或仅能够诱导正向信号传导的 PDZ 结构域截短的肝配蛋白 B1内源性肝配蛋白 B1 无效背景中的信号传导。将通过 5-CT 和组织形态计量学分析评估骨骼表型。为了检验假设 2，我们将进行免疫共沉淀实验来鉴定含有 PDZ 结构域的蛋白质，这些蛋白质与 BMS 细胞中的磷酸化肝配蛋白 B1 和 TAZ 相互作用。我们将通过蛋白质印迹检查核 TAZ 和 TAZ-GFP 蛋白的核运输来确定肝配蛋白 B1 反向信号传导的激活是否会导致 PP2A 使 TAZ 去磷酸化，以及随后的核转位。我们还将评估使用慢病毒 shRNA 对候选基因干预肝配蛋白 B1 反向信号传导对 Runx2 靶基因表达和 OB 分化的影响。该应用结果将增进我们对ephrin B1反向信号调节骨形成的分子机制的理解，并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：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节的分子途径和基因。通过基因拯救和分子实验成功完成拟议的研究应该会增进我们对肝配蛋白 B1 和/或其受体如何调节成骨细胞分化并从而调节骨形成的理解。由于肝配蛋白 B1 在小鼠和人类之间是保守的，未来对肝配蛋白 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有些人的峰值骨量减少以及纠正骨形成缺陷的治疗方案在这些患者中。