Hormetic ER Stress Regulation of Hematopoietic Stem Cell Function

项目摘要 造血包括不断产生红细胞(红细胞)、免疫细胞(白细胞)。 在受试者的整个生命周期内凝血的血小板，所有这些都来自于造血干细胞 (HSCs)位于骨髓中。造血干细胞具有两个重要特征--多能性和自我更新。一个 调节体内HSCs的血液平衡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，肝星状细胞具有低细胞内钙。 与骨髓(BM)祖细胞和谱系细胞相比。我们假设细胞外钙离子减少 环境，如低CaCl2培养基可改善HSC的功能。值得注意的是，减少了CaCl2 在长期培养中，培养基显着增加了表型HSC的数量，并表现出20倍的增长 在长期供体植入方面与经典的CaCl2介质配方进行比较，表明功能强大 肝星状细胞维持在低CaCl2状态。我们证明HSC通过高表达维持低钙水平。 和质膜钙外排泵(PMCA)和还原的骨髓间质液CaCl2的活性 级别。减少CaCl2会降低线粒体呼吸，但不会减少糖酵解，特别是在HSCs中，而 抑制糖酵解可使HSC钙离子浓度升高。这些结果证明了一种正反馈机制， 糖酵解PMCA钙外流活性降低钙，阻止线粒体呼吸，促进 糖酵解。奇怪的是，我们发现在造血干细胞中线粒体质量最高，这表明了一种重要的白化现象 不依赖呼吸，在HSCs中的作用。基于这些发现，文献表明，减少CaCl2可以诱导 急诊室压力。我们观察到在还原的CaCl2培养条件下，bc l-2呈剂量依赖性上调。 在HSCs中，这表明诱导了有利于生存的基因程序。已知的抗氧化基因的调节者 内质网应激诱导包括ATF4和Nrf2在内的抗氧化剂基因表达上调。我们 发现在低CaCl2培养条件下，ATF4和Nrf2在HSC中也有高表达。因此，我们假设 减少CaCl2诱导激动型内质网应激反应，支持HSC在体外的维持。我们建议 研究发生了哪些类型的内质网应激，以及哪些未折叠蛋白反应(UPR)信号通路分支 在低CaCl2的情况下被激活。我们建议对受PERK调控的转录程序进行表征 UPR的分支，它激活ATF4/Nrf2 ER应激反应程序，我们已经确定它是活跃的。 此外，我们建议研究在低CaCl2条件下HSCs中Bcl2的上调，以确定是否有 Bcl2的非典范作用抑制HSCs内质网钙库钙的IP3R释放。此外， 我们建议在人类脐带血造血干细胞中证实这些发现，以加速这些发现的翻译潜力。 这将建立降低钙水平和保持自我更新所需的新的正反馈循环 和造血干细胞的多能性。这些发现将扩大我们对HSC生物学的基本理解，并可能 启发改进HSC扩增的方法，用于临床应用。

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.