Nitrogen Monoxide Inhibits Heme Synthesis In Reticulocytes

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4252-4252
Author(s):  
Marc Mikhael ◽  
Sameer Apte ◽  
Shan Soe-Lin ◽  
Prem Ponka
Keyword(s):  
Iron Metabolism ◽  
Iron Uptake ◽  
Aminolevulinic Acid ◽  
Conflicts Of Interest ◽  
No Production ◽  
Erythroid Cells ◽  
Anemia Of Chronic Disease ◽  
Heme Synthesis ◽  
Eif2α Phosphorylation ◽  
Globin Synthesis

Abstract Abstract 4252 Anemia of chronic disease (ACD) is a condition that often manifests in patients with chronic immune activation due to chronic infections, autoimmune disorders, cancer and other diseases. The pathogenesis of ACD is complex and involves inefficient erythropoietin production, immune-mediated inhibition of erythropoiesis, and retention of iron in hemoglobin-processing macrophages. During their development, erythroid cells are closely associated with macrophages. In inflammatory conditions, activated macrophages generate large quantities of the gaseous molecule, nitric oxide (NO), which has numerous effects on iron metabolism. In this study, we explored the possibility that NO affects iron metabolism in erythroid cells. We treated reticulocytes with the NO donors, sodium nitroprusside (SNP) and S-Nitroso-N-acetyl-D,L-penicillamine (SNAP). We show that NO inhibits 59Fe incorporation from 59Fe-transferrin into reticulocytes and their heme. Significantly, 5-aminolevulinic acid (ALA, the product of ALA synthase, which catalyzes the first step of heme synthesis) reversed the SNP-mediated decrease in 59Fe incorporation into heme but not the cellular 59Fe uptake. In addition, SNP treatment led to an increase in eIF2α phosphorylation (which is known to occur in heme-deficient cells) and decreased globin translation. Importantly, the addition of ALA to SNP-treated reticulocytes prevented the effect of SNP on eIF2α phosphorylation and reversed globin synthesis inhibition. This indicates that in SNP treated reticulocytes, the phosphorylation of eIF2α and inhibition of globin synthesis occur indirectly, via NO's effect on erythroid-specific ALA synthase (ALA-S2). These results led us to conclude that NO has two distinct effects on reticulocytes, namely: a decrease in ALA-S2 activity and a decrease in transferrin-mediated iron uptake. The profound impact of NO on heme synthesis, iron uptake and globin translation in reticulocytes raises the possibility that NO production by macrophages could also contribute to ACD. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Single-cell analyses demonstrate that a heme–GATA1 feedback loop regulates red cell differentiation

Blood ◽  
2019 ◽  
Vol 133 (5) ◽  
pp. 457-469 ◽  
Author(s):  
Raymond T. Doty ◽  
Xiaowei Yan ◽  
Christopher Lausted ◽  
Adam D. Munday ◽  
Zhantao Yang ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Single Cell ◽  
Aminolevulinic Acid ◽  
Developmental Trajectories ◽  
Erythroid Differentiation ◽  
Surface Proteins ◽  
Red Cell ◽  
Erythroid Cells ◽  
Heme Synthesis ◽  
Globin Synthesis

Abstract Erythropoiesis is the complex, dynamic, and tightly regulated process that generates all mature red blood cells. To understand this process, we mapped the developmental trajectories of progenitors from wild-type, erythropoietin-treated, and Flvcr1-deleted mice at single-cell resolution. Importantly, we linked the quantity of each cell’s surface proteins to its total transcriptome, which is a novel method. Deletion of Flvcr1 results in high levels of intracellular heme, allowing us to identify heme-regulated circuitry. Our studies demonstrate that in early erythroid cells (CD71+Ter119neg-lo), heme increases ribosomal protein transcripts, suggesting that heme, in addition to upregulating globin transcription and translation, guarantees ample ribosomes for globin synthesis. In later erythroid cells (CD71+Ter119lo-hi), heme decreases GATA1, GATA1-target gene, and mitotic spindle gene expression. These changes occur quickly. For example, in confirmatory studies using human marrow erythroid cells, ribosomal protein transcripts and proteins increase, and GATA1 transcript and protein decrease, within 15 to 30 minutes of amplifying endogenous heme synthesis with aminolevulinic acid. Because GATA1 initiates heme synthesis, GATA1 and heme together direct red cell maturation, and heme stops GATA1 synthesis, our observations reveal a GATA1–heme autoregulatory loop and implicate GATA1 and heme as the comaster regulators of the normal erythroid differentiation program. In addition, as excessive heme could amplify ribosomal protein imbalance, prematurely lower GATA1, and impede mitosis, these data may help explain the ineffective (early termination of) erythropoiesis in Diamond Blackfan anemia and del(5q) myelodysplasia, disorders with excessive heme in colony-forming unit-erythroid/proerythroblasts, explain why these anemias are macrocytic, and show why children with GATA1 mutations have DBA-like clinical phenotypes.

