Globin Synthesis Research Articles

Oxidative Stress Signaling Pathway of Heme Regulated eIF2α Kinase in mitigating the Severity of β-Thalassemia

Maturation of erythroid precursors requires active synthesis of hemoglobin which consists of two pairs of α- and β-globin subunits with each monomer bound to a heme moiety. Heme Regulated Inhibitor (HRI) is the only eIF2αkinase responsible for the balanced synthesis of heme and globin at translational level in erythroid cells. Activation of HRI in heme deficiency leads to phosphorylation of the α-subunit of eukaryotic initiation factor (eIF2α) and inhibition of protein synthesis. HRI is also activated by denatured proteins and oxidative stress. In addition to general inhibition of protein synthesis, phosphorylation of eIF2α (eIF2αP) also leads to the induction of a stress signaling pathway. Activating transcription factor 4 (Atf4) mRNA is preferentially translated amidst global inhibition of protein synthesis. Atf4 activates transcription of stress response proteins, Chop (CCAAT/enhancer binding protein homologous protein-10) and the non-enzymatic cofactor of eIF2α phosphatase (PP1A) Gadd34. These stress response proteins help cells in mitigating the stress. While the role of HRI in translational regulation of non-nucleated reticulocytes is well established, the HRIdependent Atf4 stress signaling pathway of nucleated erythroid precursors is unknown.Sodium arsenite toxicity was used as a model system of oxidative stress to elucidate the HRI signaling pathway in Hri +/+ and −/− E14.5 mouse fetal liver erythroid precursors. In HRI deficiency, erythroid precursors were more sensitive to arsenite toxicity with decreased cell viability and increased apoptosis, by caspase 3 executed intrinsic apoptotic pathway. HRI was activated by autophosphorylation as early as 15 minutes following arsenite treatment. In addition to increased eIF2αP, there was induction of Atf4, Chop and Gadd34 in Hri+/+ fetal liver cells. Importantly, in Hri−/− cells neither the phosphorylation of eIF2α nor the expression of Atf4, Chop and Gadd34 was increased upon arsenite treatment. In addition, we also observed HRI dependent induction of Heme Oxygenase 1 (HO-1) that plays a pivotal role in adaptation to oxidative stress. These results demonstrate that HRI induces a signaling pathway for adaptive gene expression to protect the nucleated erythroid precursors from apoptosis upon oxidative stress.Iron overload, accumulation of unpaired α-globin and oxidative stress are well documented in β-thalassemia. Recently, HRI was discovered to be necessary for the survival of β-thalassemic mice. β-thalassemic mice lacking one copy of HRI (Hri+/− Hbb−/−) also manifest a more severe syndrome of the disease. We have investigated the activation of eIF2αP/Atf4 signaling pathway in Hri+/−Hbb−/− β-thalassemic erythroid cells using eIF2αP phosphatase (Gadd34) inhibitor salubrinal. Treatment of reticulocytes from Hri+/−Hbb−/− mice with salubrinal increased eIF2αP and resulted in inhibition of newly synthesized globin protein synthesis. The decreased globin protein synthesis also resulted in decreased aggregation of the unpaired α-globins. Furthermore, treatment of salubrinal in nucleated fetal liver erythroblasts also increased Chop expression and decreased apoptosis. Thus, activation of the eIF2αP/Atf4 pathway by small chemicals might be a novel pharmaceutical approach to decrease proteotoxicity and apoptosis for the treatment of β-thalassemia.

The Role of Heme Oxygenase 1 in Erythroid Differentiation.

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.

Hemin Augments Growth and Hemoglobinization of Erythroid Precursors Derived from Patients with Diamond-Blackfan Anemia (DBA).

DBA is a congenital form of pure red cell anemia characterized by a macrocytic anemia, reticulocytopenia, and a block in erythroid differentiation at the proerythroblast stage, often in association with physical anomalies and growth retardation. About 25% of the patients carry mutations in genes that encode for proteins (RPS19, RPS24 and RPS17) that bind to the 40S subunit of the ribosome. The resultant defect in ribosomal biogenesis has been proposed to impair the initiation of globin translation, leading to mismatch between intracellular levels of heme and globin chains. It has been hypothesized that the transient excess of intracellular free heme resulting from the delay in globin synthesis exerts direct toxicity to erythroid precursors and plays a major role in pathogenesis of DBA through apoptosis of proerythroblasts (Keel et al., Science319;825,2008).Free hemin, however, is not necessarily toxic to developing erythroid precursors. Exogenously supplied hemin is readily taken up by erythroid cells in culture and its iron is incorporated into hemoglobin or stored in ferritin (Fibach et al., J Cell Physiol130;460,1987). Following addition of succinylacetone, a potent inhibitor of heme synthesis, exogenously supplied hemin can replace intracellularly synthesized heme and be incorporated into de novo formed hemoglobin (Fibach et al., Blood85;2967,1995). Hemin supplementation to semi-solid cultures promotes the growth of normal erythroid precursors (e.g., Lu and Broxmeyer, Exp Hematol11;721,1983). We showed in a two-phase liquid culture that exogenous hemin promotes normal erythropoiesis by accelerating the proliferation and hemoglobinization of erythroid precursors in the presence or absence of transferrin (Fibach et al., Blood85;2967,1995). This effect was particularly prominent during the early stages of hemoglobinization, when iron-uptake and heme synthesis are rate-limiting. In the present study we show that surplus hemin (10 - 50 mM) supplemented to cultures at early stage of erythroid development is well tolerated. Although the generation of reactive oxygen species (measured by staining with dichlorofluorescein diacetate) was modestly (50 ± 15%, N=4) increased, it was not associated with increased apoptosis, as measured by binding of annexin V, nor necrosis as measured by propidium iodide staining.Having demonstrated the growth and differentiation promoting potential of exogenous hemin on normal erythroid precursors and lack of overt toxicity, we studied the effect of exogenous heme in cultures of erythroid cells derived from six patients with DBA. We show that hemin, added as heme chloride or heme arginate, circumvented the primary defect and significantly stimulated (4 - 20-fold, p<0.001) ) the growth of DBA erythroid cells and their hemoglobinization.In conclusion, our results show thatexogenous hemin is taken up by developing erythroid cells and can supplement or substitute endogenously synthesized heme;excess heme stimulates free radical generation moderately but does not cause apoptosis or necrosis;addition of hemin to cultured erythroid precursors derived from normal donors stimulates their growth and hemoglobinization, and in DBA, in contrast to the recently proposed scheme, heme can actually restore the growth and differentiation potential of the DBA-erythroid precursors.The beneficial effect of hemin on DBA erythroid precursors may be related to its effect on translation initiation factors, such as eIF-2 , and suggests a therapeutic potential.

