ABSTRACT
The production of cultured red blood cells (cRBC) for transfusion purposes requires large scale cultures and downstream processes to purify enucleated cRBC. The membrane composition, and cholesterol content in particular, are important during proliferation of (pro)erythroblasts and for cRBC quality. Therefore, we tested the requirement for cholesterol in the culture medium during expansion and differentiation of erythroid cultures with respect to proliferation, enucleation and purification by filtration. The low cholesterol level (22 µg/dl) in serum free medium was sufficient to expand (pro)erythroblast cultures. Addition of 2.0 or 5.0 mg/dL of free cholesterol at the start of differentiation induction inhibited enucleation compared to the default condition containing 3.3 mg/dl total cholesterol derived from the addition of Omniplasma to serum free medium. Addition of 5.0 mg/dl cholesterol at day 5 of differentiation did not affect the enucleation process but significantly increased recovery of enucleated cRBC following filtration over leukodepletion filters. The addition of cholesterol at day 5 increased the osmotic resistance of cRBC. In conclusion, cholesterol supplementation after the onset of enucleation improved the robustness of cRBC and increased the yield of enucleated cRBC in the purification process.
Subject(s)
Cholesterol , Culture Media , Erythrocytes , Cholesterol/metabolism , Humans , Erythrocytes/metabolism , Culture Media/chemistry , Cells, Cultured , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Cell Culture Techniques/methods , Erythroblasts/metabolism , Erythroblasts/cytology , Culture Media, Serum-FreeABSTRACT
Protective interactions with bystander cells in micro-environmental niches, such as lymph nodes (LNs), contribute to survival and therapy resistance of chronic lymphocytic leukemia (CLL) cells. This is caused by a shift in expression of B-cell lymphoma 2 (BCL-2) family members. Pro-survival proteins B-cell lymphoma-extra large (BCL-XL), BCL-2-related protein A1 (BFL-1) and myeloid leukemia cell differentiation protein 1 (MCL-1) are upregulated by LN-residing T cells through CD40L interaction, presumably via nuclear factor (NF)-κB signaling. Macrophages (Mφs) also reside in the LN, and are assumed to provide important supportive functions for CLL cells. However, if and how Mφs are able to induce survival is incompletely known. We first established that Mφs induced survival because of an exclusive upregulation of MCL-1. Next, we investigated the mechanism underlying MCL-1 induction by Mφs in comparison with CD40L. Genome-wide expression profiling of in vitro Mφ- and CD40L-stimulated CLL cells indicated activation of the phosphoinositide 3-kinase (PI3K)-V-Akt murine thymoma viral oncogene homolog (AKT)-mammalian target of rapamycin (mTOR) pathway, which was confirmed in ex vivo CLL LN material. Inhibition of PI3K-AKT-mTOR signaling abrogated MCL-1 upregulation and survival by Mφs, as well as CD40 stimulation. MCL-1 can be regulated at multiple levels, and we established that AKT leads to increased MCL-1 translation, but does not affect MCL-1 transcription or protein stabilization. Furthermore, among Mφ-secreted factors that could activate AKT, we found that induction of MCL-1 and survival critically depended on C-C motif chemokine receptor-1 (CCR1). In conclusion, this study indicates that two distinct micro-environmental factors, CD40L and Mφs, signal via CCR1 to induce AKT activation resulting in translational stabilization of MCL-1, and hence can contribute to CLL cell survival.
Subject(s)
Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Macrophages/pathology , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Receptors, CCR1/metabolism , Animals , Apoptosis/physiology , CD40 Ligand/genetics , CD40 Ligand/metabolism , Cell Line, Tumor , Cell Survival/physiology , Down-Regulation , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Macrophages/metabolism , Mice , Myeloid Cell Leukemia Sequence 1 Protein/genetics , NIH 3T3 Cells , Receptors, CCR1/genetics , Signal Transduction , T-Lymphocytes/metabolism , T-Lymphocytes/pathology , TransfectionABSTRACT
BACKGROUND: The objective of this study was to evaluate whether the red cell distribution width (RDW) is a significant risk factor for hospital mortality in critically ill patients and to investigate whether RDW is a parameter indicating inflammation, or a risk factor independent of inflammation. METHODS: We studied all patients admitted to a ten-bed mixed intensive care unit in the Netherlands between May 2005 and December 2011 for whom RDW was available, and who had not received a blood transfusion in the preceding three months. Inflammation was measured by C-reactive protein and leucocyte count. Analyses included correlation, logistic regression analysis, and receiveroperating characteristic (ROC) curves. RESULTS: We included 2915 patients, of whom 387 (13.3%) did not survive to hospital discharge. In univariate analysis higher RDW values were associated with increased hospital mortality. In multivariate analysis RDW remained an independent risk factor for mortality after correction for APACHE II score, age, admission type and mechanical ventilation (odds ratio 1.04, 95% confidence interval 1.02-1.06, for each femtolitre of RDW). Adding RDW to APACHE II, however, increased the area under the ROC curve marginally (from 0.845 to 0.849, p<0.001). RDW was not correlated with C-reactive protein and leucocyte count, refuting the hypothesis that the association between RDW and outcome is mediated through inflammation. CONCLUSION: In critically ill patients, the RDW on ICU admission was an independent predictor of mortality. Since RDW was not correlated with inflammation, the underlying mechanism of this association warrants further investigation.
