Physiologic Regulation of Hematopoiesis by Notch

描述（由申请人提供）：背景。尽管在确定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，但对骨髓稳态和应激适应性反应中这些过程的生理和分子调控知之甚少。Notch信号在正常造血过程中调控细胞命运决定中发挥重要作用，有利于干细胞自我更新，并通过促进骨髓祖细胞增殖抑制其分化。Notch信号也被证明参与了白血病的发生。初步结果。我们之前的研究表明，Notch激活抑制BM祖细胞的分化，使其保持更不成熟的表型，并通过缩短其G1期和促进过早进入s期来诱导其细胞周期的扰动。我们最近已经确定了介导这种效应的分子机制。我们发现Notch1激活诱导SKP2的转录激活，SKP2是SCFskp2复合物的F-box亚基，其靶向细胞周期抑制剂p27Kip1和p21Cip1降解，促进其下调并进入s期。值得注意的是，SKP2过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脑基反应。我们发现炎症过程中释放的细胞因子，如LPS和TNF1，诱导BM细胞Notch信号上调，导致干细胞和祖细胞数量增加4-5倍。假设。基于这些结果，我们提出了一个模型，其中BM微环境信号通过Notch信号传递给造血细胞及其细胞周期机制。我们假设SKP2/p27Kip1通路对notch介导的干细胞和祖细胞的扩增至关重要，特别是在应激条件下，并且它可能参与notch诱导的白血病发生。目标和策略。为了验证这一假设，我们提出：(1)利用SKP2或Notch信号缺失（RBP-J条件敲除）的小鼠结合移植模型，确定Notch/SKP2/CKIs通路在BM干细胞和祖细胞扩增中的作用；(2)通过分析体内缺乏SKP2时Notch过激活对造血室的影响，确定SKP2是否在Notch介导的干细胞/祖细胞扩增和Notch诱导的白血病发生中起关键作用；(3)确定不同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表达不同Notch配体的饲料共培养和增加重组Delta4配体的剂量来刺激造血细胞来解决。的相关性。我们相信，这些实验将有助于深入了解骨髓稳态生理调节和白血病发生过程的分子机制，并有助于开发新的造血疾病治疗策略。

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.