Proliferation-Independent Functions of the E2F-2 Transcription Factor during Terminal Erythroid Maturation

Abstract Lesions in the retinoblastoma (Rb) pathway induce defects in hematopoiesis that are typically thought to be due to inappropriate proliferative signaling from increased E2F transcriptional activity. Newer data demonstrate that E2F transcription factors have distinct roles in differentiating cells, but the key proliferation-independent functions of the E2Fs remain incompletely defined. We previously found that deregulated activity and expression of cyclin E induces defects in terminal erythroid cell maturation, using a mouse knock-in model (cyclin ET74A T393A). Because cyclin E-Cdk2 potentiates E2F transcriptional activity via Rb phosphorylation, we hypothesized that inhibiting E2F activity would rescue hyperactive cyclin E-associated erythroid cell defects. E2F-2 is specifically induced during terminal erythroid maturation; therefore, we crossed our cyclin E knock-in strain with E2F-2 knockout mice. Unlike cyclin E knock-in bone marrows that display obvious defects in erythroid cell maturation, cyclin E knock-in; E2F-2 knockout animals demonstrated normalized erythroid maturation by flow cytometry. However, these compound mutant mice remain anemic, suggesting red blood cell (RBC) maturation was not completely restored. We studied adult E2F-2 -/- mice further, and consistent with published data, we found that they are anemic. Interestingly, we do not detect obvious terminal erythroid maturation defects in adult E2F-2-knockout bone marrows. Using CFSE-labeled erythrocyte in vivo survival experiments, we found loss of E2F-2 results diminishes survival of adult peripheral erythroid cells within syngeneic, wild-type recipients, suggesting the anemia in the E2F-2 knockout mice is due to accelerated destruction and not solely a production defect. To study the erythroid maturation program in the absence of E2F-2 in detail, we obtained fetal liver-derived hematopoietic progenitors from E2F-2 knockout versus wild-type embryos and differentiated these towards the erythroid lineage in vitro. E2F-2 deletion results in impaired erythroid maturation as evidenced by flow cytometry-based assays of cell surface marker expression, abnormal cell morphologies, and impaired enucleation. In order to identify functionally significant E2F-2 targets during erythroid maturation, we performed microarray analyses on sorted subpopulations of fetal liver-derived erythroid cells. We found widespread gene expression changes in E2F-2 knockout cells, which included but were not limited to proliferation related pathways. Furthermore, using global histone mass spectrometry analysis, we found that loss of E2F-2 results in marked alteration of histone-H3, lysine-4 methylation during erythroid differentiation. Thus, our data demonstrate that E2F-2 has both proliferation-dependent and independent functions that include the coordination of transcriptional and epigenetic programs during terminal erythroid maturation. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Metabolic Pathways Control Normal and Beta-Thalassemic Erythroid Cell Maturation

Blood ◽

10.1182/blood.v120.21.369.369 ◽

2012 ◽

Vol 120 (21) ◽

pp. 369-369 ◽

Cited By ~ 1

Author(s):

Geníis Camprecióos ◽

Xin Zhang ◽

Valentina D'Escamard ◽

Pauline Rimmele ◽

Carolina L. Bigarella ◽

...

