Heme synthetase activity in normal human and rat erythroid cells and in sideroblastic anemia

The sideroblastic anemias are characterized by ring sideroblasts, that is, red cell precursors with mitochondrial iron accumulation. We therefore studied the expression of mitochondrial ferritin (MtF) in these conditions. Erythroid cells from 13 patients with refractory anemia with ring sideroblasts (RARS) and 3 patients with X-linked sideroblastic anemia (XLSA) were analyzed for the distribution of cytoplasmic H ferritin (HF) and MtF using immunocytochemical methods. We also studied 11 healthy controls, 5 patients with refractory anemia without ring sideroblasts (RA), and 7 patients with RA with excess of blasts (RAEB). About one fourth of normal immature red cells, mostly proerythroblasts and basophilic erythroblasts, showed diffuse cytoplasmic positivity for HF, but very few were positive for MtF (0%-10%). Similar patterns were found in anemic patients without ring sideroblasts. In contrast, many erythroblasts from patients with sideroblastic anemia (82%-90% in XLSA and 36%-84% in RARS) were positive for MtF, which regularly appeared as granules ringing the nucleus. Double immunocytochemical staining confirmed the different cellular distribution of HF and MtF. There was a highly significant relationship between the percentage of MtF+ erythroblasts and that of ring sideroblasts (SpearmanR = 0.90; P < .0001). Reverse transcription-polymerase chain reaction studies demonstrated the presence of MtF mRNA in circulating reticulocytes of 2 patients with XLSA but not in controls. These findings suggest that most of the iron deposited in perinuclear mitochondria of ring sideroblasts is present in the form of MtF and that this latter might be a specific marker of sideroblastic anemia.

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Characterization of the molecular mechanism involved in the activation of hyaluronan synthetase by platelet-derived growth factor in human mesothelial cells

Biochemical Journal ◽

10.1042/bj2830165 ◽

1992 ◽

Vol 283 (1) ◽

pp. 165-170 ◽

Cited By ~ 62

Author(s):

P Heldin ◽

T Asplund ◽

D Ytterberg ◽

S Thelin ◽

T C Laurent

Keyword(s):

Growth Factor ◽

Molecular Mechanism ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Mesothelial Cells ◽

Platelet Derived Growth Factor ◽

Synthetase Activity ◽

Normal Human ◽

Stimulation Of

The molecular mechanism involved in the stimulation of hyaluronan synthetase in normal human mesothelial cells was investigated. Exposure of mesothelial cells to platelet-derived growth factor (PDGF)-BB stimulated hyaluronan synthetase activity, measured in isolated membrane preparations, as well as hyaluronan secretion into the medium. The effect on hyaluronan synthetase was maximal after 6 h of treatment. In contrast, the stimulatory effect of transforming growth factor-beta 1 reached a maximum after 24 h. The stimulatory effect of PDGF-BB was inhibited by cycloheximide. The phosphotyrosine phosphatase inhibitor vanadate was found to stimulate hyaluronan synthetase activity, and to potentiate the effect of PDGF-BB. The protein kinase C (PKC) stimulator phorbol 12-myristate 13-acetate (PMA) also stimulated hyaluronan synthetase; furthermore, depletion of PKC by preincubation of the cells with PMA led to an inhibition of the PDGF-BB-induced stimulation of hyaluronan synthetase activity. Thus the PDGF-BB-induced stimulation of hyaluronan synthetase activity is dependent on protein synthesis and involves tyrosine phosphorylation and activation of PKC.

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MicroRNAs 155 and 451 as Possible Key Molecules for Human Erythroid Differentiation.

Blood ◽

10.1182/blood.v110.11.4071.4071 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4071-4071

Author(s):

Tsukuru Umemura ◽

Shizuka Masaki ◽

Rie Ohtsuka ◽

Yasunobu Abe ◽

Koichiro Muta

Keyword(s):

