Natural resistance-associated macrophage protein 1 (Nramp1) is a divalent metal transporter expressed exclusively in phagocytic cells. We hypothesized that macrophage Nramp1 may participate in the recycling of iron acquired from phagocytosed senescent erythrocytes. To evaluate the role of Nramp1 in vivo, the iron parameters of WT and KO mice were analyzed after acute and chronic induction of hemolytic anemia. We found that untreated KO mice exhibited greater serum transferrin saturation and splenic iron content with higher duodenal ferroportin (Fpn) and divalent metal transporter 1 (DMT1) expression. Furthermore, hepatocyte iron content and hepcidin mRNA levels were dramatically lower in KO mice, indicating that hepcidin levels can be regulated by low-hepatocyte iron stores despite increased transferrin saturation. After acute treatment with the hemolytic agent phenylhydrazine (Phz), KO mice experienced a significant decrease in transferrin saturation and hematocrit, whereas WT mice were relatively unaffected. After a month-long Phz regimen, KO mice retained markedly increased quantities of iron within the liver and spleen and exhibited more pronounced splenomegaly and reticulocytosis than WT mice. After injection of (59)Fe-labeled heat-damaged reticulocytes, KO animals accumulated erythrophagocytosed (59)Fe within their liver and spleen, whereas WT animals efficiently recycled phagocytosed (59)Fe to the marrow and erythrocytes. These data imply that without Nramp1, iron accumulates within the liver and spleen during erythrophagocytosis and hemolytic anemia, supporting our hypothesis that Nramp1 promotes efficient hemoglobin iron recycling in macrophages. Our observations suggest that mutations in Nramp1 could result in a novel form of human hereditary iron overload.

Iron is critical for life but toxic in excess because of iron-catalysed formation of pro-oxidants that cause tissue damage in a range of disorders. The Nrf2 transcription factor orchestrates cell-intrinsic protective antioxidant responses, and the peptide hormone hepcidin maintains systemic iron homeostasis, but is pathophysiologically decreased in haemochromatosis and beta-thalassaemia. Here, we show that Nrf2 is activated by iron-induced, mitochondria-derived pro-oxidants and drives Bmp6 expression in liver sinusoid endothelial cells, which in turn increases hepcidin synthesis by neighbouring hepatocytes. In Nrf2 knockout mice, the Bmp6-hepcidin response to oral and parenteral iron is impaired and iron accumulation and hepatic damage are increased. Pharmacological activation of Nrf2 stimulates the Bmp6-hepcidin axis, improving iron homeostasis in haemochromatosis and counteracting the inhibition of Bmp6 by erythroferrone in beta-thalassaemia. We propose that Nrf2 links cellular sensing of excess toxic iron to control of systemic iron homeostasis and antioxidant responses, and may be a therapeutic target for iron-associated disorders.

SUMMARY Sorting of endocytic ligands and receptors is critical for diverse cellular processes. The physiological significance of endosomal sorting proteins in vertebrates, however, remains largely unknown. Here we report that sorting nexin 3 (Snx3) facilitates the recycling of transferrin receptor (Tfrc), and thus is required for the proper delivery of iron to erythroid progenitors. Snx3 is highly expressed in vertebrate hematopoietic tissues. Silencing of Snx3 results in anemia and hemoglobin defects in vertebrates due to impaired transferrin (Tf)-mediated iron uptake and its accumulation in early endosomes. This impaired iron assimilation can be complemented with non-Tf iron chelates. We show that Snx3 and Vps35, a component of the retromer, interact with Tfrc to sort it to the recycling endosomes. Our findings uncover a role of Snx3 in regulating Tfrc recycling, iron homeostasis, and erythropoiesis. Thus, the identification of Snx3 provides a genetic tool for exploring erythropoiesis and disorders of iron metabolism.

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Transferrin receptor 1 (Tfr1) mediates uptake of circulating transferrin-bound iron to developing erythroid cells and other cell types. Its critical physiological function is highlighted by the embryonic lethal phenotype of Tfr1-knockout (Tfrc−/−) mice and the pathologies of several tissue-specific knockouts. We generated TfrcAlb-Cre mice bearing hepatocyte-specific ablation of Tfr1 to explore implications in hepatocellular and systemic iron homeostasis. TfrcAlb-Cre mice are viable and do not display any apparent liver pathology. Nevertheless, their liver iron content (LIC) is lower compared with that of control Tfrcfl/fl littermates as a result of the reduced capacity of Tfr1-deficient hepatocytes to internalize iron from transferrin. Even though liver Hamp messenger RNA (mRNA) and serum hepcidin levels do not differ between TfrcAlb-Cre and Tfrcfl/fl mice, Hamp/LIC and hepcidin/LIC ratios are significantly higher in the former. Importantly, this is accompanied by modest hypoferremia and microcytosis, and it predisposes TfrcAlb-Cre mice to iron-deficiency anemia. TfrcAlb-Cre mice appropriately regulate Hamp expression following dietary iron manipulations or holo-transferrin injection. Holo-transferrin also triggers proper induction of Hamp mRNA, ferritin, and Tfr2 in primary TfrcAlb-Cre hepatocytes. We further show that these cells can acquire 59Fe from 59Fe-transferrin, presumably via Tfr2. We conclude that Tfr1 is redundant for basal hepatocellular iron supply but essential for fine-tuning hepcidin responses according to the iron load of hepatocytes. Our data are consistent with an inhibitory function of Tfr1 on iron signaling to hepcidin via its interaction with Hfe. Moreover, they highlight hepatocellular Tfr1 as a link between cellular and systemic iron-regulatory pathways.

