Role In Erythropoiesis Research Articles

Identification of Erythroid Cell Stimulating Factor as a Unique Protein, Quiescin Q6: Role in Erythropoiesis.

We demonstrated earlier the presence of an erythroid cell stimulating factor (ESF) in mouse serum that enhanced the erythropoietin-dependent erythroid cell proliferation in vitro. Employing a highly specific monoclonal antibody generated in our laboratory, we showed further that ESF had an essential role in normal erythropoiesis in vivo [Blood 98, 296a, 2001, Exp Hematol. 31, Supp. 1, 165, 2003]. In order to clone and express this protein, ESF was purified from mouse serum and on Western blotting using the monoclonal anti-ESF antibody the purified protein was stained as bands of ~62/64 kDa. These bands were analyzed by Mass Spectrometry. The data were processed using Protein Lynx 2.1 (Waters) and the resulting peak list was submitted for database searching on Mascot. One of the peptide sequences had a highest similarity with Quiescin Q6 (Q6). This protein was then BLAST searched for mouse DNA sequences in the NCBI database for nucleotides corresponding to the mouse Q6. We designed the primers to reclone Q6 ORF (Open reading frame) having restriction sites NCO1 and ECOR1. Amplified product was sequenced to confirm DNA fragment. Fragment was cloned in pGEMT vector and subsequently re-cloned in expression vector pET30a(+). After each cloning step, the orientation of the insert was analyzed using restriction enzyme digestion. His-tagged Q6 protein was over-expressed in E.coli BL21 Codon Plus (DE3)-RP cells. Western blot analysis using anti-ESF antibody identified 62/64 kDa-bands of Q6 in the soluble fraction of the bacterial extract. Further characteristics of this recombinant protein and its CFU-E enhancing properties are being investigated and will be reported. Thus we have identified ESF to be a unique protein, quiescin Q6 and successfully cloned and expressed it. Expressed protein is similar to ESF as regards its antigenic property.

Constitutive Activation of STAT5 and Bcl-XL Overexpression Can Induce Endogenous Erythroid Colony Formation in Human Primary Cells.

In contrast with secondary erythrocytosis, progenitor cells from polycythemia vera (PV) patients can undergo in vitro erythroid differentiation despite absence of erythropoietin (EPO) and presence of such endogenous erythroid colonies (EEC) is routinely used as a diagnostic assay. Recent focus on the JAK2 mutation V617F in PV patients argue for a direct implication of JAK2 dependent signaling pathways in EEC formation. Because STAT5 is the principal target of JAK2 in erythroid cells, we investigated whether EEC formation was only dependent on STAT5 activation or required other signaling pathways that would be activated by JAK2. For this purpose, we transduced a retroviral vector coding for a constitutively active form of STAT5 (MIGR-STAT5CA) in UT7 cells, a leukemic cell line with erythroid properties. We observed in cells transduced with the MIGR-STAT5CA vector a spontaneous induction of erythroid differentiation in comparison with cells infected with the empty vector MIGR, as assessed by GPA staining. We next investigated effects of STAT5CA on erythroid differentiation of human primary progenitors. Purified CD34+ cells obtained from peripheral blood (PB) of patients treated with G-CSF were transduced with the STA5CA vector, the CD36+/GPA− erythroid progenitor cells were sorted and cultured in presence of SCF alone. When expressing STAT5CA, they both proliferate and undergo erythroid terminal differentiation despite the absence of EPO. We concluded that a phosphorylated form of STAT5 was sufficient to support in vitro erythroid differentiation of human primary cells. Because STAT5 has been shown to play a crucial role in erythropoiesis via induction of the antiapoptotic protein Bcl-xL, we next investigated whether effects of STAT5CA on erythroid maturation was dependent on Bcl-xL induction. Tansduction of human CD36+/GPA− cells with a retrovirus containing the coding sequence of human Bcl-xL progenitors allowed survival, proliferation and GPA acquisition despite the absence of EPO. We next investigated whether STAT5CA or Bcl-xL overexpression in normal primary cells could reproduce the malignant phenotype observed in PV patients, i.e. induction of EEC formation. CD36+/GPA− transduced with either the STAT5 CA or the Bcl-XL vectors were plated in methylcellulose in the absence of EPO. Bcl-xL as well as STAT5CA vectors could both induce endogenous erythroid colony formation. Regardless to these results, we hypothesized that the EEC formation observed in myeloproliferative disorders could be at least partially due to the JAK2 dependent activation of the STAT5/Bcl-XL pathway. Thus, both constitutive activation of STAT5 and Bcl-xL overexpression could substitute to EPO to induce terminal differentiation of human primary erythroid progenitors.

