Notch Signaling and Bone Fracture Healing

分离的SMA标记的祖细胞的基因表达分析显示，在骨折愈合的早期阶段，Notch信号通路显著减少。以往的研究表明，Notch信号通路在不同的成骨细胞成熟阶段表现出不同的作用。我们推测，在骨折骨痂中，Notch信号的减少调节骨膜前体细胞的扩张、迁移和分化为成熟的间充质系。我们建议使用特定阶段的遗传Notch功能增减模型来评估Notch在骨折愈合过程中的效果。我们还将用直接干扰Notch转录复合体的小肽SAHM1来评估Notch的抑制作用。这一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。通过将强制Notch 1胞内域(NICD1)的表达引导到成骨谱系的不同阶段，将实现过度表达。对于骨折发生后NICD1的定时激活，我们建议使用阶段特定的可诱导Cre转基因。？SMACreERT2小鼠将靶向Notch高表达至祖细胞阶段，而DMP1-CreERT2小鼠将在成骨细胞/骨细胞中实现高表达。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硬度的变化来评估。我们还将使用体外和体内方法研究Notch过表达对PPC增殖、迁移和分化的影响机制。在目标2中，我们将确定特定阶段的Notch抑制对骨折愈合的影响。为了干扰Notch信号，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Rbpj？在骨折发生后被删除(Rbpj？Flox)。将对使用PPC的Notch抑制进行体外和体内评估。我们会 扩大抑制研究，以评估Notch转录因子复合体抑制剂SAHM1(源自MAML1的装订α螺旋多肽)对骨折愈合的治疗作用。我们的研究结果将使我们更好地了解Notch信号在骨折愈合过程中的作用，并将评估未来对愈合过程的治疗调节。

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.