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Metabolic Pathways ◽

Research Funding ◽

Mtor Signaling ◽

Cell Maturation ◽

Erythroid Cell ◽

Beta Thalassemia ◽

Wild Type ◽

Erythroid Maturation

Abstract Abstract 369 Erythroid cell maturation requires the integration of erythropoietin receptor (EpoR)-mediated signaling pathways and transcriptional programs of erythroid cell proliferation and differentiation largely orchestrated by GATA-1 and its transcriptional partners. Although red blood cells (RBC) purely rely on glycolysis for energy production, and metabolic processes specifically autophagy have been implicated in erythroid maturation, the potential involvement of metabolic pathways in the control of erythroid cell production is not known. Foxo3 transcription factor is a direct target of GATA-1, functionally regulated by EpoR signaling and essential for the redox regulation of erythropoiesis. Mammalian target of rapamycin (mTOR) kinase is a critical regulator of metabolic processes. We have found that generation and cycling of early erythroid precursors is controlled by a redox-mediated Foxo3-mTOR signaling. Terminal erythroid maturation is specifically compromised in Foxo3-deficient mice. Terminal maturation involves nuclear condensation, enucleation, mitochondrial clearance and complete adoption (or conversion) of glycolytic pathway by erythroid cells. We found RBC to be significantly decreased in Foxo3 mutant bone marrow and peripheral blood. This was in contrast to the increase of polychromatophilic erythroblasts associated with an increase in the total TER119+ population in Foxo3 mutant bone marrow likely reflecting a compensatory mechanism. Notably, using DRAQ5, a DNA-binding fluorescent dye, we found the rate of erythroblasts' enucleation in Foxo3 mutant mice to be significantly compromised. During erythroid maturation Riok3 and Mxi1 transcripts encoding for two important regulators of fetal liver erythroid enucleation are highly upregulated in the bone marrow and robustly expressed in the adult normoblasts and reticulocytes. In agreement with defective enucleation expression of both Riok3 and Mxi1 is highly reduced in Foxo3 mutant erythroblasts and reticulocytes. Interestingly, a two-week in vivo treatment of wild type (WT) and Foxo3−/− mice with rapamycin, a specific inhibitor of mTOR complex 1 (mTORC1) activity, increased significantly the rate of enucleation in a Foxo3-dependent manner, suggesting that mTOR requires Foxo3 activity in supporting erythroid cell maturation. Importantly, targeting mTOR ameliorates beta-thalassemia as inhibition of mTOR signaling by rapamycin treatment improved erythroid cell maturation in the bone marrow, resulted in significant increase in total peripheral blood red cells and hemoglobin (1 to 1.5 g/dl increase) as well as significant reduction in reticulocyte production of beta-thalassemic intermedia (th3/+) mice. Combination of thiazole orange (an RNA and DNA probe) with DRAQ5 determined that in addition to enucleation, the relative production of reticulocytes is also decreased significantly in Foxo3 mutant bone marrow. Strikingly, 6,1% of RBC (CD71 negative) in the peripheral blood of Foxo3−/− animals contained mitochondria (CD71−Mito+) as compared to 0.7 % of wild type RBC. Autophagy is strongly implicated in late stage erythroid cell maturation and mitochondrial removal from reticulocytes. In agreement with a function for Foxo3 in control of mitochondrial removal, expression of Ulk1 (Atg1) and Nix (Bnip3l) both regulators of mitochondrial clearance via autophagy was highly downregulated in Foxo3 mutant normoblasts and reticulocytes. The expression of Nix was notable since Nix was upregulated over 40 fold in wild type but not in Foxo3 mutant reticulocytes as evaluated by the Fluidigm™ microfluidics array technology. These results are consistent with the expression pattern of Foxo3 that is highly upregulated with erythroid maturation and is the most highly expressed in normoblasts. Collectively our results indicate that Foxo3 has a key function in the regulation of terminal erythroid cell maturation. They also suggest that rapamycin may be considered for the treatment of beta-thalassemia. These results are consistent with the model of FOXO3a induction during late human erythroid cell maturation. Based on these studies we propose that Foxo3 coordinates metabolic pathways with the transcriptional program of terminal erythroid cell maturation. Understanding this metabolic program is likely to impact efficient RBC production in culture. Disclosures: Rivella: Novartis Pharmaceuticals: Consultancy; Biomarin: Consultancy; Merganser Biotech: Consultancy, Equity Ownership, Research Funding; Isis Pharma: Consultancy, Research Funding.

Download Full-text

RNA Sequencing Of Adult Bone Marrow Erythroid Populations Reveals Significant Transcriptional Deregulation In Foxo3-/- Mutant Cells

Blood ◽

10.1182/blood.v122.21.3416.3416 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3416-3416

Author(s):

Genís Campreciós ◽

Xin Zhang ◽

Yan Kou ◽

Avi Ma'ayan ◽

Saghi Ghaffari

Keyword(s):