Induction of Transient Cytopenia To Analyze Hematopoiesis in Mutant Mice.

The mouse plays an indispensable role in developing our current understanding of mammalian hematopoiesis. Most hematopoietic phenotyping assays in the mouse are non-viable techniques designed to evaluate homeostatic populations, enumerate progenitor populations, and perform functional analysis. The worldwide effort to generate mouse models of human disease and functionally annotate the mammalian genome using mouse mutagenesis (including dominant ENU screens) requires the development of robust standardized viable phenotyping tools. We have developed a phenotyping assay that induces transient cytopenias using various pharmacological agents (5-fluorouracil, phenylhydrazine, and hydroxyurea), the responses to which are monitored by tracing changes in peripheral blood levels of red blood cells, white blood cells and platelets. We have performed detailed analysis of lineage recovery kinetics, developing lineage recovery curves for various strains of mice for both males and females, which allowed us to identify appropriate testing days to identify phenodeviants. We have compared the recovery data with conventional progenitor assays and analyzed a cohort of well-studied naturally occurring and targeted hematopoietic mutants using the transient anemia assays that has yielded novel phenotypes of hemizygous mutant animals. For example, erythropoietin receptor null embryos die of severe anemia at mid-gestation; however, no defect in erythropoiesis has been reported in EpoR +/− mice. We have found that 5-fluorouracil and phenylhydrazine elicit delayed RBC recovery in EpoR +/− mice, demonstrating a critical dose-dependent role for the erythropoietin receptor in stress erythropoiesis. In addition, Stat5 has been shown to play an important role in erythropoiesis and in the regulation of steady state hematopoiesis. We have found that Stat5a/b+/− mice treated with 5-fluorouracil show altered recovery kinetics in RBC, WBC and platelets. Finally, we have adapted the transient cytopenia assay to develop a sensitized dominant ENU screen, enabling us to identify hematopoietic mutants that do not present abnormal blood cell counts in a homeostatic state. Thus, these standardized cytopenia response assays function as surrogate viable assays to analyze progenitor populations.

Role of Otx1 in the Differentiation of Myelo-Monocytic Precursors.

Homeobox-containing genes encode transcription factors implicated in the development and differentiation of multiple cell systems, including hematopoiesis. Several lines of evidence, obtained in our laboratory, indicate that Otx1, a homeobox gene of the paired class strictly required for brain morphogenesis, plays an important role also in blood cell production. It is differentially expressed in hematopoietic progenitors, particularly within the erythroid lineage and loss of Otx1 function results in a cell-autonomous erythroid impairment. Furthermore, Otx1 contributes to the control of erythropoiesis through a direct action on Scl/Tal1, a major hematopoietic regulator. In this study we have investigated whether Otx1 is also implicated in the regulation of myelo-monocytic differentiation. Analysis of Otx1 expression indicated that the gene is transcriptionally active in myelo-monocytic precursors. Moreover, using clonogenic assays we observed that Otx1−/− mice display abnormal frequencies of bone marrow myeloid subpopulations, although the total number of myeloid precursors is normal, as compared to wild type animals. Inactivation of Otx1 leads to a significant decrease of myelo-monocytic (CFU-GM) and monocytic (CFU-M) progenitors, and a concomitant increase of granulocytic precursors (CFU-G). In addition, morphological analysis of Otx1−/− bone marrow cells confirmed a high percentage of cells of the granulocytic lineage. Furthermore, at the molecular level, we detected in null mutant cells the upregulation of the gene encoding the G-CSF receptor and the modulation of other key myeloid regulators. Finally, to understand whether the action of Otx1 in myeloid cells is, like in erythoid cells, mediated by Scl we tested whether expression of a Scl transgene in Otx1−/− mice can re-establish normal ratios between the myeloid subpopulations. Results showed that, in contrast to the findings within the erythroid compartment, constitutive Scl expression does not fully rescue the alterations within the myelo-monocytic lineage. Taken together our data indicate that the brain morphogenetic gene Otx1 is an important regulator of the blood system, as it plays a relevant role in erythropoiesis, and contributes to control also myelo-monocytic differentiation, likely at the level of the commitment events of bipotential progenitor cells.

