The regulation of heme biosynthesis during erythropoietin-induced erythroid differentiation.

Heme is a prosthetic group comprising ferrous iron (Fe2+) and protoporphyrin IX and is an essential cofactor in various biological processes such as oxygen transport (hemoglobin) and storage (myoglobin) and electron transfer (respiratory cytochromes) in addition to its role as a structural component of hemoproteins. Heme biosynthesis is induced during erythroid differentiation and is coordinated with the expression of genes involved in globin formation and iron acquisition/transport. However, erythroid and nonerythroid cells exhibit distinct differences in the heme biosynthetic pathway regulation. Defects of heme biosynthesis in developing erythroblasts can have profound medical implications, as represented by sideroblastic anemia. This review will focus on the biology of heme in mammalian erythroid cells, including the heme biosynthetic pathway as well as the regulatory role of heme and human disorders that arise from defective heme synthesis.

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Heme Oxygenase 1 Plays An Unexpected Role During Erythroid Differentiation

Blood ◽

10.1182/blood.v118.21.344.344 ◽

2011 ◽

Vol 118 (21) ◽

pp. 344-344

Author(s):

Daniel Garcia Santos ◽

Matthias Schranzhofer ◽

José Artur Bogo Chies ◽

Prem Ponka

Keyword(s):

Red Blood Cells ◽

Heme Oxygenase ◽

Blood Cells ◽

Erythroid Differentiation ◽

Heme Biosynthesis ◽

Erythroid Cell ◽

Heme Oxygenase 1 ◽

Erythroid Cells ◽

Wild Type ◽

Protein Levels

Abstract Abstract 344 Red blood cells (RBC) are produced at a rate of 2.3 × 106 cells per second by a dynamic and exquisitely regulated process known as erythropoiesis. During this development, RBC precursors synthesize the highest amounts of total organismal heme (75–80%), which is a complex of iron with protoporphyrin IX. Heme is essential for the function of all aerobic cells, but if left unbound to protein, it can promote free radical formation and peroxidation reactions leading to cell damage and tissue injury. Therefore, in order to prevent the accumulation of ‘free' heme, it is imperative that cells maintain a balance of heme biosynthesis and catabolism. Physiologically, the only enzyme capable of degrading heme are heme oxyganase 1 & 2 (HO). Red blood cells contain the majority of heme destined for catabolism; this process takes place in splenic and hepatic macrophages following erythrophagocytosis of senescent RBC. Heme oxygenase, in particular its heme-inducible isoform HO1, has been extensively studied in hepatocytes and many other non-erythroid cells. In contrast, virtually nothing is known about the expression of HO1 in developing RBC. Likewise, it is unknown whether HO1 plays any role in erythroid cell development under physiological or pathophysiological conditions. Using primary erythroid cells isolated from mouse fetal livers (FL), we have shown that HO1 mRNA and protein are expressed in undifferenetiated FL cells and that its levels, somewhat surprisingly, increase during erythropoietin-induced erythroid differentiation. This increase in HO1 can be prevented by succinylacetone (SA), an inhibitor of heme synthesis that blocks 5-aminolevulinic acid dehydratase, the second enzyme in the heme biosynthesis pathway. Moreover, we have found that down-regulation of HO1 via siRNA increases globin protein levels in DMSO-induced murine erythroleukemic (MEL) cells. Similarly, compared to wild type mice, FL cells isolated from HO1 knockout mice (FL/HO1−/−) exhibited increased globin and transferrin receptor levels and a decrease in ferritin levels when induced for differentiation with erythropoietin. Following induction, compared to wild type cells, FL/HO1−/− cells showed increased iron uptake and its incorporation into heme. We therefore conclude that the normal hemoglobinization rate appears to require HO1. On the other hand, MEL cells engineered to overexpress HO1 displayed reduced globin mRNA and protein levels when induced to differentiate. This finding suggests that HO1 could play a role in some pathophysiological conditions such as unbalanced globin synthesis in thalassemias. Disclosures: No relevant conflicts of interest to declare.

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Rap-536 (Murine ACE-536/Luspatercept) Inhibits Smad2/3 Signaling and Promotes Erythroid Differentiation By Restoring GATA-1 Function in Murine b-Thalassemia

Blood ◽

10.1182/blood.v126.23.751.751 ◽

2015 ◽

Vol 126 (23) ◽

pp. 751-751 ◽

Cited By ~ 5

Author(s):

Pedro A. Martinez ◽

Rajasekhar NVS Suragani ◽

Manoj Bhasin ◽

Robert Li ◽

Robert Scott Pearsall ◽

...

