Direct Targets of Epo Receptor-JAK2-pSTAT5 Signalling in Erythropoiesis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3881-3881
Author(s):  
Kevin R. Gillinder ◽  
Hugh Tuckey ◽  
Charles Bell ◽  
Stephen Huang ◽  
Melissa Ilsley ◽  
...  
Keyword(s):  
Target Genes ◽  
Erythropoietin Receptor ◽  
Lymphoid Cells ◽  
Erythroid Cell ◽  
Polycythaemia Vera ◽  
Erythroid Cells ◽  
Rna Seq ◽  
Feedback Controls ◽  
Erythroid Progenitor ◽  
In Erythroid Cells

Abstract Erythropoietin (EPO) acts through the dimeric erythropoietin receptor (EpoR) to stimulate proliferation, survival and differentiation of colony-forming units-erythroid (CFU-e). We undertook two complimentary approaches to find pSTAT5-depenendent and independent target genes in erythroid cells. We performed RNA-seq of newly transcribed (4sU-labelled) RNA, and ChIP-seq for pSTAT5, 30 minutes after EPO stimulation of J2E cells. This is the first time genome wide pSTAT5 ChIP-seq has been successfully undertaken in hematopoietic cells. We found ~320 robust pSTAT5 occupied sites in the erythroid genome. About 15% of these reside in promoters while the rest reside in intronic enhancers or intergenic regions, some >100kb from the nearest TSS. The majority of peaks contained a central palindromic GAS element, TTCYXRGAA, and there was significant enrichment of GATA and CACCC-box elements, suggesting co-regulation of some EPO-induced genes by GATA1 and KLF1. Using 4sU RNA-seq and CAGE data, we found just 57 genes to be immediately transcribed in response to EPO within 30 minutes while other target genes had delayed responses. We suggest this has biological relevance for feed forward and feedback controls on EPO driven erythropoiesis. Some of the DEGs (e.g. Bcl2l2 and Cish) are known direct targets of pSTAT5, but many are novel and suggest new pathways by which EPO regulates erythropoiesis. These include mRNA splicing, epigenetic regulation via histone methylation, and adaptive changes in the composition of the EpoR. This could provide increased sensitivity of erythroid progenitor cells to anaemic stress; i.e. sensitization of erythroid cells to JAK2-STAT5 signalling. We found a significant overlap between direct STAT5 target genes in erythroid cells, mammary epithelial cells and lymphoid cells, which imply conserved generic effectors of JAK-STAT5 signalling in different cell types. Our results provide new insights into how EPO co-ordinates erythroid cell proliferation, survival and differentiation. Some of these DEGs could be used as biomarkers for monitoring disease activity in polycythaemia vera (PV) and responses to JAK2 inhibitors. Disclosures Perkins: Novartis Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria.

GATA-1-Dependent and -Independent Modes of Establishing the Erythroid Cell Genetic Network.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2083-2083
Author(s):  
Nathaniel James Pope ◽  
Emery H Bresnick
Keyword(s):  
Target Genes ◽  
Genetic Network ◽  
Cell Maturation ◽  
Mediator Complex ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Cell Type ◽  
Cell Type Specific ◽  
And Function ◽  
In Erythroid Cells