GATA-1 mediates auto-regulation of Gfi-1B transcription in K562 cells

Gfi-1B (growth factor independence-1B) gene is an erythroid-specific transcription factor, whose expression plays an essential role in erythropoiesis. Our laboratory has previously defined the human Gfi-1B promoter region and shown that GATA-1 mediates erythroid-specific Gfi-1B transcription. By further investigating the regulation of the Gfi-1B promoter, here we report that (i) Gfi-1B transcription is negatively regulated by its own gene product, (ii) GATA-1, instead of Gfi-1B, binds directly to the Gfi-1-like sites in the Gfi-1B promoter and (iii) Gfi-1B suppresses GATA-1-mediated stimulation of Gfi-1B promoter through their protein interaction. These results not only demonstrate that interaction of GATA-1 and Gfi-1B participates in a feedback regulatory pathway in controlling the expression of the Gfi-1B gene, but also provide the first evidence that Gfi-1B can exert its repression function by acting on GATA-1-mediated transcription without direct binding to the Gfi-1 site of the target genes. Based on these data, we propose that this negative auto-regulatory feedback loop is important in restricting the expression level of Gfi-1B, thus optimizing its function in erythroid cells.

Expression of Erythropoietin Receptor–Like Molecule in Xenopus laevis and Erythrocytopenia upon Administration of Its Recombinant Soluble Form

The regulation of hematopoiesis in non-mammalian vertebrates is poorly understood. This is partly because the structures and effects of most hematopoietic regulators have not been identified. As a first step towards studies on the key mechanism of hematopoietic regulation among phyla as well as the diversity of organisms, we have focused on amphibian hematopoiesis. A cDNA sharing the highest degree of homology with mammalian erythropoietin (EPO) receptors, tentatively named xlEPOR, was cloned from a cDNA library of Xenopus laevis immature erythrocytes. The comparative identities of the deduced entire amino acid sequence to mammalian EPO receptors were quite low, although functional domains indispensable for erythropoietic activities were found in the molecule. Northern analysis revealed that xlEPOR were expressed in peripheral blood cells. In the peripheral blood of phenylhydrazine-treated adult Xenopus, immature erythrocytes expressing xlEPOR were identified by in situ hybridization and immunostaining with polyclonal antibodies to xlEPOR. To confirm the biological functions of this molecule, the extracellular domain of xlEPOR (i.e., soluble xlEPOR) was administered to adult Xenopus by consecutive intracardiac injection. The peripheral erythrocyte counts were decreased gradually; meanwhile, immature erythrocytes appeared in the circulation, demonstrating that xlEPOR plays a significant physiological role in erythropoiesis in Xenopus laevis.