Bone Marrow ◽

Transcription Factors ◽

Expression Profiles ◽

Cell Maturation ◽

Erythroid Cell ◽

Erythroid Cells ◽

Wild Type ◽

Adult Bone Marrow ◽

Erythroid Maturation ◽

Adult Bone

Abstract Transcriptional control of last stages of erythropoiesis is a complex and well orchestrated process controlled by lineage-specific transcription factors. The precise contribution of the different transcription factors to this multistep process has not been fully elucidated. Foxo3 is a transcription factor that is required for terminal erythroid maturation and Foxo3 mutant mice exhibit ineffective erythropoiesis. In order to gain further insight into the contribution of Foxo3 to the control of adult terminal erythroid maturation we analyzed the transcriptome of three adult bone marrow erythroid precursor populations: pro-, basophilic and polychromatophilic erythroblasts from wild type and Foxo3-/- mice. Populations were FACS sorted according to their TER119 and CD44 cell surface expression and FSC properties. RNA was then isolated and sequenced using the Illumina GaII platform. Genes were grouped into 3 categories according to their expression during erythroid cell maturation using the Short Time Series Expression Miner (STEM) program: no change (4577 genes), down-regulated (2868 genes) or up-regulated (2637) (Figure 1). Enrichment analysis of groups of genes using the ChEA database identified Myb, Meis1, Runx1, Fli1 and PU.1 as the main transcription factors regulating gene repression over erythroid maturation. In contrast, ChEA identified known erythroid transcription factors like Gata1, Eklf and Tal1 to drive the up-regulation of many of the erythroid-specific genes. This analysis also enabled the identification of putative novel transcription factors implicated in erythroid cell maturation. Interestingly, the difference between WT and Foxo3-/- cells increased gradually from pro- to polychromatophilic erythroblasts in correlation with increased Foxo3 expression during these steps of maturation. Strikingly, pathway enrichment analysis detected several immune-related pathways such as Toll-like receptors, TGF-β and IL-1 signaling as expressed in maturing wild type erythroid cells and significantly deregulated in Foxo3-/- cells. The expression of a number of these immune genes in erythroid cells has been validated by qRT-PCR. In addition, among others, a cluster of genes from the autophagy pathway was noted to be significantly down-regulated in Foxo3 mutant erythroid cells. In order to better dissect Foxo3 transcriptional control during erythroid maturation, STEM analysis of Foxo3-/- samples revealed an unexpected number of differences compared to WT. Most remarkably the STEM analysis identified that 90% of the 1198 genes that are continuously up-regulated during erythroid maturation from pro- to polychromatophilic are highly compromised in their level of expression during erythroid maturation in the absence of Foxo3. Interestingly, this group was also enriched for Foxo3 direct target genes as determined by ChIP-seq studies. We also identified a subset of genes whose expression increased from pro- to basophilic erythroblasts but decreased thereafter in the absence of Foxo3 in contrast to wild type cells. Interestingly, ChEA analysis on this group identified a subset of genes that are targets of Gata1, Eklf and Tal1 that may require Foxo3 for their full expression at the last stages of erythroid cell maturation. In conclusion, we present an unbiased genome-wide approach using RNA sequencing of adult bone marrow erythroid cells to study the contribution of Foxo3 to the regulation of gene expression at the last stages of erythroid cell maturation. This analysis enabled us to identify novel genes and pathways whose function in the control of red cell generation requires further investigations.Fig. 1Genes with FPKM>2 from WT and Foxo3-/- samples analyzed with the STEM software, divided into 6 different categories according to their expression profiles during terminal erythroid cell maturation from pro- to polychromatophillic erythroblasts. Genes were then further grouped in 3 subsets: down-regulated, up-regulated and no change. The number of genes in each profile is indicated at the bottom for wild type and Foxo3-/- samples.Fig. 1. Genes with FPKM>2 from WT and Foxo3-/- samples analyzed with the STEM software, divided into 6 different categories according to their expression profiles during terminal erythroid cell maturation from pro- to polychromatophillic erythroblasts. Genes were then further grouped in 3 subsets: down-regulated, up-regulated and no change. The number of genes in each profile is indicated at the bottom for wild type and Foxo3-/- samples. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Hypoxic stress underlies defects in erythroblast islands in the Rb-null mouse

