Hormetic ER Stress Regulation of Hematopoietic Stem Cell Function

造血干细胞功能的激效内质网应激调节

• 批准号: 10624420
• 负责人: Larry L Luchsinger
• 金额: $ 49.53万
• 依托单位: NEW YORK BLOOD CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-03 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10624420
• 关键词: Acceleration; Antioxidants; Apoptotic; BCL2 gene; Biochemical; Biological Assay; Blood; Blood Cells; Blood Platelets; Blood coagulation; Bone Marrow; Bone Marrow Cells; Ca(2+)-Transporting ATPase; Calcium; Cell Count; Cell Lineage; Cell Maintenance; Cell membrane; Cell physiology; Cells; ChIP-seq; Characteristics; Culture Media; Curiosities; Data; Development; Dose; Endoplasmic Reticulum; Endowment; Engraftment; Environment; Epigenetic Process; Erythrocytes; Exhibits; Feedback; Formulation; Future; Genes; Genetic Transcription; Glycolysis; Glycolysis Inhibition; Goals; Hematologic Neoplasms; Hematological Disease; Hematopoiesis; Hematopoietic Neoplasms; Hematopoietic stem cells; Homeostasis; Human; ITPR1 gene; Immune; In Vitro; Individual; Intercellular Fluid; Knockout Mice; Leukocytes; Lipids; Literature; Longevity; Maintenance; Mediating; Methods; Mitochondria; Mitochondrial Proteins; Molecular; Mouse Strains; Mus; Nuclear; Output; Oxidation-Reduction; Pathway interactions; Patients; Persons; Phenotype; Physiological; Population; Process; Production; Proteins; Publishing; Regulation; Reporter; Reporting; Research; Respiration; Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Source; Stress; Terminal Disease; Testing; Therapeutic; Translating; Transplantation; Umbilical Cord Blood; Up-Regulation; biological adaptation to stress; cell age; cell type; clinical application; clinically relevant; cytokine; efflux pump; endoplasmic reticulum stress; experimental study; extracellular; fluorophore; gene function; hematopoietic stem cell expansion; hematopoietic stem cell self-renewal; human stem cells; improved; in vivo; insight; mitochondrial metabolism; novel; preservation; prevent; progenitor; programs; response; self-renewal; stem; stem cell biology; stem cell function; stem cell self renewal; stem cells; stemness; tissue culture; transcriptome sequencing; transcriptomics; translational potential

Project Summary Hematopoiesis involves the continuous production of red blood cells (erythrocytes), immune cells (leukocytes) and blood clotting platelets over the lifespan of the subject all of which are derived from hematopoietic stem cells (HSCs) located in bone marrow. HSCs have two critical characteristics - multipotency and self-renewal. A complete picture of the molecular mechanisms regulating homeostatic output of blood from HSCs in vivo has not yet emerged. Our published findings show HSCs are endowed with low intracellular calcium (Ca2+) compared to bone marrow (BM) progenitor and lineage cells. We hypothesized a reduced extracellular calcium environment, such as a low CaCl2 culture media, might improve HSC function. Remarkably, reduced CaCl2 media dramatically increased phenotypic HSC counts in long-term cultures and demonstrated a 20-fold increase in long-term donor engraftment compared to classical CaCl2 media formulation, suggesting functionally potent HSCs are maintained in low CaCl2. We demonstrated that HSC maintain low Ca2+ levels via high expression and activity of plasma membrane calcium efflux pumps (PMCA) and reduced bone marrow interstitial fluid CaCl2 levels. Reduced CaCl2 decreased mitochondrial respiration, but not glycolysis, specifically in HSCs, while inhibition of glycolysis elevated HSC Ca2+. These results demonstrate a positive feedback mechanism whereby glycolytic PMCA Ca2+ efflux activity reduces Ca2+ and prevents mitochondrial respiration and promotes glycolysis. Curiously, we showed mitochondrial mass is highest in HSCs suggesting an important, albiet respiration-independent, role in HSCs. Building on these findings, literature suggests reduced CaCl2 can induce ER stress. We observed Bcl-2 exhibits a dose-dependent upregulation under reduced CaCl2 culture conditions in HSCs, suggesting induction of a pro-survival gene program. Regulators of the antioxidant genes known to be induced by ER stress, including ATF4 and Nrf2, mediate upregulation of antioxidant genes including Bcl-2. We found ATF4 and Nrf2 were also high expressed in HSCs in low CaCl2 culture. Therefore, we hypothesize that reduced CaCl2 induces a hormetic ER stress response that supports HSC maintenance in vitro. We propose to study what types of ER stresses occur and which unfolded protein response (UPR) signaling path branches are activated in response low CaCl2. We propose to characterize the transcriptional program regulated by the PERK branch of UPR, which activates the Atf4/Nrf2 ER stress response programs, that we have identified to be active. Further, we propose to study the upregulation of Bcl-2 in HSCs under low CaCl2 conditions to determine if a non-canonical role for Bcl-2 inhibits IP3R release of Ca2+ from luminal ER Ca2+ stores in HSCs. Furthermore, we propose to corroborate these findings in human CB HSCs to accelerate translational potential of the findings. This would establish a novel positive feedback loop required to reduce Ca2+ levels and preserve self-renewal and multipotency in HSCs. These findings will expand our fundamental understanding of HSC biology and may inspire methods to improve HSC expansion for clinical applications.