Subject(s)
C-Reactive Protein/metabolism , Critical Illness/mortality , Erythrocyte Indices , Inflammation/blood , Leukocyte Count , APACHE , Aged , Aged, 80 and over , C-Reactive Protein/immunology , Female , Hospital Mortality , Humans , Inflammation/immunology , Intensive Care Units , Length of Stay , Leukocytes/immunology , Logistic Models , Male , Middle Aged , Netherlands/epidemiology , ROC Curve , Risk FactorsABSTRACT
Primary erythroid progenitors can be expanded by the synergistic action of erythropoietin (Epo), stem cell factor (SCF) and glucocorticoids. While Epo is required for erythropoiesis in general, glucocorticoids and SCF mainly contribute to stress erythropoiesis in hypoxic mice. This ability of normal erythroid progenitors to undergo expansion under stress conditions is targeted by the avian erythroblastosis virus (AEV), harboring the oncogenes v-ErbB and v-ErbA. We investigated the signaling pathways required for progenitor expansion under stress conditions and in leukemic transformation. Immortal strains of erythroid progenitors, able to undergo normal, terminal differentiation under appropriate conditions, were established from fetal livers of p53-/- mice. Expression and activation of the EGF-receptor (HER-1/c-ErbB) or its mutated oncogenic version (v-ErbB) in these cells abrogated the requirement for Epo and SCF in expansion of these progenitors and blocked terminal differentiation. Upon inhibition of ErbB function, differentiation into erythrocytes occurred. Signal transducing molecules important for renewal induction, i.e. Stat5- and phosphoinositide 3-kinase (PI3K), are utilized by both EpoR/c-Kit and v/c-ErbB. However, while v-ErbB transformed cells and normal progenitors depended on PI3K signaling for renewal, c-ErbB also induces progenitor expansion by PI3K-independent mechanisms.
Subject(s)
Cell Transformation, Neoplastic , ErbB Receptors/metabolism , Erythroid Precursor Cells/pathology , Erythropoiesis , Leukemia/pathology , Oncogene Proteins v-erbB/metabolism , Proto-Oncogene Proteins c-kit/metabolism , Receptors, Erythropoietin/metabolism , Animals , Cell Division , Cells, Cultured , Enzyme Activation , ErbB Receptors/genetics , Erythroblasts/cytology , Erythroid Precursor Cells/cytology , Humans , Mice , Mice, Knockout , Oncogene Proteins v-erbB/genetics , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction , Stress, Physiological , Tumor Suppressor Protein p53/geneticsABSTRACT
Stem cell factor (SCF) has an important role in the proliferation, differentiation, survival, and migration of hematopoietic cells. SCF exerts its effects by binding to cKit, a receptor with intrinsic tyrosine kinase activity. Activation of phosphatidylinositol 3'-kinase (PI3-K) by cKit was previously shown to contribute to many SCF-induced cellular responses. Therefore, PI3-K-dependent signaling pathways activated by SCF were investigated. The PI3-K-dependent activation and phosphorylation of the tyrosine kinase Tec and the adapter molecule p62Dok-1 are reported. The study shows that Tec and Dok-1 form a stable complex with Lyn and 2 unidentified phosphoproteins of 56 and 140 kd. Both the Tec homology and the SH2 domain of Tec were identified as being required for the interaction with Dok-1, whereas 2 domains in Dok-1 appeared to mediate the association with Tec. In addition, Tec and Lyn were shown to phosphorylate Dok-1, whereas phosphorylated Dok-1 was demonstrated to bind to the SH2 domains of several signaling molecules activated by SCF, including Abl, CrkL, SHIP, and PLCgamma-1, but not those of Vav and Shc. These findings suggest that p62Dok-1 may function as an important scaffold molecule in cKit-mediated signaling.