scholarly journals Iron distribution in Belgrade rat reticulocytes after inhibition of heme synthesis with succinylacetone

Blood ◽  
1993 ◽  
Vol 81 (12) ◽  
pp. 3414-3421 ◽  
Author(s):  
LM Garrick ◽  
K Gniecko ◽  
Y Liu ◽  
DS Cohan ◽  
JA Grasso ◽  
...  
Keyword(s):  
Iron Uptake ◽  
Iron Transport ◽  
Aminolevulinic Acid ◽  
Specific Inhibitor ◽  
Hypochromic Anemia ◽  
Endocytic Vesicle ◽  
Heme Synthesis ◽  
Diferric Transferrin ◽  
Rat Reticulocytes ◽  
Insight Into

Abstract We have used succinylacetone (4,6-dioxoheptanoic acid), a specific inhibitor of delta-aminolevulinic acid dehydrase, to gain insight into the defect in iron metabolism in the Belgrade anemia. The Belgrade rat has an inherited microcytic, hypochromic anemia associated with poor iron uptake into developing erythroid cells. Succinylacetone inhibits heme synthesis, leading to nonheme iron accumulation in mitochondria and cytosol of normal reticulocytes. When succinylacetone is used to inhibit Belgrade heme synthesis, iron from diferric transferrin does not accumulate in the stromal fraction that contains mitochondria, nor does 59Fe accumulate in the nonheme cytosolic fraction. Hence, the defect in the Belgrade rat reticulocyte occurs in the endocytic vesicle or in a step subsequent to iron transit from the vesicle but before the nonheme cytosolic or mitochondrial iron fractions. Therefore, the mutation affects either the release of iron from transferrin or iron transport from the vesicle to the mitochondrion.

Studies on Heme Oxygenase 1 During Erythroid Differentiation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4254-4254
Author(s):  
Daniel Garcia Santos ◽  
Jesse Eisenberg ◽  
Matthias Schranzhofer ◽  
Prem Ponka
Keyword(s):  
Aminolevulinic Acid ◽  
Conflicts Of Interest ◽  
Tissue Injury ◽  
Erythroid Differentiation ◽  
The Body ◽  
Cellular Damage ◽  
Erythroid Cell ◽  
Heme Oxygenase 1 ◽  
Erythroid Cells ◽  
Murine Erythroleukemia