Abstract Abstract 2083 The constant physiological demand to generate large numbers of red blood cells requires a complex genetic network established by the master regulatory transcription factor GATA-1, which orchestrates erythroblast survival, proliferation, and differentiation. Many questions remain regarding how GATA-1 instigates genetic networks and to what extent GATA-1-independent mechanisms regulate erythropoiesis. Med1, a component of the broadly expressed Mediator complex (Mediator), facilitates GATA-1-dependent transcriptional activation at select target genes, although its contribution to GATA-1 function in cell-based assays is considerably less than that of the cell type-specific coregulator Friend of GATA-1. Med1-nullizygous mice have hematopoietic, cardiac, and vascular defects, though the underlying mechanisms are not defined. Furthermore, whether Med1 coactivator activity is dedicated to GATA-1 in erythroid cells and whether it controls numerous or a restricted cohort of genes is also unclear. Using a genetic complementation assay in GATA-1-null erythroid cells and a functional genomics approach, we demonstrate that Med1 regulates a restricted gene ensemble in erythroid cells, consisting predominantly of genes not controlled by GATA-1. Of the 265 Med1-regulated genes and 1054 GATA-1-regulated genes, only 35 genes were regulated by Med1 and GATA-1. Given the preponderance of GATA-1-independent Med1 targets, it is attractive to propose that Med1 has important GATA-1-independent functions required to exert its crucial hematopoietic activities. Since Med1 is a Mediator subunit, it is presumed to function through Mediator to regulate target genes. However, Med1 interacts with various trans-acting factors, and therefore its gene regulatory activity may not invariably rely on Mediator or a Mediator subcomplex. As Mediator is largely unstudied in erythroid cells, we asked whether Mediator subunit expression is regulated upon primary human erythroid cell maturation ex vivo. Mining the Human Erythroblast Maturation Database revealed that Med25 is strongly up-regulated during maturation. Knockdown of Med25 significantly dysregulated all ten of the highest responding Med1 target genes. Simultaneous knockdowns of Med1 and Med25 altered expression of 9 of the 10 top Med1 target genes, resembling the individual factor knockdowns. These results support the hypothesis that Med1 and Med25 function in the erythroid Mediator complex to regulate these genes. Med1 regulated these genes in a cell type-specific manner, as 8 of the 10 top Med1 targets in G1E-ER-GATA-1 proerythroblast-like cells and Mouse Erythroleukemia Cells were not dysregulated upon Med1 knockdown in Mouse Embryonic Fibroblasts. As Med1 modulated, but was not essential for, GATA-1-dependent transcription, we reasoned that certain Med1 target genes may exert GATA-1-independent activities to control erythroid cell development and/or function. The Med1 target gene Rrad encodes a small GTPase induced during primary human erythroid cell maturation, but its regulation/function has not been described in the hematopoietic system. Loss-of-function analysis in G1E-ER-GATA-1 cells indicated that Rrad confers survival. Knocking-down Rrad increased early apoptosis 2.5 fold (p < 0.05). The Rrad requirement for survival was more pronounced when cells were deprived of Erythropoietin (Epo) and Stem Cell Factor (SCF). In cells cultured without Epo, early apoptosis increased 7.0 fold upon Rrad knockdown [from 1.0% ± 0.1% to 7.2% ± 0.5% (p < 0.05)]. Removing SCF from the media significantly increased apoptotic cells, and Rrad knockdown elevated this further from 28% ± 2.4% to 46% ± 2.8% (p < 0.01), while the number of live cells decreased 4.7 fold (p < 0.01). These studies established a dual role for Mediator in erythroid cell regulation as a context-dependent GATA-1 coregulator and a GATA-1-independent regulator of cell type-specific genes, including potentially critical regulators of erythroid cell development, survival, and function. Mechanistically, given the greater than twenty components of the canonical Mediator, it will be particularly instructive to compare our findings to that of other key Mediator components, which shall yield a comprehensive understanding of their regulation and function during the progressive transitions from erythroid precursors to the erythrocyte. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Graded Levels of GATA-1 Expression Modulate Survival, Proliferation, and Differentiation of Erythroid Progenitors

2005 ◽  
Vol 280 (23) ◽  
pp. 22385-22394 ◽  
Author(s):  
Xiaoqing Pan ◽  
Osamu Ohneda ◽  
Kinuko Ohneda ◽  
Fokke Lindeboom ◽  
Fumiko Iwata ◽  
...  
Keyword(s):  
Culture System ◽  
Target Genes ◽  
Es Cells ◽  
Embryonic Stem ◽  
Erythroid Cell ◽  
Critical Threshold ◽  
Erythroid Cells ◽  
Es Cell ◽  
Erythroid Progenitor ◽  
Proliferation And Differentiation

Transcription factor GATA-1 plays an important role in gene regulation during the development of erythroid cells. Several reports suggest that GATA-1 plays multiple roles in survival, proliferation, and differentiation of erythroid cells. However, little is known about the relationship between the level of GATA-1 expression and its nature of multifunction to affect erythroid cell fate. To address this issue, we developed in vitro embryonic stem (ES) culture system by using OP9 stromal cells (OP9/ES cell co-culture system), and cultured the mutant (GATA-1.05 and GATA-1-null) and wild type (WT)ES cells, respectively. By using this OP9/ES cell co-culture system, primitive and definitive erythroid cells were developed individually, and we examined how expression level of GATA-1 affects the development of erythroid cells. GATA-1.05 ES-derived definitive erythroid cells were immature with the appearance of proerythroblasts, and highly proliferated, compared with WT and GATA-1-null ES-derived erythroid cells. Extensive studies of cell cycle kinetics revealed that the GATA-1.05 proerythroblasts accumulated in S phase and expressed lower levels of p16INK4A than WT ES cell-derived proerythroblasts. We concluded that GATA-1 must achieve a critical threshold activity to achieve selective activation of specific target genes, thereby influencing the developmental decision of an erythroid progenitor cell to undergo apoptosis, proliferation, or terminal differentiation.

