A Closer Look at Cellular Iron Metabolism in IRP2 Deficient Erythroblasts.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3843-3843
Author(s):  
Matthias Schranzhofer ◽  
Manfred Schifrer ◽  
Bruno Galy ◽  
Matthias W. Hentze ◽  
Ernst W. Mullner ◽  
...  
Keyword(s):  
Protein Expression ◽  
Iron Metabolism ◽  
Blood Cells ◽  
Hypochromic Anemia ◽  
Erythroid Differentiation ◽  
Wild Type ◽  
Knock Out ◽  
Time Points ◽  
Cellular Iron ◽  
Irp1 And Irp2

Abstract Developing red blood cells are the major consumers of body iron which is indispensable for the enormous production of heme for hemoglobin synthesis. The uptake of iron occurs via binding of iron-loaded transferrin to its cognate receptor (TfR). Thereafter the iron is shuttled to the mitochondria where it is incorporated into protoporphyrin IX to form heme. Excess iron is enclosed within the iron storage protein ferritin. Coordinated control between iron uptake and storage is mainly achieved by the post-transcriptional regulation of TfR1 and ferritin synthesis by the iron regulatory proteins IRP1 and IRP2. Recently, two groups independently created mice lacking either IRP1 or IRP2 and showed that only IRP2 deficient mice developed microcytic hypochromic anemia. Both groups observed a reduction in TfR1 protein expression levels in the developing red blood cells of IRP2 knockout animals and suggested that the decrease in receptor levels is responsible for the development of anemia. For a more detailed analysis of how the loss of IRP2 expression influences iron metabolism and hemoglobinization during terminal erythroid differentiation, we isolated CFU-E-like erythroid cells from mouse fetal liver of wild type, IRP1 and IRP2 knock out animals. In vitro cultivation of these primary erythroid cells and their synchronous induction for differentiation allowed us to study their cellular iron metabolism at different time points. We analyzed the extent of hemoglobinization and cell size as well as the expression of ferritin and TfR1 during various stages of erythroid differentiation in IRP1, IRP2 and wild type cells. In agreement with the published phenotype of microcytic hypochromic anemia, only erythroblasts lacking IRP2 exhibited a reduction in hemoglobinization and showed a significant increase in ferritin protein levels before and after induction of differentiation. In contrast, TfR1 protein expression levels on the cell surface were significantly decreased in IRP2 deficient cells until 24h of differentiation, but converged with those of wild type cells at 48h of differentiation at the time point at which hemoglobinization is fully in progress. Moreover, measurement of 59Fe uptake and its cellular distribution showed that there is significantly more 59Fe located in cytosolic ferritin of IRP2 knock out cells at all time points compared to their wild type counterpart. In summary, these results suggest that not only the reduced expression of TfR1, but also the up-regulation of ferritin, play important roles in the development of anemic phenotype in IRP2 knock out mice. This work was supported by the Canadian Institutes of Health Research and the Canadian Blood Services.

Protoporphyrin IX Down-Regulates DMT1 Associated Protein (DAP) in K562 Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1548-1548
Author(s):  
Yasumasa Okazaki ◽  
Hong Yin ◽  
Yuxiang Ma ◽  
Mary Yeh ◽  
Kwo-yih Yeh ◽  
...  
Keyword(s):  
Iron Metabolism ◽  
Iron Content ◽  
Iron Status ◽  
Protoporphyrin Ix ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Ammonium Citrate ◽  
Ferric Ammonium Citrate ◽  
Down Regulation ◽  
Cellular Iron

