O-fucose Modified Notch as a Regulator of the Hematopoietic Stem Cell Homeostasis

描述(申请人提供)：在干细胞和造血系统内稳态中起关键作用的信号通路之一是Notch信号级联。Notch在许多发育和病理生理过程中起着关键作用。Notch通路的突变或异常激活与多种人类发育障碍和癌症有关。Notch受体被糖分子岩藻糖修饰，关键是调节Notch受体与其对应的Notch配体之间的相互作用，并对Notch信号激活起重要作用。岩藻糖与Notch受体的加成是由蛋白O-岩藻糖基转移酶1(Pofut1)介导的。在两个小鼠模型中，我们最近发现了O-岩藻糖修饰的Notch信号转导信号的作用，以及对造血干细胞(HSC)进行岩藻糖修饰以保证其在骨髓微环境中的正确位置。这些发现揭示了岩藻糖修饰的Notch在造血和HSC生物学中的独特作用。在这项研究中，为了验证岩藻糖修饰的Notch对于Notch-配体相互作用依赖的HSC稳态调控至关重要的假说，我们将首先通过转基因小鼠模型和小鼠胚胎干细胞来确定配体结合所需的Notch岩藻糖缺失对HSC稳态和血缘分化的影响。我们还将使用尖端成像技术(双光子活体显微镜)来评估Pofut1缺陷的HSCs，即缺乏已知对Notch配体结合至关重要的岩藻糖的HSCs是否被替换到适当的骨髓壁龛中。然后，我们将确定缺乏Notch O-岩藻糖导致HSC内稳态异常的机制(S)。我们将评估由于配体结合区缺乏岩藻糖而导致的Notch与其配体之间的破坏性相互作用是否是导致干细胞生态位异常和HSC功能异常的主要原因。此外，我们将评估Notch通路与其他主要黏附分子和细胞因子之间的相互作用，这些分子和细胞因子对干细胞骨髓归巢和骨髓巢倒伏非常重要。最后，我们将确定Notch的增强岩藻糖修饰在HSC与配体结合以及在HSC骨髓归巢和倒伏中的意义。在这些研究的最后，我们希望我们所获得的知识将提高我们对Notch糖基化在干细胞生物学中的作用的理解，并将为未来干细胞治疗和癌症医学中新的治疗策略的研究和开发奠定基础。

DESCRIPTION (provided by applicant): One of the signaling pathways that play pivotal roles in stem cell and hematopoietic homeostasis is the Notch signaling cascade. Notch plays a critical role in many developmental and pathophysiological processes. Mutation or abnormal activation of Notch pathway is linked to a variety of human development disorders and cancers. Notch receptor modification by a sugar molecule, fucose, critically regulates Notch receptor interaction with their counterparts, Notch ligands, and is important for Notch signaling activation. The addition of fucose to Notch receptors are mediated by protein O-fucosyltransferase 1 (Pofut1). Using two mouse models, in which mice are genetically modified to be conditionally deficient either in the expression of all fucose-carrying glycoproteins or glycolipids (FX-/-), or only O-linked fucosyl-glycans present on EGF repeats of a few proteins including Notch (conditional Pofut1-/-), we recently identified a role of O-fucose-modified Notch signaling that is essential for hematopoietic homeostasis as well as a requirement of fucose modification of hematopoietic stem cell (HSC) for their proper location in the bone marrow microenvironment, also called niche. These findings uncovered a unique role of fucose-modified Notch in hematopoiesis and HSC biology. In this study, in order to test the hypothesis that fucose-modified Notch is critical for Notch-ligand interaction- dependent control of HSC homeostasis, we will first define the impact of lack of Notch fucose moieties that are required for ligand binding on HSC homeostasis and blood lineage differentiation by using genetically modified mouse models and mouse embryonic stem cells. We will also assess if Pofut1-deficient HSCs, or HSCs that lack fucose known essential for Notch ligand binding, are displaced in the proper marrow niches by using the cutting edge imaging technology (2-photon intravital microscopy). Then, we will determine the mechanism(s) by which lack of Notch O-fucose causes aberrant HSC homeostasis. We will assess if a disruptive interaction between Notch and its ligand due to the lack of fucose in the ligand binding domain is the main reason that causes aberrant stem cell niche location and abnormal HSC function. Further, we will assess the interaction between Notch pathway with other major adhesion molecules and cytokines that are important for stem cell bone marrow homing and marrow niche lodging. Finally, we will determine the significance of the enhanced fucose modification of Notch in HSC binding with ligand and in HSC marrow homing and lodgment. At the end of these studies, we hope the knowledge we have gained will improve our understanding of the role of Notch glycosylation in stem cell biology, and will form the basis that leads to future studies and development of novel therapeutic maneuvers in stem cell therapy and cancer medicine.