Lyn kinase plays important roles in erythroid expansion, maturation and erythropoietin receptor signalling by regulating inhibitory signalling pathways that control survival

2014 ◽  
Vol 459 (3) ◽  
pp. 455-466 ◽  
Author(s):  
Neli S. Slavova-Azmanova ◽  
Nicole Kucera ◽  
Alison Louw ◽  
Jiulia Satiaputra ◽  
Adley Handoko ◽  
...  
Keyword(s):  
Cell Survival ◽  
Cell Development ◽  
Erythropoietin Receptor ◽  
Erythroid Cell ◽  
Signalling Pathways ◽  
Erythroid Cells ◽  
Src Family Kinase ◽  
Receptor Signalling ◽  
Lyn Kinase ◽  
Control Survival

In erythroid cells both positive viability signals and feedback inhibitory signalling require the Src family kinase Lyn, influencing cell survival and their ability to differentiate. This illustrates that Lyn is critical for normal erythropoiesis and erythroid cell development.

scholarly journals Possible Interactions between the NS-1 Protein and Tumor Necrosis Factor Alpha Pathways in Erythroid Cell Apoptosis Induced by Human Parvovirus B19

1999 ◽  
Vol 73 (10) ◽  
pp. 8762-8770 ◽  
Author(s):  
N. Sol ◽  
J. Le Junter ◽  
I. Vassias ◽  
J. M. Freyssinier ◽  
A. Thomas ◽  
...  
Keyword(s):  
Tumor Necrosis Factor ◽  
Tumor Necrosis ◽  
Parvovirus B19 ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Necrosis Factor Alpha ◽  
Erythroid Progenitor ◽  
Apoptotic Pathways ◽  
Induced Apoptosis ◽  
Necrosis Factor

ABSTRACT Human erythroid progenitor cells are the main target cells of the human parvovirus B19 (B19), and B19 infection induces a transient erythroid aplastic crisis. Several authors have reported that the nonstructural protein 1 (NS-1) encoded by this virus has a cytotoxic effect, but the underlying mechanism of NS-1-induced primary erythroid cell death is still not clear. In human erythroid progenitor cells, we investigated the molecular mechanisms leading to apoptosis after natural infection of these cells by the B19 virus. The cytotoxicity of NS-1 was concomitantly evaluated in transfected erythroid cells. B19 infection and NS-1 expression induced DNA fragmentation characteristic of apoptosis, and the commitment of erythroid cells to undergo apoptosis was combined with their accumulation in the G2phase of the cell cycle. Since B19- and NS-1-induced apoptosis was inhibited by caspase 3, 6, and 8 inhibitors, and substantial caspase 3, 6, and 8 activities were induced by NS-1 expression, there may have been interactions between NS-1 and the apoptotic pathways of the death receptors tumor necrosis factor receptor 1 and Fas. Our results suggest that Fas-FasL interaction was not involved in NS-1- or B19-induced apoptosis in erythroid cells. In contrast, these cells were sensitized to tumor necrosis factor alpha (TNF-α)-induced apoptosis. Moreover, the ceramide level was enhanced by B19 infection and NS-1 expression. Therefore, our results suggest that there may be a connection between the respective apoptotic pathways activated by TNF-α and NS-1 in human erythroid cells.

scholarly journals During EPO or anemia challenge, erythroid progenitor cells transit through a selectively expandable proerythroblast pool

Blood ◽  
2010 ◽  
Vol 116 (24) ◽  
pp. 5334-5346 ◽  
Author(s):  
Arvind Dev ◽  
Jing Fang ◽  
Pradeep Sathyanarayana ◽  
Anamika Pradeep ◽  
Christine Emerson ◽  
...  
Keyword(s):  
Stromal Cells ◽  
Progenitor Cell ◽  
Ex Vivo ◽  
Erythropoietin Receptor ◽  
Erythroid Cell ◽  
Short Term ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells ◽  
Stage Development ◽  
Bcl2 Expression

