LACK OF THE FERROPTOSIS INHIBITOR GPX4 IN ERYTHROID CELLS CAUSES A BLOCK IN RETICULOCYTE MATURATION AND A HYPOXIC SIGNATURE WITH IMPAIRED HEPCIDIN REGULATION.
(Abstract release date: 05/18/17)
EHA Library. Altamura S. 06/25/17; 182096; S809
Dr. Sandro Altamura
Contributions
Contributions
Abstract
Abstract: S809
Type: Oral Presentation
Presentation during EHA22: On Sunday, June 25, 2017 from 08:00 - 08:15
Location: Room N109
Background
GPX4 is a selenoprotein belonging to the family of the glutathione peroxidases, a class of enzymes involved in cellular defence against oxidative stress. This enzyme is essential for life since it is the only peroxidase able to use lipid peroxides as substrate. Mice constitutively lacking GPX4 die at embryonic stage while tissue-specific ablation in neurons and T-cells cause neurodegeneration and impaired immune response. Recent studies have identified GPX4 as the main regulator of ferroptosis, an iron-dependent ROS-mediated form of nonapoptotic cell death.
Erythrocytes are highly specialized cells that utilize a large amount of iron to bind and deliver oxygen to all tissues. Being constantly exposed to oxygen, erythroid cells need to continuously fight against oxidative stress by expressing a variety of antioxidant enzymes, including GPX4.
Iron availability for erythropoiesis depends on systemic iron levels which are regulated via the hepcidin/ferroportin regulatory system. Hepcidin binding to the iron exporter ferroportin reduces systemic iron export regulating body iron levels. In hypoxic conditions the erythroid hormone ErFe suppresses hepcidin synthesis to provide iron for the elevated erythropoietic demand.
Aims
The aim of this study is to identify how the lack of GPX4 in the hematological compartment affects iron homeostasis.
Methods
Lethally irradiated C57BL/6 female mice were reconstituted with bone marrow cells from Gpx4fl/fl; Rosa26-CreERT2 or Gpx4wt/wt; Rosa26-CreERT2 and allowed to recover for 8 to 10 weeks. Gpx4 deletion in the hematopoietic system was induced by feeding tamoxifen citrate for 3 weeks and blood and organs were drawn at 3 and 6 weeks after terminating the tamoxifen-containing diet. Erythroid cells have been analysed in FACS. Serum iron levels have been assessed using the SFBC and UIBC iron kits (Biolabo). Gene expression analysis has been performed using SYBR-green qRT-PCR. Circulating Hepcidin has been measured with a specific murine ELISA kit (Intrinsic Lifesciences). Tissue iron levels have been measured with a colorimetric assay. All animal experiments were approved by and conducted in compliance with institutional guidelines
Results
Compared to GPX4wt/wt;CreERT2 controls, GPX4fl/fl;CreERT2 transplanted mice lacking GPX4 in the haematological compartment show a decrease in the number of red blood cells, haemoglobin and haematocrit. Reticulocyte count measurement revealed a strong increase in this population, suggesting that the erythropenia could be due to a block in the reticulocyte maturation. Reticulocyte FACS characterization revealed a shift towards a more immature population while electron microscopy analysis showed an accumulation of unphagocytosed vesicles containing remnants of mitochondria. Analysis of the spleen revealed extramedullary erythropoiesis. The anemia and the erythropenia trigger a hypoxic signature hallmarked by an increase in circulating EPO and increased ErFe expression. However, both hepatic mRNA analysis and circulating protein measurement failed to show alteration in hepcidin production. Analysis of the liver showed an increase in non-heme iron content and in the lipid peroxidation causing an elevated mRNA and protein expression of heme oxygenase 1. Hepatic ferritin and ferroportin are also increased as a consequence of the increased iron content.
Conclusion
Our data show for the first time that the presence of GPX4 in the haematological compartment is essential for the proper hepcidin downregulation upon ErFe stimulation. This finding opens new insights in the mechanism that regulate hepcidin during hypoxia.
