Notch Signaling and Bone Fracture Healing

缺口信号传导和骨折愈合

• 批准号: 8815632
• 负责人: IVO Kalajzic
• 金额: $ 35.08万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-02-01 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8815632
• 关键词: Bone callus; Cells; Chondrocytes; Complex; Data; Down-Regulation; Elements; Environment; Evaluation; Exhibits; Fracture; Fracture Healing; Funding; Future; Gene Expression Profiling; Generations; Genetic; Healed; In Vitro; Label; Mesenchymal; Mesenchymal Stem Cells; Modeling; Mus; Notch Signaling Pathway; Osteoblasts; Osteocytes; Peptides; Periosteum; Phase; Process; Role; Signal Transduction; Smooth Muscle; Source; Staging; Stem cells; Therapeutic; Therapeutic Intervention; Time; Tissues; Transgenes; Transgenic Model; Work; alpha Actin; base; bone strength; design; healing; improved; in vitro Model; in vivo; inhibitor/antagonist; loss of function; migration; mouse model; notch protein; osteogenic; osteoprogenitor cell; overexpression; progenitor; public health relevance; transcription factor; treatment effect

DESCRIPTION (provided by applicant): Our recent work has shown that smooth muscle alpha actin (?SMA) is a marker of mesenchymal progenitor cells that expand rapidly following fracture, and show significant contribution to fibrous tissue, osteoblast, and chondrocyte lineages within a fracture callus. Gene expression analysis of isolated ?SMA- labeled progenitor cells revealed that the Notch signaling pathway is significantly decreased during the early stages of fracture healing. Previous studies have shown that Notch signaling exhibits different effects dependent on the stage of osteoprogenitor maturation. We hypothesize that decreases in Notch signaling regulate periosteal progenitor cells expansion, migration and differentiation into mature mesenchymal lineages in the fracture callus. We propose to evaluate the effects of Notch using stage specific genetic Notch gain- and loss-of-function models during fracture healing. We will also evaluate the inhibition of Notch using small peptide SAHM1 that directly interferes with the Notch transcriptional complex. This approach will provide evidence for potential future application to accelerate or improve fracture healing. In Aim 1 we will evaluate the effects of Notch overexpression. Overexpression will be achieved by directing forced Notch 1 intracellular domain (NICD1) expression to different stages of the osteogenic lineage. For timed activation of the NICD1 following generation of fractures, we propose to use stage- specific inducible-Cre transgenes. ?SMACreERT2 mice will be used to target Notch overexpression to progenitor stage while overexpression in osteoblasts/osteocytes will be achieved by using DMP1-CreERT2 mice. Effects of Notch modulation will be assessed by evaluating progress of callus formation, and changes in bone strength and stiffness during fracture healing. We will also examine the mechanisms of effects of Notch overexpression on PPCs using in vitro and in vivo approaches to study effects on proliferation, migration and differentiation. In Aim 2 we will determine the effects of stage-specific Notch inhibition on fracture healing. To disrupt Notch signaling, we will use a transgenic model in which a direct transcriptional effector of Notch signaling, Rbpj?, is deleted (Rbpj?flox) following generation of fracture. In vitro and in vivo evaluation of Notch inhibition using PPCs will be evaluated. We will extend the inhibition studies to evaluate the treatment with Notch transcription factor complex inhibitor SAHM1 (stapled a-helical peptides derived from MAML1) on fracture healing. Our results will provide a better understanding of the role of Notch signaling during fracture healing and will evaluate the future therapeutic modulation of the healing process.

描述（由申请人提供）：我们最近的工作表明，平滑肌α肌动蛋白（？sma）是裂缝后迅速扩展的间充质祖细胞的标记，并且对纤维组织，成骨细胞和软骨细胞谱图在骨折call虫中表现出显着贡献。分离的标记祖细胞的基因表达分析表明，在断裂愈合的早期阶段，缺口信号通路显着降低。先前的研究表明，Notch信号传导取决于骨基因生成剂的成熟阶段。 我们假设Notch信号的降低会调节骨膜祖细胞的扩展，迁移和分化为骨折愈伤组织中成熟的间充质谱系。我们建议在断裂愈合过程中使用特定于遗传学的遗传学刚口和功能丧失模型来评估Notch的效果。我们还将使用直接干扰Notch转录复合物的小肽SAHM1评估Notch的抑制作用。这种方法将提供证据，证明未来可能应用或改善断裂愈合的可能性。 在AIM 1中，我们将评估Notch过表达的效果。通过将强制性Notch 1细胞内结构域（NICD1）表达到成骨谱系的不同阶段来实现过表达。为了定时激活裂缝后的NICD1，我们建议使用阶段特异性诱导型转基因。 “ Smacreert2小鼠”将用于将缺口过表达靶向祖细胞阶段，而通过使用DMP1-Creert2小鼠，将实现成骨细胞/骨细胞中的过表达。 Notch调制的效果将通过评估愈伤组织形成的进度以及骨折愈合过程中的骨骼强度和刚度的变化来评估。我们还将使用体外和体内方法研究Notch过表达对PPC的影响的机理，以研究对增殖，迁移和分化的影响。在AIM 2中，我们将确定特异性缺口抑制对断裂愈合的影响。为了破坏Notch信号传导，我们将使用一个转基因模型，在该模型中，Notch信号的直接转录效应器RBPJ？将评估使用PPC对NOTCH抑制的体外和体内评估。我们将 扩展抑制作用研究，以评估Notch转录因子复合物抑制剂SAHM1（固定的A螺旋肽衍生自MAML1）在骨折愈合上的处理。我们的结果将更好地了解Notch信号在断裂愈合过程中的作用，并将评估愈合过程的未来治疗调节。