Subject(s)
Hematopoietic Stem Cells/metabolism , Phosphatidylinositol 3-Kinases/pharmacology , Phosphoproteins/drug effects , Protein-Tyrosine Kinases/drug effects , Stem Cell Factor/pharmacology , src-Family Kinases/drug effects , Blotting, Western , Erythroid Precursor Cells/drug effects , Erythroid Precursor Cells/metabolism , Hematopoietic Stem Cells/drug effects , Hematopoietic Stem Cells/enzymology , Humans , Phosphoproteins/metabolism , Phosphorylation/drug effects , Precipitin Tests , Protein Binding/drug effects , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins c-kit/metabolism , Signal Transduction/drug effects , Substrate Specificity , Tumor Cells, Cultured , src Homology Domains , src-Family Kinases/metabolismABSTRACT
Protein kinase C (PKC) is implied in the activation of multiple targets of erythropoietin (Epo) signaling, but its exact role in Epo receptor (EpoR) signal transduction and in the regulation of erythroid proliferation and differentiation remained elusive. We analyzed the effect of PKC inhibitors with distinct modes of action on EpoR signaling in primary human erythroblasts and in a recently established murine erythroid cell line. Active PKC appeared essential for Epo-induced phosphorylation of the Epo receptor itself, STAT5, Gab1, Erk1/2, AKT, and other downstream targets. Under the same conditions, stem cell factor-induced signal transduction was not impaired. LY294002, a specific inhibitor of phosphoinositol 3-kinase, also suppressed Epo-induced signal transduction, which could be partially relieved by activators of PKC. PKC inhibitors or LY294002 did not affect membrane expression of the EpoR, the association of JAK2 with the EpoR, or the in vitro kinase activity of JAK2. The data suggest that PKC controls EpoR signaling instead of being a downstream effector. PKC and phosphoinositol 3-kinase may act in concert to regulate association of the EpoR complex such that it is responsive to ligand stimulation. Reduced PKC-activity inhibited Epo-dependent differentiation, although it did not effect Epo-dependent "renewal divisions" induced in the presence of Epo, stem cell factor, and dexamethasone.
Subject(s)
Isoenzymes/physiology , Milk Proteins , Protein Kinase C/physiology , Receptors, Erythropoietin/metabolism , Signal Transduction/physiology , Chromones/pharmacology , DNA-Binding Proteins/metabolism , Enzyme Activators/pharmacology , Enzyme Inhibitors/pharmacology , Humans , Indoles/pharmacology , Isoenzymes/metabolism , Kinetics , Maleimides/pharmacology , Morpholines/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation , Protein Binding , Protein Kinase C/metabolism , Protein Kinase C-alpha , Proto-Oncogene Proteins c-kit/metabolism , STAT5 Transcription Factor , Trans-Activators/metabolismABSTRACT
INTRODUCTION: Anandamide (ANA) is an endogenous ligand for the cannabinoid receptors Cb1 and Cb2 that is able to synergistically stimulate the proliferation of hematopoietic growth factor-dependent blood cells in serum-free culture. To elucidate the mechanisms by which ANA enhances the proliferative responses of hematopoietic cells, we investigated the ANA-mediated effects on proliferation, cell cycling, apoptosis and intracellular signaling of erythropoietin-stimulated 32D/EPO cells. MATERIALS AND METHODS: 32D/EPO cells were cultured serum free to determine the effects of EPO and anandamide on these cells. Proliferation was analyzed by tritiated thymidine incorporation. Apoptosis as well as cell cycle analysis was carried out by flow cytometry. MAPKinase activation was determined by Western blotting, using phospho-specific MAPK antibodies. RESULTS: Simultaneous addition of erythropoietin (EPO) and ANA enhanced DNA synthesis and increased 32D/EPO cell numbers in serum-free culture. Interestingly, ANA did not alter the G1/S transition but it accelerated each of the successive cell cycle phases of EPO-stimulated 32D/EPO cells. Percentages of apoptotic 32D/EPO cells were equally low in cultures supplemented with EPO alone or a combination of EPO and ANA. Both cultures showed enhanced activation of two mitogen-activated protein kinases, namely, extracellular factor responsive kinases 1 and 2 (ERK1/2), as well as the MAPK-target gene protein c-Fos. This fully correlated with the synergistic stimulation of proliferation of 32D/EPO cells by EPO and ANA. ANA had no effect on EPO-induced STAT-5 activation of 32D/EPO cells. Experiments with the Cb2 receptor-specific antagonist SR144528 demonstrated that the synergistic stimulation of proliferation by ANA was partially Cb2 receptor-mediated. CONCLUSION: These data suggest that the positive effects of ANA on the erythropoietin-induced proliferation of 32D/EPO cells are mediated by receptor-dependent as well as receptor-independent mechanisms, both of which involve activation of the mitogen-activated protein kinases, ERK1/2.
