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成骨细胞控制HSCs至关重要的细胞和分子介质。我们先前证实甲状旁腺激素(PTH)可激活成骨细胞以增加造血干细胞(HSC)的数量，并且PTH可提高辐射损伤后HSC的存活率。这些结果为我们定义了一种新的治疗方法，通过刺激成骨细胞来增加医源性或毒性骨髓损伤后的HSCs。然而，特定的成骨细胞亚群和调控HSCs的成骨细胞依赖的关键分子事件尚不清楚。利用药理学和遗传学模型，我们已经确定Notch信号是PTH依赖的HSC调节的潜在中介。缺口激活需要细胞结合的配体与邻近细胞上的受体直接相互作用。我们证明：1)甲状旁腺素或其受体的激活刺激成骨细胞中的Notch配体Jagged1(Jag1)；2)在成骨细胞中具有固有活性的PTH受体的小鼠，HSC增加了Notch的激活；3)依赖PTH的HSC的增加被抑制β-分泌酶活性所阻断，而β-分泌酶活性是Notch激活所必需的。我们的初步研究表明，Jag1在成骨细胞中的表达是PTH依赖的HSC扩增所必需的。综上所述，这些数据表明，甲状旁腺素通过成骨细胞表达Jag1来扩大HSC，然后Jag1激活邻近骨髓细胞中的Notch信号。根据我们的数据，我们假设成骨细胞扩增的HSC需要Jag1在骨髓微环境中启动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扩增所必需的且足够的。在AIM2中，我们将确定需要Notch激活以实现成骨细胞依赖的HSC扩增的细胞群(HSC、成骨细胞和/或骨髓的其他成分)。最后，在Aim3中，我们将确定Notch信号对甲状旁腺素的骨髓保护作用的贡献，这是一种临床情景，在这种情景中，HSC生态位操作可能是一种降低发病率和死亡率的新策略。我们已经建立了微环境信号增加HSCs的体内模型，并对其进行了充分表征。既然成骨细胞Jag1已经被确定为甲状旁腺素依赖的HSC扩增的关键成分，我们就有了前所未有的机会，可以使用这里提出的体内策略来确定HSC利基的细胞和分子成分。因此，我们的实验目标的完成将为骨髓微环境中的HSC操作确定新的治疗靶点，这可以用于提高骨髓损伤后的存活率。公共卫生相关性：在这项建议中，我们研究了骨髓微环境对造血干细胞(HSC)的调节。由于HSC产生了所有的血细胞，这些调节机制可以用于治疗在特定情况下增加HSC的血细胞损伤或缺乏。

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.