Functional Characterization of Erythropoiesis in Fyn-/- mice: a Novel Role of Fyn in Stress Erythropoiesis

Erythropoiesis is a dynamic complex multistep process going from committed erythroid progenitors to erythroid precursors and circulating mature red cells. Erythroid maturation is strictly dependent on EPO signaling cascade. EPO physically interacts with its receptor (EPO-R), which expression is downregulated after the basophilic erythroblasts stage.7 Binding of EPO to EPO-R results in EPO-R conformational change and it requires the activation of Jak2, as primary kinase. STAT5 is a master of erythropoiesis and it resides in cytoplasm. In response to EPO signaling, it binds phospho-tyrosine (Tyr) residues in the nucleus, initiating the transcription of several genes important in terminal erythroid differentiation. Giving the importance of Jak2 kinase as initiator of the EPO signaling cascade, additional kinases, such as Lyn, a Src family kinase, has been described to participate to EPO pathway. Lyn is able to phosphorylate EPO-R, Jak2 itself and STAT5. The activation of EPO/Jak2 signaling pathway is associated with production of reactive oxidative species (ROS), which are also generated by a large amount of iron imported into the cells during heme biosynthesis. During erythropoiesis, ROS might function as second messenger by modulating intracellular signaling pathways. Fyn, a Src kinase, has been previously reported to participate in signaling pathways in response to ROS in various cell types. Here, we explore the potential contribution of Fyn to normal and stress erythropoiesis by studying 2-4 months-old Fyn knockout mouse strain (Fyn-/-) and C57BL/6J as wild-type controls. Fyn-/- mice showed a mild compensated microcytic anemia associated with signs of dyserythropoiesis. Increased ROS levels and Annexin-V+ cells were presented in all Fyn-/- erythroblast subpopulations compared to wild-type, suggesting a possible reduction in the efficiency of erythropoietin (EPO) signaling pathway in the absence of Fyn. Indeed, in Fyn-/- erythroblasts we observed a reduction in Tyr-phosphorylation state of EPO-R associated with a compensatory activation of Jak2 without major change in Lyn activity. A reduction in STAT5 activation resulting in down-regulation of Cish, a known direct STAT5 target gene, was noted in Fyn-/- erythroblasts. This was paralleled by a reduction in GATA1 and increased HSP70 nuclear translocation compared to wild type, supporting a higher cellular prooxidant environment in the absence of Fyn. Using the vitro cell forming colony unit assay, we found a lower CFU-E and BFU-E cells production, which once again was associated with decreased activation of EPO mediated cascade in the absence of Fyn. To explore the possible role of Fyn in stress erythropoiesis, mice were treated with recombinant EPO, phenylhydrazine (PHZ) or doxorubicin (Doxo). Fyn-/- mice showed a low response to EPO compared to wild-type animals and prolonged anemia after either PHZ or Doxo treatment with a delayed hematologic recovery compared to wildtype mice. When we analyzed the expression of a battery of ARE-genes related to oxidative response such as catalase, Gpx, heme-oxygenase 1 and peroxiredoxin-2, we noted up-regulated expression of these genes in sorted Fyn-/- erythroblasts compared to wild-type cells. In agreement, we observed increased activation of the redox-sensitive transcriptional factor Nrf2 targeting ARE-genes, whose regulation has been previously linked to Fyn. In fact, Nrf2 is switched-off by Fyn, ubiquitylated and delivered to the autophagosome by the p62 cargo protein. In Fyn-/- sorted erythroblasts, we observed (i) accumulation of p62 in large clusters; and (ii) reduction of Nrf2-p62 complex compared to wildtype cells. To address the question whether the perturbation of Nrf2-p62 system results in impairment of autophagy in the absence of Fyn, we used LysoTracker to explore late phases of autophagy. Lysosomal progression was defective in Fyn-/- reticulocytes and it was associated with accumulation of p62 during in vitro reticulocyte maturation. These data indicate that the absence of Fyn blocks the Nrf2 post-induction response to oxidation, resulting in impaired autophagy. To validate our working hypothesis, we treated Fyn-/- mice with Rapamycin, an inducer of autophagy. In Fyn-/- mice, Rapamycin treatment resulted in decrease dyserythropoiesis, ROS levels and Annexin V+ cells, associated with reduction in accumulation of p62 in Fyn-/- erythroblasts. Collectively, our data 4 enabled us to document a novel role for Fyn in erythropoiesis, contributing to EPO-R activation and harmonizing the Nrf2-p62 adaptive cellular response against oxidation. Future studies will be designed to further characterize the signaling pathways intersects by Fyn in normal and diseased erythropoiesis.