Keyword(s):

Murine Model ◽

Target Genes ◽

Erythroid Differentiation ◽

Heme Biosynthesis ◽

Ineffective Erythropoiesis ◽

Down Regulation ◽

Employment Equity ◽

Equity Ownership ◽

Terminal Erythroid Differentiation ◽

Tgfβ Superfamily

Abstract We have previously reported that Smad2/3 signaling (of the TGFβ superfamily) is elevated in myelodysplastic syndromes (MDS) and β-thalassemia, diseases that are characterized by ineffective erythropoiesis (Suragani et al. 2014). Smad2/3 pathway inhibition using RAP-536 (murine version of ACE-536/luspatercept), a modified activin receptor type IIB ligand trap, decreased ineffective erythropoiesis (IE) and alleviated disease pathology in a murine model of β-thalassemia. In this study, we investigated the a) potential role of different Smad2/3 ligands that bind to luspatercept in the regulation of erythropoiesis and b) molecular mechanism of RAP-536 therapy in the murine model of β-thalassemia. Wild-type (WT) mice were treated with neutralizing antibodies against activin B, GDF8 or GDF8/11 (10mg/kg, s.c, twice weekly for 2- weeks, N=5/group) either as a single agent or in combination, and compared with RAP-536 (10 mg/kg, s.c) treatment. β-thalassemic mice (Hbbth3/+) were administered a single bolus of vehicle (VEH) or RAP-536 (30 mg/kg, i.p) (N=2/group). At 16 hours following administration the splenic basophilic erythroblasts (CD71+ Ter119+ FSChigh) were sorted by flow cytometry and RNA was isolated and subjected to genome wide transcriptome profiling using RNA sequencing analysis. a) Surface plasmon analysis revealed that ACE-536 binds Smad2/3 signaling ligands GDF11 and GDF8 with high affinity and activin B with lower affinity. There was minimal binding detected with Activin A, TGFβ1 or TGFβ3 ligands. Wt mice treated with RAP-536 increased RBC (+19%, P<0.001) and Hgb (+15.2%, P<0.001) compared to VEH treated mice. Treatment with anti-GDF8 or anti-activin B antibodies marginally affected RBC parameters (~2-4%, N.S) where as anti-GDF8/11 treatment alone increased RBC (+6.1%, P<0.05) and Hgb (6.9%, P<0.05) compared to VEH treatment. A combination treatment of anti-GDF8/11 and activin B antibodies synergistically increased RBC (10.7%, P<0.001) and Hgb (11%, P<0.001) compared to VEH treated mice. These data suggests that in addition to GDF11 and activin B, other TGFβ superfamily ligands are possibly involved in the stimulation of erythropoiesis by luspatercept. b) Transcriptome analysis of β-thalassemic erythroblasts revealed a total of 74 genes that were differentially expressed (absolute fold change >1.5, false discovery rate adjusted P value <0.05) in RAP-536 treated samples compared to VEH treatment. To identify molecular mechanisms, we performed gene set enrichment analysis (GSEA) (Subramanian et al., 2005) on data from RAP-536 and VEH treated samples. The analysis depicted significant upregulation of target genes of multiple transcriptional regulators including GATA-1 (erythroid differentiation), NFE2 and heat shock factor (involved in globin expression and protein quality-control). Previously, multiple studies established GATA-1 as a master transcriptional regulator of terminal erythroid differentiation. The individual gene symbols based comparative analysis revealed up-regulation of 53/478 GATA-1 activators and down regulation of 9/342 GATA-1 repressors. The GATA-1 target genes that were up regulated by RAP-536 treatment are involved in heme biosynthesis (such as Ppox, Fech, Alas2 and Abcb10) and erythroid differentiation (such as Klf1, Nfe2, Gypa, Bcl2l, Bnip3l, Bach1, and Ank1). Further GSEA of GATA-1 activator and repressor signatures against RAP-536 treatment data revealed a significant up-regulation of 158/328 activated genes (Normalized Enrichment Score=2.7, P=0) involved in heme biosynthesis, and cell cycle regulation whereas there was no statistically significant down regulation of GATA-1 repressed genes. Consistent with this data, our preliminary results in differentiating mouse erythroleukemic (MEL) cells showed increased Smad2/3 phosphorylation that is correlated with reduced GATA-1 protein levels suggesting that pSmad2/3 may negatively regulate terminal erythroid differentiation by decreasing GATA-1 availability. These data provide a potential mechanistic role for luspatercept treatment in β-thalassemia, by transcriptionally upregulating genes that promote erythroid differentiation and processing of unpaired α-globins. By inhibiting SMAD2/3 signaling, luspatercept relieves the block of terminal erythroid maturation and decreases ineffective erythropoiesis in diseases such as β-thalassemia and MDS. Disclosures Martinez: Acceleron Pharma: Employment. Suragani:Acceleron Pharma Inc: Employment, Equity Ownership, Patents & Royalties: No royalties. Li:Acceleron Pharma: Employment, Equity Ownership. Pearsall:Acceleron Pharma Inc: Employment, Equity Ownership, Patents & Royalties. Kumar:Acceleron Pharma: Employment, Equity Ownership, Patents & Royalties.

