Inhibition Of Hemoglobin Synthesis Research Articles

Interleukin-6 directly impairs the erythroid development of human TF-1 erythroleukemic cells.

Anemia of inflammation or chronic disease is a highly prevalent form of anemia. The inflammatory cytokine interleukin-6 (IL-6) negatively correlates with hemoglobin concentration in many disease states. The IL-6-hepcidin antimicrobial peptide axis promotes iron-restricted anemia; however the full role of IL-6 in anemia of inflammation is not well-defined. We previously reported that chronic inflammation had a negative impact on maturation of erythroid progenitors in a mouse model. We hypothesized that IL-6 may be responsible for impaired erythropoiesis, independent of iron restriction. To test the hypothesis we utilized the human erythroleukemia TF-1 cell line to model erythroid maturation and exposed them to varying doses of IL-6 over six days. At 10 ng/ml, IL-6 significantly repressed erythropoietin-dependent TF-1 erythroid maturation. While IL-6 did not decrease the expression of genes associated with hemoglobin synthesis, we observed impaired hemoglobin synthesis as demonstrated by decreased benzidine staining. We also observed that IL-6 down regulated expression of the gene SLC4a1 which is expressed late in erythropoiesis. Those findings suggested that IL-6-dependent inhibition of hemoglobin synthesis might occur. We investigated the impact of IL-6 on mitochondria. IL-6 decreased the mitochondrial membrane potential at all treatment doses, and significantly decreased mitochondrial mass at the highest dose. Our studies indicate that IL-6 may impair mitochondrial function in maturing erythroid cells resulting in impaired hemoglobin production and erythroid maturation. Our findings may indicate a novel pathway of action for IL-6 in the anemia of inflammation, and draw attention to the potential for new therapeutic targets that affect late erythroid development.

MIR9 That Is Aberrantly Expressed in Myelodysplastic Syndrome Blocks Erythroid Differentiation Possibly Through STAT-5-Mediated Signals

Abstract 3842microRNA (miRNA) is a class of 18–24 nucleotides small non-coding RNAs that induce mRNA degradation and negatively modulate protein translation, and plays vital roles in physiological and pathological hematopoiesis. On the other hand, expressional alterations of miRNA are postulated to result in the development of hematological malignancies including myelodysplastic syndrome. RUNX1 belongs to a member of transcription factor that is essential in the development of fetal liver hematopoiesis and is critical in the regulation of adult hematopoiesis. In myelodysplastic syndrome, dysregulated functions of RUNX1 through formation of chimeric genes or point mutations are one of the major causes of their development. We speculate that aberrant expression of miRNA that negatively regulates RUNX1 synthesis could constitute novel mechanism by which RUNX1 is abrogated. To seek for the possibility, we selected 10 microRNAs (MIR27A, MIR27B, MIR9, MIR199A, MIR18A, MIR30A, MIR30B, MIR30C, MIR30D and MIR30E) among RUNX1-related miRNAs whose seed sequences reside within the 3′ UTR of human RUNX1 mRNA as analytical objects. Investigated were 16 MDS patients (RA, 4 cases; RAEB 9, cases; RAEB-t, 2 cases; CMMoL, 1 case), 11 normal controls in our hospital and 86 MDS patients registered to MDS cell bank (RA, 23 cases; RAEB 19, cases; RAEB-t, 18 cases; CMMoL, 3 cases; MDS-derived AML, 23 cases) As a result, as for all RUNX1-related miRNAs, a tendency for higher expression levels of miRNAs in MDS samples compared to control bone marrow samples was observed. Among them, MIR9 was highly expressed in the bone marrow cells of 3 MDS cases including 1 RA case and 2 RAEB cases (approximately 19%), while barely detected in other MDS samples or controls in our institute. MIR9 was also highly expressed in 5 cases of MDS registered in cell bank including 1 RA case, 1 RAEB case, 1 RAEB-t case and 2 MDS-derived AML cases (5.8%). At first, to confirm that MIR9 actually suppresses transcription of RUNX1, we constructed reporter plasmids containing luciferase gene followed by various length of 3′ UTR of RUNX1 mRNA. As a result of reporter assay, overexpression of MIR9 decreased lucifearse activities to approximately 50 % when reporter plasmids containing miR-9 binding site in 3′ UTR of RUNX1 mRNA (2219- and 2260-) were co-transfected in 293 cells. However, when reporter plasmids without MIR9 binding site (2329-, 2411- and 3245-) were co-transfected, overexpression of MIR9 didn’t suppress luciferase activities. Next, we established human hematopoietic cell line, UT-7/GM overexpressing MIR9. Evaluation by counting percentages of benzidine-positive cells, inhibition of hemoglobin synthesis by EPO treatment was observed in MIR9-overexpressing clones. Further, as a result of quantitative real-time PCR and FACS analyses, inhibition of erythroid specific gene (ALAS-E) induction and surface antigen (Glycopholin-A) expression were also observed in those clones. Finally, we analyzed protein expression levels of RUNX1 and several other intracellular signaling molecules that contribute to erythroid differentiation. There is no difference in RUNX1 protein level between mock and MIR9-overexpressing clones. Interestingly, phosphorylation of STAT-5 after EPO treatment in MIR9-overepressing clones was weaker in comparison with mock clones. All these data suggest the possibility that MIR9 functions as a negative regulator of erythroid differentiation possibly through inhibiting STAT-5 phosphorylation and that aberrant expression of MIR9 could at least partly contribute to ineffective erythropoiesis in MDS. Although, we further analyzed protein levels of JAK2 and SH-PTP2 to clarify the molecular basis of inhibition of STAT-5 phosphorylation, there was no obvious difference in expression of JAK2 or SH-PTP2 between mock and MIR9-overexpressing clones. Therefore, MIR9 must influence expression/function of unknown molecule(s) and thus may contribute to pathogenesis of MDS. Disclosures:No relevant conflicts of interest to declare.