Subject(s)
Arachidonic Acids/pharmacology , Erythropoietin/pharmacology , Milk Proteins , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinases/metabolism , Animals , Apoptosis/drug effects , Camphanes/pharmacology , Cell Cycle/drug effects , Cell Line/drug effects , Culture Media, Serum-Free , DNA Replication/drug effects , DNA-Binding Proteins/metabolism , Drug Synergism , Endocannabinoids , Enzyme Activation , Genes, fos , Mice , Mitogen-Activated Protein Kinase 3 , Polyunsaturated Alkamides , Proto-Oncogene Proteins c-fos/biosynthesis , Pyrazoles/pharmacology , RNA, Messenger/biosynthesis , Receptors, Cannabinoid , Receptors, Drug/antagonists & inhibitors , Receptors, Drug/biosynthesis , Receptors, Drug/drug effects , Receptors, Drug/genetics , STAT5 Transcription Factor , Signal Transduction/drug effects , Trans-Activators/metabolism , Up-Regulation/drug effectsABSTRACT
Although erythropoietin (Epo) is essential for the production of mature red blood cells, the cooperation with other factors is required for a proper balance between progenitor proliferation and differentiation. In avian erythroid progenitors, steroid hormones cooperate with tyrosine kinase receptors to induce renewal of erythroid progenitors. We examined the role of corticosteroids in the in vitro expansion of primary human erythroid cells in liquid cultures and colony assays. Dexamethasone (Dex), a synthetic glucocorticoid hormone, cooperated with Epo and stem cell factor to induce erythroid progenitors to undergo 15 to 22 cell divisions, corresponding to a 10(5)- to 10(6)-fold amplification of erythroid cells. Dex acted directly on erythroid progenitors and maintained the colony-forming capacity of the progenitor cells expanded in liquid cultures. The hormone delayed terminal differentiation into erythrocytes, which was assayed by morphology, hemoglobin accumulation, and the expression of genes characteristic for immature cells. Sustained proliferation of erythroid progenitors could be induced equally well from purified erythroid burst-forming units (BFU-E), from CD34(+) blast cells, and from bone marrow depleted from CD34(+) cells.
Subject(s)
Erythroid Precursor Cells/cytology , Erythropoiesis/physiology , Proto-Oncogene Proteins c-kit/physiology , Receptors, Erythropoietin/physiology , Receptors, Glucocorticoid/physiology , Adult , Age Factors , Antigens, CD34/analysis , Antigens, Differentiation/analysis , Blood Cells/cytology , Blood Cells/drug effects , Bone Marrow Cells/cytology , Bone Marrow Cells/drug effects , Cell Differentiation/drug effects , Cell Division/drug effects , Cells, Cultured , Dexamethasone/pharmacology , Drug Synergism , Erythroid Precursor Cells/drug effects , Erythroid Precursor Cells/metabolism , Erythropoiesis/drug effects , Erythropoietin/pharmacology , Fetal Blood/cytology , Humans , Infant, Newborn , Organ Specificity , Receptors, Erythropoietin/drug effects , Receptors, Glucocorticoid/drug effects , Recombinant Proteins/pharmacology , Stem Cell Factor/pharmacologyABSTRACT
The development of a non-invasive prenatal diagnostic test using fetal nucleated red blood cells (NRBCs) isolated from the maternal circulation is hampered by the low frequency of these cells in maternal blood, requiring extensive enrichment procedures before any analytical procedure can be performed. In order to improve and simplify these procedures, we have used in vitro expanded erythroid cells derived from male umbilical cord blood in a model system for the isolation of fetal NRBCs from maternal blood. Erythroblast cells were expanded in vitro to high cell numbers and were immunophenotypically identical to fetal NRBCs isolated from maternal blood. Magnetic activated cell sorting (MACS) isolation procedures were optimized using in vitro expanded male NRBCs diluted up to 1 in 400,000 with female peripheral blood mononucleated cells. The number of recovered male cells was determined using two-colour fluorescence in situ hybridization with X and Y chromosomal probes. Using this model system, an NRBC isolation technique is described. It is based on a one-step MACS enrichment protocol for CD71 positive cells, which showed a significant (Wilcoxon signed ranks test, p<0.05) two-fold higher yield of male NRBCs than previously described MACS methodologies, in which CD71 positive cells were enriched after depletion of other cell types. Application of these isolation strategies to maternal blood samples resulted in a similar improved enrichment of male fetal cells after the direct enrichment of CD71 positive cells.