Globin-methionine complexes formed during labelling studies.

In implementing globin synthetic measurements using our recently described technique of Cellogel separation of 35S-methionine-labelled globin chain we sometimes encountered aberrant results. We discovered the presence of prominent radioactive bands trailing both the alpha and beta globin chains. Such bands were not present when 3H-leucine served as a label. These bands were not due to contaminants in the 35S-methionine. Their formation could be prevented by including in the incubating mixture 1 mM cystine, cysteine or homocysteine. The bands could not be removed by trichloracetic acid or acid acetone precipitation but did disappear almost entirely after prolonged dialysis against a urea-containing buffer. The trailing bands appear to represent the result of the firm, but not covalent, binding of methionine to globin during the protein synthetic process. Since methionine is commonly used as a label for newly synthesized protein, this phenomenon may be important not only in the investigation of globin synthesis, but also in the study of the synthesis of other proteins.

Thalassemia and hypercoagulability

Thalassemia is a congenital hemolytic disease caused by defective globin synthesis resulting in decreased quantity of globin chains. Although the life expectancy of beta-thalassemia patients has markedly improved over the last few years, patients still suffer from many complications of this congenital disease. The presence of a high incidence of thromboembolic events, mainly in beta-thalassemia intermedia, has led to the identification of a hypercoagulable state in these patients. In this paper, we review the molecular and cellular mechanisms leading to hypercoagulability in beta-thalassemia, with a special focus on thalassemia intermedia being the group with the highest incidence of thrombotic events as compared to other types of thalassemias. We also discuss the recommendations for thrombosis prophylaxis in these patients.

AZT: An Old Drug with New Perspectives

The science of antiviral research was well advanced when HIV/AIDS appeared as a major new virus disease in the early 1980s. The first effective antiviral compound (AZT, azidothymidine, zidovudine) was already among the library of compounds screened and was promptly reported to be a specific inhibitor of retroviruses, including HIV. Due to the pivotal role of AZT in HIV treatment, this review summarizes the most known effects -some of which are toxic side effects- induced by AZT a drug which is still used in the combined therapy of HIV-infected patients. Among the toxic side effects, a severe bone marrow toxicity manifested as anemia, neutropenia and siderosis, and caused by inhibition of heme and globin synthesis together with a general derangement of iron supply, have been reported. In this regard, we proved that while AZT and its monophosphorylated derivative AZTMP were unable to chelate iron, the triphosphate form AZTTP displayed a significant capacity to remove iron from transferrin. Moreover, we have previously demonstrated that AZT-exposed K562 cells showed an increase of transferrin receptors located on the cell membrane without affecting their biosynthesis, but slowing down their endocytotic pathway. Interestingly, literature data report the impairement of glycosylation reactions by AZT. Indeed, we have shown that AZT-treated K562 cells exhibited a reduced sialylation of proteins and lipids, and a strong inhibition of alpha,(2-->8) sialyltransferase activity while beta,(1-->4)galactosyltransferase and beta-galactosidase activities were significantly increased. These latter observations could be of clinical relevance since alterations of intracellular and cell surface carbohydrate expression and composition, often are associated with several diseases. However, contrarily to previous reports by other authors on AZT as an inhibitor of plant and bacterial toxins activity, we have demonstrated that AZT not only did not inhibit saporin toxicity, but even increased the cytotoxic activity of this plant toxin on K562 cells. Furthermore, the review enlightens the potential utilization of AZT as a tool in proteomics since in the recent years several genes responding to this drug have been identified in different cell lines. We have shown, for the first time, an over-expression of two proteins (PDI-A3 and sthatmin), and a full repression of two others (HSP-60 and SOD1) in AZT-exposed K562 cells. At present, we are investigating if the above reported alterations are a general feature of AZT-treatment of cultured cells, or they represent a peculiar characteristic of a specific cell line. Finally, the paper reviews a number of novel methodologies aimed at enhancing the AZT plasma levels and its bioavailability in all human organs in order to improve its therapeutic efficacy against HIV infection. These new possibilities, namely the AZT prodrug strategy, the AZT transdermal delivery and the targeted brain delivery, are yet not in use for humans but they are under experimental studies.

