Search

Background: Accumulating evidence suggests the origin of juvenile myelomonocytic leukemia (JMML) is closely associated with fetal development. Nevertheless, the contribution of embryonic progenitors to JMML pathogenesis remains unexplored. We hypothesized that expression of JMML-initiating PTPN11 mutations in HSC-independent yolk sac erythromyeloid progenitors (YS EMPs) would result in a mouse model of pediatric myeloproliferative neoplasm (MPN)., Results: E9.5 YS EMPs from VavCre+;PTPN11 D61Y embryos demonstrated growth hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF) and hyperactive RAS-ERK signaling. Mutant EMPs engrafted the spleens of neonatal recipients, but did not cause disease. To assess MPN development during unperturbed hematopoiesis we generated CSF1R-MCM+;PTPN11 E76K ;ROSA YFP mice in which oncogene expression was restricted to EMPs. Yellow fluorescent protein-positive progeny of mutant EMPs persisted in tissues one year after birth and demonstrated hyperactive RAS-ERK signaling. Nevertheless, these mice had normal survival and did not demonstrate features of MPN., Conclusions: YS EMPs expressing mutant PTPN11 demonstrate functional and molecular features of JMML but do not cause disease following transplantation nor following unperturbed development. Developmental Dynamics 246:1001-1014, 2017. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

The pathophysiology of neurodegenerative diseases is poorly understood and there are few therapeutic options. Neurodegenerative diseases are characterized by progressive neuronal dysfunction and loss, and chronic glial activation. Whether microglial activation, which is generally viewed as a secondary process, is harmful or protective in neurodegeneration remains unclear. Late-onset neurodegenerative disease observed in patients with histiocytoses, which are clonal myeloid diseases associated with somatic mutations in the RAS-MEK-ERK pathway such as BRAF(V600E), suggests a possible role of somatic mutations in myeloid cells in neurodegeneration. Yet the expression of BRAF(V600E) in the haematopoietic stem cell lineage causes leukaemic and tumoural diseases but not neurodegenerative disease. Microglia belong to a lineage of adult tissue-resident myeloid cells that develop during organogenesis from yolk-sac erythro-myeloid progenitors (EMPs) distinct from haematopoietic stem cells. We therefore hypothesized that a somatic BRAF(V600E) mutation in the EMP lineage may cause neurodegeneration. Here we show that mosaic expression of BRAF(V600E) in mouse EMPs results in clonal expansion of tissue-resident macrophages and a severe late-onset neurodegenerative disorder. This is associated with accumulation of ERK-activated amoeboid microglia in mice, and is also observed in human patients with histiocytoses. In the mouse model, neurobehavioural signs, astrogliosis, deposition of amyloid precursor protein, synaptic loss and neuronal death were driven by ERK-activated microglia and were preventable by BRAF inhibition. These results identify the fetal precursors of tissue-resident macrophages as a potential cell-of-origin for histiocytoses and demonstrate that a somatic mutation in the EMP lineage in mice can drive late-onset neurodegeneration. Moreover, these data identify activation of the MAP kinase pathway in microglia as a cause of neurodegeneration and this offers opportunities for therapeutic intervention aimed at the prevention of neuronal death in neurodegenerative diseases.

A role for caspase activation in erythroid differentiation has been established, yet its precise mode of action remains elusive. A drawback of all previous investigations on caspase activation in ex vivo erythroid differentiation is the lack of an in vitro model producing full enucleation of erythroid cells. Using a culture system which renders nearly 100% enucleated red cells from human CD34(+) cells, we investigated the role of active caspase-3 in erythropoiesis. Profound effects of caspase-3 inhibition were found on erythroid cell growth and differentiation when inhibitors were added to CD34(+) cells at the start of the culture and showed dose-response to the concentration of inhibitor employed. Enucleation was only reduced as a function of the reduced maturity of the culture and the increased cell death of mature cells while the majority of cells retained their ability to extrude their nuclei. Cell cycle analysis after caspase-3 inhibition showed caspase-3 to play a critical role in cell proliferation and highlighted a novel function of this protease in erythroid differentiation, i.e. its contribution to cell cycle regulation at the mitotic phase. While the effect of caspase-3 inhibitor treatment on CD34(+) derived cells was not specific to the erythroid lineage, showing a similar reduction of cell expansion in myeloid cultures, the mechanism of action in both lineages appeared to be distinct with a strong induction of apoptosis causing the decreased yield of myeloid cells. Using a series of colony-forming assays we were able to pinpoint the stage at which cells were most sensitive to caspase-3 inhibition and found activated caspase-3 to play a signalling role in erythroid differentiation by targeting mature BFU-E and CFU-E but not early BFU-E.

Among the three types of super oxide dismutases (SODs) known, SOD2 deficiency is lethal in neonatal mice owing to cardiomyopathy caused by severe oxidative damage. SOD2 is found in red blood cell (RBC) precursors, but not in mature RBCs. To investigate the potential damage to mature RBCs resulting from SOD2 deficiency in precursor cells, we studied RBCs from mice in which fetal liver stem cells deficient in SOD2 were capable of efficiently rescuing lethally irradiated host animals. These transplanted animals lack SOD2 only in hematopoietically generated cells and live longer than SOD2 knockouts. In these mice, approximately 2.8% of their total RBCs in circulation are iron-laden reticulocytes, with numerous siderocytic granules and increased protein oxidation similar to that seen in sideroblastic anemia. We have studied the RBC deformability and oxidative stress in these animals and the control group by measuring them with a microfluidic ektacytometer and assaying fluorescent heme degradation products with a fluorimeter, respectively. In addition, the rate of hemoglobin oxidation in RBCs from these mice and the control group were measured spectrophotometrically. The results show that RBCs from these SOD2-deficient mice have reduced deformability, increased heme degradation products, and an increased rate of hemoglobin oxidation compared with control animals, indicative of increased RBC oxidative stress., (Published by Elsevier Inc.)

Aberrant JAK2 signalling plays an important role in the aetiology of myeloproliferative neoplasms (MPNs). JAK2 inhibitors, however, do not readily eliminate neoplastic MPN cells and thus do not induce patient remission. Further understanding JAK2 signalling in MPNs may uncover novel avenues for therapeutic intervention. Recent work has suggested a potential role for cellular cholesterol in the activation of JAK2 by the erythropoietin receptor and in the development of an MPN-like disorder in mice. Our study demonstrates for the first time that the MPN-associated JAK2-V617F kinase localizes to lipid rafts and that JAK2-V617F-dependent signalling is inhibited by lipid raft disrupting agents, which target membrane cholesterol, a critical component of rafts. We also show for the first time that statins, 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors, widely used to treat hypercholesterolaemia, induce apoptosis and inhibit JAK2-V617F-dependent cell growth. These cells are more sensitive to statin treatment than non-JAK2-V617F-dependent cells. Importantly, statin treatment inhibited erythropoietin-independent erythroid colony formation of primary cells from MPN patients, but had no effect on erythroid colony formation from healthy individuals. Our study is the first to demonstrate that JAK2-V617F signalling is dependent on lipid rafts and that statins may be effective in a potential therapeutic approach for MPNs., (© 2012 Blackwell Publishing Ltd.)

In red cell development, the differentiation program directed by the transcriptional regulator GATA1 requires signaling by the cytokine erythropoietin, but the mechanistic basis for this signaling requirement has remained unknown. Here we show that erythropoietin regulates GATA1 through protein kinase D activation, promoting histone deacetylase 5 (HDAC5) dissociation from GATA1, and subsequent GATA1 acetylation. Mice deficient for HDAC5 show resistance to anemic challenge and altered marrow responsiveness to erythropoietin injections. In ex vivo studies, HDAC5(-/-) progenitors display enhanced entry into and passage through the erythroid lineage, as well as evidence of erythropoietin-independent differentiation. These results reveal a molecular pathway that contributes to cytokine regulation of hematopoietic differentiation and offer a potential mechanism for fine tuning of lineage-restricted transcription factors by lineage-specific cytokines.