Abstract Investigations of bone marrow (BM) erythroblast development are important for clinical concerns but are hindered by progenitor cell and tissue availability. We therefore sought to more specifically define dynamics, and key regulators, of the formation of developing BM erythroid cell cohorts. A unique Kit−CD71highTer119− “stage E2” proerythroblast pool first is described, which (unlike its Kit+ “stage E1” progenitors, or maturing Ter119+ “stage E3” progeny) proved to selectively expand ∼ 7-fold on erythropoietin challenge. During short-term BM transplantation, stage E2 proerythroblasts additionally proved to be a predominantly expanded progenitor pool within spleen. This E1→E2→E3 erythroid series reproducibly formed ex vivo, enabling further characterizations. Expansion, in part, involved E1 cell hyperproliferation together with rapid E2 conversion plus E2 stage restricted BCL2 expression. Possible erythropoietin/erythropoietin receptor proerythroblast stage specific events were further investigated in mice expressing minimal erythropoietin receptor alleles. For a hypomorphic erythropoietin receptor-HM allele, major defects in erythroblast development occurred selectively at stage E2. In addition, stage E2 cells proved to interact productively with primary BM stromal cells in ways that enhanced both survival and late-stage development. Overall, findings reveal a novel transitional proerythroblast compartment that deploys unique expansion devices.

Regulation of SCL Expression by the Homeodomain Protein Otx-1 and the Erythroid Transcription Factor GATA-1.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1598-1598
Author(s):  
Richard Martin ◽  
Virginie Sanguin-Gendreau ◽  
Mathieu Tremblay ◽  
Elena Levantini ◽  
Christina Magli ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Fold Increase ◽  
Proximal Promoter ◽  
Physical Interaction ◽  
Homeodomain Protein ◽  
Erythroid Cells ◽  
Gain Of Function ◽  
Erythroid Progenitor ◽  
Left Right Asymmetry ◽  
In Erythroid Cells

Abstract Members of the bicoid homeodomain-containing proteins are important in establishing left-right asymmetry and the antero-posterior axis, suggesting that they could also be involved in asymmetric determination within the hematopoietic system. We have previously shown that Otx1, a member of the bicoid homeodomain-containing proteins, is co-expressed with the SCL transcription factor in hematopoietic pluripotent and erythroid progenitor cells and Otx1-deficiency impairs the erythroid compartment in mice, associated with decreased SCL levels. In the present study, we provide molecular and functional evidence that SCL is a direct transcriptional target of Otx1. First, we show by chromatin immunoprecipitation that Otx1 and GATA-1 are specifically bound to the SCL proximal promoter in erythroid cells. Second, Otx-1 synergizes with GATA-1 to activate transcription from the SCL proximal promoter and this activity depends on the integrity of the proximal GATA site of the SCL promoter 1a. At the molecular level, we show that this synergy occurs via a physical interaction between Otx-1 and GATA-1 in erythroid cells, which maps to the homeodomain of Otx-1. Furthermore, a gain of function of Otx1 in primary hematopoietic cells gives rise to a 6-fold increase in endogenous SCL levels, an increase in TER119-positive erythroid cells and a decrease in the number of CD11b-positive myeloid cells. Finally, a gain of function of SCL rescues the erythroid deficiency in Otx1−/− mice, consistent with the view that SCL operates downstream of Otx1. Taken together, our observations indicate that Otx1, GATA-1 and SCL operate within the same genetic pathway to specify the erythroid fate during hematopoiesis.

Regulation Of Erythro-Megakaryocytic Lineage Bifurcation By The Gfi1b Gene Target Rgs18

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1191-1191
Author(s):  
Ananya Sengupta ◽  
Ghanshyam Upadhyay ◽  
Asif Chowdhury ◽  
Shireen Saleque
Keyword(s):  
Conflicts Of Interest ◽  
Integration Site ◽  
Fetal Liver ◽  
Cell Types ◽  
Mapk Signaling ◽  
Erythroid Cells ◽  
Erythroid Progenitor ◽  
Gene Target ◽  
Over Expression ◽  
In Erythroid Cells

