Numb调控Notch信号通路的新机制研究

Numb is the first cell-fate determinant to be identified, and regulates cell-fate decisions by counteracting Notch signaling. At present, most researches about the mechanism of Numb in regulating Notch signaling are only focused on the Notch receptor itself. Recently, we found that Numb also regulates the intracellular trafficking of Notch ligand. Our preliminary data showed that knockdown of Numb and Numblike results in significantly increased Dll4 (a Notch ligand) protein expression, and activation of Notch signaling in cultured endothelial cells. These data indicate that Numb may target Notch signaling via Dll4. In this proposal we will investigate exactly how Numb regulates Dll4 to antagonize Notch signaling in endothelial cells. Given that the specification of endothelial tip and stalk cells is prominently regulated by Dll4-Notch signaling, we will also investigate whether Numb controls tip/stalk cell specification through the above-mentioned mechanism. The expected results not only help us fully recognize the signaling network controlling fate decisions of endothelial cells, but also understand the Numb-Notch axis more deeply.

Numb作为第一个被发现的细胞命运决定子，通过拮抗Notch信号发挥作用。关于Numb调控Notch信号通路的分子机制研究目前完全围绕Notch受体本身展开，但我们发现Numb能够调节Notch的配体。我们在血管内皮细胞中的前期研究显示敲降Numb和同源基因Numblike导致配体Dll4表达异常升高，且Notch信号激活，提示Numb可能通过Dll4调控Notch信号。本项目将以此为基础，在内皮细胞中深入研究Numb通过调节Dll4进而拮抗Notch信号的分子机制。鉴于Dll4-Notch通路在血管内皮端/茎细胞命运选择中发挥关键作用，我们将同时分析Numb是否通过上述拮抗Notch的机制参与调控端/茎细胞命运决定。预期研究结果不仅有助于全面认识血管内皮细胞命运决定的调控网络，而且将加深对Numb-Notch信号轴的理解。

Notch信号在众多生理和病理过程中发挥重要作用。近年来的研究显示Numb蛋白是Notch信号的一个关键拮抗因子，但Numb究竟如何拮抗Notch信号并不完全清楚。本项目在血管系统中对Numb拮抗Notch信号的分子细胞机制进行了详细研究。我们的研究发现，在血管内皮细胞中敲除Numb和Numlike导致Notch信号激活，并意外发现Notch的配体Dll4表达异常升高。经研究发现敲除Numb和Numblike增加Dll4的蛋白稳定性并使Dll4更多的聚集在细胞膜上。进一步分析显示Numb可作为细胞内Dll4的转运分选开关，一方面促进Dll4朝溶酶体转运，另一方面降低Dll4的循环再利用过程。我们同时证明了敲除Numb和Numblike所造成的膜上聚集的Dll4可激活相邻细胞的Notch信号。鉴于Dll4-Notch在血管生成中发挥重要作用，我们在小鼠体内分析了Numb是否参与血管生成，结果显示敲除Numb和Numblike抑制血管出芽，导致血管网络稀疏。基于这些研究结果，我们提出了一个Numb通过Dll4拮抗Notch信号的新机制。