Background: It has been reported that the phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway regulates erythropoietin (EPO)-induced survival, proliferation, and maturation of early erythroid progenitors. Erythroid cell proliferation and survival have also been related to activation of the JAK-STAT pathway. The goal of this study was to observe the function of EPO activation of JAK-STAT and PI3K/AKT pathways in the development of erythroid progenitors from hematopoietic CD34+ progenitor cells, as well as to distinguish early EPO target genes in human erythroid progenitors during ontogeny., Methods: Hematopoietic CD34+ progenitor cells, isolated from fetal and adult hematopoietic tissues, were differentiated into erythroid progenitor cells. We have used microarray analysis to examine JAK-STAT and PI3K/AKT related genes, as well as broad gene expression modulation in these human erythroid progenitor cells., Results: In microarray studies, a total of 1755 genes were expressed in fetal liver, 3844 in cord blood, 1770 in adult bone marrow, and 1325 genes in peripheral blood-derived erythroid progenitor cells. The erythroid progenitor cells shared 1011 common genes. Using the Ingenuity Pathways Analysis software, we evaluated the network pathways of genes linked to hematological system development, cellular growth and proliferation. The KITLG, EPO, GATA1, PIM1 and STAT3 genes represent the major connection points in the hematological system development linked genes. Some JAK-STAT signaling pathway-linked genes were steadily upregulated throughout ontogeny (PIM1, SOCS2, MYC, PTPN11), while others were downregulated (PTPN6, PIAS, SPRED2). In addition, some JAK-STAT pathway related genes are differentially expressed only in some stages of ontogeny (STATs, GRB2, CREBB). Beside the continuously upregulated (AKT1, PPP2CA, CHUK, NFKB1) and downregulated (FOXO1, PDPK1, PIK3CG) genes in the PI3K-AKT signaling pathway, we also observed intermittently regulated gene expression (NFKBIA, YWHAH)., Conclusions: This broad overview of gene expression in erythropoiesis revealed transcription factors differentially expressed in some stages of ontogenesis. Finally, our results show that EPO-mediated proliferation and survival of erythroid progenitors occurs mainly through modulation of JAK-STAT pathway associated STATs, GRB2 and PIK3 genes, as well as AKT pathway-coupled NFKBIA and YWHAH genes.

The JAK2V617F mutation has been identified in most patients with myeloproliferative neoplasms (MPNs), including polycythemia vera, essential thrombocythemia and primary myelofibrosis. Although JAK2V617F is the predominant allele associated with MPNs, other activating Janus kinase 2 (JAK2) alleles (such as K539L, T875N) also have been identified in distinct MPNs. The basis for the differences in the in vivo effects of different JAK2 alleles remains unclear. We have characterized three different classes of disease-associated JAK2 mutants (JAK2V617F, JAK2K539L and JAK2T875N) and found significant differences in biochemical, signaling and transforming properties among these different classes of JAK2 mutants., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

IFNγ exhibits potent antitumor effects and plays important roles in the innate immunity against cancer. However, the mechanisms accounting for the antiproliferative effects of IFNγ still remain to be elucidated. We examined the role of Mnk1 (MAPK-interacting protein kinase 1) in IFNγ signaling. Our data demonstrate that IFNγ treatment of sensitive cells results in engagement of Mnk1, activation of its kinase domain, and downstream phosphorylation of the cap-binding protein eIF4E on Ser-209. Such engagement of Mnk1 plays an important role in IFNγ-induced IRF-1 (IFN regulatory factor 1) gene mRNA translation/protein expression and is essential for generation of antiproliferative responses. In studies aimed to determine the role of Mnk1 in the induction of the suppressive effects of IFNs on primitive hematopoietic progenitors, we found that siRNA-mediated Mnk1/2 knockdown results in partial reversal of the suppressive effects of IFNγ on human CD34+-derived myeloid (CFU-GM) and erythroid (BFU-E) progenitors. These findings establish a key role for the Mnk/eIF4E pathway in the regulatory effects of IFNγ on normal hematopoiesis and identify Mnk kinases as important elements in the control of IFNγ-inducible ISG mRNA translation.

Pro-inflammatory cytokines like interferon-gamma (IFN-gamma) are considered to be important in the development of anaemia of chronic disease (ACD). Both, inhibitory and stimulatory activities of IFN-gamma on erythropoiesis have been observed in vitro earlier. IFN-gamma induces several biochemical pathways in human monocytes, among them neopterin formation by GTP-cyclohydrolase I (GTP-CH I) and tryptophan degradation by the enzyme indoleamine 2,3-dioxygenase (IDO). IDO-mediated tryptophan deprivation efficiently inhibits the growth of proliferating cells and microbes, thus we wanted to examine whether enhanced tryptophan degradation by monocytic precursor cells also suppresses erythropoiesis. Therefore, IFN-gamma-mediated pathways were investigated in human CD34(+) progenitor cells, and effects of IFN-gamma on the proliferative activity of different progenitor subpopulations were studied. Cells were either cultivated in agar-conditioned medium (ACM) or in medium containing erythroid growth factors interleukin-3 (IL-3) and stem cell factor (SCF; EGFCM). Stimulation of CD34(+) cells with IFN-gamma in different doses (either 5000U/ml once or 200 and 400U/ml every other day) induced tryptophan degradation and in parallel also neopterin formation. Unstimulated cells cultured with ACM produced higher amounts of neopterin and kynurenine (all p<0.05). IFN-gamma stimulated higher kynurenine and neopterin formation in cells cultivated in EGFCM, stimulation with 400U IFN-gamma every other day was most effective. IFN-gamma stimulated the growth and proliferation of CFU-E and BFU-E (3-8) in both media. In conclusion, stimulation of haematopoietic stem cells with IFN-gamma activates IDO and neopterin formation, and it also exerts an influence on the proliferation of various stem cell populations., (Copyright 2009 Elsevier GmbH. All rights reserved.)

Severe malarial anemia is the most common syndrome of severe malaria in endemic areas. The pathophysiology of chronic malaria is characterised by a striking degree of abnormal development of erythroid precursors (dyserythropoiesis) and an inadequate erythropoietic response in spite of elevated levels of erythropoietin. The cause of dyserythropoiesis is unclear although it has been suggested that bone-marrow macrophages release cytokines, chemokines or lipo-peroxides after exposure to hemozoin, a crystalloid form of undigested heme moieties from malarial infected erythrocytes, and so inhibit erythropoiesis. However, we have previously shown that hemozoin may directly inhibit erythroid development in vitro and the levels of hemozoin in plasma from patients with malarial anemia and hemozoin within the bone marrow was associated with reduced reticulocyte response. We hypothesized that macrophages may reduce, not enhance, the inhibitory effect of hemozoin on erythropoiesis. In an in vitro model of erythropoiesis, we now show that inhibition of erythroid cell development by hemozoin isolated from P. falciparum is characterised by delayed expression of the erythroid markers and increased apoptosis of progenitor cells. Crucially, macrophages appear to protect erythroid cells from hemozoin, consistent with a direct contribution of hemozoin to the depression of reticulocyte output from the bone marrow in children with malarial anemia. Moreover, hemozoin isolated from P. falciparum in vitro inhibits erythroid development independently of inflammatory mediators by inducing apoptotic pathways that not only involve activation of caspase 8 and cleavage of caspase 3 but also loss of mitochondrial potential. Taken together these data are consistent with a direct effect of hemozoin in inducing apoptosis in developing erythroid cells in malarial anemia. Accumulation of hemozoin in the bone marrow could therefore result in inadequate reticulocytosis in children that have adequate levels of circulating erythropoietin.