Blood ◽

10.1182/blood-2007-01-069104 ◽

2007 ◽

Vol 110 (6) ◽

pp. 2173-2181 ◽

Cited By ~ 19

Author(s):

Benjamin T. Spike ◽

Benjamin C. Dibling ◽

Kay F. Macleod

Keyword(s):

Fetal Liver ◽

Cell Maturation ◽

Null Mouse ◽

Hypoxic Stress ◽

Red Cell ◽

Exogenous Stress ◽

Erythroid Maturation ◽

Definitive Erythropoiesis

Abstract Definitive erythropoiesis occurs in islands composed of a central macrophage in contact with differentiating erythroblasts. Erythroid maturation including enucleation can also occur in the absence of macrophages both in vivo and in vitro. We reported previously that loss of Rb induces cell-autonomous defects in red cell maturation under stress conditions, while other reports have suggested that the failure of Rb-null erythroblasts to enucleate is due to defects in associated macrophages. Here we show that erythropoietic islands are disrupted by hypoxic stress, such as occurs in the Rb-null fetal liver, that Rb−/− macrophages are competent for erythropoietic island formation in the absence of exogenous stress and that enucleation defects persist in Rb-null erythroblasts irrespective of macrophage function.

Download Full-text

Hematopoietic-Specific CSNK2B Loss in Mice Causes Impaired Erythropoiesis

Blood ◽

10.1182/blood.v130.suppl_1.82.82 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 82-82

Author(s):

Laura Quotti Tubi ◽

Sara Canovas Nunes ◽

Marilena Carrino ◽

Ketty Gianesin ◽

Sabrina Manni ◽

...

Keyword(s):

Western Blot ◽

Cell Viability ◽

Knockout Mice ◽

Cell Biology ◽

Combined Treatment ◽

Conditional Knockout ◽

Erythroid Cell ◽

Red Cell ◽

Erythroid Cells ◽

Erythroid Maturation

Abstract CK2 (Csnk2, casein kinase 2) is a Ser-Thr kinase composed by two catalytic (α) and two regulatory (β) subunits and involved in the regulation of various signaling cascades, which are critical for stem cell biology and hematopoietic development. However, a direct role for CK2 during blood cell differentiation is still undefined. Here, we examined the function of CK2 in erythropoiesis by using a hematopoietic-specific conditional knockout mouse model of the β regulatory subunit (Vav1-CRE x Csnk2β f/f mice). Since CK2β knockout mice died in utero, the study was carried out during gestation collecting fetuses from 12.5 to 17.5 days post conception (dpc) and performing the analysis on fetal liver. CK2β knockout fetuses were pale and hydropic, displayed a smaller liver, disarrayed vascularization and haemorrhages. Lack of CK2β caused depletion of hematopoietic/precursor cells, in particular of common lymphoid progenitors and megakaryocyte-erythrocyte progenitors. CK2β loss resulted to affect both early and late erythroid maturation and red cell viability. CK2β knockout contained lower numbers of TER119 positive cells, which displayed a down modulation of the surface expression of transferrin receptor (CD71) and an increased spontaneous apoptosis. Erythroid cells showed alterations in morphology compatible with myelodysplastic changes. Loss of CK2β caused alterations of erythroid cell proliferation, which was different depending on the stage of erythroid maturation: indeed, BrdU and 7AAD staining showed that less mature erythroid cells (CD71+Ter119-) had a lower rate of proliferation but a normal viability; on the contrary, more mature (CD71-Ter119+) erythroid cells suffered in part of apoptosis and in part accumulated in the S phase. RNA seq analysis performed on purified Ter119+ cells revealed upregulation of TP53 -associated genes as well as of Cdkn1a (p21); on the contrary, there was a down-modulation of Stat5 (an erythropoietin receptor down-stream effector) and genes involved in red cell survival and differentiation in particular c-kit and genes associated to the PI3/Akt pathway. The expression of adhesion molecules and surface carriers for inorganic cations/anionsimportant for the osmotic equilibrium and cell membrane integrity was also found markedly dysregulated. Real time quantitative PCR and Western Blot (WB) analyses confirmed the expression data of Cdkn1a, c-Kit, Bcl-xL, Jak-Stat5 as well as of Akt-Gata-1 axis. Gata-1, the key transcription factor for definitive erythropoiesis, was reduced in CK2β knockout mice as were its downstream target genes such as Alas-2, Lrf, Eklf, Epo-R, β-globin. Immature fetal globins accumulated. In order to find a molecular mechanism, we used an in vitro model of erythroid differentiation based on G1ER cells, an estrogen inducible GATA-1 null murine erythroblast cell line; the combined treatment of β-estradiol and inhibition of CK2 through the chemical inhibitor CX-4945 or RNA interference against CK2β confirmed the negative effect on differentiation. Western blot analysis indicated a potential role of the kinase in the regulation of Akt, Gata-1 and Stat5 protein stability. Moreover, the blockade or down modulation of CK2 caused changes in Gata-1 nuclear distribution with loss of the speckled pattern induced by β-estradiol. Thus, CK2 is a likely essential controller of GATA-1 transcriptional function. Altogether, our work demonstrates that CK2 is a master regulator of erythroid development, by impinging on Stat5, Akt and Gata-1 signaling and influencing red cell viability, bioenergetics, proliferation and maturation. Disclosures No relevant conflicts of interest to declare.