Abstract The final steps of heme biosynthesis include the transport of coproporphyrin with the transport step probably mediated by the peripheral benzodiazepine receptor (PBR). Within the mitochondria copropoprhyrin is then converted to protoporphyrin IX (PPIX) which in turn is converted to hemin with insertion of iron by ferrochelatase. PBR is ubiquitously expressed and has been implicated in steriodogenesis, apoptosis, erythroid differentiation, and inflammation. Interestingly, PPIX is among several high affinity ligands for PBR. Various cytosolic proteins that interact with PBR have also been defined including PBR associated protein 7 (PAP7). The various PBR ligands including PPIX may affect the binding of these proteins to PBR. We have demonstrated (Blood, Nov 2004; 104: 53) that DAP, a protein highly homologous to PAP7, binds to the C-terminus of DMT1 and may have a role in regulation of intracellular iron transport. We, therefore, examined the effects of PPIX on the functions of DAP and other proteins that affect cellular iron metabolism. DAP is 526 amino acid protein with a nuclear localization signal domain (aa 212–229) and a Golgi localization domain (aa 380–524), and is distributed in the cytoplasm, Golgi apparatus, and nuclei of K562 cells. K562 cells were grown in the presence of 5 μM PPIX for 24 hours and then the expression of DAP, transferrin receptor 1 (TfR1), and ferritin examined by western blot analysis. In addition, cells were grown in medium of either normal iron content (3.5 μM from ferri-transferrin), high iron content (217 μM from the addition of ferric ammonium citrate), or low iron content (by the addition of 50 mM desferroxamine). Under all three iron conditions PPIX induced differentiation but down-regulated ferritin expression and up-regulated TfR1 expression. Additionally, PPIX had a striking effect on DAP expression markedly decreasing DAP levels but only in cells grown either in normal or low iron medium. In addition, PPIX affected the expression of the iron transporter DMT1in parallel with DAP. As PPIX induced erythroid differentiation of K562 cells we examined the effects of hemin which can also induce differentiation of K562 cells. In contrast to PPIX, hemin caused strong down-regulation of TfR and up-regulation of ferritin and DAP. The down-regulation of DAP induced by PPIX was restored by the addition of hemin. These results indicate that PPIX affects DAP expression and other important elements involved in cellular iron metabolism and that these effects are partially modified by the iron status of the cell.

Heme Oxygenase 1 Plays An Unexpected Role During Erythroid Differentiation

Blood ◽  
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.

Oxidative Stress and Increased Destruction of Red Blood Cells Contribute to the Pathophysiology of Anemia Caused By DMT1 Deficiency

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4027-4027 ◽  
Author(s):  
Zuzana Zidova ◽  
Daniel Garcia-Santos ◽  
Katarina Kapralova ◽  
Pavla Koralkova ◽  
Renata Mojzikova ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Fetal Liver ◽  
Erythroid Differentiation ◽  
Heme Oxygenase 1 ◽  
Wild Type ◽  
Divalent Metal Transporter 1 ◽  
Oxidative Defense

