System-Level Analysis of Two Erythroid Progenitor Survival Pathways Reveals Their Distinct Dynamical Properties

Erythropoietic rate varies through a large dynamic range. Its principal regulator is the hormone erythropoietin (Epo), which, in response to hypoxic stress increases up to 1000 fold its basal level, driving erythropoietic rate by up to ten fold. The mechanisms in erythroid progenitors that regulate large, rapid and yet precise changes in erythropoietic rate are not yet understood. It’s been suggested that survival pathways activated by the Epo receptor (EpoR) underlie its regulation of erythropoietic rate. Studies of cultured erythroid cells have identified several anti-apoptotic regulators as EpoR targets. However, their potential contribution to erythropoietic rate in vivo had not been investigated. Here we assessed the in-vivo role of two EpoR-activated survival pathways: EpoR induction of the anti-apoptotic regulator bcl-x_L, and EpoR-mediated suppression of erythroblast Fas and FasL expression. We found that these pathways differ markedly in their regulation of erythropoietic rate. We used flow-cytometric measurement of bclxL, Fas and FasL in each of four erythroblast subsets of increasing maturity (Liu et al. Blood 2006), ProE (Ter119^medCD71^highFSC^high), EryA (Ter119^highCD71^highFSC^high), EryB (Ter119^highCD71^highFSC^low) and EryC (Ter119^highCD71^lowFSC^low). Acute erythropoietic stress was induced by Epo injection or by subjecting mice to reduced atmospheric oxygen. Measurements were made on freshly explanted mouse bone-marrow and spleen, either in the basal state or at different time points following induction of stress. Acute erythropoietic stress caused a rapid but transient induction of bcl-x_L that peaked at 12 to 18 hours, principally in splenic ProE and EryA. Bcl-x_L levels returned to baseline by 24 hours, before resolution of stress. A similar time course was found for induction of the bcl-x_L mRNA. In contrast to the acute response, in mouse models of chronic erythropoietic stress, including anemic mice with beta thalassemia, bcl-x_L was not increased above baseline. However, an acute Epo injection in these mice caused transient bcl-x_L induction similar to that seen in healthy mice. The magnitude of bcl-x_L induction in acute stress was similar, regardless of the absolute change in Epo concentration. We conclude that EpoR-mediated bcl-x_L induction is designed to detect a rapid change in Epo, rather than the absolute level of Epo concentration. It undergoes rapid adaptation, and in both these properties is reminiscent of sensory pathways or bacterial chemotaxis. We suggest this pathway provides a ‘stop-gap’ that enhances erythroblast survival until slower but more permanent pathways are activated. EpoR signaling also causes suppression of erythroblast Fas and FasL, which are co-expressed in splenic ProE and EryA. The size of the EryA subset increases with erythropoietic stress over a wide range. We found that Epo-mediated suppression of Fas/FasL is inversely related to the size of the EryA subset, regardless of whether erythropoietic stress is acute or chronic. Therefore, unlike bcl-x_L induction, EpoR-mediated suppression of Fas/FasL does not undergo adaptation, is a function of the absolute degree of stress and Epo concentration, and likely responsible for long-term maintenance of EryA subset size. To investigate this further, we generated mice deficient in Fas (lpr) or FasL (gld) on an immune deficient background (rag1^−/−) in order to circumvent the autoimmune syndrome of lpr and gld mice. Both these mouse strains showed a significant increase in their CFU-e, ProE and EryA subsets, particularly in spleen, and the gld/rag1^−/− strain also showed increased basal hematocrit. This confirms a negative regulatory effect for Fas in erythropoiesis. Of note, we also found a striking increase in variance for the size of each of these subsets in the mutant mice. We conclude that, in addition to determining the size of the EryA and other erythroid precursor subsets appropriate for each stress level, the Fas-FasL interaction provides a stabilizing mechanism that filters out inappropriate variation in the number of CFU-e, ProE and EryA subsets and in erythropoietic rate. Taken together, our studies in vivo elicited system-level functions for two survival pathways which were not apparent from their investigation in vitro. In combination, these pathways endow the erythropoietic system with a fast response time and with robustness against inappropriate fluctuations in erythropoietic rate.