Ineffective Erythropoiesis Caused by Phenylhydrazine Activates the Expression of GDF15 in Maturing Erythroblasts.

Abstract 4032

Poster Board III-968

Several types of ineffective erythropoiesis (thalassemia, congenital dyserythropoietic anemia type I, and refractory anemia with ringed sideroblasts) develop anemia and eventually manifest secondary hemochromatosis even in the absence of transfusion therapy. The highly elevated levels of GDF15 in those patients may contribute to iron loading due to suppression of hepcidin. However, the underlying mechanism for high-level expression of GDF15 in association with ineffective erythropoiesis remains vague. Phenylhydrazine (PHZ), which causes hemoglobin denaturation and alpha-globin precipitation, was utilized in CD34+ cultures as an ex vivo model of ineffective erythropoiesis. All experiments were performed in triplicate with cells from three separate human donors. Initial studies were performed to determine the effects of phenylhydrazine upon reactive oxygen species (ROS) production and apoptosis in the erythroblasts. In dosed titrations (0-10 uM), PHZ was added to the culture medium before and after erythroblast hemoglobin accumulation on culture days 3-6 and 11-14, respectively. PHZ addition prior to the accumulation of hemoglobin had little effect on ROS production. In contrast, PHZ added to hemoglobinized erythroblasts resulted in significant and dosed increases in ROS production. In addition, apoptosis cascades that are downstream of ROS production were studied including activation of caspase-3, EGR-1 transcription, p53 stabilization, and phosphatidylserine on the cell surface (detected by Annexin V). There was a dose-dependent increase in caspase-3, p53 stabilization, and Annexin V positive cells that mirrored the ROS production after PHZ treatment only among the more mature erythroblasts. However, the PHZ addition did not cause a significant increase in the nuclear localization of EGR-1. Since GDF15 is a known target of p53, PHZ effects upon GDF15 expression were also measured. Production of GDF15 was not observed on culture days 3-6 in the absence or presence of PHZ. In contrast, dosed increases in GDF15 expression were detected among the hemoglobinized erythroblasts (culture days 11-14) in response to phenylhydrazine (PHZ = 0 uM, GDF15 = 16 ± 3 pg/1×10⁴cells; PHZ = 1 uM, GDF15 = 23 ± 5 pg/1×10⁴cells; PHZ = 5 uM, GDF15 = 48 ± 9 pg/1×10⁴cells [p<0.01]; PHZ = 10 uM, GDF15 = 75 ± 22 pg/1×10⁴cells [p<0.01]). Of note, GDF15 expression was not detected after enucleation in peripheral blood erythrocytes exposed to PHZ despite increased ROS. According to these data, it is proposed that ineffective erythropoiesis may be caused by increased ROS production during the later stages of erythroblast maturation that activates caspase-3, apoptosis and p53-mediated expression of GDF15.