Dissecting the Molecular Mechanisms that Govern Notch Mediated Skeletal Stem Cell Maintenance Throughout Adulthood/Aging.

项目摘要 骨科损伤是我国最重要的健康和经济负担之一。骨 由于骨质疏松症等疾病，随着年龄的增长而变得脆弱，这增加了骨折的风险， 导致延迟的骨愈合，从而导致更高的不愈合风险。随着人口老龄化的加剧， 对治疗剂的需求，以打击与年龄有关的骨质流失是美国骨科的支柱之一， 协会的“拥有骨头”倡议。目前的治疗主要集中在防止进一步的骨质流失， 而不是以实质性的方式增加骨量。皮肤干细胞和祖细胞（SSPC）是必需的 以获得适当的骨量和骨愈合。已知SSPC的频率和功能随着年龄的增长而下降。 因此，SSPC是增加骨量的最直接靶点。以前的研究和我们自己的数据 提示Notch信号在SSPC稳态和自我更新中起关键作用。我们最近发现 一种条件性Notch敲除小鼠模型，其表现出显著的骨形成增加的表型， 所有的骨骼元素。该小鼠模型，Ncstnf/f;LepR-cre小鼠，缺乏Nicastrin （NcR），LepR+中所有4种Notch受体的Notch信号转导机制的必需组分。 SSPC。该模型具有令人兴奋的治疗潜力，以对抗年龄相关的骨质流失。基于cKO模型 根据我们的初步数据，我推测Notch信号在维持骨骼发育干中是必不可少的。 在成年期和衰老期的细胞池。为了研究Notch信号转导的分子机制， 抑制导致骨小梁质量随年龄增加，我们对LepR+进行了批量RNA测序， 来自中年WT和cKO小鼠的SSPC。这种无偏测序方法将Ebf 3鉴定为 在cKO细胞中显著下调的基因，因此是Notch信号传导的潜在下游介导物 促进SSPC分化为成骨细胞。以前的工作已经将Ebf 3与抑制 然而，在骨生成中，Ebf 3的上游调节因子尚不清楚。此外，虽然其他人已经研究了 Notch在SSPC中的作用，Notch控制SSPC功能的下游机制仍然是 大部分未知。因此，该提案的目的是i）定义Notch信号在SSPC中的确切作用， 衰老/成年对增殖、自我更新和分化的影响，以及ii）通过以下方式揭示分子机制： 其中Notch信号传导对增殖、自我更新和分化具有影响。这些目标将 提供了Notch-Ebf 3信号轴维持Notch-Ebf 3信号轴的机制的新的具体机制见解， 骨骼干细胞在动物的一生中处于原始状态。

PROJECT SUMMARY Orthopedic injuries represent one of the most significant health and economic burdens in our nation. Bone fragility with age due to diseases such as osteoporosis increases the risk for fracture, while at the same time leading to delayed bone healing resulting in a higher risk for non-union. With a growing aging population, the demand for therapeutics to combat age-related bone loss is one of the pillars of the American Orthopedic Association’s “Own-the-Bone” initiative. Current treatments mainly focus on preventing further bone loss rather than increasing bone mass in a substantial manner. Skeletal stem and progenitor cells (SSPCs) are essential for proper bone mass and bone healing. SSPCs are known to decline in frequency and function with age. Therefore, SSPCs represent the most direct target to increase bone mass. Previous studies and our own data suggest that Notch signaling plays a pivotal role in SSPC homeostasis and self-renewal. We recently identified a conditional Notch knockout mouse model which exhibits a striking phenotype of increasing bone formation in all skeletal elements in aging mice. This mouse model, the Ncstnf/f;LepR-cre mouse, is deficient in Nicastrin (Ncstn), an essential component of the Notch signal transduction machinery of all 4 Notch receptors in LepR+ SSPCs. This model has exciting therapeutic potential to combat age-related bone loss. Based on this cKO model and our preliminary data I hypothesize that Notch signaling is essential in maintenance of the skeletogenic stem cell pool during adulthood and aging. In order to investigate the molecular mechanism by which Notch signal inhibition leads to increased trabecular bone mass with age, we performed bulk RNA sequencing on LepR+ SSPCs from middle-aged WT and cKO mice. This unbiased sequencing approach identified Ebf3 as a significantly downregulated gene in cKO cells and therefore potential downstream mediator of Notch signaling in promoting SSPC differentiation into osteoblasts. Previous work has linked Ebf3 to the inhibition of osteogenesis, however, the upstream regulators of Ebf3 are not yet known. Also, while others have looked into the role of Notch in SSPCs, the downstream mechanisms by which Notch is controlling SSPC function are still largely unknown. Therefore, this proposal aims to i) define the precise role of Notch signaling in SSPCs during aging/adulthood on proliferation, self-renewal and differentiation and ii) uncover the molecular mechanism by which Notch signaling is having the effects on proliferation, self-renewal and differentiation. These aims will provide novel specific mechanistic insights into the mechanism by which a Notch-Ebf3 signaling axis maintains skeletal stem cells in their primitive state throughout the lifespan of an animal.