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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：
8051630
负责人：
Weirong Xing
金额：
$ 16.39万
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 2013-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8051630
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 Deletion Mutation Diagnosis Docking Embryo Embryonic Development Enhancers Eph Family Receptors Ephrin B Receptor Ephrin-B1 Ephrins 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 Mice 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）：Ephrin配体及其受体已被证明在多种组织的生长发育中发挥关键作用，并且Ephrin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反应。人的ephrinB1基因突变导致颅额鼻综合征，而小鼠的ephrinB1基因缺失导致围产期死亡和骨骼模式缺陷，这表明骨骼发育需要ephrinB1基因。基于人、小鼠ephrin B1基因突变表型的严重程度，本课题重点研究ephrin B1介导的反向信号和Eph受体介导的正向信号在体内骨形成调控中的作用，确定ephrin B1反向信号调控成骨细胞（OB）分化和峰值骨量的分子机制。为此，基于我们的初步数据，我们在本研究中提出以下两个假设：1)OB中ephrin B1反向信号的破坏会损害OB的分化和骨形成；2)激活ephrin B1介导的反向信号通路，诱导含有NHERF、PTPN13、PP2A和14-3-3的ephrin B1支架复合物释放TAZ，转运至细胞核，结合Runx2等主转录因子，调控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，我们设计了遗传挽救实验，以表征内源性ephrin B1零背景下，过表达能够诱导正向和反向信号传导的全长ephrin B1或只能诱导正向信号传导的PDZ结构域截断的ephrin B1的转基因（Tg）小鼠的骨表型。骨骼表型将通过5-CT和组织形态学分析进行评估。为了验证假设2，我们将进行共免疫沉淀实验，以鉴定BMS细胞中含有与磷酸化ephrin B1和TAZ相互作用的PDZ结构域蛋白。我们将通过Western blot检测核TAZ和核运输TAZ- gfp蛋白来确定ephrin B1反向信号的激活是否导致PP2A对TAZ的去磷酸化，以及随后的核易位。我们还将评估使用慢病毒shRNA对候选基因干预ephrin B1反向信号传导对Runx2靶基因表达和OB分化的影响。本研究结果将有助于进一步了解ephrin B1逆转录调控骨形成的分子机制，为骨质疏松等骨病的治疗和预防提供新的治疗策略。