Impact of developmental lead exposure on splenic factors

Lead (Pb) is known to alter the functions of numerous organ systems, including the hematopoietic and immune systems. Pb can induce anemia and can lower host resistance to bacterial and viral infections. The anemia is due to Pb's inhibition of hemoglobin synthesis and Pb's induction of membrane changes, leading to early erythrocyte senescence. Pb also increases B-cell activation/proliferation and skews T-cell help (Th) toward Th2 subset generation. The specific mechanisms for many of the Pb effects are, as yet, not completely understood. Therefore, we performed gene expression analysis, via microarray, on RNA from the spleens of developmentally Pb-exposed mice, in order to gain further insight into these Pb effects. Splenic RNA microarray analysis indicated strong up-regulation of genes coding for proteolytic enzymes, lipases, amylase, and RNaseA. The data also showed that Pb affected the expression of many genes associated with innate immunity. Analysis of the microarray results via GeneSifter software indicated that Pb increased apoptosis, B-cell differentiation, and Th2 development. Direct up-regulation by Pb of expression of the gene encoding the heme-regulated inhibitor (HRI) suggested that Pb can decrease erythropoiesis by blocking globin mRNA translation. Pb's high elevation of digestive/catabolizing enzymes could generate immunogenic self peptides. With Pb's potential to induce new self-peptides and to enhance the expression of caspases, cytokines, and other immunomodulators, further evaluation of Pb's involvement in autoimmune phenomena, especially Th2-mediated autoantibody production, and alteration of organ system activities is warranted.

Bcl-XLis required for heme synthesis during the chemical induction of erythroid differentiation of murine erythroleukemia cells independently of its antiapoptotic function

Bcl-X(L) is essential for the survival and normal maturation of erythroid cells, especially at the late stage of erythroid differentiation. It remains unclear whether Bcl-X(L) serves only as a survival factor for erythroid cells or if it can induce a signal for differentiation. We have previously shown that dimethyl sulfoxide (DMSO) induction of erythroid differentiation in murine erythroleukemia (MEL) cells correlates with delay of apoptosis and specific induction of Bcl-X(L). In this study, we investigate the contribution of Bcl-2 and Bcl-X(L) to survival and erythroid differentiation by generating stable MEL transfectants expressing these antiapoptotic regulators. Overexpression of Bcl-2 completely prevented apoptosis of MEL cells before and after DMSO induction, whereas overexpression of Bcl-X(L) only delayed it. Overexpression of Bcl-2 or Bcl-X(L) neither induced spontaneous erythroid differentiation nor accelerated DMSO-induced differentiation. Inhibition of Bcl-X(L) by antisense transcripts accelerated apoptosis in DMSO-treated MEL cells and blocked the synthesis of hemoglobin without altering the growth arrest associated with terminal erythroid differentiation. An antisense oligonucleotide to Bcl-X(L) did not induce apoptosis in MEL cells overexpressing Bcl-2 but greatly decreased their hemoglobin synthesis when treated with DMSO, suggesting that Bcl-X(L) is necessary for erythroid differentiation independently of its antiapoptotic function. Importantly, Bcl-X(L) antisense transcripts prevented heme synthesis but not globin mRNA induction in DMSO-treated MEL cells. Furthermore, inhibition of hemoglobin synthesis by Bcl-X(L) antisense was reversed by addition of exogenous hemin. Finally, Bcl-X(L) localized to mitochondria during MEL erythroid differentiation, suggesting that it may mediate a critical mitochondrial transport function related to heme biosynthesis.

Effects of Lead on Electrophoretic Mobility, Membrane Sialic Acid, Deformability and Survival of Rat Erythrocytes.

The anemia frequently observed in lead poisoning is thought to result from the shortening of erythrocyte life span in combination with inhibition of hemoglobin synthesis. However, the exact mechanism by which lead shortens the life span of red blood cells (RBCs) remains unclear. In the present study, the effects of injected lead on electrophoretic mobility, membrane sialic acid content, deformability and survival of rat RBCs were investigated in order to clarify the relationships between them. As indices of lead exposure, RBC counts, hemoglobin (Hb) levels, hematocrits (Ht), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and blood lead (blood Pb) levels in the rats were also examined. Exposure to lead significantly decreased RBC counts, Hb levels, Ht, MCV and MCH. Similarly, exposure to lead significantly decreased the mobility, sialic acid content and deformability of rat RBCs. A shortening of erythrocyte survival time was also observed in the rats exposed to lead. It is speculated that decreases in membrane sialic acid content and deformability of RBCs induce a shortening of erythrocyte survival time in anemia caused by lead.