Download Full-text

The exosome complex establishes a barricade to erythroid maturation

Blood ◽

10.1182/blood-2014-04-571083 ◽

2014 ◽

Vol 124 (14) ◽

pp. 2285-2297 ◽

Cited By ~ 37

Author(s):

Skye C. McIver ◽

Yoon-A Kang ◽

Andrew W. DeVilbiss ◽

Chelsea A. O’Driscoll ◽

Jonathan N. Ouellette ◽

...

Keyword(s):

Cell Maturation ◽

Erythroid Cell ◽

Key Points ◽

Erythroid Maturation ◽

Exosome Complex ◽

Complex Components

Key Points Exosome complex components are endogenous suppressors of erythroid cell maturation. GATA-1 and Foxo3 transcriptionally repress exosome complex components, thus abrogating the erythroid maturation blockade.

Download Full-text

Integrating Enhancer Mechanisms to Establish a Hierarchical Blood Development Program

Blood ◽

10.1182/blood.v130.suppl_1.7.7 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 7-7

Author(s):

Charu Mehta ◽

Kirby D Johnson ◽

Xin Gao ◽

Irene Ong ◽

Koichi Ricardo Katsumura ◽

...

Keyword(s):

Target Gene ◽

Fetal Liver ◽

Erythroid Cell ◽

Erythroid Progenitors ◽

Hematopoietic Stem ◽

Flow Cytometric ◽

Erythroid Precursors ◽

A Cell ◽

Erythroid Maturation ◽

Myeloid Progenitors

Abstract GATA-2 levels must be stringently regulated to ensure normal hematopoiesis, and human GATA-2 mutations cause hematologic disorders. GATA-2-regulated enhancers differentially control Gata2 expression in hematopoietic stem/progenitor cells and are essential for hematopoiesis and embryonic development. Mechanisms underlying how the enhancers control Gata2 expression and GATA-2 instigated genetic networks in a cell-specific manner are not completely understood. Targeted deletion of an intronic Gata2 enhancer 9.5 kb downstream of the transcription start site (+9.5) abrogates HSC genesis in the aorta-gonad-mesonephros (AGM) region (Gao et al., JEM, 2013). By contrast, the -77 kb enhancer (-77) activates transcription in myeloid progenitors, and its deletion impairs progenitor differentiation (Johnson et al., Science Advances, 2015). To dissect relationships between the enhancers, we developed a compound heterozygous (CH) mouse model bearing +9.5 and -77 enhancer mutations on different Gata2 alleles. While the CH embryos were alive at E13.5, nearly all died by E14.5 (p = 3.58 x 10-5). Flow cytometric analyses and embryo confocal imaging demonstrated that CH embryos have modestly reduced HSC numbers in the fetal liver (2.9-fold) and the AGM (41%, p = 7.8 x 10-5), which was comparable to +9.5+/- embryos. Thus, -77 does not genetically interact with +9.5 to control HSC emergence. Flow cytometric analysis revealed that Lin-Sca1-Kit+ myelo-erythroid progenitors were 6.6-fold lower in CH vs. WT embryos (p = 1.8 x 10-11), with the difference involving disproportionate losses of GMP (8.6-fold; p = 3.7 x 10-6) and MEP (379-fold; p = 3.2 x 10-9). By contrast, +9.5+/- fetal livers had 2-fold fewer myeloid progenitors, which involved