Subject(s)
Cell Separation/methods , Erythroblasts/cytology , Fetal Blood/cytology , Antigens, CD/analysis , Antigens, Differentiation, B-Lymphocyte/analysis , Cells, Cultured , Erythroblasts/immunology , Erythropoietin/pharmacology , Female , Flow Cytometry , Gestational Age , Humans , In Situ Hybridization, Fluorescence , Infant, Newborn , Leukocyte Common Antigens/analysis , Magnetics , Male , Pregnancy , Prenatal Diagnosis/methods , Receptors, Transferrin , Stem Cell Factor/pharmacology , X Chromosome , Y ChromosomeABSTRACT
Red blood cells are of vital importance for oxygen transport in vertebrates. Thus, their formation during development and homeostasis requires tight control of both progenitor proliferation and terminal red cell differentiation. Self renewal (i.e. long-term proliferation without differentiation) of committed erythroid progenitors has recently been shown to contribute to this regulation. Avian erythroid progenitors expressing the EGF receptor/c-ErbB (SCF/TGFalpha progenitors) can be induced to long-term proliferation by the c-ErbB ligand transforming growth factor alpha and the steroids estradiol and dexamethasone. These progenitors have not yet been described in mammals and their factor requirements are untypical for adult erythroid progenitors. Here we describe a second, distinct type of erythroid progenitor (EpoR progenitors) which can be established from freshly isolated bone marrow and is induced to self renew by ligands relevant for erythropoiesis, i.e. erythropoietin, stem cell factor, the ligand for c-Kit and the glucocorticoid receptor ligand dexamethasone. Limiting dilution cloning indicates that these EpoR progenitors are derived from normal BFU-E/CFU-E. For a detailed study, mEpoR progenitors were generated by retroviral expression of the murine Epo receptor in bone marrow erythroblasts. These progenitors carry out the normal erythroid differentiation program in recombinant differentiation factors only. We show that mEpoR progenitors are more mature than SCF/TGFalpha progenitors and also do no longer respond to transforming growth factor alpha and estradiol. In contrast they are now highly sensitive to low levels of thyroid hormone, facilitating their terminal maturation into erythrocytes.
Subject(s)
Erythroid Precursor Cells/metabolism , Proto-Oncogene Proteins c-kit/metabolism , Receptors, Erythropoietin/metabolism , Animals , Cell Differentiation , Cell Division , Erythroid Precursor Cells/cytology , Erythropoietin/metabolism , Hormones/metabolism , Mice , Stem Cell Factor/metabolism , Steroids/metabolism , Transforming Growth Factor alpha/metabolism , Tretinoin/metabolismABSTRACT
v-ErbA, a mutated thyroid hormone receptor alpha (TRalpha), is thought to contribute to avian erythroblastosis virus (AEV)-induced leukemic transformation by constitutively repressing transcription of target genes. However, the binding of v-ErbA or any unliganded nuclear receptor to a chromatin-embedded response element as well as the role of the N-CoR-SMRT-HDAC co-repressor complex in mediating repression remain hypothetical. Here we identify a v-ErbA-response element, VRE, in an intronic DNase I hypersensitive site (HS2) of the chicken erythroid carbonic anhydrase II (CAII) gene. In vivo footprinting shows that v-ErbA is constitutively bound to this HS2-VRE in transformed, undifferentiated erythroblasts along with other transcription factors like GATA-1. Transfection assays show that the repressed HS2 region can be turned into a potent enhancer in v-ErbA-expressing cells by mutation of the VRE. Differentiation of transformed cells alleviates v-ErbA binding concomitant with activation of CAII transcription. Co-expression of a gag-TRalpha fusion protein in AEV-transformed cells and addition of ligand derepresses CAII transcription. Treatment of transformed cells with the histone deacetylase inhibitor, trichostatin A, derepresses the endogenous, chromatin-embedded CAII gene, while a transfected HS2-enhancer construct remains repressed. Taken together, our data suggest that v-ErbA prevents CAII activation by 'neutralizing' in cis the activity of erythroid transcription factors.
Subject(s)
Carbonic Anhydrases/genetics , Cell Transformation, Neoplastic , Leukemia/genetics , Oncogene Proteins v-erbA/metabolism , Response Elements , Alpharetrovirus , Animals , Base Sequence , Carbonic Anhydrases/biosynthesis , Cell Differentiation , DNA Footprinting , Enhancer Elements, Genetic , Erythropoiesis , Gene Expression Regulation, Neoplastic , Histone Deacetylase Inhibitors , Hydroxamic Acids/pharmacology , Introns , Molecular Sequence Data , Protein Binding , Receptors, Thyroid Hormone/metabolismABSTRACT
Disruption of the gene for transcription factor EKLF (erythroid Krüppel-like factor) results in fatal anaemia caused by severely reduced expression of the adult beta-globin gene, while other erythroid-specific genes, including the embryonic epsilon- and fetal gamma-globin genes, are expressed normally. Thus, EKLF is thought to be a stage-specific factor acting through the CACC box in the beta-gene promoter, even though it is already present in embryonic red cells. Here, we show that a beta-globin gene linked directly to the locus control region (LCR) is expressed at embryonic stages, and that this is only modestly reduced in EKLF-/- embryos. Thus, embryonic beta-globin expression is not intrinsically dependent on EKLF. To investigate whether EKLF functions in the locus control region, we analysed the expression of LCR-driven lacZ reporters. This shows that EKLF is not required for reporter activation by the complete LCR. However, embryonic expression of reporters driven by 5'HS3 of the LCR requires EKLF. This suggests that EKLF interacts directly with the CACC motifs in 5'HS3 and demonstrates that EKLF is also a transcriptional activator in embryonic erythropoiesis. Finally, we show that overexpression of EKLF results in an earlier switch from gamma- to beta-globin expression. Adult mice with the EKLF transgene have reduced platelet counts, suggesting that EKLF levels affect the balance between the megakaryocytic and erythroid lineages. Interestingly, the EKLF transgene rescues the lethal phenotype of EKLF null mice, setting the stage for future studies aimed at the analysis of the EKLF protein and its role in beta-globin gene activation.