Cell fate decisions are regulated by the coordinated activation of signalling pathways such as the extracellular signal-regulated kinase (ERK) cascade, but contributions of individual kinase isoforms are mostly unknown. By combining quantitative data from erythropoietin-induced pathway activation in primary erythroid progenitor (colony-forming unit erythroid stage, CFU-E) cells with mathematical modelling, we predicted and experimentally confirmed a distributive ERK phosphorylation mechanism in CFU-E cells. Model analysis showed bow-tie-shaped signal processing and inherently transient signalling for cytokine-induced ERK signalling. Sensitivity analysis predicted that, through a feedback-mediated process, increasing one ERK isoform reduces activation of the other isoform, which was verified by protein over-expression. We calculated ERK activation for biochemically not addressable but physiologically relevant ligand concentrations showing that double-phosphorylated ERK1 attenuates proliferation beyond a certain activation level, whereas activated ERK2 enhances proliferation with saturation kinetics. Thus, we provide a quantitative link between earlier unobservable signalling dynamics and cell fate decisions.

The daily production of 200 billion erythrocytes requires 20 mg of iron, accounting for nearly 80% of the iron demand in humans. Thus, erythroid precursor cells possess an efficient mechanism for iron uptake in which iron loaded transferrin (Tf) binds to the transferrin receptor (TfR) at the cell surface. The Tf:TfR complex then enters the endosome via receptor-mediated endocytosis. Upon endosomal acidification, iron is released from Tf, reduced to Fe(2+) by Steap3, and transported across the endosomal membrane by divalent metal iron transporter 1. Steap3, the major ferrireductase in erythrocyte endosomes, is a member of a unique family of reductases. Steap3 is comprised of an N-terminal cytosolic oxidoreductase domain and a C-terminal heme-containing transmembrane domain. Cytosolic NADPH and a flavin are predicted cofactors, but the NADPH/flavin binding domain differs significantly from those in other eukaryotic reductases. Instead, Steap3 shows remarkable, although limited homology to FNO, an archaeal oxidoreductase. We have determined the crystal structure of the human Steap3 oxidoreductase domain in the absence and presence of NADPH. The structure reveals an FNO-like domain with an unexpected dimer interface and substrate binding sites that are well positioned to direct electron transfer from the cytosol to a heme moiety predicted to be fixed within the transmembrane domain. Here, we discuss possible gating mechanisms for electron transfer across the endosomal membrane.

Polycythemia Vera (PV) is a myeloproliferative disorder (MPD) that is commonly characterized by mutant JAK2 (JAK2V617F) signaling, erythrocyte overproduction, and a propensity for thrombosis, progression to myelofibrosis, or acute leukemia. In this study, JAK2V617F expression by human hematopoietic progenitors promoted erythroid colony formation and erythroid engraftment in a bioluminescent xenogeneic immunocompromised mouse transplantation model. A selective JAK2 inhibitor, TG101348 (300 nM), significantly inhibited JAK2V617F+ progenitor-derived colony formation as well as engraftment (120 mg/kg) in xenogeneic transplantation studies. TG101348 treatment decreased GATA-1 expression, which is associated with erythroid-skewing of JAK2V617F+ progenitor differentiation, and inhibited STAT5 as well as GATA S310 phosphorylation. Thus, TG101348 may be an effective inhibitor of JAK2V617F+ MPDs in clinical trials.

K562 erythroleukemia cells have been widely used as a model for the study of globin gene regulation. A number of agents have been shown to activate or suppress globin gene expression in these cells. However, the molecular effects of these agents on the epigenetic configuration of the alpha- and gamma-globin genes that encode HbF are not known. In this report, we investigated the relationship between globin expression and histone acetylation of the human alpha- and beta-globin clusters in the fetal erythroid environment of K562 cells. Our studies suggest that acetylation of histone H3 may be important in regulating developmental stage-specific expression of the different beta-like globin genes while acetylation of both histones H3 and H4 may be important for the regulation of tissue-specific expression of these genes. In contrast, acetylation of both histones H3 and H4 at the alpha-like globin promoters appears to be important for both developmental stage- and tissue-specific expression. Interestingly, butyrate-induced activation of alpha-globin gene expression in K562 cells is associated with significant increase in histone acetylation levels while TPA-induced inhibition is associated with decreased histone acetylation at its promoters. In contrast, changes in histone acetylation and DNA methylation do not appear to be important in the regulation of gamma-globin gene expression by the same agents. These data suggest that the butyrate-mediated induction of the fetal gamma-globin genes in K562 cells is not a direct result of its histone deacetylase inhibitor activity of butyrate on the chromatin of the gamma-globin promoters, while the induction of the alpha-globin genes could be a result of a direct effect of butyrate on chromatin at its promoters. This is another example of the important differences in the molecular mechanisms of regulation of the genes of the alpha- and beta-like globin clusters.

Several cysteine proteases of the caspase family play a central role in many forms of cell death by apoptosis. Other enzymes of the family are involved in cytokine maturation along inflammatory response. In recent years, several caspases involved in cell death were shown to play a role in other cellular processes such as proliferation and differentiation. In the present review, we summarize the current knowledge of the role of caspases in the differentiation of erythroid cells and macrophages. Based on these two examples, we show that the nature of involved enzymes, the pathways leading to their activation in response to specific growth factors, and the specificity of the target proteins that are cleaved by the activated enzymes strongly differ from one cell type to another. Deregulation of these pathways is thought to play a role in the pathophysiology of low-grade myelodysplastic syndromes, characterized by excessive activation of caspases and erythroid precursor apoptosis, and that of chronic myelomonocytic leukemia, characterized by a defective activation of caspases in monocytes exposed to M-CSF, which blocks their differentiation.

The Rho GTPase Cdc42 regulates adhesion, migration, and homing, as well as cell cycle progression, of hematopoietic stem cells, but its role in multilineage blood development remains unclear. We report here that inducible deletion of cdc42 in cdc42-floxed mouse bone marrow by the interferon-responsive, Mx1-Cre-mediated excision led to myeloid and erythroid developmental defects. Cdc42 deletion affected the number of early myeloid progenitors while suppressing erythroid differentiation. Cdc42-deficient mice developed a fatal myeloproliferative disorder manifested by significant leukocytosis with neutrophilia, myeloid hyperproliferation, and myeloid cell infiltration into distal organs. Concurrently, Cdc42 deficiency caused anemia and splenomegaly accompanied with decreased bone marrow erythroid burst-forming units (BFU-Es) and colony-forming units-erythroid (CFU-Es) activities and reduced immature erythroid progenitors, suggesting that Cdc42 deficiency causes a block in the early stage of erythropoiesis. Cdc42 activity is responsive to stimulation by SCF, IL3, SDF-1alpha, and fibronectin. The increased myelopoiesis and decreased erythropoiesis of the knockout mice are associated with an altered gene transcription program in hematopoietic progenitors, including up-regulation of promyeloid genes such as PU.1, C/EBP1alpha, and Gfi-1 in the common myeloid progenitors and granulocyte-macrophage progenitors and down-regulation of proerythroid gene such as GATA-2 in the megakaryocyte-erythroid progenitors. Thus, Cdc42 is an essential regulator of the balance between myelopoiesis and erythropoiesis.

Two fast migrating, major, multiple molecular forms (MMF) of glucose-6-phosphate dehydrogenase [EC:1.1.1.49]: G-6-PDH-1 and G-6-PDH-2, and two minor forms: G-6-PDH-3 and G-6-PDH-4 were revealed in the electrophoregrams of both erythrocytes haemolisates as well in the homogenates of bone marrow cellular lines of rats at control conditions. Daily 1 cGy irradiation of rats up to a cumulative dose of 20 cGy led to a drop of G-6-PDH total activity and it caused a redistribution of the MMF of the enzyme in bone marrow cellular populations. However, G-6-PDH activity in erythrocytes exceeded the control means in all the experimental terms. The calculation of the local redistribution coefficient (l(G-6-FDH-i)) showed that these changes are mainly determined by the increase of the activity of the isoform G-6-PDH-3. Vitamin D3 administration to rats generated a correction of G-6-PDH activity in all studied cellular populations. Meanwhile, the MMF profiles were characterized by multidirectional rearrangements in the bone marrow erythroid and granulocyte-monocyte cells and in erythrocytes. The specificity of changes in the distribution of the MMF of G-6-PDH in the three studied cellular populations depends on the particularities of their energetic metabolism at irradiation conditions and on the modifying action of the natural adaptogen 1,25-dihydroxicholecalciferol.

