Hormetic ER Stress Regulation of Hematopoietic Stem Cell Function

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.