项目概要 造血涉及红细胞（红细胞）、免疫细胞（白细胞）的持续产生 受试者一生中的凝血血小板，所有这些都源自造血干细胞 （HSC）位于骨髓中。造血干细胞有两个关键特征——多能性和自我更新。一个 体内调节 HSC 血液稳态输出的分子机制的完整图景 还没有出现。我们发表的研究结果表明 HSC 细胞内钙 (Ca2+) 含量较低 与骨髓（BM）祖细胞和谱系细胞相比。我们假设细胞外钙减少 环境，例如低 CaCl2 培养基，可能会改善 HSC 功能。值得注意的是，CaCl2 减少 培养基显着增加了长期培养中的表型 HSC 计数，并显示增加了 20 倍 与经典的 CaCl2 培养基配方相比，在长期供体移植中具有显着的效果，表明其功能强大 HSC 维持在低 CaCl2 中。我们证明 HSC 通过高表达维持低 Ca2+ 水平 质膜钙外排泵 (PMCA) 的活性和减少的骨髓间质液 CaCl2 水平。 CaCl2 减少会减少线粒体呼吸，但不会减少糖酵解，特别是在 HSC 中，而 抑制糖酵解升高 HSC Ca2+。这些结果证明了一种积极的反馈机制，其中 糖酵解 PMCA Ca2+ 外流活性可减少 Ca2+ 并阻止线粒体呼吸并促进 糖酵解。奇怪的是，我们发现 HSC 中的线粒体质量最高，这表明一个重要的、尽管 不依赖呼吸，在 HSC 中发挥作用。基于这些发现，文献表明减少 CaCl2 可以诱导 急诊室压力。我们观察到 Bcl-2 在减少 CaCl2 培养条件下表现出剂量依赖性上调 在 HSC 中，表明诱导了促生存基因程序。已知的抗氧化基因的调节因子 由 ER 应激（包括 ATF4 和 Nrf2）诱导的抗氧化基因（包括 Bcl-2）上调。我们 发现 ATF4 和 Nrf2 在低 CaCl2 培养物的 HSC 中也高表达。因此，我们假设 减少的 CaCl2 会诱导激效内质网应激反应，支持体外 HSC 的维持。我们建议 研究发生什么类型的内质网应激以及哪些未折叠蛋白反应 (UPR) 信号通路分支 响应低 CaCl2 激活。我们建议表征 PERK 调节的转录程序 UPR 的分支，它激活 Atf4/Nrf2 ER 应激反应程序，我们已确定该程序处于活跃状态。 此外，我们建议研究低 CaCl2 条件下 HSC 中 Bcl-2 的上调，以确定是否 Bcl-2 的非典型作用抑制 IP3R 从 HSC 内腔 ER Ca2+ 储存中释放 Ca2+。此外， 我们建议在人类 CB HSC 中证实这些发现，以加速这些发现的转化潜力。 这将建立一个新颖的正反馈循环，以降低 Ca2+ 水平并保持自我更新 和 HSC 的多能性。这些发现将扩展我们对 HSC 生物学的基本理解，并可能 激发临床应用中改善 HSC 扩增的方法。

项目成果

Hormetic endoplasmic reticulum stress in hematopoietic stem cells.

• DOI: 10.1097/moh.0000000000000668
• 发表时间: 2021-11-01
• 期刊: Current opinion in hematology
• 影响因子: 3.2
• 作者: Luchsinger LL