Non-Canonical Notch Regulation of Cardiovascular Progenitors

心血管祖细胞（CPC）在心脏再生方面具有巨大的治疗潜力 由于它们具有独特的扩展和分化成各种心脏细胞类型的能力，因此它们在医学上具有重要意义。然而，要采取 再生疗法的优势，我们需要了解自我更新的机制， CPC的谱系特异性分化。目前的建议集中在阐明一个新的作用缺口 CPC维持和分化中的信号传导。Notch是一种进化上保守的跨膜蛋白 在决定细胞命运的过程中起着至关重要的作用典型的Notch信号传导是通过结合 Notch的细胞外配体。这导致细胞内切割和Notch易位到细胞核中 在那里它与转录介质RBP-J结合用于基因激活。我证明了Notch 1缺陷型 CPC随着增殖的增加而显著扩增，类似于用活性肽刺激的CPC的表型。 连环蛋白在RBP-J缺陷的CPC中没有观察到这种表型，表明RBP-J在CPC中的非典型作用。 缺口。Notch 1缺陷显著增加了<$-Catenin信号传导。这种增加不是由 上调<$-连环蛋白mRNA，而是通过积累活性<$-连环蛋白，这表明后， 翻译调节有趣的是，Notch对连环蛋白的调节不需要经典的配体- 依赖于Notch的膜切割或<$-连环蛋白指导的蛋白酶体降解，但它确实需要 将膜Notch运输到溶酶体的内吞蛋白。此外，膜结合Notch， 传统上被认为是生物惰性的，物理上与活性连环蛋白相关， 活性连环蛋白的积累。这些发现揭示了一个以前没有描述的作用，膜 缺口调节活性连环蛋白水平，并为探索这种作用的机制奠定了基础。 CPC的维持和分化。我建议测试Notch拮抗CPC的假设 通过配体/转录非依赖性中活性<$-连环蛋白的溶酶体降解进行自我更新/扩增 时尚.本研究的具体目的是：（1）确定Notch 1缺陷型CPC是否有利于自我更新 过度分化，以及这种效应是否需要β-连环蛋白;（2）为了测试膜结合Notch 1 影响CPC的扩展和分化以及细胞事件是否需要<$-连环蛋白;（3）为了确定CPC的分子结构， 溶酶体活性在膜结合Notch和活性<$-连环蛋白降解之间的联系中的作用。的 拟议的工作将提供基本的见解，了解控制CPC自我的机制， 再生/分化决定，CPC介导的心脏再生治疗的先决条件。的 膜Notch的生物学在CPC和干细胞领域中完全未被探索。鉴于高度 Notch和Wnt/ω-连环蛋白信号在几乎所有已知的干/祖细胞命运决定中的保守作用， 这些研究将为涉及干/祖细胞的再生医学研究开辟新的途径。

Cardiovascular progenitor cells (CPCs) hold tremendous therapeutic potential for cardiac regenerative medicine due to their unique ability to expand and to differentiate into various heart cell types. However, to take advantage of regenerative therapy, we need to understand the mechanisms underlying the self-renewal and lineage-specific differentiation of CPCs. The current proposal focuses on elucidating a novel role of Notch signaling in CPC maintenance and differentiation. Notch is an evolutionarily conserved transmembrane protein that plays critical roles in numerous cell-fate decisions. Canonical Notch signaling is initiated by binding of extracellular ligands to Notch. This leads to intracellular cleavage and translocation of Notch into the nucleus where it binds to the transcriptional mediator RBP-J for gene activation. I demonstrated that Notch1-deficient CPCs expand dramatically with increased proliferation, similar to the phenotype of CPCs stimulated with active ¿-Catenin. This phenotype is not observed in CPCs deficient for RBP-J, suggesting a non-canonical role of Notch. Notch1-deficiency significantly increased ¿-Catenin signaling. This increase was not mediated by upregulation of ¿-Catenin mRNA but rather by accumulation of active ¿-Catenin protein, suggesting post- translational regulation. Intriguingly, the Notch regulation of ¿-Catenin protein did not require classical ligand- dependent membrane cleavage of Notch or the ¿-Catenin directed proteasomal degradation, but it did require endocytic proteins that traffic membranous Notch to the lysosome. Moreover, membrane-bound Notch, conventionally considered biologically inert, physically associated with active ¿-Catenin and inhibited accumulation of active ¿-Catenin protein. These findings reveal a previously undescribed role of membrane Notch in regulating active ¿-Catenin protein levels and set the stage for a mechanistic exploration of this role in the maintenance and differentiation of CPCs. I propose to test the hypothesis that Notch antagonizes CPC self-renewal/expansion by lysosomal degradation of active ¿-Catenin in a ligand/transcription-independent fashion. The specific aims of this proposal are (1) To determine if Notch1-deficient CPCs favor self-renewal over differentiation and if ¿-Catenin is required for this effect; (2) To test whether membrane-bound Notch1 affects CPC expansion and differentiation and whether the cellular events require ¿-Catenin; (3) To identify the role of lysosomal activity in the link between membrane-bound Notch and active ¿-Catenin degradation. The proposed work will provide fundamental insights into the understanding of mechanisms controlling CPC self- renewal/differentiation decisions, a prerequisite for CPC-mediated cardiac regenerative therapeutics. The biology of membrane Notch is completely unexplored in the field of CPCs as well as in stem cells. Given highly conserved roles of Notch and Wnt/¿-Catenin signaling in nearly all known stem/progenitor cell fate decisions, these studies will open up new avenues of research for regenerative medicine involving stem/progenitor cells.