Identification of Novel Cytoprotective Systems in Normal and Pathological Erythropoiesis

Anemias are a world-wide distributed disease, affecting people of all ages and can be either hereditary or acquired. Anemias are characterized by reduction in hemoglobin levels which can be related to perturbation of either erythroid maturation and/or reduced red cell survival. In spite of the progress made in the molecular characterization of normal erythropoiesis, much still need to be investigated about the mechanism involved in maturation events in normal and pathological erythropoiesis. ß-thalassemia is a model of pathological erythropoiesis and is an inherited red cell disorder due to a mutation on ß-globin chain. This resuls in α/ß globin chains unbalance and high levels of reactive oxygen species (ROS) generated by pathological free heme/iron. The role of ROS in ß-thalassemic erythropoiesis is still under investigation. Here we carried out two studies to analyze the impact of oxidative stress in pathological erythropoiesis. We identified two novel cytoprotective systems (PART 1) and we evaluate the effects of resveratrol, as an antioxidant agent, in ß-thalassemic anemia (PART 2). PART 1. We studied in vitro erythropoiesis using peripheral CD34+ cells from normal and ß-thalassemia-intermedia subjects. We demonstrated that ROS levels were higher in ß-thalassemic erythroid cells and associated with increased heme levels in early erythropoiesis. In response to oxidative stress in ß-thalassemic cells we observed the inhibition of ALAS-2 activity, a key enzyme of the heme-biosynthetic pathway. This results in the reduction of heme levels, supporting the role of ALAS-2 as a cytoprotective mechanism against ROS in ß-thalassemic erythropoiesis. Then we showed that peroxiredoxin-2, an antioxidant enzyme, is up-regulated in response to oxidative stress in ß-thalassemia. The silencing of peroxiredoxin 2 in K562, an erythroid cell line, was associated with increased cell susceptibility to H2O2. The protective role of peroxiredoxin 2 in erythropoiesis was also supported by its ability to bind free heme. In conclusion, we demonstrate the functional relevance of two novel cytoprotective systems in ß-thalassemic erythropoiesis: ALAS-2 and peroxiredoxin 2. PART 2. We evaluated the effects of low-dose resveratrol on in vitro model of ß-thalassemic erythropoiesis. We found that resveratrol accelerates erythroid maturation with accumulation of cells in S-phase of the cell cycle together with up-regulation of p21 gene expression. In isolated CFU-E cells, resveratrol induces Akt inhibition, activation of FoxO3a with related up-regulation of antioxidant systems, such as catalase and peroxiredoxin 2. We then validated the resveratrol-induced FoxO3 activation in a mouse model for ß-thalassemia. Resveratrol supplementation (2.4 mg/Kg) ameliorated ineffective erythropoiesis of ß-thalassemic mice, with increase of Hb levels, reduction of reticulocyte count, increase of red cell survival, and reduction of red cell membrane oxidative damage. In both wt and ß-thalassemic mice resveratrol up-regulated scavenging enzymes (catalase and peroxiredoxin 2). In conclusion, we show that resveratrol inhibits Akt, resulting in FoxO3 activation, with modulation of cytoprotective systems, enabling the ß-thal erythroid precursors to resist the oxidative damage and continue to differentiate. The effects of resveratrol on erythropoiesis combined with an amelioration of oxidative stress in circulating red cells validate the use of resveratrol as a possible therapeutic agent in ß-thalassemia.