Abstract Inactivating mutations in divalent metal transporter 1 (DMT1) are associated with a severe defect in erythroid iron utilization and cause moderate to severe hypochromic microcytic anemia in human patients and two rodent models. We have previously shown that DMT1 deficiency impairs erythroid differentiation, induces apoptosis of erythroid precursors and causes the suppression of colony-forming capacity of erythroid progenitors. Using in vitro cultures of fetal liver cells we were able to recapitulate this in vivo defect. We confirmed abnormal pattern of erythroid differentiation and increased apoptosis (2.5-times) of DMT1-mutant erythroblasts when compared to wild-type (wt) fetal liver erythroblats. Determination of 2’,7’-Dichlorofluorescein diacetate-dependent intensity of fluorescence, which is proportional to the concentration of reactive oxygen species (ROS), revealed elevated levels of ROS in DMT1-mutant erythroblats when compared to wt erythroblast. This result suggests that oxidative stress contributes to the apoptosis in DMT1-mutant cells. We also observed that the defective erythroid differentiation of DMT1-mutant erythroblasts is marked by a blunted induction of heme oxygenase-1, an enzyme that co-regulates erythroid differentiation by controlling the heme regulatory pool in erythroid cells (Garcia-Santos et al., Blood, 2014, 123 (14): 2269-77). In further studies we focused on mature red blood cells (RBC), because it is known that nutritional iron deficiency and certain types of congenital hypochromic anemia are associated with increased levels of ROS and shortened life span of RBC that can be at least partially attributed to a programmed cell death of erythrocytes, so called eryptosis (Lang et al., Int J Biochem Cell Biol, 2012, 44 (8): 1236-43). Using labeling with carboxyfluorescein diacetate succinimidyl ester, we observed an accelerated clearance of DMT1-mutant RBC from circulating blood when compared to wild-type RBC. In vitro, DMT1-mutant RBC exposed to hyperosmotic shock or glucose depletion showed significantly increased levels of phosphatidylserine on the membrane detected by Annexin V binding. Together, these results confirmed eryptosis of DMT1-mutant RBC. As eryptosis is proposed to be triggered via activation of Ca2+ cation channels, we next measured the concentration of cytosolic Ca2+ using Fluo3/AM fluorescent dye and found significantly elevated content of intracellular Ca2+ in DMT1-mutant RBC when compared to wt RBC. In addition, DMT1-mutant RBC had higher levels of ROS than wt RBC despite significantly increased activity of anti-oxidative defense enzymes; glutathione peroxidase (1.6-times), catalase (1.9-times) and methemoglobin reductase (1.9-times). This indicates that exaggerated anti-oxidative defense in DMT1-mutant RBC is not sufficient to eliminate ROS effectively. Furthermore, DMT1-mutant RBC also showed accelerated anaerobic glycolysis as detected by increased activities of hexokinase (2.5-times), pyruvate kinase (2.4-times), glucose-phosphate isomerase (3.2-times). This result together with reduced ATP/ADP (1.6-times) ratio in DMT1-mutant RBC when compared to wt RBC suggests an increased demand for ATP in DMT1-mutant erythrocytes. In conclusion we propose that increased oxidative stress and accelerated destruction of RBC contribute to the pathophysiology of anemia caused by DMT1-deficiency. Grant support: Czech Grant Agency, grant No. P305/11/1745; Ministry of Health Czech Republic, grant No. NT13587, Education for Competitiveness Operational Program, CZ.1.07/2.3.00/20.0164, Internal Grant of Palacky University Olomouc, LF_2014_011 and in part by the Canadian Institutes of Health Research (D.G-S., P.P.). Disclosures No relevant conflicts of interest to declare.

scholarly journals Lentiviral Gene Therapy for the Correction of Congenital Dyserythropoietic Anemia Type II

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1994-1994
Author(s):  
Mercedes Dessy-Rodriguez ◽  
Sara Fañanas-Baquero ◽  
Veronica Venturi ◽  
Salvador Payan ◽  
Cristian Tornador ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Erythroid Differentiation ◽  
Type Ii ◽  
Wild Type ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
Knock Out ◽  
Cd34 Cells ◽  
Cda Ii