The Erythroid Phenotype of EKLF-Null Mice: Defects in Hemoglobin Metabolism and Membrane Stability

Development of red blood cells requires the correct regulation of cellular processes including changes in cell morphology, globin expression and heme synthesis. Transcription factors such as erythroid Kruppel-like factor EKLF (Klf1) play a critical role in erythropoiesis. Mice lacking EKLF die around embryonic day 14 because of defective definitive erythropoiesis, partly caused by a deficit in beta-globin expression. To identify additional target genes, we analyzed the phenotype and gene expression profiles of wild-type and EKLF null primary erythroid progenitors that were differentiated synchronously in vitro. We show that EKLF is dispensable for expansion of erythroid progenitors, but required for the last steps of erythroid differentiation. We identify EKLF-dependent genes involved in hemoglobin metabolism and membrane stability. Strikingly, expression of these genes is also EKLF-dependent in primitive, yolk sac-derived, blood cells. Consistent with lack of upregulation of these genes we find previously undetected morphological abnormalities in EKLF-null primitive cells. Our data provide an explanation for the hitherto unexplained severity of the EKLF null phenotype in erythropoiesis.

Erythropoietin and Erythropoietin Receptor Expression after Experimental Spinal Cord Injury Encourages Therapy by Exogenous Erythropoietin

Erythropoietin (EPO) is a pleiotropic cytokine originally identified for its role in erythropoiesis. Recent studies have demonstrated that EPO and its receptor (EPO-R) are expressed in the central nervous system, where EPO exerts neuroprotective functions. Because the expression of the EPO and EPO-R network is poorly investigated in the central nervous system, the aim of the present study was to investigate whether the resident EPO and EPO-R network is activated in the injured nervous system. A well-standardized model of compressive spinal cord injury in rats was used. EPO and EPO-R expression was determined by immunohistochemical analysis at 8 hours and at 2, 8, and 14 days in the spinal cord of injured and noninjured rats. In noninjured spinal cord, weak immunohistochemical expression of EPO and EPO-R was observed in neuronal and glial cells as well as in endothelial and ependymal cells. In injured rats, a marked increase of expression of EPO and EPO-R was observed in neurons, vascular endothelium, and glial cells at 8 hours after injury, peaking at 8 days, after which it gradually decreased. Two weeks after injury, EPO immunoreactivity was scarcely detected in neurons, whereas glial cells and vascular endothelium expressed strong EPO-R immunoreactivity. These observations suggest that the local EPO and EPO-R system is markedly engaged in the early stages after nervous tissue injury. The reduction in EPO immunoexpression and the increase in EPO-R staining strongly support the possible usefulness of a therapeutic approach based on exogenous EPO administration.

Erythropoietin after Focal Cerebral Ischemia Activates the Janus Kinase–Signal Transducer and Activator of Transcription Signaling Pathway and Improves Brain Injury in Postnatal Day 7 Rats

Erythropoietin (Epo) plays a central role in erythropoiesis but also has neuroprotective properties. Recently, Epo-related neuroprotective studies used a hypoxic-ischemic neonatal model, which is different from focal stroke, a frequent cause of neonatal brain injury. We report on the effects of Epo treatment given after focal stroke and its potential neuroprotective mechanisms in postnatal day 7 rats with focal cerebral ischemia (FCI) achieved by occlusion of the middle cerebral artery. The experimental groups included sham operation, FCI plus vehicle, and FCI plus Epo. In the Epo-treated group, pups received a single intraperitoneal injection of 1000 U/kg 15 min after FCI or three injections of 100, 1000, or 5000 U/kg, starting at 15 min and repeated at 1 and 2 d after FCI. Epo treatment produced significant reductions in the mean infarct area and volume at 1 and 3 d after FCI, demonstrated by 2,3,5-triphenyltetrazolium chloride staining. Terminal deoxynucleotidyltransferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling (TUNEL) staining showed a markedly reduced number of TUNEL-positive cells in the Epo-treated group when compared with the vehicle control 3 d after FCI (p<0.01). The most effective dose after FCI was 1000 U/kg for 3 d. Immunoanalyses showed that Epo induced a significant increase in phosphorylated Janus kinase 2 and signal transducer and activator of transcription-5 expressions at 1 and 3 d and up-regulated Bcl-xL expression by 24 h after FCI but did not affect Epo receptor or NF-kappaB expression. In conclusion, Epo given after FCI in neonatal rats provides significant neuroprotection, mediated possibly by activation of the Janus kinase-signal transducer and activator of transcription-Bcl-xL signaling pathways.

