Hormetic ER Stress Regulation of Hematopoietic Stem Cell Function

项目摘要 造血涉及红细胞（红细胞），免疫细胞（白细胞） 和凝血血小板，所有这些都来源于造血干细胞 （HSC）位于骨髓中。HSC具有两个关键特征-多能性和自我更新。一 在体内调控造血干细胞血液稳态输出的分子机制的完整图景， 尚未出现。我们发表的研究结果表明，HSC具有低细胞内钙（Ca 2+） 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 低CaCl 2培养液等环境可能改善HSC功能。值得注意的是， 培养基显着增加了长期培养物中的表型HSC计数，并显示增加了20倍 与经典CaCl 2培养基配方相比，在长期供体植入中，表明功能强大 HSC维持在低CaCl 2中。我们证明HSC通过高表达维持低的Ca 2+水平， 和质膜钙外排泵（PMCA）的活性和骨髓间质液CaCl 2的减少 程度.减少CaCl 2减少线粒体呼吸，但不影响糖酵解，特别是在HSC中， 抑制糖酵解升高HSC Ca 2+。这些结果证明了正反馈机制， 糖酵解PMCA Ca 2+流出活性减少Ca 2+，阻止线粒体呼吸，促进线粒体呼吸。 糖酵解令人好奇的是，我们发现HSC中线粒体质量最高，这提示了一个重要的白细胞分化机制。 不依赖于呼吸，在HSC中的作用。基于这些发现，文献表明减少CaCl 2可以诱导 急诊室压力。我们观察到Bcl-2在减少CaCl 2培养条件下表现出剂量依赖性上调 在HSC中，提示促存活基因程序的诱导。抗氧化剂基因的调节因子， 由ER应激诱导，包括ATF 4和Nrf 2，介导包括Bcl-2在内的抗氧化基因的上调。我们 发现低CaCl 2培养的HSC中也有ATF 4和Nrf 2的高表达。因此，我们假设， 减少的CaCl 2诱导支持HSC体外维持的激素样ER应激反应。我们建议 研究什么类型的内质网应激发生，以及哪些未折叠蛋白反应（UPR）信号通路分支 在低CaCl 2浓度下激活。我们建议描述由PERK调控的转录程序 UPR的分支，它激活Atf 4/Nrf 2 ER应激反应程序，我们已经确定是活跃的。 此外，我们建议研究低CaCl 2条件下HSC中Bcl-2的上调，以确定是否存在Bcl-2的上调。 Bcl-2的非典型作用抑制HSC中IP 3R从腔ER Ca 2+库释放Ca 2+。此外，委员会认为， 我们建议在人CB HSC中证实这些发现，以加速这些发现的转化潜力。 这将建立一个新的正反馈回路，以降低Ca 2+水平并保持自我更新 和多能性。这些发现将扩大我们对HSC生物学的基本理解， 为临床应用改进HSC扩增的启发性方法。

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.