Study of Expression of FLVCR in Childhood Thalassemic Patients Demonstrate the Importance of FLVCR in Effective Erythropoiesis.

Feline Leukemia Virus, Subgroup C (FLVCR) is a newly found cytoplasmic heme exporter. Retroviral interference with FLVCR display results in a loss of erythroid cells after the CFU-E/proerythroblast stage of differentiation and severe anemia in cats. We studied expression of FLVCR in thalassemia with relatively redundant free cytoplasmic heme, evaluated the correlation of FLVCR expression with clinical parameters, and explore the importance of FLVCR for erythropoiesis in human cells. Samples of bone marrow from 50 children with thalassemia (n=21), other hemolytic anemia(AIHA, G6PD Deficiency, n=17) and non-anemia controls (n=12) were collected. Expression of FLVCR protein and mRNA in mononuclear red cells was quantitated in CFU-E/proerythroblasts (CD71 bright, CD117+) and later erythroid precursors (CD71 bright, CD117 negative cells) by 3 color flow cytometry and fluorescence quantitative Real-time PCR. Expression of FLVCR is higher in early precursors than later erythroid cells: Specifically, the percentage of specimens with high levels of FLVCR protein expression in proerythroblasts and the mean fluorescence intensities(MFI) of thalassemia, other hemolytic anemia and non-anemias were 78.9%, 84.2%,74.85% and 306,347, 291.5 respectively; while these values in later erythroid precursors were 8.8%,10.6%,6.9% and 150,122,119 respectively, P<0.01. Expression of FLVCR tended to be higher in proerythroblasts of thalassemia and other hemolytic anemia:Positive rate and MFI of FLVCR protein expression in thess two groups were 78.9%,306 and 84.2%,347 respectively, as compared with cells of non-anemias (74.85%,291.5), with no statistical differences, P>0.05. Expression of FLVCR is significantly higher in later erythroid precursors of thalassemia:Positive rate and MFI of FLVCR protein expression in later erythroid precursors of thalassemia were 8.8%, and 150 respectively, with significantly higher levels of expression compared with non-anemias (6.9%,119) P<0.05. Expression of FLVCR mRNA in proerythroblasts of thalassemia, other hemolytic anemia and non-anemias were 3.6,4.85,3.7 respectively; while in later erythroblast were 4.13,6.06,3.92 respectively, P<0.01. Hemolytic anemia patients with higher expression of FLVCR had higher reticulocyte count, higher percentage of later erythroblast and lower LDH, P<0.05. Thalassemic patients with higher expression of FLVCR had higher percentage of sideroblasts and serum ferritin, P<0.05. Expression of FLVCR protein was remarkably upregulated in proerythroblasts, combined with previous studies in vitro and identification of FLVCR as a human heme exporter, supporting FLVCR plays an important role in developing erythroid cells to protect them from heme toxicity. There was no significantly up-regulation of FLVCR expression in proerythroblasts of thalassemia, indicating probably other pathways exist in regulating heme metabolic balance combined with FLVCR; Expression of FLVCR in later erythroid precursors of thalassemia was higher than that of non-anemias, which implies FLVCR up-regulates with compensation, this may help counteract the ineffectiveness of late erythropoiesis that results from the poor coordination of heme and globin synthesis. The disparity between FLVCR protein and mRNA may reflect posttranscriptional regulation of protein level. Clinical results also support effect of FLVCR on erythropoiesis. FLVCR expression has close relation with iron metabolic parameters.

A Humanized Cooley's Anemia Mouse Model.

Cooley's Anemia (CA) is a heterogeneous group of inherited disorders all marked by the complete absence of adult β-globin chains in red blood cells (RBCs). Newborns with CA are healthy because of the high level of fetal hemoglobin (HbF) present at birth. As HbF levels decline during the first year of life, the absence of functional adult β-globin genes results in a severe anemia that necessitates the initiation of regular blood transfusions for the remainder of life. CA has been difficult to study in murine models due to the lack of a human fetal hemoglobin equivalent in the mouse. This study reports a novel preclinical animal model of CA that survives solely on human fetal hemoglobin at birth and is blood transfusion dependent for life upon completion of the hemoglobin switch after birth. These humanized CA mice were generated by targeted gene replacement in embryonic stem cells of the adult mouse α-globin genes with human α-globin and the adult mouse β-globin genes with a delayed switching γδβ0-globin gene cassette. The nonfunctional human β0-globin knock-in allele contains a single G to A nucleotide mutation in the first base of intervening sequence 1. Both wild-type and hereditary persistence of fetal hemoglobin (HPFH) promoter mutations were tested in the human γ-globin knock-in allele. Heterozygous knock-in mice exhibit β thalassemia intermedia. Newborn homozygous knock-in mice express 100% human hemoglobin in their RBCs, suffer from ineffective erythropoiesis, survive from one to ten days after birth, and are blood transfusion dependent for adult life. This is the first CA mouse model that recapitulates the temporal onset of the disease in human patients. This CA disease model is useful for the study of the regulation of globin gene expression, synthesis, and switching; development of transfusion and iron chelation therapies; induction of fetal hemoglobin synthesis; and the testing of novel genetic and cell-based therapies for the correction of thalassemia.

Normal Initial Response to Erythropoietin but Impaired Proliferation in 2-Phase Erythroid Culture in Diamond Blackfan Anemia.