Notch Signals Inhibit the Development of Erythroid/Megakaryocytic Cells by Suppressing GATA-1 Activity through the Induction of HES1

The effects of Notch signals on the erythroid/megakaryocytic differentiation of hematopoietic cells were examined. Activation of Notch signals by the intracellular Notch1 or an estradiol-inducible form of Notch1/ER suppressed the expression of the erythroid marker glycophorin A in an erythroid/megakaryocytic cell line K562. Although Mock-transfected K562 cells underwent megakaryocytic differentiation in response to 12-O-tetradecanoylphorbol-13-acetate (TPA), estradiol-activated Notch1/ER induced apoptosis during TPA treatment in the transfectant, which was accompanied by the reduced expression of an antiapoptotic molecule Bcl-XL. Even when apoptosis was prevented by the overexpression of Bcl-XL, activated Notch signals still inhibited TPA-induced megakaryocytic differentiation. As for this mechanism, Notch1/recombination signal binding protein J-kappa-induced HES1 but not HES5 was found to inhibit the function of an erythroid/megakaryocytic lineage-specific transcription factor GATA-1. Although HES1 did not affect the DNA binding activity of GATA-1 in gel shift and chromatin immunoprecipitation assays, it directly bound to GATA-1 and dissociated a critical transcriptional cofactor, p300, from GATA-1. Furthermore, overexpressed HES1 inhibited the development of erythroid and megakaryocytic cells in colony assays. Also, the Notch ligand Jagged1 expressed on NIH3T3 cells suppressed the development of erythroid and megakaryocytic cells from cocultured Lin-Sca-1+ hematopoietic stem/progenitor cells. These results suggest that Notch1 inhibits the development of erythroid/megakaryocytic cells by suppressing GATA-1 activity through HES1.

Erythropoietin treatment improves neurological functional recovery in EAE mice

Erythropoietin (EPO), originally recognized for its central role in erythropoiesis, has been shown to improve neurological outcome after stroke. Here, we investigated the treatment of experimental autoimmune encephalomyelitis (EAE) in mice with EPO. Mice were treated with recombinant human EPO (rhEPO) upon onset of paresis. Neurological functional tests were scored daily by grading of clinical signs (score 0–5). Hematoxylin and eosin (HE) staining of cerebral tissue was performed to detect inflammatory infiltrates. Double staining for Luxol fast blue and Bielshowsky was used to demonstrate myelin and axons, respectively. Immunohistochemistry was performed to measure the expression of bromodeoxyuridine (BrdU, a marker for cell proliferation), NG2 (a marker for oligodendrocyte progenitor cells) and brain-derived neurotrophic factor (BDNF). Treatment with rhEPO significantly improved neurological functional recovery, reduced inflammatory infiltrates and demyelination, and increased oligodendrocyte progenitor cell proliferation and BDNF + cells compared to the EAE controls. These data indicate that rhEPO treatment improved functional recovery after EAE in mice, possibly, via stimulating oligodendrogenesis, downregulating proinflammatory infiltrates and by elevating BDNF expression.

Human growth hormone and insulin-like growth factor-I inhibit erythropoietin secretion from the kidneys of adult rats

