Notch Signaling and Bone Fracture Healing

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）是间充质祖细胞的标志物，其在骨折后迅速扩增，并且显示出对骨折骨痂内的纤维组织、成骨细胞和软骨细胞谱系的显著贡献。基因表达分析分离？SMA标记的祖细胞揭示了Notch信号通路在骨折愈合的早期阶段显著降低。先前的研究表明，Notch信号传导根据骨祖细胞成熟的阶段表现出不同的作用。 我们推测Notch信号的减少调节骨折骨痂中骨膜祖细胞的扩增、迁移和分化为成熟的间充质谱系。我们建议在骨折愈合过程中使用阶段特异性遗传Notch获得和丧失功能模型来评估Notch的作用。我们还将评估使用直接干扰Notch转录复合物的小肽SAHM 1对Notch的抑制。这种方法将为未来加速或改善骨折愈合的潜在应用提供证据。 在目标1中，我们将评估Notch过表达的影响。将通过将强制Notch 1胞内结构域（NICD 1）表达引导至成骨谱系的不同阶段来实现过表达。对于骨折产生后NICD 1的定时激活，我们提出使用阶段特异性诱导Cre转基因。? SMACreERT 2小鼠将用于将Notch过表达靶向至祖细胞阶段，而成骨细胞/骨细胞中的过表达将通过使用DMP 1-CreERT 2小鼠实现。将通过评价骨痂形成的进展以及骨折愈合期间骨强度和刚度的变化来评估Notch调节的效果。我们还将使用体外和体内方法研究Notch过表达对PPC的影响机制，以研究其对增殖，迁移和分化的影响。在目标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信号传导，我们将使用转基因模型，其中Notch信号传导的直接转录效应子Rbpj？删除（Rbpj？flox）之后产生裂缝。将评价使用PPC的Notch抑制的体外和体内评价。我们将 扩展抑制研究以评估Notch转录因子复合物抑制剂SAHM 1（源自MAML 1的钉合α-螺旋肽）对骨折愈合的治疗。我们的研究结果将提供一个更好的理解Notch信号在骨折愈合过程中的作用，并将评估未来的治疗调节愈合过程。

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.