The clinical phenotype of RPS19-mutated and non-mutated DBA is indistinguishable, suggesting a common function for all DBA genes. The recent identification of mutations affecting other ribosomal protein genes in DBA provides powerful support for this common function being ribosomal biogenesis. However, the molecular basis for the characteristic steroid responsive red cell aplasia of DBA remains elusive. Using a 2-phase culture system, in which the phases of erythropoietin dependence are separated, we have demonstrated a consistent and profound deficiency in EPO/SCF-induced in vitro expansion of peripheral blood erythroid progenitor cells in DBA. To further localise the defect in DBA we have studied early cell cycle, proliferation and transcriptional events of enriched erythroid cells on transfer to phase II. Phase II cultures were established in 10μM BrdU for bivariate analysis of BrdU uptake and cell cycle status by FACS. Propidium iodide staining showed a reduction of cells in G0/G1 from 67.09±4.5% at t=0 to 58.44±3.8% at 24hrs post EPO in control cultures (n=6). Cumulative BrdU uptake after 4 hours of exposure to EPO showed 51.5±8.5% of cells to have entered S phase in control (n=4) and 57.5±3.5% in DBA (n=2). Erythroid progenitors at the end of phase I in DBA are thus responsive to the EPO-induced transition to phase II, with apparently normal kinetics of entry into the first cell cycle, consistent with the previously observed similarity in phase II colony numbers in control and DBA cultures. EPO-dose dependence was not apparent in the first cycle, with BrdU uptake similar at limiting (0.2U/ml) and saturating (2U/ml) EPO (65.4 vs 66.1%) at 9hrs post EPO, again consistent with observation of colony numbers. However, by 72hrs post EPO, the proportion of cells in S phase was lower in DBA cultures (n=3) than controls (n=6) (31.16±1.99% vs 38.4±1.05%; p<0.05), with proportionately more in G0/G1 (59.88±3.6 vs 50.78±2.4%). MTS assay showed a detectable onset of proliferation from day 3 in both control and DBA cultures, with a lower proliferation rate in DBA. In cultures supplemented with 10−7 M dexamethasone, there was a higher proliferation rate in both control and DBA cultures. RNA extracted from harvested pre- and at 24 hours post-EPO exposure was subjected to one round of linear amplification and labelled with Cy3/Cy5 for hybridisation to an in-house microarray comprising 395 genes. There was little change in the transcriptional profile at 24 hrs post-EPO in comparison with pre-EPO exposure, or between control and DBA cultures. The time course of expression of selected genes was studied by quantitative RT-PCR at 0–96hrs following EPO exposure. In both control and DBA cultures, there was no change in the level of expression of the selected genes at 24hrs, consistent with microarray data. An increase in expression of α-globin mRNA was first apparent at 48hrs post-EPO, observed in both DBA and control cultures. Erythroid cells at the end of the pre-EPO phase I are thus predicted to be quiescent until exposure to EPO on transfer to phase II, with EPO acting in an initial permissive role, enabling cells to re-enter cycle, followed by synchronous EPO-dose dependent proliferation coincident with active globin synthesis, when the erythroid defect in DBA becomes apparent. We propose that the 2-phase culture thus imposes significant translational stress, analogous to starvation/refeeding in hepatocytes, exposing a proliferation/cell cycle defect to explain our observation of a consistent severe in vitro erythroid defect in DBA irrespective of clinical severity.

Alpha Hemoglobin Stabilizing Protein (AHSP) Optimizes Hemoglobin A Synthesis by Maintaining a Pool of Viable Alpha Globin Subunits.

AHSP binds alpha hemoglobin (Hb) to maintain its structure and limit its prooxidant activities. In addition, AHSP binds and stabilizes apo-alpha globin, which lacks heme. Previously, we demonstrated that Ahsp−/− mice exhibit hemolytic anemia with Hb precipitation in erythroid cells. Through interbreeding of mutant strains, we also showed that loss of AHSP exacerbates beta thalassemia. Together, these studies indicate that AHSP participates in Hb homeostasis and may act to neutralize potentially toxic excess alpha globin that is known to accumulate in normal erythroid precursors, and to a greater extent, in beta thalassemic ones. However, additional functions for AHSP may exist. In particular, AHSP-alpha globin complexes may also promote HbA synthesis. To test this, we depleted the pool of excess alpha globin in Ahsp−/− mice by interbreeding with alpha thalassemic ones. Compared to mice with either mutation alone, compound mutants missing both AHSP and one alpha globin allele (genotype Ahsp−/− //alpha globin*α/αα) exhibited more severe erythroid defects, including worsened anemia, hypochromia, Hb instability and ineffective erythropoiesis. Pulse-labeling of double-mutant reticulocytes showed that alpha to beta globin synthetic ratios were unaffected by loss of AHSP, but precipitation of both alpha and beta nascent chains into cell membranes was strongly enhanced. These data indicate that AHSP is important for erythropoiesis and Hb production even when alpha globin is not produced in excess. In vitro translation studies using wheat germ extracts showed that recombinant AHSP present during the synthesis of alpha globin improved its ability to become incorporated into HbA. Moreover, AHSP conferred protease resistance to nascent alpha globin, suggesting enhanced folding into the native state. Further supporting this interpretation, circular dichroism studies showed that AHSP accelerated refolding of purified denatured alpha Hb. Finally, through pulse labeling of reticulocytes followed by isoelectric focusing of soluble cytosolic fractions, we identified transient pools of free alpha Hb and AHSP-alpha Hb in vivo and showed that Ahsp gene ablation fully depleted both pools. Together, our studies indicate that AHSP acts as a molecular chaperone to promote alpha globin folding and stability prior to its incorporation into HbA. In addition, it is possible that alterations in AHSP gene function or expression could modulate alpha thalassemia severity in patients.