Growth hormone (GH) and insulin-like growth factor-I (IGF-I) play important roles in erythropoiesis and erythro-poietin (EPO) secretion. We examined the effects of GH and IGF-I on EPO production in adult rat kidney and liver in vivo and in vitro. Male Wistar rats aged 8-10 weeks were used. Recombinant human GH (hGH) was continuously infused (20 mug/kg per h) subcutaneously for 48 h using a micro-osmotic infusion pump. Octreotide (10 mug/kg) was subcutaneously injected every 12 h beginning 12 h before the hGH treatment. GH increased plasma EPO levels earlier than it increased plasma IGF-I levels. At 24 h, the IGF-I content in the liver and kidney was increased from 172.8+/-14.6 to 232.6+/-17.8 ng/g tissue (means+/-S.E.) and from 53.8+/-3.1 to 112.8+/-7.2 ng/g tissue, respectively. The EPO content in the liver was increased from 7.5+/-1.2 to 15.1+/-1.4 mIU/g tissue at 48 h, whereas the EPO content in the kidney was decreased at 12, 24, and 48 h after the start of hGH treatment. When the kidneys were organ-cultured, hGH considerably decreased EPO levels in the culture medium in a dose-related manner. The addition of anti-hGH IgG blunted the GH-induced inhibition of EPO secretion from the kidneys. IGF-I also decreased EPO levels in the medium in a dose-related manner. The addition of anti-IGF-I IgG blunted the IGF-I-induced inhibition of EPO secretion from the kidneys, whereas the GH-induced inhibition of EPO secretion was not affected. These findings suggest that both hGH and IGF-I have direct inhibitory effects on EPO secretion from adult rat kidneys.

Expression of Erythropoietin Receptor-Like Molecule in Xenopus laevis (xeEPOR) and the Development of Anemia by the Administration of Its Recombinant Soluble Form.

The regulation of hematopoiesis in non-mammalian vertebrates is poorly understood. This is partly because the structures and effects of most hematopoietic regulators have not been identified. As a first step towards studies on the key ingredient of hematopoietic regulation among phyla as well as the diversity of organisms, we have focused on amphibian hematopoiesis. In this study, a cDNA sharing the highest degree of homology with mammalian erythropoietin (EPO) receptors, named xeEPOR tentatively, was cloned from cDNA library of Xenopus immature erythrocytes. The identities of the deduced entire amino acid sequence to human and murine EPO receptors were 24% and 25%, respectively; whereas transmembrane region and motifs of WSXWS and Box1/2 domains were found in the molecule. The northern analysis revealed that two types of xeEPOR RNAs were expressed in normal peripheral blood cells. In addition, by in situ hybridization and immunostaining with monoclonal antibodies raised against the extracellular domain of xeEPOR (soluble xeEPOR), immature basophilic erythrocytes expressing xeEPOR appeared in peripheral blood of phenylhydrazine-treated adult Xenopus. The fulllength xeEPOR cDNA was introduced into murine FDC/P2 cells and the signaling for the cellular proliferation and differentiation was examined in the presence of serum derived from anemic Xenopus as a stimulator. To further understanding the contribution of the xeEPOR gene expression to primitive and definitive hematopoiesis on Xenopus development, whole mount in situ hybridization was performed. As the binding motif of GATA-1, the hematopoietic specific transcription factor, was located at −24 to −15 base upstream of the translation initiation sequence, the correlated expressions of xeEPOR and Xenopus GATA-1 on developing embryo were evaluated with RT-PCR. The xeEPOR RNA was abundantly expressed at Nieuwkoop stage 30 (blood island formation) and thereafter, and temporally followed the expression of GATA-1, suggesting that the functional expression of xeEPOR was upregulated by GATA-1 in Xenopus as reported in studies on mammalian erythropoiesis. To confirm biological functions of the molecule, soluble xeEPOR was administered into adult Xenopus by intracardiac consecutive injections. The peripheral erythrocyte counts were gradually decreased; meanwhile immature erythrocytes were emerged in the circulation, demonstrating that this molecule plays a significant physiological role in erythropoiesis in Xenopus.

Cell-intrinsic requirement for pRb in erythropoiesis

AbstractRetinoblastoma (Rb) and family members have been implicated as key regulators of cell proliferation and differentiation. In particular, accumulated data have suggested that the Rb gene product pRb is an important controller of erythroid differentiation. However, current published data are conflicting as to whether the role of pRb in erythroid cells is cell intrinsic or non–cell intrinsic. Here, we have made use of an in vitro erythroid differentiation culture system to determine the cell-intrinsic requirement for pRb in erythroid differentiation. We demonstrate that the loss of pRb function in primary differentiating erythroid cells results in impaired cell cycle exit and terminal differentiation. Furthermore, we have used coculture experiments to establish that this requirement is cell intrinsic. Together, these data unequivocally demonstrate that pRb is required in a cell-intrinsic manner for erythroid differentiation and provide clarification as to its role in erythropoiesis.