Abstract Abstract 4254 Heme is indispensable for the function of all aerobic cells as a prosthetic group of innumerable proteins. However, “free heme” (uncommitted) can initiate the formation of free radicals and cause lipid peroxidation, which can lead to cellular damage and tissue injury. Therefore, the rate of heme biosynthesis and catabolism must be well balanced by tight control mechanisms. 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 one order of magnitude higher (on the per cell basis) than those in the liver – the second most active heme producer in the body. The degradation of heme is exclusively carried out by heme oxygenases 1 and 2 (HO1 and HO2), which catalyze the rate-limiting step in the oxidative degradation of heme. 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 HO1 plays any role in erythroid cell development under physiological or pathophysiological conditions. Using both a murine erythroleukemia cell line (MEL) and primary erythroid cells isolated from mouse fetal livers, we have demonstrated that during erythroid differentiation HO1 is up-regulated at both mRNA and protein levels. This increase in HO1 can be prevented by succinylacetone (SA), an inhibitor of heme synthesis that blocks 5-aminolevulinic acid dehydratase. These data suggest that in developing RBC, in addition to the continuous assembly of heme with globin chains, there is an increase in levels of uncommitted heme, which upregulates HO1 expression. Additionally, we have shown that down-regulation of HO1 via siRNA increased hemoglobinization in differentiating MEL cells. In contrast, induction of HO1 expression by NaAsO2 reduced the hemoglobinization of MEL cells. This effect could be reversed to control levels by the addition of HO1 inhibitor tin-protophorphyrin (SnPP). These results show that in differentiating erythroid cells the balance between levels of heme and HO1 have to be tightly regulated to maintain hemoglobinization at appropriate levels. Our results lead us to propose that disturbances in HO1 expression could play a role in some pathophysiological conditions such as thalassemias. Disclosures: No relevant conflicts of interest to declare.

Fam210b Is Required for Optimal Cellular and Mitochondrial Iron Uptake during Erythroid Differentiation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 405-405
Author(s):  
Yvette Y Yien ◽  
Caiyong Chen ◽  
Jiahai Shi ◽  
Liangtao Li ◽  
Daniel E. Bauer ◽  
...  
Keyword(s):  
Iron Uptake ◽  
Iron Transport ◽  
Aminolevulinic Acid ◽  
Genetic Modifier ◽  
Terminal Differentiation ◽  
Erythroid Differentiation ◽  
Hemoglobin Synthesis ◽  
Heme Synthesis ◽  
Mitochondrial Iron ◽  
Murine Erythroleukemia

Abstract Red cells synthesize large quantities of heme during terminal differentiation. Central to erythropoiesis is the transport and trafficking of iron within the cell. Despite the importance of iron transport during erythroid heme synthesis, the molecules involved in intracellular trafficking of iron are largely unknown. In a screen for genes that are up-regulated during erythroid terminal differentiation, we identified FAM210B, a predicted multi-pass transmembrane mitochondrial protein as an essential component of mitochondrial iron transport during erythroid differentiation. In zebrafish and mice, Fam210b mRNA is enriched in differentiating erythroid cells and liver (fetal and adult), which are tissues that require large amounts of iron for heme synthesis. Here, we report that FAM210B facilitates mitochondrial iron import during erythroid differentiation and is essential for hemoglobin synthesis. Zebrafish are anemic when fam210b is silenced using anti-sense morpholinos (Fig. A). CRISPR knockout of Fam210b caused a heme synthesis defect in differentiating Friend murine erythroleukemia (MEL) cells. PPIX levels in Fam210b deficient cells are normal, demonstrating that Fam210b does not participate in synthesis of the heme tetrapyrrole ring. Consistent with this result, supplementation of Fam210b deficient MEL cells with either aminolevulinic acid, the first committed substrate of the heme synthesis pathway or a chemical analog of protoporphyrin IX failed to chemically complement the heme synthesis defect. While Fam210b was not required for basal housekeeping heme synthesis, Fam210b deficientcells showed defective total cellular and mitochondrial iron uptake during erythroid differentiation (Fig. B). As a result, Fam210b deficient cells had defective hemoglobinization. Supplementation of Fam210b-/- MEL cells with non-transferrin iron chelates restored erythroid differentiation and hemoglobin synthesis; whereas, similar chemical complementation could not be achieved in the Tmem14c-/- cells, which have a primary defect in tetrapyrrole transport. (Fig. C). Our findings reveal that FAM210B is required for optimal mitochondrial iron import during erythroid differentiation for hemoglobin synthesis. It may therefore function as a genetic modifier for mitochondriopathies, anemias or porphyrias. Figure 1. Figure 1. Disclosures Bauer: Biogen: Research Funding; Editas Medicine: Consultancy. Orkin:Editas Inc.: Consultancy.

