Fyn Kinase Is Involved in EPO Receptor Signaling and Is Required to Harmonize the Response to Oxidation

Erythropoiesis is a complex multistep process during which committed erythroid progenitors undergo terminal differentiation to produce circulating mature red cells. Erythroid differentiation is characterized by the production of reactive oxygen species (ROS) both in response to erythropoietin (EPO) and to the large amount of iron imported into the cells for heme biosynthesis. During erythropoiesis, ROS might function as second messenger by modulating intracellular signaling pathways. Fyn, an 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 C57B6/2J 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 pro-oxidant environment in the absence of Fyn. Using the vitro cell forming colony unit assay, we found a lower in 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 either phenylhydrazine (PHZ) or doxorubicin (Doxo). Fyn^-/- mice showed prolonged anemia after either PHZ or Doxo treatment with a delayed hematologic recovery compared to wild-type animals. 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 wild-type cells. To address the question whether the perturbation of Nrf2-p62 system results in impairment of autophagy in the absence of Fyn, we used Lysotrack 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. As a proof of concept, we treated both mouse strains with PHZ with or without Rapamycin. This latter worsened PHZ induced acute anemia in wild-type mice but not in Fyn^-/- animals. Collectively, our data 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 during normal and more importantly in stress erythropoiesis.