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）在心脏再生方面具有巨大的治疗潜力 然而，由于它们具有扩展和分化成各种心脏细胞类型的独特能力，因此可以作为医学药物。 再生疗法的优势，我们需要了解自我更新和再生背后的机制 CPC 谱系特异性分化 目前的提案重点是阐明 Notch 的新作用。 Notch 是一种进化上保守的跨膜蛋白。 在许多细胞命运决定中发挥关键作用的经典Notch信号传导是通过结合来启动的。 Notch 的细胞外配体这导致 Notch 的细胞内裂解和易位到细胞核中。 我证明了 Notch1 缺陷，它与转录介质 RBP-J 结合。 CPC 随着增殖的增加而急剧扩增，类似于活性物质刺激的 CPC 的表型 ¿ -Catenin 在缺乏 RBP-J 的 CPC 中未观察到这种表型，表明其具有非典型作用。 Notch1 缺陷显着增加 ¿ -连环蛋白信号传导不介导。 的上调-连环蛋白 mRNA 而是通过活性 ¿ 的积累-连环蛋白，表明后- 有趣的是，Notch 调节。 -连环蛋白不需要经典配体- Notch 或 ¿ 的依赖性膜裂解-连环蛋白指导蛋白酶体降解，但它确实需要 将膜Notch运输至溶酶体的内吞蛋白。 通常认为具有生物惰性，物理上与活性相关 ¿ -连环蛋白和抑制 积极积累 ¿ -连环蛋白。这些发现揭示了膜先前未描述的作用。 调节活性 ¿ -连环蛋白水平并为探索这一作用的机制奠定了基础 我建议检验 Notch 对抗 CPC 的假设。 通过活性 ¿ 的溶酶体降解进行自我更新/扩张- 配体/转录独立的连环蛋白 该提案的具体目标是（1）确定Notch1缺陷的CPC是否有利于自我更新。 过度微分和 if ¿ -Catenin是该作用所必需的；（2）测试是否膜结合Notch1； 影响 CPC 扩展和分化以及细胞事件是否需要 ¿ -连环蛋白；(3) 鉴定 溶酶体活性在膜结合Notch和活性之间的联系中的作用 ¿ -连环蛋白降解。 拟议的工作将为理解控制中共自我的机制提供基本见解 更新/分化决策，这是 CPC 介导的心脏再生治疗的先决条件。 膜Notch 的生物学在CPC 领域以及干细胞领域中完全未被探索。 Notch 和 Wnt/¿ 的保守角色-几乎所有已知的干细胞/祖细胞命运决定中的连环蛋白信号传导， 这些研究将为涉及干/祖细胞的再生医学研究开辟新途径。