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Mitochondrial Heme Export Through FLVCR1b Controls Erythroid Differentiation.

Blood ◽

10.1182/blood.v120.21.2090.2090 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2090-2090

Author(s):

Deborah Chiabrando ◽

Carlotta Giorgi ◽

Lorenzo Silengo ◽

Fiorella Altruda ◽

Paolo Pinton ◽

...

Keyword(s):

Cell Membrane ◽

Sodium Butyrate ◽

Hela Cells ◽

K562 Cells ◽

Erythroid Differentiation ◽

Heme Biosynthesis ◽

Heme Oxygenase 1 ◽

Agonist Stimulation

Abstract Abstract 2090 Feline Leukemia Virus subgroup C Receptor 1 (FLVCR1) is a cell membrane heme exporter that contributes to maintain the balance between heme level and globin synthesis in erythroid precursors. Consistently, FLVCR1-null mice died in utero due to a failure of erythropoiesis1. We previously reported the identification of a mitochondrial isoform of FLVCR1, named FLVCR1b, that was able to support fetal murine erythroid differentiation in the absence of the cell membrane isoform (herein called FLVCR1a)2. The aim of this work was to investigate the role of FLVCR1b during erythroid differentiation. To this end, overexpression and silencing experiments were performed. FLVCR1b overexpression promotes in vitro erythroid differentiation of K562 cells, as indicated by the increased transcription of globin mRNA and a higher percentage of benzidine positive cells, compared to control cells. On the contrary, FLVCR1a-overexpressing K562 cells were not able to differentiate. Interestingly, shRNA-mediated down-regulation of FLVCR1a led to the elevation of the percentage of benzidine positive cells compared to controls that further increased upon the stimulation of erythroid differentiation using sodium butyrate. A decrease of the percentage of benzidine positive cells was observed in K562 cells lacking both FLVCR1 isoforms compared to FLVCR1a-deficient cells. Moreover these cells were not able to differentiate upon stimulation with sodium butyrate. These results suggest a fundamental role of FLVCR1b during erythroid differentiation in vitro, supporting previous data obtained in the mouse model2. To understand the molecular mechanisms in which FLVCR1b is involved, we investigate whether FLVCR1b could affect heme export from mitochondria. The overexpression of FLVCR1b in HeLa cells led to increased intracellular heme content together with a strong induction of the heme degrading enzyme heme oxygenase 1 (HO-1) mRNA. Inhibition of the heme biosynthetic pathway using succinylacetone, completely prevented intracellular accumulation of heme observed when FLVCR1b was overexpressed. So, increased heme biosynthesis rate is responsible for the elevation of heme content. Accordingly, HeLa cells overexpressing FLVCR1b showed an alteration of heme biosynthesis enzymes and transporters. On the contrary, the specific loss of FLVCR1b using siRNA causes heme accumulation in mitochondria and a subsequent block of heme biosynthesis. These data are consistent with a role of FLVCR1b as a mitochondrial heme exporter. Similar results were also obtained in K562 cells thus suggesting that loss of FLVCR1b reduces the availability of heme for haemoglobin synthesis, a process essential during erythroid differentiation. To assess whether mitochondrial heme accumulation due to the loss of FLVCR1b affect mitochondrial functionality, the mitochondrial Ca2+ response after agonist stimulation was monitored as a highly sensitive readout of mitochondrial state. It is well known that mitochondrial alterations cause defects in Ca2+ uptake by the organelle. The silencing of FLVCR1a and FVLCR1b in HeLa cells caused a significant reduction of Ca2+spike in the mitochondrial matrix evoked by agonist stimulation. These data suggested that when FLVCR1b is lost heme accumulates in mitochondria resulting in the alterations of mitochondrial functionality. All together these data indicate that the impairment of erythroid differentiation observed in the absence of FLVCR1b is due to the block of heme export from mitochondria and a consequent impairment of mitochondrial functionality, which is essential for cell survival. These results, linking heme biosynthesis pathway to mitochondrial Ca2+signaling, will have broad implications in cellular metabolism. Disclosures: No relevant conflicts of interest to declare.