Effects of lead on sialic acid content and survival of rat erythrocytes

The anemia frequently observed in lead poisoning is thought to result from a shortening of erythrocyte survival in combination with inhibition of hemoglobin synthesis. However, the exact mechanism by which lead shortens erythrocyte survival remains unclear. In the present study, the effects of lead, injected intraperitoneally, on sialic acid content and survival of rat erythrocytes were investigated in order to study the relationship between them. As indices of lead exposure, hemoglobin (Hb) levels, hematocrits (Ht) and blood lead (blood Pb) levels in the injected rats were also examined. Exposure to lead significantly decreased the sialic acid content of the erythrocyte membrane. The decreases in sialic acid content were evident to some extent below a blood Pb level of 100 μg/100 ml and generally present at a level of 100 μg/100 ml and higher. In the rats exposed to lead a significant negative correlation was found between sialic acid content and the logarithm of blood Pb level. A shortening of erythrocyte survival was also observed in the rats exposed to lead.

Inhibitors of protein glycosylation inhibit the differentiation of Friend erythroleukemia cells

Tunicamycin, 2-deoxy- d-glucose and 2-deoxy-2-fluoro- d-glucose inhibit dimethyl sulfoxide-induced differentiation of Friend cells. This inhibition, characterized by inhibition of hemoglobin synthesis, is accompanied by a specific inhibition of protein glycosylation. The results of cloning experiments indicate that this inhibition specifically affects cells in the period preceding their commitment. These results suggest that glycoprotein synthesis is a requirement for Friend erythroleukemia cells in order to initiate the expression of the terminal differentiation program.

The dependence of erythroid differentiation on cell replication in dimethyl sulfoxide-treated friend leukemia-virus-infected cells

The dimethyl sulfoxide (Me 2SO)-mediated induction of hemoglobin synthesis in Friend leukemia cells (a murine erythroblastoid cell line) is coupled with the number of cell replications occurring in the presence of inducer. Varying concentrations of proflavine increase the generation time of these cells from 24 hours to over 50 hours, and in each case the induction of hemoglobin synthesis follows the completion of two cell doublings. Once the induction is initiated, the rate of hemoglobin accumulation is not affected by proflavine. These data indicate that proflavine does not affect the transcription or translation of globin mRNA and that the delay in induction of hemoglobin synthesis is due to its effect on the rate of cellular replication. In experiments using high concentrations of thymidine to block replication, hemoglobin accumulation is prevented only if the cells are blocked prior to 36 hours after Me 2SO addition. If the cells have completed two generations in the presence of Me 2SO, there is no effect upon their ability to synthesize hemoglobin even though their growth is arrested. Thus, the inhibition of hemoglobin synthesis by proflavine is not merely the result of a toxic effect on newly subcultured cells but is due to its effect on cellular replication. These experiments confirm that, after addition of Me 2SO, Friend leukemia cells require more than one complete cell cycle in order to synthesize hemoglobin.

Inhibition of hemoglobin synthesis by pancreatic deoxyribonuclease in rabbit reticulocyte lysates

A pancreatic deoxyribonuclease preparation, shown to be free of significant ribonuclease activity, inhibits hemoglobin synthesis in a rabbit reticulocyte lysate. Preincubation with DNAase (15 min at 25°C) is required to obtain a marked inhibitory effect (nearly 70%). It has been shown that DNAase does not significantly interfere with the elongation or termination steps of translation, but it seems to prevent reinitiation of globin polypeptide chains allowing polysome run-off. In particular, the formation of the 40 S met-tRNA met f complex is greatly reduced in the DNAase-treated lysate. At the moment the mechanism by which DNAase inhibits initiation is not clear.

Inhibition of Hemoglobin Synthesis by Cyanate In Vitro

Abstract Cyanate inhibits sickling and prolongs red cell survival in sickle cell anemia. However, cyanate markedly inhibits hemoglobin synthesis in vitro. Incorporation of radioactive amino acid into hemoglobin by human sickle reticulocytes or bone marrow and by rabbit reticulocytes (whole cell or cell-free lysate) was inhibited by as little as 2 mM cyanate and abolished by 50 mM. Both alpha- and beta-S chains were equally affected. The inhibition was only partially reversible by washing the cells after exposure to cyanate. Transport of radioactive amino acid into the cell was not impaired, and free intracellular amino acid was not carbamylated. Aminoacylation of transfer RNA was not inhibited; the acylated amino acid was not carbamylated. Examination of polysome patterns by sucrose density gradient centrifugation revealed degradation of polysomes to monosomes, suggesting inhibition of initiation of protein synthesis by cyanate. In a cell-free lysate, cyanate prevented hemin stimulation of initiation. We conclude that cyanate profoundly inhibits initiation of hemoglobin synthesis in vitro.