Page 96 - Haematologica April 2020

S. Altamura et al.
Introduction
Glutathione peroxidase 4 (GPX4) is unique in its ability to reduce lipid peroxidation products in biological mem- branes in vivo. Lipid peroxidation is brought about either enzymatically by lipoxygenases at specific sites or unse- lectively by non-enzymatic mechanisms, usually through Fe2+-driven Fenton chemistry.3 Lipoxygenases are dioxyge- nases that catalyze the incorporation of molecular oxygen into polyunsaturated fatty acids (PUFA) in a site- and stereospecific manner thus yielding the respective hydrox- yperoxides. Lipoxygenases become activated by low con- centrations of peroxides (the so called “peroxide tone”) that oxidize Fe2+ to Fe3+ in the catalytic site.4 The peroxide tone and the activity of lipoxygenases is under the control of GPX4. 12/15-Lipoxygenase and GPX4 act antagonisti- cally as far as oxidation (lipoxygenases) and reduction (GPX4) of substrates, and induction of cell death and cell survival are concerned. 12/15-Lipoxygenase induces cell death in murine fibroblasts, whereas GPX4 rescues cells from lipoxygenase-induced cell death.5 But in other set- tings, lipoxygenases and GPX4 biochemically cooperate: lipoxygenases generate highly reactive peroxidation prod- ucts of unsaturated fatty acids (P-O-O-H) that are prone to further uncontrolled lipid membrane peroxidation. GPX4 reduces these peroxides to stable hydroxyl-derivatives (P-O-H).6-9 Thereby, 15-lipoxygenase in humans or 12/15- lipoxygenase, its functional homolog in mice, and GPX4 constitute a pair of enzymes whose activities are tightly interconnected. In the interplay of lipoxygenases and GPX4 the role of vitamin E also has to be considered. Vitamin E intercalates into membranes, acts as a chain breaker of lipid peroxidation through its high affinity for unpaired electrons and thus antagonizes peroxide produc- tion.
GPX4 is 1 of 24 (25 in man) selenoproteins in mam- mals10 and is positioned at the top of the hierarchy of selenoproteins, i.e. Gpx4 expression is maintained even under severe selenium-deficiency when the synthesis of most other selenoproteins has ceased.11 GPX4 has evolved to carry the 21st amino acid selenocysteine rather than its functional counterpart cysteine in the active site which renders the enzyme highly resistant to irreversible overox- idation through peroxides.12 Dietary selenium is known to be required for stress erythropoiesis in mice and blockage of the synthesis of all selenoproteins in hematopoietic cells by selective deletion of the selenocysteine-specific t-RNA Trsp in the BM of chimeric mice severely impairs stress erythropoiesis.13
Mitochondria are removed from reticulocytes by a par- ticular form of autophagy, called mitophagy, and oxidized lipids are considered to play a crucial role in triggering autophagy in various cell types. Furthermore, macrophages from 12/15-lipoxygenase knockout (k.o.) mice exhibit abnormal mitochondria, cytoplasmic vac- uoles and an altered phospholipidomics pattern indicative of impaired autophagy.19 In addition, the 12/15-lipoxyge- nase oxidation product 12-hydroxyeicosatetraenoic acid- phosphatidylethanolamine (12-HETE-PE) was shown to be a better substrate for yeast Atg8 than native PE, where- as native as well as oxidized PE were both effective sub- strates for LC3 lipidation. With regard to reticulocyte mat- uration current data suggest that elimination of mitochon- dria through mitophagy may be stimulated by oxidation products of polyunsaturated membrane phospholipids.
15-lipoxygenase is highly expressed in reticulocytes and was reported to be involved in the elimination of mito- chondria by Rapoport and his coworkers.20-23 The initial work of the Rapoport group was confirmed and extended by van Leyen et al. who reported a similar 12/15-lipoxyge- nase-driven mechanism that degrades organelles in the eye lens.24,25 Yet, the great interest in this early work decreased with the findings that erythrocyte and reticulo- cyte counts were normal in 12/15-lipoxygenase ko mice.26 Definitive genetic proof for a role of lipid oxidation during reticulocyte maturation is thus still lacking. Several rea- sons may account for the fact that 12/15-lipoxygenase k.o. mice exhibit normal red blood and reticulocyte counts: (i) lipoxygenases other than 12/15-lipoxygenase may compen- sate for the targeted loss of reticulocyte 12/15-lipoxyge- nase; (ii) lipoxygenases may become dispensable if non- enzymatic mechanisms of lipid oxidation prevail;27-29 or (iii) lipid oxidation events are dispensable during reticulo- cyte maturation.
Our present work was conceived to definitively answer if lipid oxidation is indeed critically involved in mitophagy in reticulocytes using a well-defined genetic approach. Whereas many different enzymatic and non-enzymatic mechanisms may account for the initial lipid oxidation step, GPX4 stands out as the only enzyme that effectively prevents detrimental lipid peroxidation and allows lipid oxidation to proceed in a highly controlled manner. Thus, if lipid oxidation is an essential step in the elimination of mitochondria in reticulocytes, ablation of Gpx4 should result in uncontrolled lipid peroxidation and perturbation of reticulocyte maturation. Since GPX4 is essential for early embryonic development and the survival of adult mice,30,31 Gpx4 had to be deleted specifically in hematopoi- etic cells. To this end, we took advantage of the Tamoxifen-inducible Cre/lox system,32 which is of invalu- able help when the side effects of Cre33-37 and Cre activa- tors38 or inducers39 are properly controlled. We show that GPX4 is required for stress erythropoiesis. Deletion of Gpx4 in adult mice causes anemia and ineffective erythro- poiesis due to impaired reticulocyte maturation, a pheno- type dramatically aggravated by depleting vitamin E from the diet. As a consequence hepatic iron overload develops despite the continuous iron demand for red blood cell pro- duction.
Methods
Mice
Mice were bred under SPF conditions. Gpx4fl/fl;CreERT2 mice were backcrossed for at least ten generations onto C57BL/6J mice (Taconic Biosciences, Köln, Germany). All animal experiments were performed according to the institutional guidelines and were approved by the local ethic committees on animal experimenta- tion and by the Government of Upper Bavaria and Kantonales Veterinäramt in Zürich, respectively.
Generation of chimeric mice with Gpx4-proficient and Gpx4-deficient hematopoietic cells and analysis of blood parameters
Female wild-type (wt) recipient mice of 10 to 12 weeks (Taconic Biosciences, Köln) were lethally irradiated with 850 cGy and reconstituted with 106 BM cells from Gpx4fl/fl;CreERT2 or Gpx4wt/wt;CreERT2 donor mice. BM cells had been collected by flushing the leg bones and crushing the pelvic bone.
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haematologica | 2020; 105(4)