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（由申请人提供）：已证明ephrin配体及其受体在多个组织的生长和发育中起关键作用，以及ephrin配体与其受体的相互作用会导致双向信号的激活，从而激活受体和配体的下游信号群信号壳体。人类源代磷脂B1的突变会导致颅面综合征，而小鼠ephrinb1基因的缺失会导致骨骼模式的围产致死性和缺陷，从而表明骨骼发育需要以骨蛋白B1。基于在人类和小鼠中具有Ephrin B1基因突变的表型的严重程度，我们在此授予中的重点是检查Ephrin B1介导的Ephrin B1反向信号传导和EPH受体介导的正向信号在调节体内骨形成中的正向信号传导，并通过eppersement b1 reake and ob ob ob ob ob）识别分子机制（均可构想的跨越峰值）。为此，我们根据我们的初步数据提出了以下2个假设：1）obs中ephrin B1反向信号的破坏会损害OB分化和骨形成； 2）Ephrin B1介导的反向信号的激活诱导TAZ从Ephrin B1的脚手架复合物中释放，其中含有NHERF，PTPN13，PP2A和14-3-3，以便随后转运到核转运到核以与Runx2（例如Runx2）结合，例如将基因转移（例如，将基因转移）转移到基因的转录中，以调节骨骼Marrows的转录（将其转移到基因的转录中）。为了检验假设1，设计了遗传救援实验，以表征转基因（TG）小鼠的骨表型，其全长ephrin B1的过度表达能够诱导向前和反向信号传导或PDZ域截断的Ephrin B1能够仅诱导内源性Ephrin ephrin B1 Null Broggrtment truncing Ephrin B1。骨骼表型将通过5-CT和组织形态计分析进行评估。为了检验假设2，我们将执行共免疫沉淀实验，以鉴定含有与BMS细胞中与磷酸化的Ephrin B1和TAZ相互作用的蛋白质的PDZ结构域。我们将确定以弗林B1反向信号的激活是否导致PP2A的TAZ去磷酸化，以及随后通过Western blot检查核TAZ和TAZ-GFP蛋白的核运输核TAZ的后续核易位。我们还将评估使用慢病毒shRNA对runx2靶基因和OB分化的表达的鼻蛋白B1反向信号干预的后果。该应用的结果将促进我们对调节骨形成中ephrin B1反向信号的分子机制的理解，并为治疗和预防骨骼疾病（例如骨质疏松症）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将需要对分子途径和涉及骨形成过程调节的基因有深入的了解。通过遗传救援和分子实验成功完成拟议的研究，应促进我们对ephrin B1和/或其受体如何调节成骨细胞分化的理解，从而使骨形成。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最终将更好地理解为什么某些人降低了峰值骨骼质量以及治疗方案以纠正这些患者的骨骼形成缺乏。