Recently, a unique recurrent somatic mutation was identified as a major molecular event in polycythemia vera, essential thrombocythemia and idiopathic myelofibrosis. Expression of this mutant in cytokine-dependent hematopoietic cell lines induces autonomous growth. This effect is enhanced by overexpression of cytokine receptors, and can be inhibited by co-expression at higher levels of the wild type JAK2, which may compete for a limited pool of receptors. In JAK2-deficient cells, we showed that JAK2 V617F can transmit signals from ligand-activated TpoR or EpoR. Furthermore, the mutant JAK2 can be demonstrated to stimulate traffic of the EpoR. Thus, JAK2 V617F mutant must be able to interact via its intact FERM-SH2 domains with the cytosolic domains of cytokine receptors. A synergy between JAK2 V617F and insulin-like growth factor 1 receptor (IGF1R) can be detected in cytokine-dependent cell proliferation. Once cells are rendered autonomous by expression of JAK2 V617F, IGF1 acquires the ability to activate the JAK-STAT pathway. Thus, expression of JAK2 V617F may explain the described hypersensitivity of PV erythroid progenitors to IGF1. The V617 is conserved in two other mammalian JAKs, JAK1 and Tyk2. The homologous mutants JAK1 V658F and Tyk2 V678F are also active in proliferation and transcriptional assays. Such mutants may be found in human cancers or autoimmune diseases. In contrast, the JAK3 M592F does not lead to activation of JAK3. Current hypotheses on how JAK2 V617F contributes to three myeloproliferative diseases, and which other events may favor one disease versus another, are discussed.

In this work the attempt to estimate a nitric oxide (NO*) role in regulation of the number of pool haemopoietic stem cells at the irradiated mice was made. With this purpose the number of new compounds from dihydrothiazine-thiazoline line was synthesized, their NO-inhibiting activity was investigated in vivo by the method of ESR-spectroscopy of spin trap and their influence on an output endogenous spleen colonies (CFU-S-8) after the total sublethal y-irradiation of mice in a doze of 6 Gy was also investigated. Was shown, that the tested compounds reduced the contents of NO* in a liver tissue of mice which have received an injection of nitric oxide synthesis inductor - lipopolysaccharide, and also increased an output CFU-S-8 forming endogenous colonies in the spleen of the irradiated mice. Received data testify to perceptivity of search radioprotective agents among NO* synthesis inhibitors.

Glycosphingolipids and glycoproteins play pivotal roles in the complex series of events governing cell adhesion and signal transduction. Aberrant glycosilation, typical of tumor cells, represents a key event in the induction of invasion and metastasis. Sialidases remove sialic acid residues from sialoconjugates and, in mammals, these enzymes have been proved to be involved in several cellular phenomena, including cell proliferation and differentiation, membrane function, and malignant transformation. Herein we show that only the lysosomal sialidase Neu1 and the plasma membrane-associated sialidase Neu3 are expressed in CFU-E erythroid precursors and K562 erythroleukemic cells. Tumour cells show much higher expression levels than CFU-E cells and, during differentiation, the content of the two enzymes progressively decreases. The sialoglycoconjugate pattern is different in the two cell types. In fact, the differentiating erythroid precursors show an increase of the typical erythrocyte sphingolipids, whereas K562 cells treated with butyrate show a marked increase of GD1a, GM2, PE, and ceramide. Finally, during differentiation the sialoglycoprotein content of erythroid cells shows a marked increase, and in K562 cells the process induces the synthesis of some sialoglycoprotein typical of the erythroid membrane. Overall, these results point out the great differences in sialoglycoconjugate and sialidase patterns exhibited by normal and tumour cells.

Acute myeloid leukemia (AML) is a clonal disease characterized by heterogeneous involvement of hematopoietic stem cell/progenitor cell populations. Using FLT3 internal tandem duplication (FLT3/ITD) as a molecular marker, we tested the hypothesis that clinical outcome in AML correlates with disease involvement of CD34(+)/CD33(-) precursors. Diagnostic specimens from 24 children with FLT3/ITD-positive AML were sorted by fluorescence-activated cell sorting (FACS), and resultant CD34(+)/CD33(-) and CD34(+)/CD33(+) progenitors were analyzed directly and after colony-forming cell (CFC) assay for the presence of FLT3/ITD. FLT3/ITD was present in all CD34(+)/CD33(+) patient samples. In contrast, FLT3/ITD was detected in CD34(+)/CD33(-) progenitors in only 19 of 24 samples. A bipotent progenitor was affected in a subset of patients, as evidenced by the presence of FLT3/ITD in both granulocyte-macrophage colony-forming unit (CFU-GM) and erythroid burst-forming unit (BFU-E) colonies. Those patients in whom CD34(+)/CD33(-) precursors harbored the FLT3/ITD had worse clinical outcome; actuarial event-free survival (EFS) at 4 years from study entry for those patients with and without FLT3/ITD detection in CD34(+)/CD33(-) progenitors was 11% +/- 14% versus 100% +/- 0%, respectively (P = .002). This study suggests that FLT3/ITD involvement in CD34(+)/CD33(-) precursors is heterogeneous and that detection of the mutation in the less-mature progenitor population may be associated with disease resistance.

Although a large proportion of patients with polycythemia vera (PV) harbor a valine-to-phenylalanine mutation at amino acid 617 (V617F) in the JAK2 signaling molecule, the stage of hematopoiesis at which the mutation arises is unknown. Here we isolated and characterized hematopoietic stem cells (HSC) and myeloid progenitors from 16 PV patient samples and 14 normal individuals, testing whether the JAK2 mutation could be found at the level of stem or progenitor cells and whether the JAK2 V617F-positive cells had altered differentiation potential. In all PV samples analyzed, there were increased numbers of cells with a HSC phenotype (CD34+CD38-CD90+Lin-) compared with normal samples. Hematopoietic progenitor assays demonstrated that the differentiation potential of PV was already skewed toward the erythroid lineage at the HSC level. The JAK2 V617F mutation was detectable within HSC and their progeny in PV. Moreover, the aberrant erythroid potential of PV HSC was potently inhibited with a JAK2 inhibitor, AG490.

The deficiency of methylthioadenosine phosphorylase (MTAP) in T-cell acute lymphoblastic leukemia (T-ALL) and other cancers, while constitutively expressed in normal cells, allows for selective therapy using L-alanosine, an inhibitor of de novo AMP synthesis. We demonstrate that MTAP- T-ALL cells obtained at relapse are as sensitive to L-alanosine toxicity as diagnosis samples. The therapeutic index of L-alanosine can be increased by the use of a MTAP substrate, which protects MTAP+ normal cells. Since MTAP substrates MTA and 5'deoxyadenosine are prone to toxicities associated with adenosine, we synthesized and evaluated a potentially nontoxic MTAP substrate, 9-beta-D-erythrofuranosyladenine (EFA). The cytotoxicity of EFA to hematopoietic progenitors erythroid burst-forming units (BFU-Es) and granulocyte-macrophage colony-forming units (CFU-GMs) was at least 26- to 41-fold less than that of MTA. In addition, EFA selectively rescued MTAP+ MOLT-4 cells from L-alanosine toxicity at 25 microM with negligible toxicity even at 100 microM. As for MTA, significant, albeit incomplete, rescue was achieved at 12.5 microM, but higher concentrations were toxic. EFA at 20 microM or less rescued primary MTAP+ T-ALL cells and normal lymphocytes from L-alanosine toxicity. Collectively, these data indicate that EFA is an effective agent for salvaging MTAP+ cells from L-alanosine toxicity and is superior to MTA due to lower cytotoxicity.

