Abstract
The rate of red blood cell production increases up to ten-fold during stress erythropoiesis. We have recently identified stress-responsive CD71highTer119positive early erythroblast subsets in freshly-isolated mouse hematopoietic tissue by flow cytometry. Both the absolute number and relative frequency of these early erythroblast subsets increase dramatically during stress. We have shown that this erythroblast expansion is associated with enhanced erythroblast viability, which is at least in part due to down-regulation of the death-receptor Fas, and its ligand, FasL from early erythroblasts by erythropoietin-receptor (EpoR) signaling (Liu et al., Blood 2006).
The anti-apoptotic protein bcl-xL is induced in differentiating erythroid cells in vitro by EpoR and Stat5 signaling (Socolovsky et al., Cell 1999). Bcl-xL is essential for erythroid cell viability and is required for the maintenance of the normal basal hematocrit (Motoyama et al., Science 1995). However, it is unclear whether bcl-xL plays a role in enhancing erythroblast viability during the stress response. Serum factors other than Epo may modulate erythroid bcl-xL levels (Dolznig et al., Oncogene 2006), complicating the interpretation of bcl-xL measurements in cultured erythroid cells in vitro. Therefore, we examined the potential role of bcl-xL in stress erythropoiesis by measuring bcl-xL mRNA directly in CD71highTer119positive early erythroblasts in vivo in a mouse model of stress.
We mimicked the effect of acute erythropoietic stress by injecting adult Balb/C mice with a single dose of Epo (50 mg/kg subcutaneously). Control mice were injected with an equal volume of saline. Spleen cells were harvested at 3, 16, 24, 48 and 72 hours post injection, and CD71highTer119positive early erythroblasts were immediately sorted by flow-cytometry. RNA was extracted from these freshly sorted cells and used in quantitative real-time PCR to measure bcl-xL mRNA expression. We normalized the level of bcl-xL mRNA in each sample by expressing it relative to beta-actin mRNA. At least 3 independent experiments were conducted for each time point.
In parallel, we measured serum Epo concentration following Epo injection by ELISA. This showed that Epo increased approximately 100 fold by 40 minutes post-injection, reaching a peak by 6 hours and returning to basline levels by 48 hours. We found that bcl-xL mRNA began to increase in spleen early erythroblasts by 3 hours following Epo injection. By 16 hours, bcl-xL mRNA in Epo-injected mice was three-fold higher than in mice injected with saline. Bcl-xL mRNA continued to be elevated, by 2.5 fold, at 24 hours, but declined back to baseline levels by 48 hours.
The time course of the increase in splenic early erythroblast bcl-xL mRNA therefore closely parallels the time course of serum Epo. The induction of early erythroblat bcl-xL mRNA suggests it is likely to contribute to the viability of stress-responsive CD71highTer119positive early erythroblasts, and therefore to the increased erythropoietic rate during the stress response.
WrpA Is an Atypical Flavodoxin Family Protein under Regulatory Control of the Brucella abortus General Stress Response System