Red Blood Cells ◽

Internal Control ◽

Blood Cells ◽

Human Peripheral Blood ◽

Erythroid Differentiation ◽

Erythroid Cells ◽

Expression Levels ◽

Final Maturation ◽

Normal Human ◽

U6 Rna

Abstract MicroRNAs (miRNAs) are 18–25-nucleotide noncoding RNAs which play important roles for cell death, proliferation, development and differentiation. MiRNA is an important molecule to regulate genes by suppressing the translation or inducing instability of miRNAs, and is consist of the network system to regulate gene functions in combination with transcription factors. Many recent works demonstrated that some of miRNAs are playing key roles for hematopoiesis and leukemogenesis. In this study, we analyzed the expression of miRNAs(miRNA-155, miRNA-221, miRNA-223, miRNA-451) during differentiation of purified normal human eryhroid progenitors in the liquid culture system. Cells increased almost 500-folds in a number, and differentiated to benzidine-positive mature erythroblasts after days 7 to 9 which were partly red blood cells on days 12 to 14. Since mature erythroid cells loose cellular nucleic acids at the final maturation stages, we measured changes in U6 RNA contents as the internal control for assays of miRNA. Each expression levels of miRNAs were normalized using U6 RNA contents. Analyses of miRNA expressions using quantitative real-time reversetranscriptase polymerase chain reaction have shown that the expression level of miRNA-155 decreased about 200-folds from day 3 to day 12 with almost 87.5% reduction between days 3 and 5. On the other hand, the expression levels of miRNA-451 increased about 270-folds by day 12 in parallel to an increase in benzidine-positive cell numbers. To extend our observation on the up-regulation of miRNA-451 in mature blood cells, we analyzed the miRNA-451 levels in each mature blood cells (red blood cells, granulocytes, lymphocytes and monocytes, platelets) purified from normal human peripheral blood by using a density centrifugation method. miRNA-451 was expressed in red blood cells about 104 folds more than in granulocytes, about 102 folds more than in platelets. Moderate down-regulations of miRNAs 221 and 223 were observed. In conclusion, our observations suggest that the down-regulation of miRNA-155 and the up-regulation of miRNA-451 are key events for normal erythroid differentiation, and that quantitative assays of the two miRNAs may be useful tools for specifying the differentiation stage of each erythroid cells.

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A Genetic Study of Erythrocyte Arginine-tRNA Synthetase Activity in Man

Acta geneticae medicae et gemellologiae ◽

10.1017/s0001566000010151 ◽

1977 ◽

Vol 26 (1) ◽

pp. 21-27 ◽

Cited By ~ 1

Author(s):

Sylvia A. McCune ◽

P. L. Yu ◽

Walter E. Nance

Keyword(s):

Enzyme Activity ◽

Trna Synthetase ◽

Synthetase Activity ◽

Trna Synthetases ◽

Automated Assay ◽

Aminoacyl Trna Synthetases ◽

Assay Procedure ◽

Normal Human ◽

Genetically Determined ◽

Mz Twins

To search for evidence of genetic variation among the aminoacyl-tRNA synthetases, a semi-automated assay procedure employing a Technicon Auto Analyzer was used to measure erythrocyte arginine-tRNA synthetase activity in samples obtained from normal human twins of various ages. Variation in enzyme activity within the older DZ twins was five times that of the MZ twins suggesting the existence of genetically determined variation in enzyme activity. Higher enzyme activity was observed in newborn DZ unlike-sexed twins than in like-sexed twins of either zygosity. Possible explanations for this observation are discussed.

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Generation of Patient-Derived Human Induced Pluripotent Stem Cells in X-Linked Sideroblastic Anemia and Novel Therapeutic Strategies

Blood ◽

10.1182/blood.v130.suppl_1.938.938 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 938-938

Author(s):

Yuki Morimoto ◽

Kazuhisa Chonabayashi ◽

Masayuki Umeda ◽

Hiroshi Kawabata ◽

Akifumi Takaori-Kondo ◽

...