Erythroid differentiation involves the transcription factor GATA-1 that positively regulates promoters of erythroid genes (including haemoglobin, glycophorin, erythropoietin receptor) and of erythropoietin. Terminal erythroid differentiation is characterized by major morphological changes that include chromatin condensation and cell size reduction. The morphological changes are partially similar at least to those observed during apoptosis. The production of red cells depends on the apoptosis rate of erythroid progenitors and precursors. Upon erythropoietin starvation or engagement of the death receptor Fas, caspases are activated in erythroid precursors and cleave GATA-1, thus inducing maturation arrest and apoptosis of immature erythroblasts. We have recently demonstrated that, upon erythropoietin stimulation, caspase-3 was also activated, an event required for human terminal erythroblast maturation. Proteins cleaved by caspases in erythroid cells undergoing terminal differentiation include Lamin B and Acinus, which are involved in chromatin condensation. In contrast, despite caspase-3 activation neither GATA-1 degradation nor apoptosis was observed. Thus, the fate of erythroid precursors is determined downstream of caspase activation by the pattern of cleaved targets. Therefore, there are some mechanisms underlying the selective protection of caspase-3 targets during erythropoiesis. This model in which caspases activation is required for differentiation may apply to other haematopoietic or non haematopoietic cellular systems which are described in this review.

The molecular mechanism of anemia that is hyporesponsive to recombinant human erythropoietin (rHuEPO) in hemodialysis patients without underlying causative factors has not been investigated fully in hematopoietic stem cell system. Circulating CD34+ cells (1 x 10(4)) were isolated from rHuEPO hyporesponsive hemodialysis patients (EPO-H; n = 9), patients who were responsive to rHuEPO (EPO-R; n = 9), and healthy control subjects (n = 9). The patients with known causes of EPO hyporesponsiveness were eliminated from the current study. The cells were cultured in STEM PRO 34 liquid medium, supplemented with rHuEPO, IL-3, stem cell factor, and granulocyte-macrophage colony stimulating factor for 7 d and then transferred to a semisolid methylcellulose culture medium for performing burst forming unit-erythroid (BFU-E) colony assay. Expression of src homology domain 2 (SH2)-containing tyrosine phosphatase-1 (SHP-1), phosphorylated Janus kinase 2 (p-JAK2), and phosphorylated signal transducer and activator of transcription 5 (p-STAT5) was assessed with Western blot analysis. In EPO-H patients, SHP-1 antisense or scrambled S-oligos were included in the culture medium, and its effects were evaluated. The number of circulating CD34+ cells was not statistically different among the three groups, and their proliferation rates were similar for 7 d in culture. However, BFU-E colonies were significantly decreased in EPO-H patients compared with EPO-R and control groups. The mRNA and protein expression of SHP-1 and p-SHP-1 was significantly increased, whereas that of p-STAT5 was reduced in EPO-H patients. The inclusion of SHP-1 antisense S-oligo in culture suppressed SHP-1 protein expression associated with p-STAT5 upregulation, increase in p-STAT5-regulated genes, and partial recovery of BFU-E colonies. In EPO-H hemodialysis patients, the EPO signaling pathway is attenuated as a result of dephosphorylation of STAT5 via upregulation of SHP-1 phosphatase activity, and SHP-1 may be a novel target molecule to sensitize EPO action in these patients.

Objective: A substantial body of evidence implicates the cytoplasmic protein tyrosine kinase Fps/Fes in regulation of myeloid differentiation and survival. In this study we wished to determine if Fps/Fes also plays a role in the regulation of erythropoiesis., Methods: Mice tissue-specifically expressing a "gain-of-function" mutant fps/fes transgene (fps(MF)) encoding an activated variant of Fps/Fes (MFps), were used to explore the in vivo biological role of Fps/Fes. Erythropoiesis in these mice was assessed by hematological analysis, lineage marker analysis, bone-marrow colony assays, and biochemical approaches., Results: fps(MF) mice displayed reductions in peripheral red cell counts. However, there was an accumulation of immature erythroid precursors, which displayed increased survival. Fps/Fes and the related Fer kinase were both detected in early erythroid progenitors/blasts and in mature red cells. Fps/Fes was also activated in response to erythropoietin (EPO) and stem cell factor (SCF), two critical factors in erythroid development. In addition, increased Stat5A/B activation and reduced Erk1/2 phosphorylation was observed in fps(MF) primary erythroid cells in response to EPO or SCF, respectively., Conclusions: These data support a role for Fps/Fes in regulating the survival and differentiation of erythroid cells through modulation of Stat5A/B and Erk kinase pathways induced by EPO and SCF. The increased numbers and survival of erythroid progenitors from fps(MF) mice, and their differential responsiveness to SCF and EPO, implicates Fps/Fes in the commitment of multilineage progenitors to the erythroid lineage. The anemic phenotype in fps(MF) mice suggests that downregulation of Fps/Fes activity might be required for terminal erythroid differentiation.

Oncogenic mutations in ras genes frequently occur in patients with myeloid disorders, and in these patients erythropoiesis is often affected. Previously, we showed that expression of oncogenic H-ras in purified mouse primary fetal liver erythroid progenitors blocks terminal erythroid differentiation and supports erythropoietin (Epo)-independent proliferation. As a first step in understanding the underlying molecular mechanisms we examined the signaling pathways downstream of Ras in primary erythroid cells. We found that 3 major pathways are abnormally activated by oncogenic H-ras: Raf/ERK (extracellular signal-regulated kinase), phosphatidyl inositol 3 (PI3)-kinase/Akt, and RalGEF/RalA. However, only constitutive activation of the MEK (MAPK [mitogen-activated protein kinase]/ERK kinase)/ERK pathway alone could recapitulate all of the effects of oncogenic H-ras expression in blocking erythroid differentiation and inducing Epo-independent proliferation. Although expression of a constitutively active Akt kinase (ca.Akt) in erythroid progenitors does not significantly affect erythroid differentiation in the presence of Epo, coexpression of ca.Akt together with a constitutively active MEK causes prolonged Epo-independent proliferation of erythroid progenitors in addition to a block in differentiation. Moreover, the effects of oncogenic H-ras expression on primary erythroid cells are blocked by the addition of U0126, a specific inhibitor of MEK1 and MEK2, allowing normal terminal erythroid proliferation and differentiation. Our data suggest that the interruption of constitutive MEK/ERK signaling is a potential therapeutic strategy to correct impaired erythroid differentiation in patients with myeloid disorders.

Background: Erythropoietin is a multifunctional cytokine which regulates the number of erythrocytes circulating in mammalian blood. This is crucial in order to maintain an appropriate oxygen supply throughout the body. Stimulation of primary human erythroid progenitors (PEPs) with erythropoietin (Epo) leads to the activation of the mitogenic kinases (MEKs and Erks). How this is accomplished mechanistically remained unclear., Results: Biochemical studies with human cord blood-derived PEPs now show that Ras and the class Ib enzyme of the phosphatidylinositol-3 kinase (PI3K) family, PI3K gamma, are activated in response to minimal Epo concentrations. Surprisingly, three structurally different PI3K inhibitors block Ras, MEK and Erk activation in PEPs by Epo. Furthermore, Erk activation in PEPs is insensitive to the inhibition of Raf kinases but suppressed upon PKC inhibition. In contrast, Erk activation induced by stem cell factor, which activates c-Kit in the same cells, is sensitive to Raf inhibition and insensitive to PI3K and PKC inhibitors., Conclusions: These unexpected findings contrast with previous results in human primary cells using Epo at supraphysiological concentrations and open new doors to eventually understanding how low Epo concentrations mediate the moderate proliferation of erythroid progenitors under homeostatic blood oxygen levels. They indicate that the basal activation of MEKs and Erks in PEPs by minimal concentrations of Epo does not occur through the classical cascade Shc/Grb2/Sos/Ras/Raf/MEK/Erk. Instead, MEKs and Erks are signal mediators of PI3K, probably the recently described PI3K gamma, through a Raf-independent signaling pathway which requires PKC activity. It is likely that higher concentrations of Epo that are induced by hypoxia, for example, following blood loss, lead to additional mitogenic signals which greatly accelerate erythroid progenitor proliferation.