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Ferrochelatase forms an oligomeric complex with mitoferrin-1 and Abcb10 for erythroid heme biosynthesis

Blood ◽

10.1182/blood-2009-12-259614 ◽

2010 ◽

Vol 116 (4) ◽

pp. 628-630 ◽

Cited By ~ 99

Author(s):

Wen Chen ◽

Harry A. Dailey ◽

Barry H. Paw

Keyword(s):

Affinity Purification ◽

Protoporphyrin Ix ◽

Erythroid Differentiation ◽

Heme Biosynthesis ◽

Hek293 Cells ◽

Heterologous Proteins ◽

Interacting Protein ◽

Mitochondrial Iron ◽

Oligomeric Complex ◽

Endogenous Proteins

AbstractIn erythroid cells, ferrous iron is imported into the mitochondrion by mitoferrin-1 (Mfrn1). Previously, we showed that Mfrn1 interacts with Abcb10 to enhance mitochondrial iron importation. Herein we have derived stable Friend mouse erythroleukemia (MEL) cell clones expressing either Mfrn1-FLAG or Abcb10-FLAG and by affinity purification and mass spectrometry have identified ferrochelatase (Fech) as an interacting protein for both Mfrn1 and Abcb10. Fech is the terminal heme synthesis enzyme to catalyze the insertion of the imported iron into protoporphyrin IX to produce heme. The Mfrn1-Fech and Abcb10-Fech interactions were confirmed by immunoprecipitation/Western blot analysis with endogenous proteins in MEL cells and heterologous proteins expressed in HEK293 cells. Moreover, Fech protein is induced in parallel with Mfrn1 and Abcb10 during MEL cell erythroid differentiation. Our findings imply that Fech forms an oligomeric complex with Mfrn1 and Abcb10 to synergistically integrate mitochondrial iron importation and use for heme biosynthesis.

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IDH1 mutation contributes to myeloid dysplasia in mice by disturbing heme biosynthesis and erythropoiesis

Blood ◽

10.1182/blood.2020007075 ◽

2020 ◽

Author(s):

Yu Gu ◽

Risheng Yang ◽

Ying Yang ◽

Yuanlin Zhao ◽

Andrew Wakeham ◽

...

Keyword(s):

Erythroid Differentiation ◽

Genetic Alterations ◽

Idh1 Mutation ◽

Heme Biosynthesis ◽

Heme Oxygenase 1 ◽

Erythroid Cells ◽

Hematopoietic Stem ◽

Idh Mutations ◽

Mutant Idh1 ◽

New Treatments

Isocitrate dehydrogenase (IDH) mutations are common genetic alterations in myeloid disorders, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Epigenetic changes, including abnormal histone and DNA methylation, have been implicated in the pathogenic build-up of hematopoietic progenitors, but it is still unclear whether and how IDH mutations themselves affect hematopoiesis. Here, we show that IDH1-mutant mice develop myeloid dysplasia in that these animals exhibit anemia, ineffective erythropoiesis, increased immature progenitor and erythroblast. In erythroid cells of these mice, D-2-hydroxyglutarate (D-2HG), an aberrant metabolite produced by the mutant IDH1 enzyme, inhibits oxoglutarate dehydrogenase (OGDH) activity and diminishes succinyl-CoA production. This succinyl-CoA deficiency attenuates heme biosynthesis in IDH1-mutant hematopoietic cells, thus blocking erythroid differentiation at the late erythroblast stage and the erythroid commitment of hematopoietic stem cells (HSC), while the exogenous succinyl-CoA or 5-ALA rescues erythropoiesis in IDH1-mutant erythroid cells. Heme deficiency also impairs heme oxygenase-1 (HO-1) expression, which reduces levels of important heme catabolites such as biliverdin and bilirubin. These deficits result in accumulation of excessive reactive oxygen species (ROS) that induce the cell death of IDH1-mutant erythroid cells. Our results clearly demonstrate the essential role of IDH1 in normal erythropoiesis and show how its mutation leads to myeloid disorders. Our data thus have important implications for the devising of new treatments for IDH-mutant tumors.

