Page 105 - Haematologica April 2020

GPX4 controls reticulocyte maturation
We next measured iron-related plasma parameters. Plasma iron levels were not increased upon Gpx4 dele- tion, but were increased when Gpx4 was deleted and vitamin E simultaneously depleted (Figure 6N). Despite this, hepcidin levels in the plasma were not altered (Figure 6R). Bilirubin levels appeared to increase with the ablation of Gpx4 under both conditions (± vitamin E), but the standard deviation (SD) was high so that differences became barely significant (Figure 6S-U). Consistent with elevated liver iron levels, plasma ferritin was highly increased under combined Gpx4- and vitamin E-deficien- cy (Figure 6O).
In mouse models with β-thalassemia plasma erythro- poietin (EPO) levels are increased. EPO stimulates the expression of the blood hormone erythroferrone (ERFE) in erythroid precursor cells, which then down regulates hepcidin levels. Similarly, EPO and ERFE levels appeared to be increased in mice with Gpx4-deficient hematopoiesis, but only EPO levels reached significance. Under combined Gpx4- and vitamin E-deficiency plasma EPO and ERFE levels increased to very high and highly significant levels (Figure 6P,Q), apparently mirroring the severity of erythropenia under combined Gpx4- and vita- min E-deficiency.52 Taken together, the defect in reticulo- cyte maturation by loss of the Gpx4 gene causes ineffec- tive erythropoiesis with simultaneous iron overload in the liver and iron-deficiency in the hematopoietic system and this phenotype is to a large part masked by vitamin E in the normal diet.
Discussion
Based on biochemical studies Schewe et al. reported in 1975 that mitochondrial clearance during reticulocyte maturation is initiated by enzymatic lipid peroxidation.20 These authors purified the enzyme 15-lipoxygenase from rabbit reticulocytes and showed that it oxidizes polyun- saturated fatty acids in mitochondrial membranes as an initiating event in the disposal of mitochondria. Even though the work has been confirmed and extended by others, these studies fell into oblivion due to the fact that erythropoiesis including erythrocyte and reticulocyte counts appeared to be normal in mice lacking 12/15-lipoxygenase.26
However, for a few years the work of the Rapoport group has been regaining attention due to several impor- tant novel findings: (i) GPX4 was shown to be an antago- nist of a novel 12/15-lipoxygenase-induced, non-apoptotic cell death pathway in murine fibroblasts,5 (ii) this mode of cell death initiated by ablation of Gpx4 has been shown to be driven by iron-induced lipid peroxidation and is now known as ferroptosis, (iii) distinct phospholipid hydroper- oxide species have been identified that act as death signals and inducers of ferroptosis in various cell types,54 (iv) vita- min E synergizes with GPX4 in antagonizing the action of lipid hydroxyperoxides in vitro and in vivo, (v) 12/15- lipoxygenase and its hydroxylated oxidation products are involved in the regulation of autophagy in murine macrophages,19 (vi) autophagy is inhibited by oxidation of enzymes like ATG3 and ATG7 that catalyze ATG8/LC3 lipidation,56 (vii) lipoxygenases are not necessarily the decisive source of lipid hydroperoxides, they may rather sensitize cells to iron-mediated non-enzymatic autoxida- tion.29
It was the aim of our study to identify a missing link between membrane lipid oxidation and reticulocyte mat- uration by genetic means. Based on the finding that GPX4 is unique in antagonizing lipid membrane hydroperoxides and stabilizing the hydroxylated oxidation products, we hypothesized that deletion of Gpx4 in hematopoietic cells would perturb mitophagy and thus reticulocyte matura- tion.
The role of GPX4 in hematopoietic cells had already been studied by Canli et al. using Gpx4fl/fl;Mx1-Cre mice.55 These authors primarily focused, however, on the mode of cell death in hematopoietic precursor cells and not on a comprehensive view on Gpx4-deficient erythropoiesis at a quantitative level.
As an inducible k.o. model for Gpx4 exclusively in hematopoietic cells, we have reconstituted lethally irradi- ated wt mice with Gpx4fl/fl;Cre-ERT2 or Gpx4wt/wt;CreERT2 BM cells. We now show that activation of Cre by feeding a tamoxifen-containing diet induces an aplastic anemia affecting RBC as well as WBC not only in mice reconsti- tuted with Gpx4-deficient, but also in control mice with Gpx4wt/wt;CreERT2 BM cells thus confirming the acute toxic effect of Cre activation on the hematopoietic system described by Higashi et al.33 There is, however, a clear dif- ference between mice harboring or lacking the Gpx4 gene in hematopoietic cells: mice reconstituted with Gpx4wt/wt;CreERT2 BM cells recovered within three to six weeks whereas mice reconstituted with cells lacking the Gpx4 gene failed to fully recover and remained partially anemic. The erythropenic phenotype could be stably transmitted into lethally irradiated wt mice by two con- secutive rounds of transplantation. It is noteworthy that Cre activation by tamoxifen and Gpx4 ablation cause dif- ferent types of anemia. Cre activation leads to an aplastic anemia with cessation of RBC (Online Supplementary Figure S1F) and WBC formation (Online Supplementary Figure S1L-P), whereas ablation of Gpx4 in the hematopoietic system causes ineffective erythropoiesis with increased formation of reticulocytes (Figure 1H). As recovery from anemia as well as from hematopoietic reconstitution is driven by stress erythropoiesis,43 stress erythropoiesis is perturbed by Gpx4-deficiency in hematopoietic cells. Depletion of vitamin E in the diet strongly aggravated the anemic phenotype. To exclude unintended side effects of vitamin E depletion with the well-known toxicity of Cre and Cre inducers and activators, the Gpx4 gene was delet- ed prior to vitamin E depletion. The dynamics of erythro- poiesis under the various conditions including the matura- tion state of reticulocytes was assessed by FACS staining of proerythroblasts and erythroblasts in the BM and spleen and of reticulocytes in the peripheral blood. A more comprehensive picture emerged (i) by calculating the total number of proerythroblasts and erythroblasts in the BM and spleen, (ii) by extrapolating to the total number of reticulocytes and erythrocytes per mouse, and (iii) by quantifying the fraction of mature, immature and highly immature reticulocytes.
In mice with Gpx4-deficient hematopoiesis the total number of proerythroblasts was increased, mainly due to an increase in extramedullary erythropoiesis. Total num- bers of mature, immature as well as highly immature reticulocytes were also increased. The differentially higher increase in immature and highly immature reticulocytes led to a shift towards immature reticulocytes. This indi- cates that under hematopoietic Gpx4-deficiency the rate
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