Session topic: 28. Iron metabolism, deficiency and overload
Keyword(s): hepcidin, Erythroid cells, Iron Metabolism
Abstract: S809
Type: Oral Presentation
Presentation during EHA22: On Sunday, June 25, 2017 from 08:00 - 08:15
Location: Room N109
Background
GPX4 is a selenoprotein belonging to the family of the glutathione peroxidases, a class of enzymes involved in cellular defence against oxidative stress. This enzyme is essential for life since it is the only peroxidase able to use lipid peroxides as substrate. Mice constitutively lacking GPX4 die at embryonic stage while tissue-specific ablation in neurons and T-cells cause neurodegeneration and impaired immune response. Recent studies have identified GPX4 as the main regulator of ferroptosis, an iron-dependent ROS-mediated form of nonapoptotic cell death.
Erythrocytes are highly specialized cells that utilize a large amount of iron to bind and deliver oxygen to all tissues. Being constantly exposed to oxygen, erythroid cells need to continuously fight against oxidative stress by expressing a variety of antioxidant enzymes, including GPX4.
Iron availability for erythropoiesis depends on systemic iron levels which are regulated via the hepcidin/ferroportin regulatory system. Hepcidin binding to the iron exporter ferroportin reduces systemic iron export regulating body iron levels. In hypoxic conditions the erythroid hormone ErFe suppresses hepcidin synthesis to provide iron for the elevated erythropoietic demand.
Aims
The aim of this study is to identify how the lack of GPX4 in the hematological compartment affects iron homeostasis.
Methods
Lethally irradiated C57BL/6 female mice were reconstituted with bone marrow cells from Gpx4fl/fl; Rosa26-CreERT2 or Gpx4wt/wt; Rosa26-CreERT2 and allowed to recover for 8 to 10 weeks. Gpx4 deletion in the hematopoietic system was induced by feeding tamoxifen citrate for 3 weeks and blood and organs were drawn at 3 and 6 weeks after terminating the tamoxifen-containing diet. Erythroid cells have been analysed in FACS. Serum iron levels have been assessed using the SFBC and UIBC iron kits (Biolabo). Gene expression analysis has been performed using SYBR-green qRT-PCR. Circulating Hepcidin has been measured with a specific murine ELISA kit (Intrinsic Lifesciences). Tissue iron levels have been measured with a colorimetric assay. All animal experiments were approved by and conducted in compliance with institutional guidelines
Results
Compared to GPX4wt/wt;CreERT2 controls, GPX4fl/fl;CreERT2 transplanted mice lacking GPX4 in the haematological compartment show a decrease in the number of red blood cells, haemoglobin and haematocrit. Reticulocyte count measurement revealed a strong increase in this population, suggesting that the erythropenia could be due to a block in the reticulocyte maturation. Reticulocyte FACS characterization revealed a shift towards a more immature population while electron microscopy analysis showed an accumulation of unphagocytosed vesicles containing remnants of mitochondria. Analysis of the spleen revealed extramedullary erythropoiesis. The anemia and the erythropenia trigger a hypoxic signature hallmarked by an increase in circulating EPO and increased ErFe expression. However, both hepatic mRNA analysis and circulating protein measurement failed to show alteration in hepcidin production. Analysis of the liver showed an increase in non-heme iron content and in the lipid peroxidation causing an elevated mRNA and protein expression of heme oxygenase 1. Hepatic ferritin and ferroportin are also increased as a consequence of the increased iron content.
Conclusion
Our data show for the first time that the presence of GPX4 in the haematological compartment is essential for the proper hepcidin downregulation upon ErFe stimulation. This finding opens new insights in the mechanism that regulate hepcidin during hypoxia.
Session topic: 28. Iron metabolism, deficiency and overload
Keyword(s): hepcidin, Erythroid cells, Iron Metabolism
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