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Characterization of Congenital Sideroblastic Anemia Model Due to ABCB7 Defects: How Do Defects in Iron-Sulfur Cluster Metabolism Lead to Ring Sideroblast Formation?

Blood ◽

10.1182/blood-2019-123918 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2232-2232

Author(s):

Tohru Fujiwara ◽

Chie Suzuki ◽

Tetsuro Ochi ◽

Koya Ono ◽

Kei Saito ◽

...

Keyword(s):

Bone Marrow ◽

Research Funding ◽

Erythroid Differentiation ◽

Heme Biosynthesis ◽

Atp Binding ◽

Chugai Pharmaceutical ◽

Iron Sulfur ◽

Causative Link ◽

Atp Binding Cassette

Backgroun d: The sideroblastic anemias (SAs) are a group of congenital and acquired bone marrow disorderscharacterized by bone marrow ring sideroblasts (RSs). The disease commonly presents as myelodysplastic syndrome with RS (MDS-RS), known as an acquired clonal SA that is strongly correlated with a specific somatic mutation inSF3B1 (splicing factor 3b subunit 1), which is involved in RNA splicing machinery. Thus far, several studies have consistently revealed compromised splicing and/or expression of ABCB7 (ATP-binding cassette subfamily B member 7) in MDS-RS harboring the SF3B1 mutation. ABCB7 encodes an ATP-binding cassette family transporter localizing to the inner mitochondrial membrane, and its loss-of-function mutation causes a syndromic form of congenital SA, which is associated with cerebellar ataxia. The substrates transported by ABCB7 are predicted to be iron-sulfur clusters (ISCs), which are essential for the function of multiple mitochondrial and extramitochondrial proteins, such as ferrochelatase and aconitase (its apo-form without ISC is called IRP1; iron regulatory protein 1). However, the detailed molecular mechanisms by which defects in ISC metabolism resulting from ABCB7 defects contribute to RS formation remains to be fully elucidated. Methods: Endogenous ABCB7 was depleted based on pGIPZ lentiviral shRNAmir (Dharmacon) in human umbilical cord blood-derived erythroid progenitor (HUDEP)-2 cells (Kurita et al., PLoS ONE, 2013). Puromycin (Sigma) was used for the selection of transduced cells. To induce terminal erythroid differentiation, HUDEP-2 cells were co-cultured with OP9 stromal cells (ATCC) in Iscove's modified Dulbecco's medium supplemented with fetal bovine serum, erythropoietin, dexamethasone, monothioglycerol, insulin-transferrin-selenium, ascorbic acid, and sodium ferrous citrate (Saito and Fujiwara et al., MCB, 2019). For transcription profiling, Human Oligo Chip 25K (Toray) was used. Results: We first conducted ABCB7 knockdown in HUDEP-2 cells based on two independent shRNA plasmids. When the knockdown cells were induced to undergo erythroid differentiation,the majority of the erythroblasts exhibited aberrant mitochondrial iron deposition. Thus, we sought to clarify the potential causative link between ABCB7 defects and RS formation. Expression profiling revealed >1.5-fold up- and down-regulation of 33 and 44 genes, respectively, caused by the ABCB7 knockdown. Intriguingly, 43% of the downregulated gene ensemble (19/44 genes) included multiple ribosomal genes, such as RPS2, RPL11,and RPS12. The downregulated genes also included HMOX1 (heme oxygenase 1), implying that heme biosynthesis would be compromised by the knockdown. Gene ontology (GO) analysis revealed significant (p< 0.01) enrichment of genes associated with nuclear-transcribed mRNA catalytic process, cytoplasmic translation, and cellular iron ion homeostasis. Whereas the mRNA expression for ALAS2 (erythroid-specific 5-aminolevulinate synthase), encoding a rate-limiting enzyme of heme biosynthesis and one of the responsible genes for congenital SA, was not affected, its protein expression was noticeably decreased by ABCB7 knockdown, indicating that compromised transport of ISC from mitochondria to the cytosol may result in decreased ALAS2 translation by the binding of IRP1 to the iron-responsive element located in the 5'-UTR of ALAS2 mRNA.We are currently conducting detailed biological analyses to elucidate the causative link between defects in ISC metabolism due to ABCB7 defects and RS formation. Conclusion: We have first demonstrated the emergence of RS by ABCB7 depletion in human erythroblasts. Further characterization of the established SA model would aid in the clarification of its molecular etiology and the establishment of novel therapeutic strategies. Furthermore, our results may lead to a better understanding of the role of ISC in affecting cerebellar symptoms. Disclosures Fukuhara: Gilead: Research Funding; Nippon Shinkyaku: Honoraria; Zenyaku: Honoraria; AbbVie: Research Funding; Takeda Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Mundi: Honoraria; Ono Pharmaceutical Co., Ltd.: Honoraria; Bayer: Research Funding; Celgene Corporation: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria; Eisai: Honoraria, Research Funding; Janssen Pharma: Honoraria; Kyowa-Hakko Kirin: Honoraria; Mochida: Honoraria; Solasia Pharma: Research Funding. Onishi:Novartis Pharma: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Honoraria; Astellas Pharma Inc.: Honoraria; ONO PHARMACEUTICAL CO., LTD.: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Janssen Pharmaceutical K.K.: Honoraria; MSD: Honoraria, Research Funding; Sumitomo Dainippon Pharma: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Takeda Pharmaceutical Co., Ltd.: Research Funding; Nippon Shinyaku: Honoraria; Pfizer Japan Inc.: Honoraria; Kyowa-Hakko Kirin: Honoraria; Celgene: Honoraria. Yokoyama:Astellas: Other: Travel expenses.

