Non-Canonical Notch Regulation of Cardiovascular Progenitors

心血管祖细胞的非典型Notch调节

• 批准号: 8218455
• 负责人: Chulan Kwon
• 金额: $ 40.5万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8218455
• 关键词: Affect; Binding; Biology; Cardiac; Cardiovascular system; Cell Maintenance; Cell Nucleus; Cells; Cellular biology; Complex; Development; Embryo; Endocytosis; Event; Future; Gene Activation; Genes; Genetic Transcription; Goals; Heart; Integral Membrane Protein; Investigation; Ligands; Link; Lysosomes; Maintenance; Mediating; Mediator of activation protein; Membrane; Membrane Biology; Messenger RNA; Molecular; Natural regeneration; Pathway interactions; Phenotype; Play; Population; Post-Translational Regulation; Process; Protein Binding; Proteins; Proteolysis; Regenerative Medicine; Regulation; Research; Role; Signal Pathway; Signal Transduction; Signaling Protein; Staging; Stem cells; Testing; Therapeutic; Up-Regulation; Work; base; cell type; embryonic stem cell; extracellular; heart cell; in vivo; insight; mutant; notch protein; novel; progenitor; protein transport; regenerative; regenerative therapy; self-renewal; stem

DESCRIPTION (provided by applicant): 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 2-Catenin. This phenotype is not observed in CPCs deficient for RBP-J, suggesting a non-canonical role of Notch. Notch1-deficiency significantly increased 2-Catenin signaling. This increase was not mediated by upregulation of 2-Catenin mRNA but rather by accumulation of active 2-Catenin protein, suggesting post- translational regulation. Intriguingly, the Notch regulation of 2-Catenin protein did not require classical ligand- dependent membrane cleavage of Notch or the 2-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 2-Catenin and inhibited accumulation of active 2-Catenin protein. These findings reveal a previously undescribed role of membrane Notch in regulating active 2-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 2-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 2-Catenin is required for this effect; (2) To test whether membrane-bound Notch1 affects CPC expansion and differentiation and whether the cellular events require 2-Catenin; (3) To identify the role of lysosomal activity in the link between membrane-bound Notch and active 2-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/2-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. PUBLIC HEALTH RELEVANCE: Understanding the biology of the cells that develop into the heart, called multipotent cardiovascular progenitor cells (CPCs), is key to realizing the promise of future cell-mediated cardiac therapeutics. The proposed research aims to elucidate the ligand/transcription-independent role of a protein called Notch in CPC self- renewal and differentiation. Our work will provide mechanistic insights into understanding the self-renewal- differentiation processes of multipotent CPCs regulated by Notch, which will facilitate future cell-based cardiac therapeutics and open new avenues of investigation for non-canonical Notch biology.

描述（由申请人提供）：心血管祖细胞（CPC）具有巨大的心脏再生医学治疗潜力，因为它们具有独特的扩展和区分为各种心脏细胞类型的能力。但是，要利用再生疗法，我们需要了解CPC的自我更新和谱系特异性分化的基础机制。当前的提案着重于阐明Notch信号在CPC维持和分化中的新作用。 Notch是一种进化保守的跨膜蛋白，在许多细胞命运决定中起着关键作用。通过细胞外配体与缺口结合来启动典型的Notch信号传导。这导致了细胞内裂解和Notch转移到核中，它与转录介质RBP-J结合以进行基因激活。我证明，缺乏notch1的CPC随着增殖的增加而大大扩展，类似于用活性2-catenin刺激的CPC的表型。在RBP-J缺乏的CPC中未观察到这种表型，这表明Notch的非典型作用。 Notch1缺乏显着提高了2-catenin信号传导。这种增加不是通过上调2-catenin mRNA的介导的，而是通过积累活性2-catenin蛋白的介导的，这表明转化后调节。有趣的是，2-catenin蛋白的Notch调节不需要Notch或2-catenin的定向蛋白酶体降解的经典配体依赖性膜裂解，但是它确实需要内吞蛋白，使膜膜凹槽传递到溶酶体。此外，膜结合的缺口常规视为生物学惰性，与活性2-catenin物理相关，并抑制活性2-catenin蛋白的积累。这些发现揭示了膜缺口在调节活性2-catenin蛋白水平上的先前未描述的作用，并为对CPC的维持和分化而进行机械探索奠定了基础。我建议测试以下假设，即通过配体/不依赖的方式，通过活性2-catenin的溶酶体降解来拮抗CPC自我更新/扩展。该提案的具体目的是（1）确定缺乏notch1的CPC是否支持自我更新而不是分化，以及是否需要2-catenin才能进行这种效果； （2）测试膜结合的Notch1是否影响CPC的扩展和分化以及细胞事件是否需要2-catenin； （3）确定溶酶体活性在膜结合凹槽与活性2-catenin降解之间的联系中的作用。拟议的工作将提供对控制CPC自我更新/分化决策机制的理解的基本见解，这是CPC介导的心脏再生治疗学的先决条件。在CPC和干细胞领域，膜缺口的生物学完全没有探索。鉴于Notch和Wnt/2-catenin信号在几乎所有已知的茎/祖细胞命运决策中的高度保守作用，这些研究将为涉及茎/祖细胞的再生医学研究开辟新的研究途径。 公共卫生相关性：了解发展为心脏的细胞的生物学，称为多能性心血管祖细胞（CPC），是实现未来细胞介导的心脏疗法的希望的关键。拟议的研究旨在阐明称为Notch在CPC自我更新和分化中的蛋白质的配体/转录独立的作用。我们的工作将为理解由Notch调节的多能CPC的自我更新 - 分化过程提供机理见解，该过程将促进未来的基于细胞的心脏疗法和开放新的非经典Notch生物研究途径。