Notch Signaling and Bone Fracture Healing

描述（由申请人提供）：我们最近的研究表明，平滑肌α -肌动蛋白（αSMA）是骨折后快速扩张的间充质祖细胞的标志物，对骨折愈伤组织内的纤维组织、成骨细胞和软骨细胞谱系有重要贡献。α - sma标记祖细胞的基因表达分析显示，Notch信号通路在骨折愈合早期显著减少。先前的研究表明Notch信号在成骨细胞成熟的不同阶段表现出不同的作用。我们假设Notch信号的减少调节骨膜祖细胞在骨折愈伤组织中的增殖、迁移和向成熟间充质细胞系的分化。我们建议在骨折愈合过程中使用特定阶段的基因Notch获得和功能丧失模型来评估Notch的影响。我们还将评估使用直接干扰Notch转录复合体的小肽SAHM1对Notch的抑制作用。该方法将为加速或改善骨折愈合提供潜在的未来应用证据。在目的1中，我们将评估Notch过表达的影响。通过将Notch 1细胞内结构域（NICD1）的强制表达引导到成骨谱系的不同阶段，可以实现过表达。对于骨折后NICD1的定时激活，我们建议使用阶段特异性诱导的cre转基因。α - smacreert2小鼠将靶向Notch过表达至祖细胞阶段，而DMP1-CreERT2小鼠将在成骨细胞/骨细胞中实现过表达。Notch调节的效果将通过评估骨痂形成的进展，以及骨折愈合过程中骨强度和刚度的变化来评估。我们还将通过体外和体内方法研究Notch过表达对PPCs的影响机制，以研究其对增殖、迁移和分化的影响。在目的2中，我们将确定阶段特异性Notch抑制对骨折愈合的影响。为了破坏Notch信号，我们将使用一种转基因模型，在这种模型中，Notch信号的直接转录效应因子Rbpjκ在发生骨折后被删除（Rbpjκflox）。我们将评估PPCs对Notch抑制作用的体内和体外评价。我们将

DESCRIPTION (provided by applicant): Our recent work has shown that smooth muscle alpha actin (αSMA) 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 αSMA- 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. αSMACreERT2 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, Rbpjκ, is deleted (Rbpjκflox) 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.