Non-Canonical Notch Regulation of Cardiovascular Progenitors

心血管祖细胞的非典型Notch调节

• 批准号: 8602525
• 负责人: Chulan Kwon
• 金额: $ 39.69万
• 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/8602525
• 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; 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; cardiac regeneration; cell type; embryonic stem cell; extracellular; heart cell; in vivo; insight; mutant; notch protein; novel; progenitor; protein transport; regenerative; regenerative therapy; self-renewal; stem

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.

心血管祖细胞（CPC）具有巨大的心脏再生治疗潜力 医学由于其独特的扩展能力和分化为各种心脏细胞类型的能力。但是，要服用 再生疗法的优势，我们需要了解自我更新和 CPC的谱系特异性分化。当前的提案重点是阐明Notch的新作用 CPC维护和分化中的信号传导。 Notch是一种进化构成的跨膜蛋白 这在众多细胞命运决策中起着关键作用。通过结合开始 细胞外配体至缺口。这导致了细胞内切割和凹槽的易位 它与转录介质RBP-J结合以进行基因激活。我证明了Notch1缺陷 随着增殖的增加，CPC急剧扩展，类似于用活动刺激的CPC的表型 - - 蛋白酶。在RBP-J缺乏的CPC中未观察到这种表型，这表明 缺口。 Notch1缺乏显着提高 - - 蛋白质信号传导。这种增加不是由 - 钙蛋白mRNA的上调，而是通过积累活性�-蛋白质蛋白的积累，表明 翻译法规。有趣的是， - 钙蛋白蛋白的缺口调节不需要经典配体 缺口或 - 帕宁的蛋白酶体降解的依赖性膜切割，但确实需要 溶酶体交通膜凹槽的内吞蛋白质。而且，膜结合的凹槽， 传统上认为是生物学上的惰性，与活性� -catenin物理相关并抑制 活性�-蛋白质蛋白的积累。这些发现揭示了膜的先前未描述的作用 在控制主动» - 蛋白质蛋白水平的缺点，并为对该作用的机械探索奠定了基础 CPC的维护和差异。我建议测试Notch拮抗CPC的假设 通过溶酶体降解的自我更新/扩展活性» - 与配体/转录无关 时尚。该提案的具体目的是（1）确定Notch1缺陷CPC是否有利于自我更新 差异化，如果需要ood -catenin，则需要这种效果； （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 - 蛋白酶； （3）确定 溶酶体活性在膜结合凹槽与活性 - 帕宁蛋白降解之间的联系中的作用。这 拟议的工作将提供对控制CPC自我机制的理解的基本见解 更新/分化决策，这是CPC介导的心脏再生疗法的先决条件。这 在CPC和干细胞领域，膜缺口的生物学是完全出乎意料的。给予高度 Notch和Wnt/® -Catenin信号在几乎所有已知的茎/祖细胞脂肪决策中的保守作用， 这些研究将为再生医学参与茎/祖细胞的研究开辟新的研究途径。