Regulatory Cell Signals in the Bone Marrow

描述（由申请人提供）：本提案的长期目标是确定对造血干细胞成骨细胞控制至关重要的细胞和分子介质。我们之前已经证实，甲状旁腺激素（PTH）激活成骨细胞，增加造血干细胞（HSC）的数量，并且PTH可以提高放射损伤后造血干细胞的存活率。这些结果为我们提供了一个模型来定义一种新的治疗方法，通过刺激成骨细胞来增加医源性或毒性骨髓损伤后的造血干细胞。然而，特定的成骨细胞亚群和调节造血干细胞的关键成骨细胞依赖分子事件尚不清楚。使用药理学和遗传学模型，我们已经确定Notch信号作为pth依赖性HSC调节的潜在介质。Notch激活需要细胞结合配体与邻近细胞上的受体直接相互作用。我们证明了1)PTH或其受体的激活刺激成骨细胞中的Notch配体Jagged1 (Jag1)；2)在成骨细胞中PTH受体具有组成性活性的小鼠中，造血干细胞增加了Notch的激活；3) pth依赖性HSC的增加可通过抑制？-分泌酶活性，这是Notch激活所必需的。我们现在的初步研究表明，成骨细胞中Jag1的表达是pth依赖性HSC扩增所必需的。综上所述，这些数据表明PTH通过成骨细胞表达Jag1来扩展HSC， Jag1随后激活邻近骨髓细胞中的Notch信号。基于我们的数据，我们假设成骨细胞的HSC扩增需要骨髓微环境中jag1启动的Notch激活。为了验证这一假设，在Aim1中，我们将定义成骨细胞亚群，其中Jag1是介导HSC扩增的必要和充分条件。在Aim2中，我们将确定需要Notch激活来实现成骨依赖的HSC扩增的细胞群（HSC、成骨细胞和/或骨髓的其他成分）。最后，在aims中，我们将确定Notch信号对PTH骨髓保护作用的贡献，这是一种临床情况，在这种情况下，HSC生态位操作可能是一种降低发病率和死亡率的新策略。我们已经建立并充分表征了微环境信号增加造血干细胞的体内模型。既然成骨细胞Jag1已被确定为pth依赖性HSC扩增的关键因素，我们就有了前所未有的机会，可以使用本文提出的体内策略来定义HSC生态位的细胞和分子成分。因此，完成我们的实验目标将确定在骨髓微环境中操作HSC的新治疗靶点，这可以用来提高骨髓损伤后的存活率。公共卫生相关性：在本提案中，我们研究了造血干细胞（HSC）通过其骨髓微环境的调节。由于造血干细胞产生所有的血细胞，这些调节机制可以在治疗上用于在血细胞损伤或缺乏的特定情况下增加造血干细胞。

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to define the cellular and molecular mediators crucial for osteoblastic control of HSCs. We previously established that parathyroid hormone (PTH) activates osteoblastic cells to increase hematopoietic stem cell (HSC) numbers and that PTH improves HSC survival after radiation injury. These results give us a model to define a novel therapeutic approach to increase HSCs following iatrogenic or toxic injury to the bone marrow by stimulating osteoblastic cells. However, the specific osteoblastic cell subsets and the key osteoblastic-dependent molecular events regulating HSCs are unknown. Using pharmacologic and genetic models, we have identified Notch signaling as a potential mediator of PTH-dependent HSC regulation. Notch activation requires direct interaction of cell-bound ligands with receptors on neighboring cells. We demonstrated that 1) PTH or activation of its receptor stimulate the Notch ligand Jagged1 (Jag1) in osteoblastic cells; 2) in mice with constitutively active PTH receptors in osteoblastic cells, HSCs have increased Notch activation; 3) the PTH-dependent HSC increase is blocked by inhibition of ?-secretase activity, which is required for Notch activation. Our preliminary studies now demonstrate that expression of Jag1 in osteoblastic cells is required for the PTH-dependent HSC expansion. Together, these data suggest that PTH expands HSC through osteoblastic expression of Jag1, which then activates Notch signaling in neighboring bone marrow cells. Based on our data, we hypothesize that HSC expansion by osteoblasts requires Jag1-initiated Notch activation in the bone marrow microenvironment. To test this hypothesis, in Aim1 we will define the osteoblastic cell subset in which Jag1 is necessary and sufficient to mediate HSC expansion. In Aim2, we will identify the cell population (HSC, osteoblastic cells and/or other components of the bone marrow) in which Notch activation is required to achieve osteoblastic-dependent HSC expansion. Finally in Aim3 we will determine the contribution of Notch signaling to the myeloprotective effects of PTH, a clinical scenario in which HSC niche manipulation could be a novel strategy to reduce morbidity and mortality. We have already established and fully characterized in vivo models in which microenvironmental signals increase HSCs. Now that osteoblastic Jag1 has been identified as a key element of PTH-dependent HSC expansion, we have the unprecedented opportunity of defining the cellular and molecular components of the HSC niche using the in vivo strategies proposed here. Completion of our experimental aims will thus define novel therapeutic targets for HSC manipulation in the bone marrow microenvironment, which can be exploited to improve survival after bone marrow injury. PUBLIC HEALTH RELEVANCE: In this proposal, we study the regulation of hematopoietic stem cells (HSC) by their bone marrow microenvironment. Since HSC give rise to all blood cells, these regulatory mechanisms could be therapeutically exploited to increase HSC in specific situations of blood cell injury or deficiency.