Abstract The molecular basis for the divergence of the erythroid (red blood cell) and megakaryocyte (platelet) lineages from a common bipotent MEP (megakaryocyte-erythroid progenitor) remains undefined. We now demonstrate that Rgs18 (regulator of G protein signaling 18), a GAP (GTPase activating protein) factor and a transcriptional gene target of the Gfi1b transcriptional repressor complex, likely arbitrates this critical lineage decision downstream of Gfi1b. Rgs18 was identified in a chromatin immunoprecipitation (ChIP on chip) screen for Gfi1b/LSD1/Rcor1 targets in erythroid cells. Accordingly, Rgs18 expression was found to be up-regulated in LSD1 inhibited, and Gfi1b deficient erythroid cells confirming repression of this gene by Gfi1b and its co-factors in this lineage. In contrast, Rgs18 expression was comparable in megakaryocytic cells derived from wild type and gfi1b-/-hematopoietic progenitors indicating Gfi1b independent expression of Rgs18 in these cells. Manipulation of Rgs18 expression produced opposite effects in the erythroid and megakaryocytic lineages. Rgs18 inhibition retarded megakaryocytic differentiation while its ectopic over-expression promoted differentiation at the expense of proliferation. The reverse was observed in erythroid cells where Rgs18 inhibition produced an enhancement of differentiation while over-expression impaired erythropoiesis. Since Rgs signaling regulates the activity of downstream MAPK pathways we determined the status of these pathways in Rgs18 manipulated cells. Inhibition of Rgs18 stimulated ERK phosphorylation in megakaryocytes but diminished it in erythroid cells. In contrast, Rgs18 inhibition in erythroid cells elevated p38MAPK protein and phosphorylation levels. The opposite effects of Rgs18 manipulation on MAPK signaling in erythroid versus megakaryocytic cells while intriguing are consistent with the changes in differentiation and proliferation observed in each lineage, respectively. Although Rgs18 manipulation produced opposite effects in erythroid and megakaryocytic cells, the level and activity of this factor correlated similarly with those of the mutually antagonistic transcription factors Fli1 (Friend leukemia integration [site] 1) and KLF1/EKLF (Kruppel like factor1) in both cell types. In both lineages, Rgs18 protein levels correlated directly with Fli1, and inversely with KLF1, message levels. Since Fli1 promotes megakaryocytic, and KLF1 erythroid, development; these results demonstrate that Rgs18 promotes the emergence of megakaryocytic cells from bipotent MEPs by modulating MAPK signaling and altering Fli1/KLF1 stoichiometries. Although it is unclear why Gfi1b mediated repression of Rgs18 is erythroid specific even though the former is expressed in both lineages, these results demonstrate that repression of Rgs18 by Gfi1b in fetal liver MEPs limits megakaryopoiesis and augments erythropoiesis. However following megakaryocytic commitment, robust Gfi1b independent expression of Rgs18 drives differentiation of this lineage while continued repression of Rgs18 by Gfi1b in erythroid cells ensures their proper maturation. Disclosures: No relevant conflicts of interest to declare.

Diagnosis and Treatment of Erythrocytosis

2010 ◽  
Vol 00 (04) ◽  
pp. 55 ◽  
Author(s):  
Mary Frances McMullin ◽  
Keyword(s):  
Cell Mass ◽  
Erythropoietin Receptor ◽  
Congenital Defects ◽  
Intrinsic Defect ◽  
Haematocrit Level ◽  
Polycythaemia Vera ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells ◽  
Number Of Patients ◽  
Secondary Causes

An erythrocytosis arises when the red cell mass is increased. This can be due to a primary intrinsic defect in the erythroid progenitor cells or secondary to erythropoietin production from some source. Primary and secondary causes can be congenital or acquired. Rare, primary congenital defects are due to mutations leading to truncation of the erythropoietin receptor. The main acquired, primary erythrocytosis is polycythaemia vera. Among the congenital secondary causes, a number of defects in the genes in the oxygen-sensing pathway have recently been described, which lead to a secondary erythrocytosis. An extensive list of acquired secondary causes needs to be considered. A number of patients do not have an identifiable cause of erythrocytosis and are therefore described as having idiopathic erythrocytosis. Investigation should commence with careful clinical evaluation. Determination of the erythropoietin level is then a first step to guide the further direction of investigation. In those with congenital defects, a number of serious thromboembolic events have been described, but there is little information available about outcomes in these individuals and, therefore, no evidence to guide management. In this group, consideration should be given to the use of venesection to attain an achievable haematocrit level, and also low-dose aspirin therapy.

scholarly journals Antagonistic Regulation of β-Globin Gene Expression by Helix-Loop-Helix Proteins USF and TFII-I

