Dynamics of Erythropoietic Survival Pathways In Vivo: A Dissertation

Erythropoiesis maintains stable tissue oxygenation in the basal state, while accelerating red cell production in anemia, blood loss or high altitude. The principal regulator of erythropoiesis is the hormone erythropoietin (Epo). In response to hypoxic stress, Epo can increase a 1000-fold, driving erythropoietic rate by up to 10-fold. It’s been suggested that survival pathways activated by the Epo receptor (EpoR) underlie its regulation of erythropoietic rate. A number of apparently redundant EpoR survival pathways were identified in vitro, raising the possibility of their functional specialization in vivo. Here I assessed the roles of three survival pathways activated by EpoR in erythroblasts in-vivo: the suppression of cell-surface Fas and FasL, the suppression of the pro-apoptotic regulator Bim, and the induction of the anti-apoptotic regulator Bcl-xL. I used the novel CD71/Ter119 flow-cytometric method of identifying erythroblast maturation stages in vivo to measure these apoptotic pathways in fetal liver and adult erythropoietic tissues. I found that these pathways differ markedly in their regulation of erythropoietic rate. Using mouse genetic models, I found that apoptosis mediated by interaction between erythroblasts that co-express cell-surface Fas and FasL plays a key autoregulatory role in stabilizing the size of the erythroblast pool in the basal state. Further, mice mutant for Fas or FasL showed a delayed erythropoietic response to hypoxia or high Epo. This suggests that Fas and FasL accelerate the stress response by providing an apoptotic ‘cell reserve’ that can be rescued by Epo in stress. I also examined the in-vivo behavior of two cell-intrinsic apoptotic regulators, Bcl-xL and Bim, previously unexamined in stress. The induction of Bcl-xL was rapid but transient, whilst the suppression of Bim was slower but persistent. My data suggest that Bcl-xL is a key mediator of EpoR’s anti-apoptotic signal very early in the stress response, before Bim and Fas are suppressed. Bcl-xL adaptation to high Epo occurs through inhibition of Stat5 activation, and resets it for the next acute stress. My findings suggest that in vivo, Epo regulates erythropoietic rate through erythroblast apoptosis, and that various apoptotic regulators play distinct and unique roles in this process. My work provides new molecular insights into erythropoiesis that are relevant to cytokine biology and to clinical approaches of disease treatment