Abstract Congenital dyserythropoietic anemias (CDAs) are a group of inherited anemias that affect the development of the erythroid lineage. CDA type II is the most common one: it accounts for around 60% of all cases, and more than 600 cases have been reported so far. CDA II is caused by biallelic mutations in the SEC23B gene and is characterized by ineffective erythropoiesis with morphologic abnormalities of erythroblasts, hemolysis, and secondary iron overload, which is the most frequent complication. Patients usually suffer from variable degrees of jaundice, splenomegaly, and absolute reticulocyte count inadequate depending on the degree of anemia. Hydrops fetalis, aplastic crisis and gallstones are other associated clinical signs. CDA II bone marrow is characterized by the presence of more than 10% mature binucleated erythroblasts. Another distinctive feature of CDA II erythrocytes is hypoglycosylation of membrane proteins. The management of CDA II is generally limited to blood transfusion and iron chelation. Splenectomy has proved to reduce the number of transfusions in CDA II patients. However, allogenic hematopoietic stem cell transplant (HSCT) represents the only curative option for this disease. Autologous HSCT of genetically corrected cells will mean a definitive treatment for CDA II, overcoming the limitations of allogeneic HSCT, such as limited availability of HLA-matched donors, infections linked to immunosuppression or development of graft versus host disease. This strategy has been used to treat many inherited hematological diseases, including red blood cell diseases such as β-thalassemia, sickle cell disease or pyruvate kinase deficiency. Therefore, we have addressed a similar strategy to be applied to CDAII patients. Two different lentiviral vectors carrying either wild type or codon optimized versions of SEC23B cDNA (wtSEC23B LV or coSEC23B LV, respectively) under the control of human phosphoglycerate kinase promoter (PGK) have been developed. Taking advantage of a CDA II model, in which SEC23B knock-out was done in human hematopoietic progenitors through gene editing, we have determined the most effective SEC23B LV version and the most suitable multiplicity of infection (MOI) to compensate protein deficiency. SEC23B knock out human hematopoietic progenitors (CD34 + cells; 80% frame shift mutations; SEC23BKO) showed a sharp reduction in SEC23B protein level. Those SEC23BKO hematopoietic progenitors were transduced with both lentiviral vectors at MOIs ranged from 3 to 25. We observed that SEC23B protein reached physiological or even supraphysiological levels. In addition, the reduction in the number of erythroid colony forming units (CFUs) identified in SEC23BKO CD34 + cells, was partially restored in the LV transduced SEC23BKO progenitors. Significantly, we observed a clear correlation between the used MOI and the vector copy number (VCN) in the CFUs derived from transduced SEC23BKO CD34 + cells. Furthermore, SEC23BKO hematopoietic progenitors were subjected to an in vitro erythroid differentiation protocol. A sharp decrease in the cell growth throughout erythroid differentiation was observed in SEC23BKO condition. However, the transduction with any of SEC23B LVs at MOIs above 10 was able to recover cell expansion to values equal to wild type cells. Interestingly, total level of protein glycosylation during erythroid differentiation was enhanced after SEC23B LV transduction. Glycosylation level in wtSEC23B LV transduced SEC23BKO cells was most similar to the level in wild type cells. Then, we transduced peripheral blood-derived hematopoietic progenitors (PB-CD34 + cells) from a CDA II patient with wtSEC23B LV at MOI 25 and differentiated in vitro to erythroid cells. A complete restauration of SEC23B protein expression and a cell growth increase of wtSEC23B transduced CDAII was observed with vector copy numbers of 0.3 after 14 days under erythroid conditions. More importantly, we could find a decrease in the percentage of bi-/multinucleated erythroid cells generated in vitro after wtSEC23B LV transduction. In summary, SEC23B LV compensate the SEC23B deficiency in SEC23BKO and in CDAII hematopoietic progenitor cells, paving the way for gene therapy of autologous hematopoietic stem and progenitor cell as an alternative and feasible treatment for CDA II. Disclosures Bianchi: Agios pharmaceutics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Sanchez: Bloodgenetics: Other: Co-Founder and promoter; UIC: Current Employment. Ramirez: VIVEBiotech: Current Employment. Segovia: Rocket Pharmaceuticals, Inc.: Consultancy, Research Funding. Quintana Bustamante: Rocket Pharmaceuticals, Inc.: Current equity holder in publicly-traded company.

RAP-536 Promotes Terminal Erythroid Differentiation and Reduces Anemia in Myelodysplastic Syndromes

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 610-610 ◽  
Author(s):  
Rajasekhar NVS Suragani ◽  
Aaron Mulivor ◽  
R. Scott Pearsall ◽  
Ravindra Kumar
Keyword(s):  
Bone Marrow ◽  
Mouse Model ◽  
Supportive Care ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Significant Proportion ◽  
Erythroid Differentiation ◽  
Wild Type ◽  
Erythroid Progenitor ◽  
Hematopoietic Stem