Keyword(s):

X Chromosome ◽

Pluripotent Stem Cells ◽

Molecular Mechanisms ◽

Aminolevulinic Acid ◽

Ips Cells ◽

Erythroid Cells ◽

Wild Type ◽

Sideroblastic Anemia ◽

Induced Pluripotent ◽

In Erythroid Cells

Abstract Sideroblastic anemias consist of a heterogeneous group of inherited and acquired disorders. The most common hereditary type is X-linked sideroblastic anemia (XLSA), which is associated with mutations in the erythroid-specific δ-aminolevulinic acid synthase (ALAS2) gene. Heme synthesis starts with the polymerization of glycine and succinyl CoA polymerization and synthesis of δ-aminolevulinic acid (ALA) in the mitochondria. ALAS2 encodes the enzyme that catalyzes these first steps in the heme synthetic pathway in erythroid cells, steps that require pyridoxal 5'-phosphate (PLP) as a cofactor. It has been found that treatment with PLP is effective for a small fraction of XLSA patients, but there are no effective treatments for the other fraction. The aim of this study is to explore the molecular mechanisms of XLSA and to develop new effective therapies. We used episomal methods to generate induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells (PBMCs) of three late-onset XLSA female patients in one family. The cells harbored the heterozygous mutation (R227C) in the ALAS2 gene. Because ALAS2 is located in the X-chromosome, either wild-type or mutant ALAS2 gene is inactivated in the erythroid cells of female heterozygotes. All three patients showed severe anemia and their PBMCs showed skewed X-chromosome inactivation with preferential inactivation of the X chromosome carrying wild-type ALAS2, indicating a condition associated with unbalanced lyonization. From each patient, we successfully established iPSC lines with the active mutant ALAS2 allele and with the active wild-type ALAS2 allele. We assessed the hematopoietic differentiation potential of these two types of iPSC lines derived from the same patient. Differentiation into hematopoietic progenitor cells (HPCs) using embryoid body formation was comparable in the two groups. However, further differentiation in erythroid culture was significantly impaired in iPSC lines harboring the active mutant ALAS2 allele compared with those harboring the active wild-type ALAS2 allele (CD235a+ cells: 59.20±12.16% with the active wild-type ALAS2 allele vs. 3.95±4.71% with the active mutant ALAS2 allele, p<0.01). Only mutant ALAS2 expression was observed in erythroid cells differentiated from iPS cells harboring the active mutant ALAS2 allele, and only wild-type ALAS2 expression was observed in erythroid cells differentiated from iPS cells harboring the active wild-type ALAS2 allele. Hematopoietic maturation capacity was assessed by performing colony-forming unit (CFU) assays of HPCs (CD34+CD38-CD43+lineage marker-) from iPSC lines derived from the same XLSA patient. Erythroid colony count was significantly less in HPCs from iPSC lines with the active mutant ALAS2 allele, but there was no difference in total colony count between the two types of iPSC lines (erythroid colony numbers: 9.66±10.69 vs. 0±0 per 7,500 HPCs, p<0.01; mixed erythroid colony numbers: 15.00±11.26 vs. 0.66±0.57 per 7,500 HPCs, p<0.01; HPCs with the active wild-type ALAS2 allele vs. HPCs with the active mutant ALAS2 allele). We examined the effect of ALA on the erythroid differentiation of the HPCs. The CD235a-positive erythroid cell ratio of HPCs with the active wild-type ALAS2 allele did not increase following administration of ALA. By contrast, the ratio reached normal levels for HPCs with the active mutant ALAS2 allele (CD235a+ cells: 6.10± 5.61% vs. 85.34± 11.05%, p<0.01; without vs. with administration of ALA). Our data suggest that our iPSC-based system could be useful for studying the precise molecular mechanisms of XLSA and drug testing. Figure Figure. Disclosures Morimoto: Grant-in-Aid for JSPS Research Fellow: Research Funding. Takaori-Kondo: celgene: Honoraria, Research Funding; Bristol-Myers Squibb, Novartis, Janssen pharma, Pfizer: Honoraria.

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Exploring the Potential Usefulness of 5-Aminolevulinic Acid for X-Linked Sideroblastic Anemia

Blood ◽

10.1182/blood.v124.21.215.215 ◽

2014 ◽

Vol 124 (21) ◽

pp. 215-215 ◽

Cited By ~ 2

Author(s):

Tohru Fujiwara ◽

Ryoyu Niikuni ◽

Koji Okamoto ◽

Yoko Okitsu ◽

Noriko Fukuhara ◽

...