p38alpha, p38beta, p38gamma, and p38delta are four isoforms of p38 mitogen-activated protein (MAP) kinase (MAPK) involved in multiple cellular functions such as cell proliferation, differentiation, apoptosis, and inflammation response. In the present study, we examined the mRNA expression pattern of each of the four isoforms during erythroid differentiation of primary erythroid progenitors. We show that p38alpha and p38gamma transcripts are expressed in early hematopoietic progenitors as well as in late differentiating erythroblasts, whereas p38delta mRNA is only expressed and active during the terminal phase of erythroid differentiation. On the other hand, p38beta is minimally expressed in early CD34(+) hematopoietic progenitors but not expressed in lineage-committed erythroid progenitors. We also determined the phosphorylation/activation of p38alpha, MAPK kinase 3/6, and MAPKAP-2 in response to erythropoietin and stem cell factor. We found that phosphorylation of p38alpha, MAPK kinase kinase 3/6 and MAPKAP-2 occurs only upon growth factor withdrawal in primary erythroid progenitors. Moreover, our data indicate that activation of p38alpha does not induce apoptosis or promote proliferation of erythroid progenitors. On the other hand, under steady-state culture conditions, both p38alpha and p38delta isoforms are increasingly phosphorylated activated in the terminal phase of differentiation. This increased phosphorylation/activity was accompanied by up-regulation of heat shock protein 27 phosphorylation. Finally, we demonstrate that tumor necrosis factor alpha, an inflammatory cytokine that is modulated by p38alpha, is expressed by differentiating erythroblasts and inhibition of p38alpha or tumor necrosis factor alpha results in reduction in differentiation. Taken together, our data demonstrate that both p38alpha and delta isoforms function to promote the late-stage differentiation of primary erythroid progenitors and are likely to be involved in functions related to erythrocyte membrane remodeling and enucleation.

Polycythemia vera (PV) is a human clonal hematologic disorder. Previously we demonstrated that erythroid colony-forming cells (ECFCs) from PV patients contained a hyperactive membrane-associated tyrosine phosphatase. We now show that this phosphatase corresponded to protein tyrosine phosphatase (PTP)-MEG2, an intracellular enzyme with a putative lipid-binding domain. The increased activity of PTP-MEG2 in PV cells is due to its elevated distribution in the membrane fraction. With the development of ECFCs to mature red cells, the protein level of PTP-MEG2 decreased gradually, but membrane-associated PTP-MEG2 was sustained for a longer period of time in PV cells, which correlated with an enhanced colony-forming capability of the cells. Importantly, expression of dominant-negative mutant forms of PTP-MEG2 suppressed in vitro growth and expansion of both normal and PV ECFCs. The data indicate that PTP-MEG2 has an important role in the development of erythroid cells.

It is proposed that a locally active, intrinsic renin-angiotensin system (RAS) exists in the bone marrow (BM) and plays a role in regulating haematopoiesis. Angiotensin II type I receptor has been detected on erythroid burst-forming unit-derived cells; its antagonist losartan and angiotensin I-converting enzyme (ACE) inhibitors can suppress erythropoiesis. The possible role of ACE/RAS in BM was investigated by evaluating ACE expression in normal BM, several myeloproliferative disorders and myelodysplasia. Immunohistochemical studies showed that erythroid elements expressed ACE protein in both normal and disturbed haematopoiesis. The presence of ACE in erythroid cells suggests another mechanism for direct ACE inhibitor activity in erythropoiesis.

Evidence of increased oxidative stress is a hallmark of many chronic diseases associated with anemia. The current study was undertaken to evaluate the effects of oxidative stress on erythroid colony formation in vitro by bone marrow light density mononuclear cells (LDMN) and by peripheral blood derived cells enriched for erythroid colony forming units (CFU-E), and how these effects can be modified by a cytokine implicated in the anemia of chronic disease. When blood-derived and marrow cells were cultured with 50 microM H(2)O(2), CFU-E colony formation by blood-derived cells but not by marrow cells was significantly inhibited, suggesting a protective effect of marrow accessory cells. This inhibitory effect on peripheral blood-derived CFU-E was shown to be caspase-dependent. rhgammaIFN at concentrations which did not inhibit CFU-E colony formation sensitized LDMN marrow cells to inhibition by H(2)O(2). Exposure of LDMN marrow cells to rhgammaIFN at concentrations of 10 U/mL or higher significantly decreased the concentration of thioredoxin (Trx) in cell supernatant. Addition of recombinant Trx to LDMN marrow cells cultured with H(2)O(2) and rhgammaIFN partially (although not completely) reversed inhibition of CFU-E colony formation. These findings suggest that inflammatory cytokines implicated in the pathogenesis of the anemia of chronic disease may exert their effects at least in part through modulation of oxidative stress.

The production of red blood cells is tightly regulated by erythropoietin (Epo). The phosphoinositide 3-kinase (PI 3-kinase) pathway was previously shown to be activated in response to Epo. We studied the role of this pathway in the control of Epo-induced survival and proliferation of primary human erythroid progenitors. We show that phosphoinositide 3 (PI 3)-kinase associates with 4 tyrosine-phosphorylated proteins in primary human erythroid progenitors, namely insulin receptor substrate-2 (IRS2), Src homology 2 domain-containing inositol 5'-phosphatase (SHIP), Grb2-associated binder-1 (Gab1), and the Epo receptor (EpoR). Using different in vitro systems, we demonstrate that 3 alternative pathways independently lead to Epo-induced activation of PI 3-kinase and phosphorylation of its downstream effectors, Akt, FKHRL1, and P70S6 kinase: through direct association of PI 3-kinase with the last tyrosine residue (Tyr479) of the Epo receptor (EpoR), through recruitment and phosphorylation of Gab proteins via either Tyr343 or Tyr401 of the EpoR, or through phosphorylation of IRS2 adaptor protein. The mitogen-activated protein (MAP) kinase pathway was also activated by Epo in erythroid progenitors, but we found that this process is independent of PI 3-kinase activation. In erythroid progenitors, the functional role of PI 3-kinase was both to prevent apoptosis and to stimulate cell proliferation in response to Epo stimulation. Finally, our results show that PI 3-kinase-mediated proliferation of erythroid progenitors in response to Epo occurs mainly through modulation of the E3 ligase SCF(SKP2), which, in turn, down-regulates p27(Kip1) cyclin-dependent kinase (CDK) inhibitor via proteasome degradation.

Erythropoietin (EPO) can rescue erythroid cells from apoptosis during erythroid development, leading to red cell production. However, the detailed mechanism of how EPO protects erythroid cells from apoptosis is still open to question. To address this problem, we used a human EPO-dependent leukemia cell line UT-7/EPO and normal erythroid progenitor cells. After deprivation of EPO, UT-7/EPO cells underwent apoptosis, accompanied by down-regulation of the Bcl-xL protein. In addition, the cleaved products of caspase-3, p11 and p21, and a few cleaved forms of inhibitor of caspase-activated DNase (ICAD) were detected in these cells. When the cells were pre-treated with the pancaspase inhibitor Z-VAD-FMK, the ratio of apoptotic cells was significantly reduced, suggesting that EPO protects the UT-7/EPO cells from apoptosis via inhibition of caspase activities. When an MEK 1/2 inhibitor U0126 inhibited activities of extracellular signal-regulated kinases (ERKs), the expression of Bcl-xL protein was down-regulated and subsequently apoptosis was induced. Interestingly, Z-VAD-FMK blocked U0126-induced down-regulation of Bcl-xL protein and apoptosis, strongly suggesting that Bcl-xL expression is regulated by caspases which lies downstream of ERK activation pathway in EPO signaling. Importantly, these findings were also observed in normal erythroid progenitor cells. In conclusion, the activation of ERKs by EPO up-regulates Bcl-xL expression via inhibition of caspase activities, resulting in the protection of erythroid cells from apoptosis., (Copyright 2003 Wiley-Liss, Inc.)