Abstract Abstract 610 Myelodysplatic syndromes (MDS) are a heterogeneous group of hematopoietic stem cell disorders characterized by ineffective hematopoiesis. Patients develop peripheral blood cytopenias; however, the bone marrow shows increased proliferation and apoptosis. In addition to bone marrow apoptosis, a failure of differentiation contributes to reduced terminally differentiated blood cells. A significant proportion of patients with MDS will develop anemia that are refractory to treatment with recombinant human erythropoietin (EPO) and must rely on transfusions as supportive care. The use of blood transfusions as supportive care is associated with iron overload and significant morbidity. Therefore, alternative therapies to treat anemia in MDS patients are needed. Members of the TGFβ super family of signaling molecules have been implicated in erythropoiesis and represent alternative, EPO-independent targets for the treatment of anemia. ACE-536 is a soluble receptor fusion protein consisting of a modified Activin Receptor Type IIB extracellular domain linked to a human Fc domain. ACE-536 acts as a ligand trap to modulate the activity of TGFβ ligands and promote erythroid differentiation in an EPO independent manner. Subcutaneous administration of ACE-536 to C57BL/6 mice resulted in significant increases in hematocrit, hemoglobin and red blood cells compared to vehicle treated controls within four days. These effects were observed with concurrent treatment of an EPO neutralizing antibody, indicating that EPO is not directly responsible for the initial RBC response of ACE-536. BFU-E or CFU-E colony formation assays from bone marrow or spleen of mice 48 hours after ACE-536 were normal, indicating no effect on the erythroid progenitor population. Differentiation profiling of bone marrow and splenic erythroblasts by FACS analysis following 72 hours after RAP-536 (murine version of ACE-536) treatment revealed a decrease in basophilic erythroblasts and an increase in late stage poly-, ortho-chromatophilic and reticulocytes in bone marrow and spleen compared to vehicle treated mice. The data demonstrate that while EPO treatment increases proliferation of erythroid progenitors, ACE-536 promotes maturation of terminally differentiating erythroblasts. The efficacy of ACE-536 has been demonstrated in various animal models of acute and chronic anemia. In this study we investigated the effect of ACE-536 on anemia in mouse model of MDS. The NUP98-HOXD13 (NHD13) transgenic mouse carries a common translocation found in MDS patients. NHD13 mice develop anemia, neutropenia and lymphopenia at 4–7 months of age, with normal or hypercellular bone marrow. Starting at 4 months of age, mice were treated with RAP-536 (murine homolog of ACE-536) at 10 mg/kg or vehicle control twice per week for 8 months. Wild-type littermate controls were also dosed on the same schedule. As expected, at study baseline (mice 4 months of age), NHD13 mice had reduced RBC, Hb and HCT compared to wild-type littermates. The progression of anemia over the study period was reduced by treatment with RAP-536 compared to vehicle (HCT: −8.3% v. −22%, RBC: −13% v. −30%). Based on blood smear analyses, there was no indication of increased leukemic cells with ACE-536 treatment. Our data demonstrate that RAP-536 can increase hematology parameters through enhancing maturation of terminally differentiated red blood cells and can serve as a therapeutic molecule for the treatment of anemia. As anemia contributes significantly to the morbidity of patients with MDS, a mouse model was used to test the therapeutic efficacy of ACE-536 in this disease. We have shown that systemic administration of RAP-536 to MDS mice promotes increases in red blood cell mass without enhanced progression to AML. Therefore ACE-536 may represent a novel treatment for anemia associated with MDS, particularly in patients that are refractory to EPO therapy. Disclosures: Suragani: Acceleron Pharma Inc: Employment. Mulivor:Acceleron Pharma Inc: Employment. Pearsall:Acceleron Pharma Inc: Employment. Kumar:Acceleron Pharma Inc: Employment.

The Regulation of Hepcidin and Its Effects on Systemic and Cellular Iron Metabolism

Hematology ◽  
2008 ◽  
Vol 2008 (1) ◽  
pp. 151-158 ◽  
Author(s):  
Mark D. Fleming
Keyword(s):  
Iron Metabolism ◽  
Blood Cells ◽  
Iron Homeostasis ◽  
Iron Absorption ◽  
Peptide Hormone ◽  
Intestinal Cells ◽  
Cellular Physiology ◽  
Cellular Iron ◽  
Regulated Expression ◽  
Total Body

Abstract Systemic iron homeostasis depends on the regulated expression of hepcidin, a peptide hormone that negatively regulates iron egress from intestinal cells and macrophages by altering the expression of the cellular iron exporter ferroportin. In doing so, hepcidin can control both the total body iron by modulating intestinal iron absorption as well as promote iron available for erythropoiesis by affecting the efficiency with which macrophages recycle iron from effete red blood cells. This review focuses on the systemic and cellular physiology of hepcidin regulation in relation to iron stores, erythropoiesis, inflammation, and hypoxia and how hepcidin regulation and dysregulation contributes to normal iron homeostasis and iron metabolism disorders.