Subject(s)
DNA-Binding Proteins/metabolism , Erythroid Precursor Cells/metabolism , Globins/genetics , Transcription Factors/metabolism , Transcriptional Activation , Animals , DNA-Binding Proteins/genetics , Erythropoiesis , Gene Expression Regulation , Humans , Kruppel-Like Transcription Factors , Mice , Mice, Transgenic , Phenotype , Transcription Factors/geneticsABSTRACT
Earlier work demonstrated that an activated estrogen receptor (ER) is required for long-term self-renewal of c-ErbB-expressing avian erythroid progenitors. Here, we demonstrate that activation of the ER does not only arrest or retard differentiation of early progenitors but that it affects erythroid differentiation at all stages of erythroid maturation. A search for genes whose expression is affected by the ER showed that the 17beta-estradiol-activated receptor suppressed the differentiation-associated up-regulation of Gata-1, SCL-1, and globin genes in partially mature cells. In the same cells, the expression of carbonic anhydrase II (CAII) and histone H5 was enhanced. This led to premature expression of CAII, a possible explanation for the toxic effects of overexpressed ER. Repression specifically required the transactivation domain AF-2, but neither an intact DNA-binding domain (DBD) nor the AF-1 domain. The transcriptional activation of CAII, however, required both an intact AF-2 and a functional DBD. The requirement for the AF-2, but not the DBD, suggested that the ER may compete with other nuclear hormone receptors for transcriptional coactivators that bind AF-2, a domain well conserved within this family of transcription factors. We show, however, that this model does not apply for the most likely candidate, the avian thyroid hormone receptor.
Subject(s)
DNA-Binding Proteins/physiology , Erythroid Precursor Cells/cytology , Receptors, Estrogen/physiology , Transcriptional Activation , Animals , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cell Line , Chickens , DNA-Binding Proteins/metabolism , Erythroid Precursor Cells/metabolism , Erythroid Precursor Cells/physiology , Estradiol/pharmacology , Gene Expression Regulation , Humans , Mice , Protein Structure, Tertiary , Receptors, Estrogen/metabolism , Receptors, Thyroid Hormone/physiology , Transcription, Genetic , Transcriptional Activation/drug effectsABSTRACT
V-ErbA, a mutated thyroid hormone receptor (TR) alpha cooperates with tyrosine kinase oncoproteins to induce fatal erythroleukemia in chicks. In vitro, v-ErbA employs a similar cooperation to induce sustained proliferation and arrest differentiation of committed erythroid progenitors. V-ErbA has been proposed to function as a dominant-negative c-ErbA/TR alpha, since it lacks an AF-2 transactivation domain and cannot be activated by hormone but retains the capacity to bind corepressors. However, v-ErbA fails to heterodimerize with the coreceptor RXR, exhibits an altered DNA binding specificity and fails to suppress the action of coexpressed TR alpha/c-ErbA in erythroblasts. In this paper, we identify a novel mechanism by which v-ErbA contributes to leukemogenesis. Recently, the glucocorticoid receptor (GR) was identified as a key regulator of proliferation and differentiation in normal erythroid progenitors. For this, the GR required to cooperate with endogenous receptor tyrosine kinases (c-Kit) and with the estrogen receptor (ER). Here, we demonstrate that v-ErbA can substitute for the ligand-activated GR and ER, inducing proliferation and arresting differentiation in the presence of specific GR and ER antagonists. Like the GR, v-ErbA required to cooperate with c-Kit for both proliferation induction and differentiation arrest, being devoid of biological activity in the absence of an active c-Kit. In self-renewing erythroblasts, v-ErbA not only repressed known v-ErbA target genes but also maintained high expression of c-myb. These biological activities of v-ErbA depended on distinct mutations in the DNA-binding domain. Additionally, v-ErbA acted as a partial, weak repressor of c-ErbA/TR alpha function in normal erythroblasts. It could be converted into a truly dominant-negative receptor by restoring its ability to heterodimerize with RXR.