5-Aminolevulinic acid synthase (ALAS), the first enzyme of the heme biosynthesis pathway, catalyses the pyridoxal 5'-phosphate-dependent condensation between glycine and succinyl-CoA to yield 5-aminolevulinic acid (5-amino-4-oxopentanoate). A three-dimensional structural model of Rhodobacter spheroides ALAS has been constructed and used to identify amino acid residues at the active site that are likely to be important for the recognition of glycine, the only amino acid substrate. Several residues have been investigated by site-directed mutagenesis and enzyme variants have been generated that are able to use alanine, serine or threonine. A three-dimensional structure model of 5-aminolevulinic acid synthase from human erythrocytes (ALAS 2) has also been constructed and used to map a range of naturally occurring human mutants that give rise to X-linked sideroblastic anemia. A number of these anemias respond favourably to vitamin B(6) (pyridoxine) therapy, whereas others are either partially responsive or completely refractory. Detailed investigations with selected human mutants have highlighted the importance of arginine-517 that is implicated in glycine carboxyl group binding.

We have shown that Fv2, the Friend virus susceptibility 2 locus, encodes a naturally occurring amino-terminally truncated form of the STK receptor tyrosine kinase (Sf-Stk). Sf-Stk appears to interact with the viral glycoprotein gp55 and drive erythropoietin (Epo)-independent expansion of Friend virus-infected erythroblasts. Presumably, Sf-Stk provides signals that cooperate with EpoR signaling to induce the polyclonal expansion of infected cells. In this report, we show that macrophage-stimulating protein (MSP), the ligand for full-length STK, can also cooperate with Epo to enhance burst-forming units-erythroid (BFU-E) formation. To evaluate the signals induced by MSP/STK in primary erythroid progenitor cells, we adapted a method for the expansion of murine bone marrow mononuclear cells. The expanded progenitor cells express STK and respond to MSP in a colony assay. Furthermore, we demonstrate that low doses of MSP and Epo stimulation of the expanded cells cooperate to induce the phosphorylation of MAP kinase. Using the MEK inhibitor PD98059, we show that the activation of ERK is required for the enhanced BFU-E formation in response to MSP. These findings suggest that MSP has the ability to enhance erythroid colony formation in response to Epo, and that this response is dependent on the ability of MSP to induce the MAP kinase pathway.

There have been conflicting reports regarding the role of p38 mitogen-activated protein kinase (MAPK) in the regulation of differentiation, proliferation and apoptosis in erythroid cell lines. We have, therefore, examined the functions of this kinase in primary human erythroid progenitors. Cells in steady-state culture showed low-level p38 MAPK activity, which decreased further within 1 h of growth factor withdrawal and increased over a limited range within minutes of re-exposure of cells to erythropoietin or stem cell factor, demonstrating the link between low-level p38 MAPK activity and the prevailing growth factor milieu. Use of the p38 MAPK-specific inhibitor SB203580 demonstrated that this level of activity was necessary for (1) optimal proliferation, (2) erythroid burst-forming unit migration and (3) full upregulation of E-cadherin and CD36 expression, but not haemoglobin A or glycophorin A expression, during human erythroid differentiation. In contrast, cells deprived of growth factors for an 8-h period, following a transient decrease in p38 MAPK activity, demonstrated sustained, substantial and caspase-independent increases in p38 MAPK activity, and its blockade using SB203580 reduced the proportion of erythroblasts undergoing apoptosis by 40 +/- 7%, demonstrating a role for p38 MAPK in apoptosis induction in human erythroblasts. Thus, in primary human erythroblasts, different environmental conditions induce different levels of p38 MAPK activity, which have distinct functions.

We studied the effects of cyclosporin A (CsA) on the erythroid differentiation of human erythroid leukemia cell line K562. After K562 was treated with CsA for 4 days, the percentage of hemoglobinized cells was increased by 3.3 times. Because it was reported p38 MAPK (p38) and ERK are involved in erythropoietin-induced erythroid differentiation, we studied their roles using specific inhibitors. p38 inhibitor (SB203580) prevented CsA-induced hemoglobin synthesis in K562 cells, although MEK/ERK inhibitor (U0126) enhanced it by 3.3 times in K562 cells. These results indicate activation of p38 and inactivation of ERK are involved in CsA-induced erythroid differentiation of K562 cells., (Copyright 2002 Wiley-Liss, Inc.)

The Friend virus susceptibility gene 2 (Fv2) controls the polyclonal expansion of infected cells that occurs early during Friend erythroleukemia virus infection. Fv2 has recently been shown to encode a truncated form of the Stk receptor tyrosine kinase (Sf-Stk). This observation, coupled with earlier work, suggested that Sf-Stk drives the expansion of infected cells by forming a complex with the Friend virus envelope glycoprotein, gp55, and the erythropoietin receptor. Fv2 has also been implicated in the control of cell cycling in early erythroid progenitors (erythroid blast-forming units [BFU-Es]). Mouse strains that are homozygous for the resistant allele of Fv2 (Fv2(rr)) have few actively cycling BFU-Es. In this report, we demonstrate that the control of BFU-E cycling is encoded by a gene linked to, but distinct from, Fv2, and suggest that this gene is the dual-specific protein phosphatase Cdc25A, which regulates the G1- to S-phase transition of the cell cycle. We show that a naturally occurring allele of Cdc25A, which increases Cdc25A phosphatase activity and promotes cell-cycle progression, segregates in mouse strains that exhibit high levels of BFU-E cell cycling. In wild-type mice, this allele of Cdc25A does not overtly affect erythropoiesis; however, when this allele is combined with a mutation of the Kit receptor (Kit(WV)), the anemia of the mice is enhanced. Furthermore, overexpression of Cdc25A in bone marrow cells causes a defect in the BFU-E colony formation. These results suggest that proper regulation of the cell cycle through Cdc25A is required for normal erythropoiesis.

Previous analyses have shown that the phosphorylation state of chicken erythroid ankyrin regulates its association with the spectrin cytoskeleton in vivo. Treatment of erythroid cells with serine and threonine phosphatase inhibitors stimulates the hyperphosphorylation of ankyrin and its dissociation from spectrin. In this study, we demonstrate that a kinase that directs the phosphorylation of ankyrin in vivo coprecipitates with ankyrin-containing complexes and has properties identical to CK2. Studies using CK2-specific inhibitors have indicated that all of the phosphorylation events associated with erythroid ankyrin in vivo are CK2 dependent. Furthermore, inhibitor studies combined with in vitro binding analyses have indicated that the phosphorylation of erythroid ankyrin by CK2 regulates its ability to associate with spectrin. Additional analyses revealed that CK2 coprecipitates with ankyrin-3-containing complexes isolated from Madin Darby canine kidney epithelial cells and phosphorylates this epithelial ankyrin isoform in vivo. These results are the first demonstration of a kinase constitutively associating with the ankyrin-spectrin cytoskeleton in erythroid and kidney epithelial cells. This association provides a mechanism for rapidly reorganizing the membrane cytoskeleton in these cell types through the phosphorylation of ankyrin.

This study is aimed at identifying the Na pump isoform composition of human erythroid precursor cells and mature human erythrocytes. We used purified and synchronously growing human erythroid progenitor cells cultured for 7-14 days. RNA was extracted from the progenitor cells on different days and analyzed by RT-PCR. The results showed that only the alpha1, alpha3, beta2, and beta3 subunit isoforms and the gamma modulator were present. Northern analysis of the erythroid progenitor cells again showed that beta2 but not beta1 or alpha2 isoforms were present. The erythroid cells display a unique beta subunit expression profile (called beta-profiling) in that they contain the message for the beta2 isoform but not beta1, whereas leukocytes and platelets are known to have the message for the beta1 but not for the beta2 isoform. This finding is taken to indicate that our preparations are essentially purely erythroid and free from white cell contamination. Western analysis of these cultured progenitor cells confirmed the presence of alpha1, alpha3, (no alpha2), beta2, beta3, and gamma together now with clear evidence that beta1 protein was also present at all stages. Western analysis of the Na pump from mature human erythrocyte ghosts, purified by ouabain column chromatography, has also shown that alpha1, alpha3, beta1, beta2, beta3, and gamma are present. Thus, the Na pump isoform composition of human erythroid precursor cells and mature erythrocytes contains the alpha1 and alpha3 isoforms of the alpha subunit, the beta1, beta2, and beta3 isoforms of the beta subunit, and the gamma modulator.