Iron regulatory protein-1 and -2: transcriptome-wide definition of binding mRNAs and shaping of the cellular proteome by iron regulatory proteins

Blood ◽  
2011 ◽  
Vol 118 (22) ◽  
pp. e168-e179 ◽  
Author(s):  
Mayka Sanchez ◽  
Bruno Galy ◽  
Bjoern Schwanhaeusser ◽  
Jonathon Blake ◽  
Tomi Bähr-Ivacevic ◽  
...  
Keyword(s):  
Iron Metabolism ◽  
Rna Binding ◽  
Isotope Labeling ◽  
Rna Binding Proteins ◽  
Regulatory Protein ◽  
Regulatory Proteins ◽  
Iron Regulatory Proteins ◽  
Ribonucleoprotein Complexes ◽  
Cellular Iron ◽  
Irp1 And Irp2

Abstract Iron regulatory proteins (IRPs) 1 and 2 are RNA-binding proteins that control cellular iron metabolism by binding to conserved RNA motifs called iron-responsive elements (IREs). The currently known IRP-binding mRNAs encode proteins involved in iron uptake, storage, and release as well as heme synthesis. To systematically define the IRE/IRP regulatory network on a transcriptome-wide scale, IRP1/IRE and IRP2/IRE messenger ribonucleoprotein complexes were immunoselected, and the mRNA composition was determined using microarrays. We identify 35 novel mRNAs that bind both IRP1 and IRP2, and we also report for the first time cellular mRNAs with exclusive specificity for IRP1 or IRP2. To further explore cellular iron metabolism at a system-wide level, we undertook proteomic analysis by pulsed stable isotope labeling by amino acids in cell culture in an iron-modulated mouse hepatic cell line and in bone marrow-derived macrophages from IRP1- and IRP2-deficient mice. This work investigates cellular iron metabolism in unprecedented depth and defines a wide network of mRNAs and proteins with iron-dependent regulation, IRP-dependent regulation, or both.

scholarly journals Coordinated Mis-Splicing of Multiple Mitochondrial Iron Metabolism Genes Causes Ring Sideroblast Formation in SF3B1-Mutant MDS

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 4-4 ◽  
Author(s):  
Courtnee Clough ◽  
Joseph Pangallo ◽  
Martina Sarchi ◽  
Stephanie Busch ◽  
Janis L. Abkowitz ◽  
...  
Keyword(s):  
Iron Metabolism ◽  
Erythroid Differentiation ◽  
Causal Connection ◽  
Wild Type ◽  
Conditional Expression ◽  
Ring Sideroblasts ◽  
Mitochondrial Iron ◽  
Mutant Lines ◽  
Mutant Cells