Keyword(s):

Aminolevulinic Acid ◽

K562 Cells ◽

Pcr Analysis ◽

Heme Oxygenase 1 ◽

Globin Genes ◽

Erythroid Cells ◽

Rt Pcr ◽

Sideroblastic Anemia ◽

Erythroid Progenitor ◽

In Erythroid Cells

Abstract (Backgroun d) Congenital sideroblastic anemia (CSA) is an inherited disease; it is a microcytic type of anemia characterized by bone marrow sideroblasts with excess iron deposition in the mitochondria. The most common form of CSA is XLSA (X-linked sideroblastic anemia), which is attributed to mutations in the X-linked gene ALAS2 (erythroid-specific 5-aminolevulinate synthase). ALAS2 encodes the first and rate-limiting enzyme involved in heme biosynthesis in erythroid cells, which utilizes glycine and acetyl-coenzyme A to form 5-aminolevulinic acid (ALA) and also requires pyridoxal 5'-phosphate (PLP, vitamin B6) as a cofactor. Based on the evidence that half of the XLSA cases were unresponsive to PLP (Ohba et al. Ann Hematol 2013), ALA supplementation could emerge as an alternative therapeutic strategy to restore heme synthesis in CSA caused by ALAS2 defects. As a preclinical study, we focused our study on the effect of ALA on human erythroid cells. Furthermore, we investigated the molecular mechanism by which ALA is transported into erythroid cells. (Method ) Human K562 erythroid cells as well as human induced pluripotent stem-derived erythroid progenitor (HiDEP) cells (Kurita et al. PLoS ONE 2013) were used for the analysis. We investigated the effects of ALA (0.01, 0.1, and 0.5 mM for 72 h) on heme content, hemoglobinization, and erythroid-related gene expression. Heme content was determined fluorometrically at 400 nm (excitation) and 662 nm (emission). Small interfering RNA (siRNA)-mediated knockdown of ALAS2 was conducted using Amaxa Nucleofector™ (Amaxa Biosystems, Koln, Germany). For transcription profiling, Human Oligo chip 25K (Toray, Tokyo, Japan) was used for control and ALAS2 siRNA-treated HiDEP cells. Gamma-aminobutyric acid (GABA) (Sigma, St. Louis, MO, USA) was used at concentrations of 10 and 20 mM. (Results) First, we demonstrated that ALA treatment resulted in significant dose-dependent accumulation of heme in K562 cells. Concomitantly, the treatment substantially induces erythroid differentiation as assessed using hemoglobin (benzidine) staining. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis confirmed significant upregulation of heme-regulated genes such as the globin genes (HBA and HBG) and the heme oxygenase 1 (HMOX1) gene in K562 cells. To investigate the mechanism by which ALA was transported into erythroid cells, we conducted quantitative RT-PCR analysis for previously reported ALA transporters, including SLC15A1, SLC15A2, SLC36A1, and SLC6A13 (Frølund et al. Br J Pharmacol 2010; Ahlin et al. Drug Metab Dispos 2009; Moretti et al. Br J Cancer 2002). The analysis revealed that SLC36A1 was abundantly expressed in K562 and HiDEP cells. Thus, GABA was added to K562 cells to competitively inhibit SLC36A1-mediated transport (Frølund et al. Br J Pharmacol 2010). GABA treatment significantly impeded the ALA-mediated increase in the number of hemoglobinized cells. Next, siRNA-mediated knockdown of ALAS2 in HiDEP cells resulted in a significant decrease in the expression of globin genes as well as HMOX1; however, ringed sideroblasts were not observed. Microarray analysis revealed >2-fold up- and down-regulation of 38 and 68 genes caused by ALAS2 knockdown, respectively. The downregulated gene ensemble included globins (HBZ, HBG, HBE, HBD, and HBM) as well as genes involved in iron metabolism (ferritin heavy chain 1: FTH1, transferrin receptor: TFRC and glutaredoxin-1: GLRX5). Gene ontology analysis revealed significant enrichment of cellular iron ion homeostasis (p = 0.000076), cell division (p = 0.00062), DNA repair (p = 0.0006) and translation (p = 0.018), implying that heme was involved in various biological processes in erythroid cells. Interestingly, ALA treatment significantly improved the consequences of ALAS2 knockdown-mediated downregulation of HBA, HBG, and HMOX1. (Conclusion) ALA appears to enter into erythroid cells mainly by SLC36A1 and utilized to generate heme precursor. Thus,ALA may represent a novel therapeutic option for CSA, particularly for cases harboring ALAS2 mutations. Disclosures Fujiwara: Chugai Pharmaceutical, CO., LTD.: Research Funding.