Transcriptional Regulation of Transferrin Receptor by Heme in Erythroid Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2049-2049
Author(s):  
Matthias Schranzhofer ◽  
Nam-Chun Lok ◽  
Manfred Schifrer ◽  
Ernst W Muellner ◽  
Prem Ponka
Keyword(s):  
Mrna Stability ◽  
Aminolevulinic Acid ◽  
Conflicts Of Interest ◽  
Erythroid Cells ◽  
Knock Out ◽  
Iron Regulatory Proteins ◽  
Iron Incorporation ◽  
Knock Out Mice ◽  
Feedback Regulator ◽  
In Erythroid Cells

Abstract Abstract 2049 Erythroid cells are the largest consumers of iron which is delivered to them by tansferrin (Tf) by its cognate receptor (TfR). In contrast to other cells, developing red blood cells (RBC) regulate TfR expression not only at the level of mRNA stability via the iron regulatory proteins (IRP) 1 and 2, but also by transcription (Lok & Ponka, J Biol Chem 275:24185-90, 2000). Here we provide evidence that TfR expression and cellular uptake of iron from Tf is stimulated by enhanced heme synthesis. Incubation of erythroid cells with 5-aminolevulinic acid (ALA) increased TfR expression accompanied by increased iron incorporation into heme. This effect of ALA can be completely prevented by the inhibitors of heme biosynthesis (succinylacetone [blocks ALA dehydratase] or N-methylprotoporphyrin [blocks ferrochelatase]), indicating that the effect of ALA requires its metabolism to heme. The induction of TfR mRNA expression by ALA is mainly a result of increased mRNA synthesis since the effect of ALA can be abolished by actinomycin D. Recently, IRP2 was proposed to play a role in maintaining TfR mRNA stability in developing RBC (Cooperman et al., Blood 106:1084-91; 2005; Galy et al., Blood 106:2580-9, 2005). Importantly, we have demonstrated that ALA added to cultures of erythroid cells derived from IRP2 knock out mice restores the expression of TfR to levels observed in cells obtained from wild type mice. In conclusion, our results indicate that in erythroid cells heme serves as a positive feedback regulator that maintains high TfR levels thus ensuring adequate iron availability for hemoglobin synthesis. Disclosures: No relevant conflicts of interest to declare.

Tmem14c Plays An Essential Role In Mitochondrial Heme Metabolism

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 427-427 ◽  
Author(s):  
Barry H. Paw ◽  
Yvette Y. Yien ◽  
Raymond F Robledo ◽  
Iman J. Schultz ◽  
Naoko Takahashi-Makise ◽  
...  
Keyword(s):  
De Novo ◽  
Conflicts Of Interest ◽  
Transmembrane Protein ◽  
Protoporphyrin Ix ◽  
Terminal Differentiation ◽  
Gene Trap ◽  
Erythroid Cells ◽  
Coproporphyrinogen Oxidase ◽  
Heme Synthesis ◽  
Murine Erythroleukemia

Abstract Red cells synthesize large amounts of heme during terminal differentiation. Central to this process is the transport and trafficking of heme synthesis intermediates within the cell. Despite the importance of transport during heme synthesis, the molecules involved in this process are largely unknown. In a screen for genes that are upregulated during erythroid terminal differentiation, we identified Tmem14c, a predicted multi-pass transmembrane protein as an essential component of the porphyrin metabolism pathway. Here, we report that Tmem14c facilitates the synthesis of mitochondrial protoporphyrin IX from coproporphyrinogen III and is thus required for heme synthesis. Tmem14c is a mitochondrial inner-membrane protein enriched in vertebrate hematopoietic tissues and is required for terminal erythropoiesis. Tmem14c gene-trap mouse embryos are severely anemic and mostly die by E13.5 (Fig. A). Fetal liver erythroid cells derived from gene-trap embryos experience maturation arrest. shRNA silencing of Tmem14c in Friend murine erythroleukemia (MEL) cells results in a significant decrease in de-novo heme synthesis. The biochemical defect is due to a decrease in mitochondrial protoporphyrin IX synthesis, while cytoplasmic porphyrin levels remain normal (Fig. B). The heme synthesis defect in Tmem14c-silenced MEL cells is complemented with a protoporphyrin IX analog. These data show the role of Tmem14c in regulating the terminal steps in mitochondrial porphyrin trafficking. Our findings collectively demonstrate that Tmem14c is required for the transport of mitochondrial porphyrins in developing erythroid cells. Due to its inner-mitochondrial localization and its relative proximity to heme synthetic enzymes coproporphyrinogen oxidase and protoporphyrinogen oxidase (Rhee et al., 2013 Science), Tmem14c can function as a molecular adaptor that facilitates the interaction of proteins involved in porphyrin transport, or as a protoporphyrinogen IX transporter (Fig. C). The identification of Tmem14c as an essential regulator of porphyrin transport and heme synthesis provides a novel genetic tool for exploring erythropoiesis and disorders of heme synthesis such as porphyria and anemia. Disclosures: No relevant conflicts of interest to declare.