Splicing is a fundamental process by which introns are removed from primary RNA transcripts. Alternative splicing is a major mechanism of gene regulation by which eukaryotic cells expand their transcriptional repertoire. By contrast, aberrant splicing generates novel transcripts not found in normal cells. Heterozygous gain-of-function mutations in a core U2 spliceosome factor SF3B1 are present in ~25% of myelodysplastic syndrome (MDS) patients. Strikingly, SF3B1 mutations are present in ~80% of MDS with ring sideroblasts (MDS-RS) patients suggesting a causal connection between mutant SF3B1 and ring sideroblasts (RS), erythroid precursors with iron-laden mitochondria (Yoshida et al. Nature 2011, Papaemmanuil et al.NEJM 2011). However, the mechanism by which SF3B1 mutations cause RS formation remains poorly understood since existing models of SF3B1-mutant MDS do not recapitulate RS formation in vitro. We have established an induced pluripotent stem cell (iPSC) model of MDS-RS that recapitulates mutant SF3B1-mediated mis-splicing and in vitro ring sideroblast formation. We reprogrammed SF3B1-mutant and SF3B1-wild-type iPSCs from individual MDS-RS patients enabling internal normalization to the isogenic normal clone (Hsu et al. Blood 2019). We established expandable multipotential HPC lines by conditional expression of five transcription factors (5F-HPC), followed by an 18-day erythroid differentiation. We monitored RS formation during the erythroid differentiation of 5F-HPCs using Prussian blue histological staining. RS formation increased during terminal erythropoiesis and peaked at 25-40% on day 18 in SF3B1-mutant lines, whereas SF3B1-wild-type lines showed no detectable RS formation. The frequency of RS was similar in isogenic SF3B1-only and SF3B1/EZH2 co-mutant cells suggesting that the mutant SF3B1 is sufficient to drive RS formation. To identify mis-splicing events that contribute to RS formation, we performed RNA-sequencing and splicing analysis of SF3B1-mutant and SF3B1-wild-type iPSCs at three stages of erythroid differentiation: CD34+ progenitor, CD71+ early erythroblast, and CD71+Glycophorin A+ erythroblast. Global isoform usage was dramatically altered during erythropoiesis, but was more similar in stage-matched SF3B1-mutant and SF3B1-wild-type cells suggesting that mutant SF3B1 selectively mis-splices a subset of transcripts. We identified 2300 transcripts with >10% mis-splicing in SF3B1-mutant lines, and only 120 transcripts with more significant >40% mis-splicing. Of these, TMEM14C and PPOX, inner mitochondrial membrane components of the heme synthesis pathway were strongly mis-spliced throughout erythroid differentiation, consistent with previous studies (Conte et al.BJHaem 2015, Shiozawa et al. Nat Commun 2018). The transcript levels of PPOX but not TMEM14C were reduced in SF3B1-mutant lines as a result of mis-splicing. The expression of ABCB7, a mitochondrial iron sulfur cluster biogenesis component mutated in inherited X-linked sideroblastic anemia (Allikmets et al. Hum Mol Genet 1999), was also reduced in SF3B1-mutant cells as expected (Shiozawa et al. Nat Commun 2018, Dolatshad et al. Leukemia 2016). To investigate the role of these mis-splicing events in ring sideroblast formation, we performed lentiviral overexpression of TMEM14C, PPOX, and ABCB7, in SF3B1-mutant 5F-HPCs and quantified RS formation during late stages of erythroid differentiation. Overexpression of TMEM14C and ABCB7 in SF3B1-mutant cells partially but not completely rescued RS formation compared to luciferase control. These findings confirm the long-standing hypothesis that mis-splicing of mitochondrial iron metabolism genes causes RS formation. Furthermore, these findings suggest that RS formation in MDS is a multigenic event caused by coordinated but incomplete mis-splicing of several critical iron metabolism genes. Disclosures No relevant conflicts of interest to declare.

The Role of Heme Oxygenase 1 in Erythroid Differentiation.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3847-3847
Author(s):  
Daniel Garcia dos Santos ◽  
Matthias Schranzhofer ◽  
Jose Artur Bogo Chies ◽  
Prem Ponka
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Tissue Injury ◽  
Erythroid Differentiation ◽  
The Body ◽  
Erythroid Cell ◽  
Heme Oxygenase 1 ◽  
Transferrin Receptors ◽  
Erythroid Cells ◽  
Wild Type