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Biological Significance of the Different Erythropoietic Factors Secreted by Normal Human Early Erythroid Cells

Leukemia & Lymphoma ◽

10.1080/1042819021000046967 ◽

2003 ◽

Vol 44 (5) ◽

pp. 767-774 ◽

Cited By ~ 9

Author(s):

Janina Ratajczak ◽

Jacek Kijowski ◽

Marcin Majka ◽

Kacper Jankowski ◽

Ryan Reca ◽

...

Keyword(s):

Biological Significance ◽

Erythroid Cells ◽

Normal Human

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Mitochondrial Ferritin Expression and Clonality of Hematopoiesis in Patients with Refractory Anemia with Ringed Sideroblasts.

Blood ◽

10.1182/blood.v106.11.3444.3444 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3444-3444 ◽

Cited By ~ 7

Author(s):

Matteo G. Della Porta ◽

Luca Malcovati ◽

Anna Galli ◽

Sabrina Boggi ◽

Erica Travaglino ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Early Stage ◽

Refractory Anemia ◽

Specific Marker ◽

Erythroid Cells ◽

Prognostic Information ◽

Sideroblastic Anemia ◽

Erythroid Progenitor ◽

Ringed Sideroblasts

Abstract Sideroblastic anemias are a heterogeneous group of disorders that have in common the presence of erythroblasts with iron-loaded mitochondria defined as ringed sideroblasts. We have previously demonstrated that mitochondrial iron deposition in these disorders is in the form of mitochondrial ferritin (MtF), suggesting that this latter may be a specific marker of sideroblastic anemia (Cazzola et al, Blood2003;101:1996–2000). The most common type of acquired sideroblastic anemia is the myelodysplastic syndrome (MDS) defined as refractory anemia with ringed sideroblasts, which is generally associated with a relatively benign clinical course. In the present work, we studied the relationship between MtF expression and clonality of hematopoiesis in 55 consecutive female patients with low-risk MDS, including 20 cases with ringed sideroblasts and 35 cases without ringed sideroblasts. The expression of MtF, as well as that of cytosolic ferritin (H and L subunits) and of transferrin receptor (CD71), was evaluated by flow cytometry in bone marrow erythroid cells; in selected cases, these immunophenotypic investigations were also performed on liquid cultures of purified CD34-positive cells. X-chromosome inactivation patterns (XCIPs) were assessed in peripheral blood granulocytes and in bone marrow CD34-positive cells by analysis of both DNA methylation at the HUMARA and PGK loci and of IDS gene expression. Within informative females, 11 out of 12 patients with ringed sideroblasts displayed clonal XCIPs in granulocytes; by contrast, only 9 out of 22 patients without ringed sideroblasts displayed clonal XCIPs. Purified CD34-positive cells showed clonal XCIPs in 6 out of 7 patients with ringed sideroblasts but had polyclonal XCIPs in 4 out of 5 individuals without ringed sideroblasts. Flow cytometry evaluation of bone marrow erythroid cells showed that MtF expression was restricted to MDS patients with ringed sideroblasts. A close positive relationship was found between MtF and CD71 expression (r=.49, P=.001); this association may reflect the cytosolic iron deprivation induced by MtF overexpression. Analysis of cultured erythroid progenitor cells showed that MtF was detectable at a very early stage of differentiation from CD34-positive cells. Addition of erythropoietin to the culture system sustained the appearance of a polyclonal erythroid population in 2 out of 4 patients with ringed sideroblasts and clonal CD34-positive cells. These observations suggest that refractory anemia with ringed sideroblasts is a truly clonal stem cell disorder, while more than 50% of patients with refractory anemia without ringed sideroblasts have evidence of polyclonal hematopoiesis. The clonal pattern of CD34-positive cells and the early appearance of MtF during erythroid differentiation suggest that - despite be benign natural history of refractory anemia with ringed sideroblasts - the initial pathogenetic event in this condition occurs in multipotent stem cells. Although the mechanisms responsible for overexpression of MtF are still unclear, flow cytometry evaluation of this protein is a useful diagnostic tool that also provides helpful prognostic information.

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