New insights into erythropoiesis: the roles of folate, vitamin B12, and iron.

Erythropoiesis is the process in which new erythrocytes are produced. These new erythrocytes replace the oldest erythrocytes (normally about one percent) that are phagocytosed and destroyed each day. Folate, vitamin B12, and iron have crucial roles in erythropoiesis. Erythroblasts require folate and vitamin B12 for proliferation during their differentiation. Deficiency of folate or vitamin B12 inhibits purine and thymidylate syntheses, impairs DNA synthesis, and causes erythroblast apoptosis, resulting in anemia from ineffective erythropoiesis. Erythroblasts require large amounts of iron for hemoglobin synthesis. Large amounts of iron are recycled daily with hemoglobin breakdown from destroyed old erythrocytes. Many recently identified proteins are involved in absorption, storage, and cellular export of nonheme iron and in erythroblast uptake and utilization of iron. Erythroblast heme levels regulate uptake of iron and globin synthesis such that iron deficiency causes anemia by retarded production rates with smaller, less hemoglobinized erythrocytes.

Targeted disruption of the ribosomal protein S19 gene is lethal prior to implantation.

The ribosomal protein S19 (RPS19) is located in the small (40S) subunit and is one of 79 ribosomal proteins. The gene encoding RPS19 is mutated in approximately 25% of patients with Diamond-Blackfan anemia, which is a rare congenital erythroblastopenia. Affected individuals present with decreased numbers or the absence of erythroid precursors in the bone marrow, and associated malformations of various organs are common. We produced C57BL/6J mice with a targeted disruption of murine Rps19 to study its role in erythropoiesis and development. Mice homozygous for the disrupted Rps19 were not identified as early as the blastocyst stage, indicating a lethal effect. In contrast, mice heterozygous for the disrupted Rps19 allele have normal growth and organ development, including that of the hematopoietic system. Our findings indicate that zygotes which are Rps19(-/-) do not form blastocysts, whereas one normal Rps19 allele in C57BL/6J mice is sufficient to maintain normal ribosomal and possibly extraribosomal functions.

Role of Ras signaling in erythroid differentiation of mouse fetal liver cells: functional analysis by a flow cytometry–based novel culture system

AbstractRas signaling plays an important role in erythropoiesis. Its function has been extensively studied in erythroid and nonerythroid cell lines as well as in primary erythroblasts, but inconclusive results using conventional erythroid colony-forming unit (CFU-E) assays have been obtained concerning the role of Ras signaling in erythroid differentiation. Here we describe a novel culture system that supports terminal fetal liver erythroblast proliferation and differentiation and that closely recapitulates erythroid development in vivo. Erythroid differentiation is monitored step by step and quantitatively by a flow cytometry analysis; this analysis distinguishes CD71 and TER119 double-stained erythroblasts into different stages of differentiation. To study the role of Ras signaling in erythroid differentiation, different H-ras proteins were expressed in CFU-E progenitors and early erythroblasts with the use of a bicistronic retroviral system, and their effects on CFU-E colony formation and erythroid differentiation were analyzed. Only oncogenic H-ras, not dominant-negative H-ras, reduced CFU-E colony formation. Analysis of infected erythroblasts in our newly developed system showed that oncogenic H-ras blocks terminal erythroid differentiation, but not through promoting apoptosis of terminally differentiated erythroid cells. Rather, oncogenic H-ras promotes abnormal proliferation of CFU-E progenitors and early erythroblasts and supports their erythropoietin (Epo)–independent growth.