A Heme Export Protein Is Required for Red Cell Differentiation and May Facilitate Iron Homeostasis.

Heme is a component of cytochromes, catalases, glutathione peroxidase, hydroxylases, and nitric oxide synthase, as well as myoglobin and hemoglobin, and thus is critical to all aerobic cells. It is also a transcriptional and translational initiator of globin synthesis. However, free heme is toxic so that cells must balance synthesis with use. We recently determined that the cell surface receptor for Feline Leukemia Virus, subgroup C, (FLVCR), which causes pure red cell aplasia in cats, is a heme export protein (Cell; 118:6, 2004). To understand its physiologic significance, we generated constitutive (Flvcr+/−) and inducible (Flvcrflox/flox;Mx-cre) FLVCR null mice by targeted deletion of exon 3, and confirmed this produced a nonfunctional protein by zinc mesoporphyrin (ZnMP) and 55Fe-heme export studies. Intercrossing Flvcr+/− mice produced 0 null animals among 109 progeny genotyped. FLVCR null embryos die either before E8.5 or at E14.5, suggesting two separate mechanisms for their demise. In situ hybridization studies in normal mouse embryos show highest Flvcr transcript levels in the ectoplacental cone which forms placenta (E8.5), the placenta (E9.5), and high levels in the fetal liver (E12.5). These data plus studies with Flvcrflox/floxx Meox2-Cre mice (Cre inactivates Flvcr in all embryonic tissue, but not extraembryonic visceral endoderm and placenta) suggest that the early lethality of null mice is due to impaired maternal-embryo heme-iron transport. At E14.5, residual null mice were small with deformed limb buds and craniofacial abnormalities resembling the congenital anomalies of Diamond-Blackfan anemia. Flow cytometric analyses of fetal liver cells from deleted animals with Ter119 and CD71 demonstrated a block in erythroid differentiation at the CFU-E/proerythroblast stage, implying that their death results from a stage-specific failure in definitive erythropoiesis. Heterozygous null animals (Flvcr+/−) were viable with normal hematologic parameters. These mice had 30.9% of normal Flvcr mRNA by q RT-PCR, however, they demonstrated normal protein by western blot analysis and normal heme export function by ZnMP studies. Thus, heterozygous null mice compensate at the protein level, confirming FLVCR is critical in vivo. We next utilized our inducible mutant line (Flvcrflox/flox;Mx-cre) to generate a viable null mouse for study. Flvcr deleted mice developed pure red cell aplasia characterized by macrocytic anemia (deleted: HCT 13.8±1.2, MCV 68.35± 1.6; control: HCT 50.24 ± 2.0, MCV 51.3±1.5), reticulocytopenia, and reactive thrombocytosis. Marrow and spleen had excess cells with a proerythroblast morphology and lacked maturing red cells. Flow cytometry confirmed maturation arrest at the CFU-E/proerythroblast stage. CFU-E from FLVCR null mice were undetectable in culture. Deleted mice also demonstrated iron loading of hepatocytes. FLVCR is highly expressed in multiple human tissues with high heme-flux by western blot analyses, including liver, placenta, duodenum, and uterus, in addition to the bone marrow. Our studies prove that FLVCR is required for the survival or differentiation of proerythroblasts (that stage where heme synthesis intensifies). These results, plus additional data not shown, suggest that FLVCR may also protect nonerythroid tissues with high free heme exposure (uterus, duodenum), may mediate maternal-embryo heme-iron transport, and may mediate the transport of heme from hepatocyte to bile which would imply a novel mechanism for iron export from the body.

Proliferative Defect but Normal Transcriptional Response to Erythropoietin in Diamond Blackfan Anemia Erythroid Progenitor Cells in 2-Phase Erythroid Culture.

Diamond Blackfan anaemia (DBA) is a rare congenital red cell aplasia, characterised by a steroid responsive intrinsic erythropoietic failure. Mutations affecting RPS19 are found in up to 25% of DBA, but the pathophysiological link between ribosomal gene haploinsufficiency and the specific erythroid phenotype of DBA remains elusive. One potential mechanism is that ribosomal insufficiency might affect the level of expression of critical erythroid proteins, resulting in a block in terminal erythroid differentiation and expansion. The aim of this study was to elucidate the cellular and molecular events occurring in response to erythropoietin (EPO) in DBA. We applied a 2-phase erythroid culture system in which we have previously demonstrated a consistent and profound failure of erythroid expansion in DBA cultures, localised to early in the second EPO-dependent phase of culture. The timing of the onset of proliferation in response to EPO was studied by tetrazolium dye assay of enriched erythroid progenitor cells in phase II of culture. An increase in cell number was detectable following 72hrs of EPO exposure in DBA (n=5) as well as control cultures, but with a subsequently slower proliferation rate in DBA. The proliferation rate of both DBA and control cells was enhanced by the addition of dexamethasone. Transcriptional profiles of control and DBA erythroid progenitors were then compared by microarray analysis after 24hrs exposure to EPO. Cy3/Cy5 labelled aRNA was competitively hybridised on an in-house array containing 395 transcript exon identifiers in ENSEMBL. The results showed little difference in expression between control and DBA after 24hrs of EPO exposure, the only significant changes being in the expression of just 4 of the 251 genes expressed above background (CDKN2D, TNFRSF6, MAPKK and CXCR), which were upregulated in DBA (p<0.0001 on t-test analysis). The gene expression profile from 0–96 hrs following EPO exposure was then studied in control and DBA (n=3) erythroid progenitors by real time PCR. There was little change in the first 48hr of exposure to EPO. At 72hrs the expression of both EPOR and α-GLOBIN was increased 3–6 fold in both DBA and controls in comparison with pre-EPO, confirming the occurrence of an erythroid-specific transcriptional response to EPO in DBA erythroid progenitors. These results are consistent with a qualitatively normal onset of terminal erythroid maturation in DBA, rather than a block in differentiation, and tend to favour an alternative hypothesis that the erythroid failure in DBA is the result of ribosomal insufficiency at the simultaneous onset of both rapid cell proliferation and a high rate of globin synthesis. In keeping with this, the expression of RPS19 showed 1.5–2 fold increase at 72hrs after EPO exposure, with 3.5 fold increase in the presence of dexamethasone, with equivalent results in both DBA and controls. We now aim to study translational and cell cycle events during this important wave of erythroid proliferation to further elucidate the molecular basis for the specific erythroid defect in DBA, despite the ubiquitous cellular requirement for ribosomal biogenesis.