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Study of Uncoupling Protein 2 (UCP2) Function during Erythroid Differentiation and Maturation.

Blood ◽

10.1182/blood.v106.11.3535.3535 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3535-3535

Author(s):

Alvaro A. Elorza ◽

Sarah E. Haigh ◽

Hanna K. Mikkola ◽

Orian S. Shirihai

Keyword(s):

Oxidative Stress ◽

Oxidative Damage ◽

Peripheral Blood ◽

Biosynthetic Pathway ◽

Erythroid Differentiation ◽

Heme Biosynthesis ◽

Erythroid Cell ◽

Primary Mouse ◽

Wide Range ◽

Stimulation Of

Abstract Mitochondrial oxidative stress is thought to play a key role in sideroblastic anemia and the myelodysplastic syndrome. Potential sources of reactive radicals reside in the heme biosynthetic pathway involving the import and production of pro-oxidant agents, such as ALA and iron and in the respiratory chain. Antioxidant mechanisms are, therefore, expected to be an integral function in erythroid differentiation and their impairment is expected to affect hemoglobinization and maturation. The mitochondrial uncoupling proteins have been shown to reduce oxidative stress through the generation of proton leak across the inner membrane of the mitochondria. They have been implicated in a wide range of physiological and pathological states, including obesity, diabetes, aging neurodegenerative, and immunological diseases. Here we report that UCP2 is induced during erythroid differentiation and that UCP2 deficient mice have a delayed recovery from anemia. We hypothesized that erythroid heme biosynthesis is accompanied by oxidative stress, which results in the induction of UCP2, and that UCP2 plays a role in erythroid maturation by preventing oxidative stress and damage. We found that UCP2 transcripts and protein are induced following the activation of GATA-1 in G1ER cells and during DMSO, butyrate and heme -induced differentiation of murine erythroleukemic (MEL) and K652 erythroid cell lines. Similarly, differentiation of primary mouse c-kit+ / Ter119− erythroid progenitors to Ter119+ is accompanied by induction of UCP2 transcripts. To test the functional significance of UCP2 in erythroid differentiation we studied a UCP2 null mouse. Peripheral blood analysis from UCP2 KO mice revealed a mild elevation of the reticulocyte index as compared to wild type (WT C57BL/6J) mice, which may be related to mild anemia. To test the role of UCP2 in recovery from anemia, we treated WT and UCP2 KO mice with phenylhydrazine for 3 days and studied erythropoiesis using FACS analysis of Ter119 and CD71 surface markers in cells isolated from bone marrow. Stimulation of erythropoiesis was more rapid in the WT mice as compared to the UCP2 KO. The delay in the mutant is more pronounced at the stage of the proerythroblast and is also reflected in the peripheral blood where a higher level of reticulocytes was transiently observed. By 9 days the UCP2 KO mice peripheral blood count was identical to the WT. Analysis of oxidative damage confirmed that UCP2 acts to reduce oxidative damage of mitochondrial proteins. The delayed reticulocytosis could not however be explained by cell death or by reduced hemoglobinization. The increased oxidative damage present in the UCP2 null cells during erythroid differentiation and maturation did not result in the stimulation of apoptosis as revealed by identical Annexin V staining profile of UCP2 KO and WT mice. Remarkably, iron incorporation and hemoglobin content assays ruled out a function of UCP2 in the process of heme biosynthesis per-se. We therefore conclude that UCP2 deficiency regulates maturation in the erythroid lineage independent of the heme biosynthetic pathway.