Role of Ceruloplasmin in Macrophage Iron Efflux during Hypoxia

The reticuloendothelial system has a central role in erythropoiesis and iron homeostasis. An important function of reticuloendothelial macrophages is phagocytosis of senescent red blood cells. The iron liberated from heme is recycled for delivery to erythrocyte precursors for a new round of hemoglobin synthesis. The molecular mechanism by which recycled iron is released from macrophages remains unresolved. We have investigated the mechanism of macrophage iron efflux, focusing on the role of ceruloplasmin (Cp), a copper protein with a potent ferroxidase activity that converts Fe2+ to Fe3+ in the presence of molecular oxygen. As shown by others, Cp markedly increased iron binding to apotransferrin at acidic pH; however, the physiological significance of this finding is uncertain because little stimulation was observed at neutral pH. Introduction of a hypoxic atmosphere resulted in marked Cp-stimulated binding of iron to apotransferrin at physiological pH. The role of Cp in cellular iron release was examined in U937 monocytic cells induced to differentiate to the macrophage lineage. Cp added at its normal plasma concentration increased the rate of 55Fe release from U937 cells by about 250%. The stimulation was absolutely dependent on the presence of apotransferrin and hypoxia. Cp-stimulated iron release was confirmed in mouse peritoneal macrophages. Stimulation of iron release required an intracellular "labile iron pool" that was rapidly depleted in the presence of Cp and apotransferrin. Ferroxidase-mediated loading of iron into apotransferrin was critical for iron release because ferroxidase-deficient Cp was inactive and because holotransferrin could not substitute for apotransferrin. The extracellular iron concentration was critical as shown by inhibition of iron release by exogenous free iron, and marked enhancement of release by an iron chelator. Together these data show that Cp stimulates iron release from macrophages under hypoxic conditions by a ferroxidase-dependent mechanism, possibly involving generation of a negative iron gradient.

Chemotherapy-Induced Cognitive Dysfunction: A Clearer Picture

Chemotherapy-associated cognitive dysfunction occurs in a subset of patients treated with adjuvant chemotherapy. Recent data suggest that development of chemotherapy-related anemia predisposes patients to cognitive dysfunction. Endogenous erythropoietin (EPO) is well recognized for its central role in erythropoiesis, and recombinant human EPO (epoetin alfa) is established as a safe and effective treatment for chemotherapy- related anemia. Treatment with epoetin alfa also improves health-related quality of life in anemic cancer patients undergoing chemotherapy, and several controlled studies have documented increases in quality-of-life scores correlated with increases in hemoglobin. Erythropoietin also plays a role in neuroprotection, presumably by activation of antiapoptotic genes. Erythropoietin and its receptor are expressed in neural cells of the human brain, and their expression is upregulated after hypoxic or ischemic injury. In animal models, systemic administration of epoetin alfa protects against such neural injury. Ongoing and future studies will determine whether epoetin alfa can provide neuroprotection with respect to the development of cognitive dysfunction in patients undergoing adjuvant chemotherapy treatment for breast cancer.

High expression of YB-1 gene in erythroid cells in patients with refractory anemia.

It has been shown that aberrant expression of a transcription factor, GATA-1, leads to maturation arrest and transformation of erythroid cells. We previously reported that a multifunctional protein, YB-1, was expressed strongly in the spleen of a GATA-1 mutant mouse, which was filled with transformed erythroblasts. This finding suggested that YB-1 has roles in erythropoiesis. In this study, we examined in vivo expression of YB-1 messenger RNA (mRNA) in bone marrow erythroid cells and erythroid leukemic cell lines. During erythroid differentiation of erythroid leukemic cell lines, the expression level of YB-1 mRNA was highest at the early phase of differentiation and then decreased. In human bone marrow cells, the in vivo expression level of YB-1 mRNA was higher in glycophorin A-positive cells than in glycophorin A-negative cells. An interesting finding was that expression of YB-1 was higher in erythroblasts in myelodysplastic syndrome-refractory anemia (MDS-RA) than in normal cells. The findings suggested that YB-1 functions in the early stage of erythropoiesis and that aberrant expression of this protein may induce hematological diseases such as MDS.