Translational control of ceruloplasmin gene expression: Beyond the IRE

Translational control is a common regulatory mechanism for the expression of iron-related proteins. For example, three enzymes involved in erythrocyte development are regulated by three different control mechanisms: globin synthesis is modulated by heme-regulated translational inhibitor, erythroid 5-aminolevulinate synthase translation is inhibited by binding of the iron regulatory protein to the iron response element in the 5'-untranslated region (UTR); and 15-lipoxygenase is regulated by specific proteins binding to the 3'-UTR. Ceruloplasmin (Cp) is a multi-functional, copper protein made primarily by the liver and by activated macrophages. Cp has important roles in iron homeostasis and in inflammation. Its role in iron metabolism was originally proposed because of its ferroxidase activity and because of its ability to stimulate iron loading into apo-transferrin and iron efflux from liver. We have shown that Cp mRNA is induced by interferon (IFN)-gamma in U937 monocytic cells, but synthesis of Cp protein is halted by translational silencing. The silencing mechanism requires binding of a cytosolic inhibitor complex, IFN-Gamma-Activated Inhibitor of Translation (GAIT), to a specific GAIT element in the Cp 3'-UTR. Here, we describe our studies that define and characterize the GAIT element and elucidate the specific trans-acting proteins that bind the GAIT element. Our experiments describe a new mechanism of translational control of an iron-related protein and may shed light on the role that macrophage-derived Cp plays at the intersection of iron homeostasis and inflammation.

Antagonistic Roles of ERK1/2 and p38 MAP Kinases in Hemoglobin Synthesis.

Murine erythroleukemia (MEL) cells provide a valuable model to study the molecular events leading to erythroid differentiation. Maturing erythroid cells synthesize large quantities of hemoglobin, a process requiring the coordinated synthesis of heme and globin. Here, we investigated the role of the ERK and p38 mitogen-activated protein kinase (MAPK) signaling pathways in the differentiation of MEL cells. We determined the effect of the MEK1/2 inhibitor U0126 that blocks the ERK1/2 pathway, and the p38 inhibitor SB202190 on the differentiation potential of MEL cells induced by hexamethylene bisacetamide (HMBA). We found that treatment of HMBA induced MEL cells with the ERK1/2 pathway inhibitor U0126 results in higher hemoglobin levels. Using a fluorometric assay, we determined that intracellular heme levels also increased. Immunoblot studies showed an increase in globin protein levels. In contrast, treatment of MEL cells with the p38 inhibitor SB202190 has the opposite effect, leading to decreased amounts of heme and hemoglobin. In addition, inhibition of the p38 pathways results in lower transferrin receptor levels. Our results suggest that the ERK1/2 and p38 pathways play antagonistic roles in HMBA induced erythroid differentiation in MEL cells. This data also provides a novel link between MAPK signaling and the regulation of heme biosynthesis and iron uptake in erythroid cells.

Identification of a Gain-of-Function SNP Causing a New Model of α-Thalassaemia.