Subject(s)
Oncogene Proteins v-erbA/physiology , Transformation, Genetic , Animals , Cell Differentiation/genetics , Cell Division/genetics , Cells, Cultured , Chick Embryo , Chickens , Erythroblasts , Fibroblasts , Gene Expression Regulation, Neoplastic , Genetic Vectors/genetics , Kinetics , Oncogene Proteins v-erbA/genetics , Oncogene Proteins v-erbA/metabolism , Phosphotyrosine/analysis , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-kit/metabolism , Proto-Oncogene Proteins c-kit/physiology , Proto-Oncogene Proteins c-myb , Receptors, Estrogen/antagonists & inhibitors , Receptors, Estrogen/metabolism , Receptors, Estrogen/physiology , Receptors, Glucocorticoid/antagonists & inhibitors , Receptors, Glucocorticoid/metabolism , Receptors, Glucocorticoid/physiology , Receptors, Thyroid Hormone/genetics , Receptors, Thyroid Hormone/metabolism , Receptors, Thyroid Hormone/physiology , Retroviridae/genetics , Trans-Activators/genetics , Trans-Activators/metabolismABSTRACT
In the bone marrow, multipotent and committed hematopoietic progenitors have to closely regulate their balance between sustained proliferation without differentiation (self renewal) and entering a terminal differentiation pathway. A useful model to analyze this regulation at the molecular level is committed avian erythroid progenitors. These are induced to undergo long-term self renewal by the ligand-activated receptor tyrosine kinase (RTK) c-ErbB, in cooperation with steroid hormone receptors. This self-renewal induction by c-ErbB even occurs in the presence of differentiation factors (erythropoietin and insulin). Under the same conditions, the RTK c-Kit is unable to sustain erythroid progenitor self renewal, stimulating cell proliferation without arresting terminal differentiation. Two mechanisms are involved in these differential activities of c-Kit and c-ErbB. The first one, differential regulation of receptor expression, proved to be of minor importance, because c-Kit was unable to induce self renewal, even if exogenously expressed from a retrovirus at high levels. Rather our results support the second mechanism, i.e., that receptor-specific signal transduction is responsible for the differential biological activity of c-Kit and c-ErbB: (a) specific tyrosine kinase inhibitors (tryphostins) were found which selectively inhibited the biological function of either c-Kit or c-ErbB in erythroblasts but did not affect ligand-induced autophosphorylation of either RTK; and (b) c-ErbB selectively induced SHC phosphorylation and STAT5 activation. The Ras pathway was similarly activated by c-Kit and c-ErbB. The c-ErbB-specific tyrphostin AG30 specifically blocked STAT5 activation, implicating this signal transducer in c-ErbB-induced self renewal.
Subject(s)
Erythroblasts/cytology , Proto-Oncogene Proteins c-kit/physiology , Receptor, ErbB-2/physiology , Signal Transduction , Animals , Cell Differentiation , Cell Division , Cell Line , Chick Embryo , Erythroblasts/physiologyABSTRACT
Stem cell factor (SCF) is a polypeptide growth factor active on multiple cell types, mainly of hematopoietic origin. We studied the effects of avian SCF on the differentiation of chicken osteoclasts from their putative progenitors as well as on the bone-resorbing activity of terminally differentiated osteoclasts. Osteoclast formation was analyzed in long-term cocultures of osteoblasts and nonadherent, osteoclast-depleted bone marrow cells. Osteoclast activity was studied in short-term (48 h) cultures of bone marrow cell populations enriched for osteoclasts, on dentine slices. SCF strongly enhanced osteoclast differentiation. The IL-6-related chicken myelomonocytic growth factor (cMGF) had a similar effect, and the effects of SCF and cMGF were additive. SCF, but not cMGF, also stimulated the bone-resorbing activity of existing osteoclasts. As osteoblasts have been found to regulate osteoclast activity and formation, chicken osteoblasts were tested for their ability to express and secrete SCF. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that osteoblasts express SCF mRNA and that parathyroid hormone increases expression levels about fourfold. SCF did not accumulate in the culture medium, but remained cell (osteoblasts) surface associated.
Subject(s)
Avian Proteins , Intercellular Signaling Peptides and Proteins , Osteoclasts/drug effects , Stem Cell Factor/pharmacology , Acid Phosphatase/isolation & purification , Animals , Biological Assay , Bone Resorption , Cell Communication , Cell Differentiation/drug effects , Cell Membrane/chemistry , Cells, Cultured , Chick Embryo , Culture Media, Conditioned/pharmacology , Cytokines , Dose-Response Relationship, Drug , Drug Interactions , Growth Substances/pharmacology , Histocytochemistry , Immunohistochemistry , Isoenzymes/isolation & purification , Osteoblasts/physiology , Osteoclasts/physiology , Polymerase Chain Reaction , Stem Cell Factor/isolation & purification , Tartrate-Resistant Acid PhosphataseABSTRACT
During development and in regenerating tissues such as the bone marrow, progenitor cells constantly need to make decisions between proliferation and differentiation. We have used a model system, normal erythroid progenitors of the chicken, to determine the molecular players involved in making this decision. The molecules identified comprised receptor tyrosine kinases (c-Kit and c-ErbB) and members of the nuclear hormone receptor superfamily (thyroid hormone receptor and estrogen receptor). Here we identify the glucocorticoid receptor (GR) as a key regulator of erythroid progenitor self-renewal (i.e. continuous proliferation in the absence of differentiation). In media lacking a GR ligand or containing a GR antagonist, erythroid progenitors failed to self-renew, even if c-Kit, c-ErbB and the estrogen receptor were activated simultaneously. To induce self-renewal, the GR required the continuous presence of an activated receptor tyrosine kinase and had to cooperate with the estrogen receptor for full activity. Mutant analysis showed that DNA binding and a functional AF-2 transactivation domain are required for proliferation stimulation and differentiation arrest. c-myb was identified as a potential target gene of the GR in erythroblasts. It could be demonstrated that delta c-Myb, an activated c-Myb protein, can functionally replace the GR.