The Interplay of GATA1 with Heme Regulates an Erythroid Cell's Differentiation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 541-541
Author(s):  
Raymond T Doty ◽  
Xiaowei Yan ◽  
Christopher Lausted ◽  
Zhantao Yang ◽  
Li Liu ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Aminolevulinic Acid ◽  
Macrocytic Anemia ◽  
Erythroid Differentiation ◽  
Gene Set Enrichment Analysis ◽  
Erythroid Cells ◽  
Ineffective Erythropoiesis ◽  
P53 Pathway ◽  
Heme Synthesis ◽  
Marrow Cells

Abstract GATA1 promotes the transcription of ALAS2, the first and rate limiting step of heme synthesis, and the transcription of many other erythroid-specific genes. It also increases its own transcription while silencing proliferation genes active in early progenitors and thus assures that erythroid differentiation correctly initiates. Heme then transcriptionally and translationally upregulates globin to guarantee adequate hemoglobin production in each cell as it matures. In mice lacking the heme exporter, FLVCR1, excess heme and ROS accumulate and erythropoiesis fails at the CFU-E/proerythroblast stage, resulting in a severe macrocytic anemia (HGB 4.4±0.97 vs 14.8±0.57 g/dL; MCV 66.9±6.2 vs 48.4±0.65 fL). To determine how excess heme causes ineffective erythropoiesis and whether heme is key to terminating differentiation in normal erythroid cells, we performed RNA sequencing of single early erythroid cells (BFU-E to basophilic erythroblasts) from wildtype control and Flvcr1-deleted mice and linked this transcription data to the total quantity of Ter119 on that cell. Principal component analysis (PCA) identified 4 transcriptionally unique clusters A, B, C, & D, which contained cells with negative, low, intermediate, and high Ter119 levels respectively. α- and β-globin transcription were highly correlated (r=0.975), occurred in all cells, increased as Ter119 expression increased, and upregulated in Flvcr1-deleted cells. Gene set enrichment analysis (GSEA) comparing control cells to Flvcr1-deleted cells revealed excess heme results in significant downregulation of the hallmark heme metabolism pathway genes (heme biosynthesis and erythroid differentiation genes), upregulation of the ribosome pathway genes, and no alteration of the P53 pathway genes. All eight heme biosynthetic enzyme genes were expressed equivalently in cluster A cells from control and Flvcr1-deleted mice; however expression in Flvcr1-deleted cells was significantly reduced in clusters B-D. Of the 181 erythroid differentiation genes in the hallmark heme pathway, Gata1 had the greatest reduction (67%) in Flvcr1-deleted cells. Coupled two-way clustering analysis (CTWC) identified 150 genes co-regulated with Gata1 including 106 known GATA1 target genes which were all poorly upregulated in Flvcr1-deleted cells in clusters B-D. Independent microarray analysis of mRNA from control and Flvcr1-deleted CD71+ erythroid cells confirmed low Gata1 mRNA and low GATA1-dependent gene expression in the Flvcr1-deleted cells. To determine if excess heme was directly responsible for Gata1 downregulation, we treated K562, HEL-R, and primary human erythroid marrow cells with aminolevulinic acid (ALA) and iron to increase endogenous heme synthesis. In the primary cells, GATA1 protein decreased by 30-43% (p=0.03) within 15 minutes and 66% by 90 minutes (similar decreases observed in cell lines), suggesting that heme disrupts GATA1 protein function resulting in the loss of autoregulation and reduced GATA1 mRNA. Of 88 genes in the ribosome pathway, 73 were significantly upregulated in Flvcr1-deleted cells, including 16 of the 17 ribosomal protein genes linked to Diamond-Blackfan anemia (DBA) or del(5q) myelodysplastic syndrome (MDS). When heme synthesis was induced in primary human erythroid marrow cells with ALA and iron, the transcription of ribosome protein genes such as Rps19, Rps14, and Rpl35 increased, further supporting the concept that heme assures sufficient ribosome production for globin protein synthesis. While P53 activation is a key factor in ineffective erythropoiesis caused by ribosomal protein imbalance (i.e., DBA and del(5q) MDS), GSEA did not reveal any increased activation of the P53 pathway in Flvcr1-deleted cells. To confirm that P53 was not involved in the ineffective erythropoiesis caused by excess heme, we generated mice lacking both P53 and FLVCR1. These double mutant mice had severe macrocytic anemia (HGB 2.4±0.70 g/dL; MCV 56.5±4.3 fL) comparable to mice lacking just FLVCR1. Thus, GATA1 turns on heme synthesis and initiates the erythroid differentiation program. GATA1 with heme assure each cell's appropriate progression. Then heme turns off GATA1 to end differentiation. By linking excess heme to prematurely low GATA1, our data may also explain the ineffective (early termination of) erythropoiesis in DBA and reconcile the observations of Sci Transl Med 8:338ra67, 2016 and Nat Med 20:748, 2014. Disclosures No relevant conflicts of interest to declare.