similar reductions of CMP (2.1-fold; p = 1 x 10-6), GMP (2.6-fold; p = 0.0007) and MEP (1.9-fold; p = 0.002). Consistent with the myelo-erythroid progenitor reductions and MEP depletion, CH fetal livers lacked BFU-E (p < 0.001) and CFU-GEMM (p < 0.001) in a colony assay. These results illustrate a genetic interaction between +9.5 and -77 in progenitors, but not HSCs, and a new paradigm in which both enhancers must reside on a single allele to generate MEPs. As erythroid precursor cells express GATA-2, we tested whether the -77 deletion impairs erythroid maturation due to a reduction in myelo-erythroid progenitors or due to a cell-autonomous requirement of the enhancer in erythroid precursors. -77-/- E14.5 fetal livers were pale and smaller than WT counterparts, and -77-/- fetal liver cellularity was reduced 7.2-fold (5.3 x 10-4). When liver size was taken into account, there was little difference in the number of E14.5 R1 cells in -77-/- liver vs. WT littermates (p = 0.31). However, -77-/- R2-R5 cells declined sharply (R2, 8.2-fold, p = 0.004; R3, 14-fold, p < 10-5; R4, 9.7-fold, p = 0.002; R5, 14-fold, p = 0.087). The mutant R1 cells were defective in forming BFU-Es and CFU-Es. Analysis of transcriptomes of purified 77-/- and WT R1 cells from E14.5 fetal livers revealed 2805 and 2519 upregulated and downregulated (p < 0.05) genes, respectively, in -77-/- R1 cells. The -77 enhancer conferred GATA-2 expression, which strongly upregulated GATA-1 and therefore a large GATA-1 target gene cohort. A comparison of WT and -77-/- R1 cell transcriptomes with those of early (Tgbfr3low) and late (Tgbfr3high) BFU-Es (Gao et al., Blood, 2016) revealed a -77-/- R1 signature that correlated with the early BFU-E signature (r = 0.73, p < 10-4) and negatively correlated with the late BFU-E signature (r = -0.42, p = 4 x 10-4) differing from WT cells. In addition to GATA-1 target gene alterations, 253 of the -77-activated genes were not GATA-1-regulated in the G1E-ER-GATA-1 system. These genes included Ryk, which encodes a non-canonical Wnt receptor, and had not been studied in erythroid cells. Two Ryk shRNAs significantly decreased BFU-Es and CFU-GMs in lineage-depleted fetal liver cells. Ongoing studies are integrating Ryk function into signaling circuits that control erythroid maturation and analyzing other -77-regulated targets predicted to constitute new regulators of erythroid cell maturation/function. Thus, loss of the -77 enhancer creates multi-faceted defects in erythroid precursors, involving deficiencies of constituents of signaling and transcriptional circuitry required to enable and drive erythroid maturation. Figure Figure. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Activation of JAK/STAT Signaling in Megakaryocytes Is Necessary and Sufficient for Myeloproliferation In Vivo

Blood ◽

10.1182/blood.v128.22.949.949 ◽

2016 ◽

Vol 128 (22) ◽

pp. 949-949

Author(s):

