Physiologic Regulation of Hematopoiesis by Notch

简介(申请人提供)：背景。尽管在识别在成人造血过程中协调自我更新、增殖和分化的关键分子方面取得了进展，但对这些过程在骨髓内稳态和适应应激反应过程中的生理和分子调控知之甚少。Notch信号在正常造血过程中对细胞命运的决定起着至关重要的调节作用，它促进干细胞的自我更新，并通过促进人骨髓前体细胞的扩张来抑制其分化。Notch信号也被证明参与了白血病的发生。初步结果。我们以前的研究表明，Notch激活抑制了BM前体细胞的分化，使其保持在更不成熟的表型，并通过缩短其G1期并促进其过早进入S期而诱导其细胞周期紊乱。我们最近已经确定了介导这一效应的分子机制。我们发现Notch1的激活诱导了Skp2的转录激活，Skp2是SCFskp2复合体的F-box亚单位，针对细胞周期抑制物p27Kip1和p21Cip1的降解，促进它们的下调并进入S期。值得注意的是，Skp2的过度表达在白血病和淋巴瘤中经常发生改变。为了解决这些结果的生理学相关性，我们研究了炎症应激期间的骨髓反应。我们发现，炎症过程中释放的细胞因子，如内毒素和肿瘤坏死因子，可以诱导骨髓细胞Notch信号的上调，并导致干细胞和祖细胞增加4-5倍。假设。基于这些结果，我们提出了一个模型，在该模型中，骨髓微环境信号通过Notch信号向造血细胞及其细胞周期机制发出信号。我们推测，Skp2/p27Kip1通路对于Notch介导的干细胞和祖细胞的扩张至关重要，特别是在应激条件下，它可能参与了Notch诱导的白血病的发生。目标和战略。为了验证这一假说，我们建议：(1)通过使用Skp2或Notch信号缺失的小鼠(RBP-J条件基因敲除)并结合移植模型，确定Notch/Skp2/CKIs通路在骨髓干/祖细胞扩增中的作用；(2)通过分析在没有Skp2的情况下，Notch过度激活对造血室的影响，确定Skp2是否在Notch介导的干祖细胞扩张和Notch诱导的白血病发生中起关键作用；(3)确定不同的Notch配体和Notch信号强度在Skp2/CKIs通路募集中的作用。这个问题将通过与过度表达不同Notch配体的饲养层共培养刺激造血细胞来解决，并增加重组Delta4配体的剂量。关联性。我们相信，这些实验将深入了解骨髓内稳态的生理调节和白血病发生过程的分子机制，并将有助于开发新的造血疾病治疗策略。 项目简介：本提案旨在确定在正常条件下和在应激条件下，如炎症、感染或骨髓移植时，骨髓调节血细胞产生的机制。特别是，我们专注于了解Notch受体在维持和扩增骨髓造血祖细胞和干细胞方面的作用。更好地理解在正常造血过程中协调细胞增殖和分化的事件对于理解白血病的发生机制至关重要，因此对于开发新的治疗策略是至关重要的。该项目的长期目标是评估Notch信号通路在正常骨髓重建和白血病中的作用，并确定新的治疗干预靶点。

DESCRIPTION (provided by applicant): Background. Despite the progress made in identifying key molecules that coordinate self-renewal, proliferation and differentiation during adult hematopoiesis, little is known about the physiologic and molecular regulation of these processes during bone marrow (BM) homeostasis and during its adaptive response to stress. Notch signaling plays an essential role in the regulation of cell fate decisions in normal hematopoiesis, favoring stem cell self-renewal and inhibiting differentiation of human bone marrow progenitors by promoting their expansion. Notch signaling has also been shown to participate in leukemogenesis. Preliminary results. Our previous studies showed that Notch activation inhibits differentiation of BM progenitors, preserving them in a more immature phenotype, and induces perturbation of their cell cycle by shortening their G1 phase and promoting premature entry into S-phase. We have recently identified the molecular mechanism that mediates this effect. We discovered that Notch1 activation induces the transcriptional activation of SKP2, the F-box subunit of the SCFskp2 complex that targets the cell cycle inhibitors p27Kip1 and p21Cip1 for degradation, promoting their downregulation and the entry into S-phase. Of note, SKP2 overexpression, is frequently altered in leukemias and lymphoma. To address the physiological relevance of these results we investigated the BM response during inflammatory stress. We found that cytokines released during inflammation, such as LPS and TNF1, induce upregulation of Notch signaling in BM cells and result in 4-5 fold increase in stem cells and progenitors. Hypothesis. Based on these results, we propose a model in which BM microenvironmental cues signal to hematopoietic cells and their cell cycle machinery via Notch signaling. We hypothesize that the SKP2/p27Kip1 pathway is critical for Notch-mediated stem cells and progenitor expansion, in particular during conditions of stress, and that it may be involved in Notch-induced leukemogenesis. Aims and Strategy. To test this hypothesis we propose to: (1) Determine the role of the Notch/SKP2/CKIs pathway in the expansion of BM stem and progenitor cells by using mice deficient for SKP2 or Notch signaling (RBP-J conditional knock out) in combination with transplantation models; (2) Define whether SKP2 is critical for Notch-mediated effects on stem/progenitor expansion and Notch-induced leukemogenesis by analyzing the in vivo effects of Notch iperactivation on the hematopoietic compartment in the absence of SKP2; (3) Determine the role of different Notch ligands and of intensity of Notch signaling in the recruitment of the SKP2/CKIs pathway. This question will be addressed by stimulating hematopoietic cells through co-culture with feeders overexpressing different Notch ligands and with increasing doses of recombinant Delta4 ligand. Relevance. We believe that these experiments will yield insight into the molecular mechanisms underlying the physiologic regulation of bone marrow homeostasis and the process of leukemogenesis and that they will contribute to the development of novel therapeutic strategies in hematopoietic disorders. PROJECT NARRATIVE: The present proposal aims at defining the mechanisms that regulate blood cell production by the bone marrow in normal conditions and in conditions of stress, such as inflammation, infections or bone marrow transplantation. In particular, we are focused on understanding the role of the Notch receptors in the maintenance and expansion of bone marrow hematopoietic progenitors and stem cells. A better comprehension of the events that coordinate cell proliferation and differentiation during normal hematopoiesis is crucial for the understanding of the mechanisms of leukemogenesis and, therefore, is essential for the development of novel therapeutic strategies. The long term goal of this project is to evaluate the role of the Notch signaling pathway both in normal bone marrow reconstitution and in leukemias and to identify novel targets of therapeutic intervention.