Abstract Heme is a complex of iron with protoporphyrin IX that is essential for the function of all aerobic cells. However, if left unguarded, non-protein-bound heme promotes free radical formation, resulting in cell damage and tissue injury. The highest amounts of organismal heme (75–80%) are present in circulating red blood cells (RBC) whose precursors synthesize heme with rates that are at least 1 order of magnitude higher than those in the liver (on the per cell basis), which is the second most active heme producer in the body. The only physiological mechanism of heme degradation is performed by heme oxygenases (HO1 and HO2), which catalyze the rate-limiting step in the oxidative degradation of heme and are, therefore, involved in the control of cellular heme levels. 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. Although the heme-inducible HO isoform, HO1, has been extensively studied in hepatocytes and many other non-erythroid cells, virtually nothing is known about the expression of HO1 in developing RBC. Similarly, it is unknown whether HO-1 plays any role in erythroid cell development under physiological or pathophysiological conditions. In this study we have shown that HO1 protein is expressed in uninduced murine erythroleukemic (MEL) cells and that its levels, somewhat surprisingly, do not decrease during DMSO-induced erythroid differentiation. Moreover, we demonstrated that heme significantly induces HO1 in both uninduced and induced MEL cells. Additionally, we investigated the effect of overexpressed HO1 on heme and iron metabolism in stably transfected MEL cells (MEL-HO1) and their non-transfected counterparts. Compared to wild type cells, DMSO-treated MEL-HO1 cells displayed a reduction in heme stability (measured by the incorporation of 59Fe into heme) in addition to impairment of erythroid differentiation. Moreover, although wild type and transfected cells expressed similar levels of transferrin receptors in the uninduced state, MEL-HO1 cells, as compared to wild type MEL cells, showed only a small increase in transferrin receptors upon treatment with DMSO. Finally, we measured apoptosis using annexin-V and observed an increase in the number of apoptotic cells in HO1 transfectants, but not in wild type MEL cells. These results suggest that an as yet unknown mechanism exists to protect heme against endogenous HO1 action during physiological erythroid differentiation. In addition, our results showing that high levels of HO1 in erythroid cells cause heme catabolism and a defect in erythroid differentiation raise the possibility that HO1 could play a role in some pathophysiological conditions such as unbalanced globin synthesis in thalassemias.

Hfe deficiency increases susceptibility to cardiotoxicity and exacerbates changes in iron metabolism induced by doxorubicin

Blood ◽  
2003 ◽  
Vol 102 (7) ◽  
pp. 2574-2580 ◽  
Author(s):  
Carlos J. Miranda ◽  
Hortence Makui ◽  
Ricardo J. Soares ◽  
Marc Bilodeau ◽  
Jeannie Mui ◽  
...  
Keyword(s):  
Iron Metabolism ◽  
Chemotherapeutic Agent ◽  
Genetic Disorder ◽  
Hereditary Hemochromatosis ◽  
Wild Type ◽  
Knock Out ◽  
Liver And Pancreas ◽  
Iron Deposits ◽  
Acute Cardiotoxicity ◽  
Deficient Mice

Abstract The clinical use of doxorubicin (DOX), an anthracycline chemotherapeutic agent, is limited by cardiotoxicity. The possible involvement of iron in DOX-induced cardiotoxicity became evident from studies in which iron chelators were shown to be cardioprotective. Iron overload is found in hereditary hemochromatosis, a genetic disorder prevalent in individuals of European descent. We hypothesized that Hfe deficiency may increase susceptibility to DOX-induced toxicity. Acute cardiotoxicity and iron changes were studied after treatment with DOX in Hfe knock-out (Hfe-/-) mice and wild-type mice. DOX-induced iron metabolism changes were intensified in Hfe-/- mice, which accumulated significantly more iron in the heart, liver, and pancreas, but less in the spleen compared with wild-type mice. In addition, Hfe-deficient mice exhibited significantly greater sensitivity to DOX-induced elevations in serum creatine kinase and aspartate aminotransferase. Increased mortality after chronic DOX treatment was observed in Hfe-/- mice and Hfe+/-mice compared with wild-type mice. DOX-treated Hfe-/- mice had a higher degree of mitochondrial damage and iron deposits in the heart than did wild-type mice. These data demonstrate that Hfe deficiency in mice increases susceptibility to DOX-induced cardiotoxicity and suggest that genetic mutations related to defects in iron metabolism may contribute to its cardiotoxicity in humans. (Blood. 2003;102:2574-2580)

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