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

描述（由申请人提供）：在干细胞和造血稳态中起关键作用的信号传导途径之一是Notch信号传导级联。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。通过糖分子岩藻糖修饰Notch受体，关键性地调节Notch受体与其对应物Notch配体的相互作用，并且对于Notch信号传导激活是重要的。岩藻糖加入Notch受体是由蛋白质O-岩藻糖基转移酶1（Pofut 1）介导的。使用两种小鼠模型，其中小鼠被遗传修饰为在所有携带岩藻糖的糖蛋白或糖脂（FX-/-）的表达中有条件地缺陷，或者仅在包括Notch在内的一些蛋白的EGF重复序列上存在O-连接的岩藻糖基聚糖（条件Pofut 1-/-），我们最近发现了O-岩藻糖的作用修饰的Notch信号传导，其对于造血稳态以及造血干细胞（HSC）的岩藻糖修饰的需要是必需的因为它们在骨髓微环境中的适当位置，也称为龛。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。在本研究中，为了检验岩藻糖修饰的Notch对于Notch-配体相互作用依赖性控制HSC稳态至关重要的假设，我们将首先通过使用遗传修饰的小鼠模型和小鼠胚胎干细胞来定义配体结合所需的Notch岩藻糖部分的缺乏对HSC稳态和血液谱系分化的影响。我们还将评估Pofut 1缺陷型HSC或缺乏已知Notch配体结合所必需的岩藻糖的HSC是否通过使用尖端成像技术（双光子活体显微镜）在适当的骨髓龛中被置换。然后，我们将确定Notch O-岩藻糖缺乏导致异常HSC稳态的机制。我们将评估由于配体结合结构域中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龛位置异常和HSC功能异常的主要原因。此外，我们将评估Notch途径与其他主要粘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龛寄宿很重要。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HSC骨髓归巢和沉积中的意义。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，并将成为未来研究和开发干细胞治疗和癌症医学新治疗策略的基础。

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.