Subject(s)
Erythroid Precursor Cells/cytology , Receptors, Glucocorticoid/physiology , Animals , Blotting, Northern , Cell Differentiation , Cell Division , Chick Embryo , Chickens , DNA Mutational Analysis , DNA-Binding Proteins/physiology , Dexamethasone/metabolism , ErbB Receptors/physiology , Furylfuramide/metabolism , Kinetics , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/physiology , Proto-Oncogene Proteins c-myb , Receptors, Estrogen/physiology , Stem Cell Factor/physiology , Trans-Activators/physiologySubject(s)
Avian Leukosis Virus , Avian Leukosis , Cell Transformation, Viral , Erythropoiesis , Leukemia, Erythroblastic, Acute , Animals , Avian Leukosis/genetics , Avian Leukosis/pathology , Avian Leukosis/virology , Cell Differentiation/genetics , Cell Differentiation/physiology , Cell Transformation, Viral/genetics , ErbB Receptors/genetics , ErbB Receptors/physiology , Erythropoiesis/genetics , Erythropoiesis/physiology , Genes, erbA/physiology , Genes, erbB-1/physiology , Leukemia, Erythroblastic, Acute/genetics , Leukemia, Erythroblastic, Acute/pathology , Leukemia, Erythroblastic, Acute/virology , Oncogene Proteins v-erbA/genetics , Oncogene Proteins v-erbA/physiology , Receptors, Glucocorticoid/genetics , Receptors, Glucocorticoid/physiology , Signal TransductionABSTRACT
The emergence of leukemic cells in Friend virus complex-induced erythroleukemia is associated with two recurrent genetic alterations, namely the inactivation of the p53 tumor suppressor gene and the overexpression of Spi-1, a member of the Ets family of transcriptional regulators. In order to determine the role of these genetic alterations on the proliferation and differentiation control of erythroblasts, we expressed Spi-1 and the temperature sensitive mutant p53(V135A) in avian primary erythroid progenitors. We show that enforced expression of Spi-1 in erythroblasts obtained from bone marrow cells by expression of the ts-Sea tyrosine kinase inhibits the execution of the differentiation program normally induced in these cells in response to Epo and insulin and following inactivation of ts-Sea function. In contrast, overexpression of p53(V135A) is without effect on the ability of these cells to differentiate into erythrocytes. However, expression of p53(V135A) in erythroid progenitors obtained from bone marrow cells in the presence of SCF, TGF alpha and estradiol, was found to relieve these cells from their absolute TGF alpha requirement for long term proliferation. This phenotype is dependent upon the expression of the mutant form of p53(V135A) as it is not observed at a temperature at which p53(V135A) regains wild type p53 function. Our results show that each of the genetic alterations which characterize Friend erythroleukemic cells affect in a distinct manner the proliferation and differentiation control of primary erythroid progenitors.
Subject(s)
Cell Differentiation/genetics , Cell Division/genetics , DNA-Binding Proteins/genetics , Erythroid Precursor Cells/cytology , Genes, p53 , Animals , Base Sequence , Cells, Cultured , Chickens , DNA Primers , Estradiol/pharmacology , Molecular Sequence Data , Mutation , Retroviridae Proteins, Oncogenic , Stem Cell Factor/pharmacology , Transforming Growth Factor alpha/pharmacology , Tumor Cells, CulturedABSTRACT
Fusion genes encoding the 3' part of the can gene are implicated in two types of leukemia. The dek-can fusion gene is present in t(6;9) acute myeloid leukemia and the set-can fusion gene is present in one case of acute undifferentiated leukemia. In order to obtain leads towards the molecular basis of these diseases, we have studied the cellular localization of the DEK-CAN and SET-CAN fusion proteins and their normal counterparts. DEK-CAN and SET-CAN were localized exclusively in the nucleus, and also DEK and SET were found to be nuclear proteins. However, CAN was mainly located at the nuclear and cytoplasmic face of the nuclear envelope. This observation is in accordance with the presence of an amino acid repeat in the C-terminal part of CAN, common to the family of nucleoporins. The C-terminal part also contains a nuclear location domain as shown by deletion analysis. This domain may be important for the presence of CAN at the nucleoplasmic side of the nuclear envelope. The relocation of the carboxyterminal part of CAN due to DEK-CAN and SET-CAN may reinforce a nuclear function of the CAN protein.