2006 ◽  
Vol 26 (18) ◽  
pp. 6832-6843 ◽  
Author(s):  
Valerie J. Crusselle-Davis ◽  
Karen F. Vieira ◽  
Zhuo Zhou ◽  
Archana Anantharaman ◽  
Jörg Bungert
Keyword(s):  
Gene Expression ◽  
Rna Polymerase Ii ◽  
Control Region ◽  
Adult Stage ◽  
Globin Gene ◽  
Locus Control Region ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Globin Gene Expression ◽  
In Erythroid Cells

ABSTRACT The human β-globin genes are expressed in a developmental stage-specific manner in erythroid cells. Gene-proximal cis-regulatory DNA elements and interacting proteins restrict the expression of the genes to the embryonic, fetal, or adult stage of erythropoiesis. In addition, the relative order of the genes with respect to the locus control region contributes to the temporal regulation of the genes. We have previously shown that transcription factors TFII-I and USF interact with the β-globin promoter in erythroid cells. Herein we demonstrate that reducing the activity of USF decreased β-globin gene expression, while diminishing TFII-I activity increased β-globin gene expression in erythroid cell lines. Furthermore, a reduction of USF activity resulted in a significant decrease in acetylated H3, RNA polymerase II, and cofactor recruitment to the locus control region and to the adult β-globin gene. The data suggest that TFII-I and USF regulate chromatin structure accessibility and recruitment of transcription complexes in the β-globin gene locus and play important roles in restricting β-globin gene expression to the adult stage of erythropoiesis.

Role of Atypical p50:p50:Bcl-3 NFκlB Complex in Differentiation of Erythroid Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 568-568 ◽  
Author(s):  
Stephen Sawyer ◽  
Jingchun Chen
Keyword(s):  
Dna Binding ◽  
Transcriptional Control ◽  
Biological Significance ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Necrosis Factor Alpha ◽  
Erythroid Progenitor ◽  
Tnf Α

Abstract We recently reported that mouse and human primary erythroid progenitor cells and erythroid cell lines synthesize and respond to Tumor Necrosis Factor-alpha (TNF-α). The nuclear transcriptional control complex, NFκlB is central in signaling downstream from TNF-α; so we began to study the function of NFκlB in erythroid cells. We made three very interesting initial findings: 1) first we found that NFκlB binding to DNA increased very slowly in HCD57 erythroid cells treated with erythropoietin (EPO, the hormone required for red blood cell development). An inhibitory effect of adding a neutralizing antibody to TNF-α on EPO-stimulated NFκlB DNA suggested this increase in NFκlB was due to TNF-α rather than direct EPO signaling. 2) We also found that NFκlB binding to DNA increased 10-fold or greater during erythroid differentiation. We found greatly increased NFκlB DNA binding in HCD57 cells that differentiated due to over-expression of JunB, F-MEL induced by DMSO, or human UT7-EPO or murine HCD57 cells induced to differentiate with hemin. 3) Surprisingly, we found that the NFκlB DNA binding complex in mouse primary erythroid cells and the erythroid cells lines tested was almost exclusively composed of the atypical p50/p50/Bcl3 NFκlB rather than the canonical p65/p50 or the non-canonical p65/p52 NFκlB. When we begin to study the biological significance of this atypical NFκlB in EPO-mediated erythroid differentiation in vivo using genetic tools, we found marked deficiencies in the development of erythroid cells in either the nfkb1−/− mice (p50−/−) or the bcl3 −/− mice. The nfkb1−/− mice were mildly anemic. The number of red blood cells in the circulation of these mice was statistically lower than in control mice. The number of CFU-e was also reduced in nfkb1−/− mice. Using the Ter-119 and CD71 staining method, we noted that proerythroblasts and immature erythroid cells increased and mature erythroblasts decreased in either non-anemic bone marrow or anemic spleens of nfkb1−/− mice. Forward scatter of Ter-119+ cells also showed an increased size of the average immature erythroid cell in the bone a marrow of nfkb1 −/− mice, suggesting a block in differentiation and continued cell cycling of the immature erythroblasts. Similar erythroid defects were observed in the spleens of anemic bcl-3−/− mice. nfkb1−/− mice and bcl-3−/− mice are also apparently unable to produce new reticulocytes as effectively as wild type mice after induction of anemia. Our working hypothesis is without expression of either p50 or Bcl-3 NFκlB proteins, immature erythroid cells continue to proliferate and ineffectively differentiate. In summary, the atypical p50/p50/Bcl-3 NFκlB complex appears necessary for maximal differentiation of immature erythroid cells.

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