Page 104 - Haematologica April 2020

S. Altamura et al.
Figure 7. Proposed model for the role of 12/15-lipoxygenase, GPX4 and vitamin E during reticulocyte maturation.
reticulocytes when vitamin E was additionally depleted (Figure 5D).
To further study potential subcellular alterations in response to Gpx4 loss, erythrocytes and reticulocytes of the peripheral blood were subjected to ultrastructural analysis by electron microscopy (Figure 5G-L). Reticulocytes in blood of wt mice and in blood of mice lacking Gpx4 in hematopoietic cells were characterized by remnants of ribosomes (fine granular structure) and mito- chondria. An accumulation of unphagocytosed mitochon- dria in large vacuoles was evident when vitamin E was depleted in mice with Gpx4-deficient hematopoiesis in vivo (white arrows in Figure 5G-L) indicating that GPX4 and vitamin E physiologically contribute to mitochondrial clearance during reticulocyte maturation.
Iron overload and oxidative stress in the liver of mice with Gpx4-deficient hematopoiesis
As shown above, Gpx4-deficiency in the hematopoietic system is characterized by ineffective erythropoiesis that is severely aggravated by additional vitamin E deprivation. Ineffective erythropoiesis is also a hallmark of β-tha- lassemia, and β-thalassemia is associated with iron over- load that is known to sustain and to aggravate the anemia in mouse models of β-thalassemia.51 To address whether similar mechanisms are operating in Gpx4-deficient ery- thropoiesis, iron-related parameters were analyzed in the liver, spleen, and plasma of mice with Gpx4-deficient hematopoiesis. Non-heme iron content in the liver was significantly increased upon deletion of Gpx4 (Figure 6D). At the molecular level iron overload in the liver was con- firmed by elevated levels of the iron storage protein
Ferritin L (FTL) (Figure 6V). An excess of free iron triggers the formation of reactive oxygen species (ROS) and lipid peroxidation via the Fenton reaction.3 Consistently, mark- ers of oxidative stress such as increased lipid peroxidation products (as revealed by TBARS production) (Figure 6G) and elevated heme oxygenase-1 mRNA and protein levels (Figure 6F,V) were detected in the liver. Combined Gpx4- and vitamin E-deficiency further increased heme oxygenase-1 mRNA and protein levels as well as TBARS production in the liver.
Unaltered hepcidin levels despite higher erythropoietic iron demand
Systemic iron homeostasis is maintained by the hep- cidin/ferroportin regulatory system. Hepcidin regulates the amount of iron exported into systemic circulation by modulating cell surface expression of the iron exporter fer- roportin on iron exporting cells. Remarkably, despite increased liver non-heme iron levels in mice with Gpx4-deficient hematopoietic cells, hepatic hepcidin mRNA expression was not affected, regardless whether mice were kept on a normal or on a vitamin E-depleted diet (Figure 6H). Likewise, target genes of the iron-sensing BMP/SMAD signaling pathway in the liver (SMAD6, SMAD7, and ID1) were not significantly altered by Gpx4- and vitamin E-deficiency (Figure 6I-K). Hepatic fer- roportin mRNA and protein levels were increased upon Gpx4 ablation with or without vitamin E-deficiency (Figure 6E,V), a finding explained by the oxidative stress that occurs in the liver (Figure 6G). Ferroportin expression in the duodenum was unaltered (Online Supplementary Figure S7).
946
haematologica | 2020; 105(4)