Erythropoietin (Epo)-induced glycosylphosphatidylinositol (GPI) hydrolysis was previously described to be correlated with phospholipase C-gamma 2 (PLC-gamma2) activation. Here, we analyzed the involvement of phosphatidylinositol (PtdIns) 3-kinase in GPI hydrolysis through PLC-gamma2 tyrosine phosphorylation in response to Epo in FDC-P1 cells transfected with a wild type (WT) erythropoietin-receptor (Epo-R). We showed that phosphatidylinositol 3-kinase (PtdIns 3-kinase) inhibitor LY294002 inhibits Epo-induced hydrolysis of endogenous GPI and Epo-induced PLC-gamma2 tyrosine phosphorylation in a dose-dependent manner. Wortmannin, another PtdIns 3-kinase inhibitor, also suppressed Epo-induced PLC-gamma2 tyrosine phosphorylation. We also present evidence that PLC-gamma2 translocation to the membrane fraction on Epo stimulation is completely inhibited by LY294002. Upon Epo stimulation, the tyrosine-phosphorylated PLC-gamma2 was found to be associated with the tyrosine-phosphorylated Grb2-associated binder (GAB)2, SHC and SHP2 proteins. LY294002 cell preincubation did not affect GAB2, SHC and SHP2 tyrosine phosphorylation but inhibited the binding of PLC-gamma2 to GAB2 and SHP2. Taken together, these results show that PtdIns 3-kinase controls Epo-induced GPI hydrolysis through PLC-gamma2., (Copyright 2002 Elsevier Science Inc.)

To determine whether erythropoietin (EPO) affects haem biosynthesis and iron transport, we studied the effects of EPO on the expression of erythroid 5-aminolevulinate synthase (eALAS), ferrochelatase and divalent metal transporter 1 (DMT-1) in human erythroid progenitor cells, and in the murine and human erythroid cell lines MEL and K562. Cytoplasmic e-ALAS mRNA levels were significantly increased after incubation of cells with EPO for at least 24 h, which could be the result of a transcriptional mechanism. In contrast, ferrochelatase or DMT-1 mRNA expression were not affected. Moreover, EPO also increased e-ALAS enzyme activity after only 4 h of stimulation, when mRNA levels were unchanged. The underlying mechanism was an effect of EPO on e-ALAS mRNA translation, which was under the control of iron regulatory proteins (IRP) 1 and 2. Thereby, EPO weakened the binding affinity of IRP-2 to the iron responsive element (IRE) within e-ALAS mRNA which resulted in the increased expression of e-ALAS IRE-controlled reporter gene constructs, following EPO stimulation. Our results show that EPO directly affected haem biosynthesis by stimulating the transcriptional and post-transcriptional expression of the key enzyme e-ALAS. These data provide new insights into the complex biochemical interaction between iron metabolism, haem biosynthesis and EPO biology.

Objectives: Murine erythroblasts infected with the anemia-inducing strain of Friend virus (FVA cells) terminally differentiate to the reticulocyte stage after 48 hours of culture in vitro in response to erythropoietin (EPO). The objective of this study was to determine the possible role of proteasome-mediated proteolysis during the terminal differentiation of FVA cells., Materials and Methods: The proteasome inhibitors MG132 and lactacystin were used to perturb the normal function of proteasomes during terminal differentiation. Effects of proteasome inhibitors on terminal differentiation were quantitated by evaluation of cellular morphology after benzidine staining and by Western blot analyses., Results: Treatment of EPO-stimulated FVA cells with lactacystin or MG132 at later periods of culture increased accumulations of nuclear and cytosolic ubiquitinated proteins and decreased nuclear extrusion to less than 40% of controls., Conclusions: Our results suggest that the proteasomal degradation of ubiquitinated proteins plays an important role in the enucleation of mammalian erythroblasts.

D-3 phosphorylated inositides are a peculiar class of lipids, synthesized by phosphatidylinositol 3-kinase (PtdIns 3-K), which are also present in the nucleus. In order to clarify a possible role for nuclear D-3 phosphorylated inositides during human erythroid differentiation, we have examined the issue of whether or not, in K562 human erythroleukemia cells, erythropoietin (EPO) may generate nuclear translocation of an active PtdIns 3-K. Immunoprecipitation with an anti-p85 regulatory subunit of PtdIns 3-K, revealed that both the intranuclear amount and the activity of the kinase increased rapidly and transiently in response to EPO. Enzyme translocation was blocked by the specific PtdIns 3-K pharmacological inhibitor, LY294002, which also inhibited erythroid differentiation. In vivo, intranuclear synthesis of phosphatidylinositol (3,4,5) trisphosphate (PtdIns (3,4,5)P(3)) was stimulated by EPO. Almost all PtdIns 3-K that translocated to the nucleus was highly phosphorylated on tyrosine residues of the p85 regulatory subunit. These findings strongly suggest that an important step in the signaling pathways that mediate EPO-induced erythroid differentiation may be represented by the intranuclear translocation of an active PtdIns 3-K.

Previous studies have demonstrated that SH2-containing inositol phosphatase (SHIP) is involved in the control of B cell, myeloid cell and macrophage activation and proliferation. The goal of the present study was to examine the role of SHIP during proliferation and apoptosis in cells of the erythroid lineage. Wild-type and catalytically inactive SHIP proteins were overexpressed in the erythropoietin (EPO)-dependent cell line AS-E2. Stable overexpression of catalytically inactive SHIP decreased proliferation and resulted in prolonged activation of the extracellular signal-regulated protein kinases ERK1/2 and protein kinase B (PKB), while wild-type SHIP did not affect EPO-mediated proliferation or phosphorylation of ERK and PKB. When AS-E2 cells were EPO deprived a significant increase in apoptosis was observed in clones overexpressing wild type. Mutational analysis showed that this increase in apoptosis was independent of the enzymatic activity of SHIP. The enhanced apoptosis due to overexpression of SHIP was associated with an increase in caspase-3 and -9 activity, without a distinct effect on caspase-8 activity or mitochondrial depolarization. Moreover, in cells overexpressing SHIP apoptosis could be reduced by a caspase-3 inhibitor. These data demonstrate that in the erythroid cell line AS-E2 overexpression of catalytically inactive SHIP reduced proliferation, while overexpression of wild-type SHIP had no effect. Furthermore, overexpression of SHIP enhanced apoptosis during growth factor deprivation by inducing specific caspase cascades, which are regulated independently of the 5-phosphatase activity of SHIP.

The prevention of apoptosis is a key function of growth factors in the regulation of erythropoiesis. This study examined the role of the constitutively active serine/threonine kinase glycogen synthase kinase-3 (GSK3), a target of the phosphoinositide-3-kinase (PI3K)/Akt pathway, in the regulation of apoptosis in primary human erythroid progenitors. GSK3 phosphorylation at its key regulatory residues S21 (alpha isoform) and S9 (beta isoform) was high in steady-state culture, disappeared on growth factor withdrawal, and returned in response to treatment of cells with either erythropoietin or stem cell factor. Phosphorylation correlated with a PI3K-dependent reduction of 25% to 30% in measured GSK3 activity. LY294002, a specific inhibitor of PI3K, induced apoptosis in growth factor-replete erythroid cells to a degree similar to growth factor deprivation, whereas the Mek1 inhibitor U0126 had no effect, implicating PI3K and not mitogen-activated protein kinase in survival signaling. Growth factor-deprived erythroblasts, which undergo apoptosis rapidly, were protected from apoptosis by both lithium chloride, a GSK3 selective inhibitor, and inhibition of caspase activity. However, the clonogenic potential of single cells, which more accurately reflects cell survival, was maintained by lithium chloride, but not by caspase inhibition. Furthermore, lithium chloride, but not caspase inhibition, prevented the appearance of the conformational form of Bax associated with apoptosis induction. In summary, GSK3 activity is suppressed by erythropoietin and stem cell factor in human erythroid progenitor cells, and increased GSK3 activity, brought about by growth factor withdrawal, may regulate commitment to cell death through a caspase-independent pathway that results in a conformational change in Bax.