Iron availability for erythropoiesis and its dysregulation in β-thalassemia are incompletely understood. We previously demonstrated that exogenous apotransferrin leads to more effective erythropoiesis, decreasing erythroferrone (ERFE) and derepressing hepcidin in β-thalassemic mice. Transferrin-bound iron binding to transferrin receptor 1 (TfR1) is essential for cellular iron delivery during erythropoiesis. We hypothesize that apotransferrin's effect is mediated via decreased TfR1 expression and evaluate TfR1 expression in β-thalassemic mice in vivo and in vitro with and without added apotransferrin. Our findings demonstrate that β-thalassemic erythroid precursors overexpress TfR1, an effect that can be reversed by the administration of exogenous apotransferrin. In vitro experiments demonstrate that apotransferrin inhibits TfR1 expression independent of erythropoietin- and iron-related signaling, decreases TfR1 partitioning to reticulocytes during enucleation, and enhances enucleation of defective β-thalassemic erythroid precursors. These findings strongly suggest that overexpressed TfR1 may play a regulatory role contributing to iron overload and anemia in β-thalassemic mice. To evaluate further, we crossed TfR1 mice, themselves exhibiting iron-restricted erythropoiesis with increased hepcidin, with β-thalassemic mice. Resultant double-heterozygote mice demonstrate long-term improvement in ineffective erythropoiesis, hepcidin derepression, and increased erythroid enucleation in relation to β-thalassemic mice. Our data demonstrate for the first time that TfR1 haploinsufficiency reverses iron overload specifically in β-thalassemic erythroid precursors. Taken together, decreasing TfR1 expression during β-thalassemic erythropoiesis, either directly via induced haploinsufficiency or via exogenous apotransferrin, decreases ineffective erythropoiesis and provides an endogenous mechanism to upregulate hepcidin, leading to sustained iron-restricted erythropoiesis and preventing systemic iron overload in β-thalassemic mice.

5-Fluorouracil (5-FU) is an antineoplasic drug widely used to treat cancer. Its cytotoxic effect has been principally ascribed to the misincorporation of fluoronucleotides into DNA and RNA during their synthesis, and the inhibition of thymidylate synthase (TS) by FdUMP (one of the 5-FU active metabolites), which leads to nucleotide pool imbalance. In the present study, we compared the ability of 5-FU and FdUMP to induce apoptosis and to influence the cell cycle progression in human colon SW620 adenocarcinoma cells in regards to their genotoxic and clastogenic activities. Our study demonstrates that 5-FU induces SSB, DSB and apoptosis earlier than FdUMP. Interestingly, while both drugs are able to induce apoptosis, their effect on the cell cycle progression differed. Indeed, 5-FU induces an arrest in G1/S while FdUMP causes an arrest in G2/M. Independently of the temporal difference in strand breaks and apoptosis induction, as well as the differential cell cycle modulation, both drugs presented similar clastogenic effects. The different pattern of cell cycle arrest suggests that the two drugs induce different types of primary DNA lesions that could lead to the activation of different checkpoints and recruit different DNA repair pathways.

Key Points• Heme oxygenase-1 levels increase during erythroid differentiation.• Heme oxygenase-1 actively participates in maintaining appropriate hemoglobinization rates.Heme is essential for the function of all aerobic cells. However, it can be toxic when it occurs in a non-protein-bound form; cells maintain a fine balance between heme synthesis and catabolism. The only physiological mechanism of heme degradation is by heme oxygenases (HOs). The heme-inducible isoform, HO-1, has been extensively studied in numerous nonerythroid cells, but virtually nothing is known about the expression and potential significance of HO-1 in developing red blood cells. We have demonstrated that HO-1 is present in erythroid cells and that its expression is upregulated during erythroid differentiation. Overexpression of HO-1 in erythroid cells impairs hemoglobin synthesis, whereas HO-1 absence enhances hemoglobinization in cultured erythroid cells. Based on these results, we conclude that HO-1 controls the regulatory heme pool at appropriate levels for any given stage of erythroid differentiation. In summary, our study brings to light the importance of HO-1 expression for erythroid development and expands our knowledge about the fine regulation of hemoglobin synthesis in erythroid cells. Our results indicate that HO-1 plays an important role as a coregulator of the erythroid differentiation process. Moreover, HO-1 expression must be tightly regulated during red blood cell development. (Blood.