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Remodeling the regulation of iron metabolism during erythroid differentiation to ensure efficient heme biosynthesis

Blood ◽

10.1182/blood-2005-05-1809 ◽

2006 ◽

Vol 107 (10) ◽

pp. 4159-4167 ◽

Cited By ~ 54

Author(s):

Matthias Schranzhofer ◽

Manfred Schifrer ◽

Javier Antonio Cabrera ◽

Stephan Kopp ◽

Peter Chiba ◽

...

Keyword(s):

Iron Metabolism ◽

Aminolevulinic Acid ◽

Fetal Liver ◽

Mrna Translation ◽

Erythroid Differentiation ◽

Heme Biosynthesis ◽

Iron Storage ◽

Iron Regulatory Proteins ◽

Heme Synthesis ◽

Mrna Binding

Terminal erythropoiesis is accompanied by extreme demand for iron to ensure proper hemoglobinization. Thus, erythroblasts must modify the “standard” post-transcriptional feedback regulation, balancing expression of ferritin (Fer; iron storage) versus transferrin receptor (TfR1; iron uptake) via specific mRNA binding of iron regulatory proteins (IRPs). Although erythroid differentiation involves high levels of incoming iron, TfR1 mRNA stability must be sustained and Fer mRNA translation must not be activated because iron storage would counteract hemoglobinization. Furthermore, translation of the erythroid-specific form of aminolevulinic acid synthase (ALAS-E) mRNA, catalyzing the first step of heme biosynthesis and regulated similarly as Fer mRNA by IRPs, must be ensured. We addressed these questions using mass cultures of primary murine erythroid progenitors from fetal liver, either undergoing sustained proliferation or highly synchronous differentiation. We indeed observed strong inhibition of Fer mRNA translation and efficient ALAS-E mRNA translation in differentiating erythroblasts. Moreover, in contrast to self-renewing cells, TfR1 stability and IRP mRNA binding were no longer modulated by iron supply. These and additional data stemming from inhibition of heme synthesis with succinylacetone or from iron overload suggest that highly efficient utilization of iron in mitochondrial heme synthesis during normal erythropoiesis alters the regulation of iron metabolism via the IRE/IRP system.

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The nuclear 3,5,3'-triiodothyronine receptor in human leukaemic cell lines

Acta Endocrinologica ◽

10.1530/acta.0.1050429 ◽

1984 ◽

Vol 105 (3) ◽

pp. 429-432 ◽

Cited By ~ 7

Author(s):

Juan Bernal ◽

Leif C. Andersson

Keyword(s):

Growth Hormone ◽

Cell Line ◽

Cell Lines ◽

Rat Liver ◽

Association Constant ◽

Erythroid Differentiation ◽

Leukaemic Cell ◽

Cells In Culture ◽

Gh Cells ◽

High Concentrations

Abstract. The 3,5,3'-triiodothyronine (T3) receptor has been studied in a series of continuously growing human leukaemic cell lines. High concentrations of receptor were found in the erythroblastoid cell line K-562. T3 was bound to the nuclei of these cells with an association constant of 3.4 × 109 m−1, and capacity 104 fmol/100 μg DNA, or 8700 molecules/nucleus. This capacity is comparable to that of rat liver or growth hormone producing cells (GH cells) in culture, and suggests that the K-562 cell line could be a useful model for the study of T3 action on erythroid differentiation.

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