The human α globin cluster includes an embryonic gene ζ and 2 fetal/adult genes (α2 and α1) arranged along the chromosome in the order in which they are expressed in development (5′-ζ-pseudoζ- αD- α2-α1-θ-3′). Fully activated expression of these genes in erythroid cells depends on upstream regulatory elements of which HS-40, located 40kb upstream of the cluster, appears to exert the greatest effect. We have recently shown that during terminal differentiation, key transcription factors (GATA-2, GATA-1, NF-E2, SCL complex) sequentially bind the α promoters and their regulatory elements and a domain of histone acetylation develops which eventually encompasses the entire α globin cluster including the upstream regulatory sequences.α-thalassemia most frequently results from deletions or point mutations affecting the structural α globin genes, but may also result from rare sporadic deletions which remove the upstream regulatory sequences. In a single family α globin expression was silenced by a mutation which drives an anti-sense RNA through the α gene. Alpha thalassemia may also result from inherited and acquired mutations in a trans-acting factor called ATRX. Over the past few years we have continued to screen for new mechanisms which lead to α thalassemia and thereby elucidate new principles underlying the regulation of gene expression in hemopoiesis.Here we describe a new mechanism of α thalassemia occurring in Pacific Islanders in whom we could detect no mutations or rearrangements in the α globin gene locus. Despite this, extensive genetic analysis showed unequivocally that the causative mutation is linked to the terminal 169kb of chromosome 16 (Viprakasit et al accompanying abstract). Analysis of globin synthesis, steady state RNA levels and detection of RNA in situ demonstrated that the mutation downregulates α globin transcription. To identify the mutation, we constructed a new BAC library from an affected homozygote, isolated and re-sequenced the candidate region and focussed further analysis on 8 SNPS within the α globin cluster, one of which creates a new GATA-1 binding site (GACA>GATA). Using primary erythroblasts from normal individuals and patients with this form of thalassemia, together with interspecific hybrids containing either the normal or abnormal copy of chromosome 16, we have shown that this SNP creates a new binding site in vivo for GATA-1 and the SCL complex. Furthermore, the chromatin at this site becomes activated as judged by acetylation of histone H3 and H4 (H3ac2 and H4ac4) and methylation of histone H3 (H3K4me2).Based on these data we postulate that an active transcriptional complex binding this new GATA site created by the SNP-mutation, could distract the upstream regulatory regions, which normally interact with the α globin promoter, and silence α globin gene expression. This model thus represents a new example of α globin gene down-regulation and a new mechanism by which gene expression can be perturbed during hemopoiesis.

Microcytosis in Myelofibrosis with Myeloid Metaplasia: Prevalence and Clinical Correlates.

Background:Microcytosis is a characteristic laboratory feature for both iron deficiency anemia and thalassemia. Microcytosis is also infrequently seen with “anemia of chronic disease”. A partial list of the latter include rheumatoid arthritis, polymyalgia rheumatica, diabetes mellitus, connective tissue disease, chronic infection, Hodgkin's lymphoma, Castleman's disease, and renal cell carcinoma. In the current study, we show that microcytosis is a frequent laboratory feature in myelofibrosis with myeloid metaplasia (MMM) and investigate its clinical relevance in the particular setting.Methods:In order to minimize the confounding effects of nutritional deficiencies, red blood cell transfusion, and drug therapy on mean corpuscular volume (MCV), the current study was limited to young patients (age < 60 years) that had not received either red blood cell transfusion or drug therapy for MMM during the time of their initial laboratory investigation.Results:The study population consisted of 102 consecutive patients with MMM (median age 51 years, range 18–60) including 60 males. At diagnosis, the median (range) values for hemoglobin, leukocyte count, and platelet count were 11 gm/dL (7.4–14.9), 8.3 x 109/L (1–40) and 357 x 109/L (14–1512), respectively. Spleen measurements were documented in 78 patients (median 6 cm below the left costal margin, range 0–24) and the incidence of hypercatabolic symptoms was 18% (12 of 67 evaluable patients). The Dupriez prognostic score distribution included low-risk (n=73), intermediate-risk (n=19), and high-risk (n=10) disease categories.The overall incidence of microcytosis (MCV < 80 fL) was 28%; median MCV was 84.3 fL (range, 60.6–99.1). Among the 28 patients with microcytosis, MCV was less than 70 fL in 4 patients. Concomitant serum ferritin levels were available in 44 patients (median, 92 μg/L; range, 4–6440) and the levels were below 30 μg/L in 7 patients including 4 with microcytosis. Bone marrow iron stains were sporadically performed and showed normal iron stores in the majority of evaluable cases. The study cohort was followed for a median of 70 months (range 0–300). Multivariate analysis of variable that were measured in all study patients identified anemia, thrombocytopenia, and age but not MCV as independent prognostic indicators of inferior survival. The addition of variables that were measured in a variable proportion of the study population disclosed additional prognostic value for hypercatabolic symptoms and abnormal cytogenetics. Interestingly, microcytosis was significantly associated with the absence of cytogenetic abnormalities (p=0.04); among the 10 patients with abnormal cytogenetic findings, 9 had an MCV of ≥ 80 fL. Microcytosis did not correlate with several other clinical and laboratory features including gender, presence of hypercatabolic symptoms, spleen size, hemoglobin level, platelet count, leukocyte count, serum lactate dehydrogenase level, and the Dupriez prognostic score.Conclusion:Microcytosis with a normal or elevated serum ferritin level is a frequent laboratory feature in MMM but does not appear to influence survival. The observation regarding the low incidence of cytogenetic abnormalities in MMM patients with microcytosis is intriguing but requires validation by a prospective study. Whether or not MMM-associated microcytosis represents an acquired defect of globin synthesis, as might be the case in some patients with myelodysplastic syndrome, remains to be elucidated.

Thalassemia and related hemoglobinopathies

Hemoglobinopathies are the most common single gene disorders in man. There are several hundred of these disorders though the thalassemias -- alpha and beta and the sickling disorders make up the vast majority. Recent advances in the understanding of the hemoglobin structure and the genetics of its synthesis has contributed significantly to the understanding of these diseases. Disorders include those with reduced globin synthesis, abnormal globin chains and failure to switch globin chain synthesis at the appropriate age. This review focuses on the clinical features, diagnosis and management strategies of the alpha and beta thalassemias, the sickling disorders and touches on a few rarer hemoglobinopathies. It also emphasizes prevention strategies and chronic transfusion safety in countries like India where there are limited resources.