scholarly journals Regulation of heme synthesis in erythroid cells: hemin inhibits transferrin iron utilization but not protoporphyrin synthesis

Blood ◽  
1985 ◽  
Vol 65 (4) ◽  
pp. 850-857 ◽  
Author(s):  
P Ponka ◽  
HM Schulman
Keyword(s):  
Biosynthetic Pathway ◽  
Aminolevulinic Acid ◽  
Cell Uptake ◽  
Erythroid Cell ◽  
Transferrin Receptors ◽  
Erythroid Cells ◽  
Heme Synthesis ◽  
High Concentrations ◽  
In Erythroid Cells

Abstract The inhibition of delta-aminolevulinic acid (ALA) synthase activity by heme is commonly thought to regulate the overall rate of heme synthesis in erythroid cells. However, since heme inhibits erythroid cell uptake of iron from transferrin, we have tested the hypothesis that in reticulocytes heme regulates its own synthesis by controlling the cellular acquisition of iron from transferrin rather than by controlling the synthesis of ALA. We found that hemin added to reticulocytes in vitro inhibits not only the total cell incorporation of 59Fe from transferrin but also the incorporation of [2–14C]-glycine and transferrin-bound 59Fe into heme. However, hemin did not inhibit [2 –14C]-glycine incorporation into protoporphyrin. Furthermore, cycloheximide, which increases the level of non-hemoglobin heme in reticulocytes, also inhibited [2–14C]-glycine into heme but not into protoporphyrin. With high concentrations of ferric pyridoxal benzoylhydrazone (Fe-PBH), which, independent of transferrin and transferrin receptors, can be used as a source of iron for heme synthesis in reticulocytes, significantly more iron is incorporated into heme than from saturating concentrations of Fe-transferrin. This suggests that some step (or steps) in the pathway of iron from extracellular transferrin to protoporphyrin limits the overall rate of heme synthesis in reticulocytes. In addition, hemin in concentrations that inhibit the utilization of transferrin-bound iron for heme synthesis has no effect on the incorporation of iron from Fe-PBH into heme. Our results indicate that in reticulocytes heme inhibits and controls the utilization of iron from transferrin but has no effect on the enzymes of porphyrin biosynthesis and ferrochelatase. This mode of regulation of heme synthesis may be a specific characteristic of the hemoglobin biosynthetic pathway.

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