Effect of coenzyme Q10 as an antioxidant in β‐thalassemia/Hb E patients

Thalassemia is a group of genetic disorders resulting from different mutations in the globin gene complex and leading to an imbalance in globin synthesis. Unmatched globin chains are less stable and susceptible to oxidation. Patients with beta-thalassemia/HbE are prone to increased oxidative stress as indicated by increased lipid peroxidation product, malondialdehyde (MDA), partly because of the presence of iron in the form of heme and hemichromes released from excess globin chains and excess iron deposition in various tissues. The level of antioxidant such as glutathione is markedly decreased while activities of antioxidant enzymes including superoxide dismutase (SOD), catalase, and glutathione peroxidase (GSH-Px) are increased. We have recently found that the levels of coenzyme Q(10) (CoQ(10)) are also very low in thalassemia. We therefore evaluated the oxidative stress and the antioxidants in these patients before and after supplementation with 100 mg CoQ(10) daily for 6 months. The results showed that the plasma level of CoQ(10) significantly increased and the oxidative stress decreased as the level of MDA declined. The administration of CoQ(10) led to significant improvement of biochemical parameters of antioxidant enzymes. The antioxidant supplementation will be beneficial for thalassemia patients as adjunct therapy to increase their quality of life.

Hb Bristol-Alesha Presenting Thalassemia-Type Hyperunstable Hemoglobinopathy

Hemoglobin (Hb) Bristol-Alesha is caused by a GTG --> ATG mutation at codon 67 in the Hb beta chain, resulting in abnormal beta globin chains with mutated molecules from normal beta67 valine (Val) to beta67 methionine (Met) or beta67 aspartate (Asp). We describe a Japanese child with this rare hemoglobinopathy and a very unstable Hb molecule phenotype. The diagnosis of hemolytic anemia was made when the patient was 6 months of age. Development of marked splenomegaly necessitated red blood cell transfusions twice a month. After splenectomy when the patient was 4 years of age, laboratory findings of hemolytic anemia became more prominent. Specific abnormal Hb molecules initially were not detected, and the alpha/beta globin synthesis ratio was abnormal at 2.22. After splenectomy, we identified the presence of abnormal beta-globin chains with a beta67Val:beta67Met:beta67Asp molecule ratio of 74:11:15. We speculate that the high fraction of the beta67Met molecule in this patient, compared with that in previously reported cases, caused extreme Hb instability, which resulted in thalassemic hyperunstable hemoglobinopathy and very severe clinical findings.

Suppression of Erythroid-Specific 5-Aminolevulinate Synthase Using Short-Interfering RNA Alters Iron Metabolism and Inhibits Terminal Differentiation of Human Erythroleukemia Cells.

Erythroid-specific 5-aminolevulinate synthase (ALAS2) is the first and the rate limiting enzyme for heme biosynthesis in erythroid cells. ALAS2 plays a critical role in hemoglobin synthesis and erythrocyte maturation, since targeting the ALAS2 gene results in embryonic death in mice because of severe anemia. In humans, heritable mutations of the ALAS2 gene are responsible for X-linked sideroblastic anemia (XLSA). However, the effect of suppressed expression of ALAS2 on erythroid cell differentiation has not been examined in human cells. We therefore addressed this question, by stably suppressing ALAS2 mRNA with short-interfering RNA (siRNA) in a human erythroleukemia cell line, YN-1. After cloning of cells expressing low ALAS2 (ALAS2low cells), cells were induced to undergo erythroid differentiation by treatment with transforming growth factor beta1 (TGF-β1). Gene expression profiles of induced and uninduced cells were examined, including genes involved in globin synthesis and iron metabolism. Hemoglobin production, as judged by o-dianisidine staining, was significantly lower in ALAS2low cells than in control cells both before and after erythroid differentiation. Both alpha and gamma globin mRNA levels were also reduced in ALAS2low cells, compared with control cells. Decreased heme synthesis as well as reduced globin production in ALAS2low erythroid cells are consistent with our previous findings in murine erythroleukemia cells studied by antisense technology (Meguro K, et al. Blood 86:940–948, 1995), and extends our previous conclusion on the critical role of ALAS2 in heme and globin formation to human erythroid cells. Transferrin receptor (TFR) mRNA level was decreased in ALAS2low cells, and remained low following TGF-β1 treatment, whereas its level was increased in control cells during erythroid differentiation, which reflects enhanced iron uptake by differentiated control cells. Decreased TFR mRNA level in ALAS2low cells may suggest iron accumulation, since TFR mRNA is known to be unstable when intracellular iron level is increased. Notably, mitochondrial ferritin (MtF) mRNA level was decreased in control cells after differentiation, reflecting utilization of mitochondrial iron for heme synthesis, but it did not change in ALAS2low cells following TGF-β1 treatment. As accumulation of MtF protein is known to occur in iron-overloaded erythroid cells of patients with XLSA, our finding also suggests that there may be intramitochondrial iron accumulation in ALAS2low cells even after differentiation. In contrast to MtF mRNA, the level of cytosolic ferritin heavy chain mRNA was similar both in ALAS2low cells and control cells. These findings suggest that MtF levels, rather than cytosolic ferritin levels, may be a sensitive and specific indicator for iron accumulation in mitochondria. This study shows the critical role of ALAS2 not only in heme synthesis and hemoglobin formation, but also in iron metabolism in erythroid cells during their cell differentiation. An ALAS2low erythroid cell line, such as ALAS2-suppressed YN-1, will provide a good model for the study of relationship between heme biosynthesis and iron metabolism during terminal differentiation of human erythroid cells.