Q. Jeremy Wen ◽

Brittany Woods ◽

Qiong Yang ◽

Chiu Sophia ◽

Gu Lillu ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Diphtheria Toxin ◽

Bone Marrow Cells ◽

Erythroid Cell ◽

Spleen Weight ◽

Wild Type ◽

Myeloid Progenitor

Abstract Aberrant megakaryopoiesis is a hallmark of the myeloproliferative neoplasms (MPN). It is has been long known that abnormal megakaryocytes secrete elevated levels of cytokines such as TGFβ, resulting in pathologies including bone marrow fibrosis. Two recent studies showed that megakaryocytes regulate the quiescence of HSCs, raising the possibility that megakaryocytes may promote the MPNs by influencing the biology of non-malignant HSCs. However, the mechanism by which megakaryocytes regulate the initiation and progression of MPNs is largely unknown. To study the role of megakaryocytes in the MPNs, we analyzed the phenotype of PF4-Cre/Jak2V617F mice in which Jak2 is expressed in the megakaryocyte lineage from the endogenous locus, in contrast to previous studies, which used transgenic models. Selective activation of Jak2V617F was confirmed by allele-specific qPCR. CD41+ cells were positive for mutant Jak2, whereas sorted stem/progenitor cells and erythroid cells were Jak2 wild-type. Furthermore, flow cytometry showed that Stat5 activation was present in megakaryocytes, but not in erythroid or myeloid cells. Activation of JAK-STAT signaling caused an expansion of megakaryocytes in the bone marrow and spleen and a modest increase in the platelet count. Surprisingly, PF4-Cre/Jak2V617F mice also displayed a robust expansion of TER119(low)/CD71(high) and TER119(high)/CD71(high) red cells in the spleen, increased hematocrit and splenomegaly. Histological examination of the spleen revealed expansion of the erythroid lineage coupled with disrupted splenic architecture and fibrosis. This PV-like phenotype was fully penetrant and comparable to that of Vav-Cre/Jak2V617F mice, which express mutant Jak2 in all hematopoietic lineages. Profiling of hematopoietic progenitors by flow cytometry demonstrated that myeloid progenitor populations were expanded and skewed toward the erythroid-megakaryocyte lineage with a significant increase in Pre Meg-E, Pre CFU-E and MKPs in the PF4Cre/Jak2V617F mice. In addition, LSK cells were increased in both the bone marrow and spleen. Cytokine profiling of the plasma revealed increased levels of several cytokines, including Il-6, which is known to be upregulated in human JAK2 mutant PV megakaryocytes. Significant increases in Cxcl1, Cxcl2, and Ccl11 were also detected. Real-time qPCR analysis confirmed increased expression of these cytokines/chemokines in Jak2V617F-mutant CD41+ cells. Furthermore, IL6 treatment increased EPO-dependent colony formation of wild type LSKs and MEPs, and also enhanced expression of the erythroid cell markers CD71 and Ter119. To further explore the role of megakaryocytes in the MPNs, we used a strategy in which expression of the diphtheria toxin receptor (DTR) sensitizes cells to diphtheria toxin (DT). We transduced c-Kit+ cells from PF4-Cre/iDTR+/- mice with MPLW515L and transplanted the cells to irradiated mice. As expected, both iDTR+/- and PF4-Cre/iDTR+/- mice developed a PMF-like phenotype, including leukocytosis, thrombocytosis, splenomegaly and myelofibrosis (Fig 1). Treatment of these animals with DT caused significant reductions in megakaryocytes in the bone marrow and spleen as well as a decrease in the platelet count of PF4-Cre/iDTR+/- mice. Of note, DT also significantly reduced the white count and spleen weight, while restoring splenic architecture. PF4Cre/iDTR+/- mice also showed significant reduction of c-Kit+ myeloid progenitor cells. Therefore, depletion of megakaryocytes significantly attenuated the disease phenotype of MPLW515L induced MPN in vivo. Together, these two model systems reveal that JAK2 activation in megakaryocytes is sufficient and necessary for MPNs and support the development of megakaryocyte differentiation therapy in the disease. Moreover our data resonate with studies in MPN patients in which a JAK2V617F low allele burden in the setting of full-blown, clinical MPN. figure 1 Depletion of megakaryocytes attenuated the MPN phenotype induced by MPLW515L. c-Kit+ bone marrow cells of IDTR+/- mice with or without PF4Cre were transduced with retroviruses carrying MPLW515L. Injection of diphtheria toxin (DT) was initiated on day 28 post-transplant. Depletion of megakaryocytes by DT reduced platelet and white count (A, B), decreased spleen weight (C) and reduced megakaryocyte and erythroid cell infiltration in the spleen (D). *, p<0.05, **, p<0.01. figure 1. Depletion of megakaryocytes attenuated the MPN phenotype induced by MPLW515L. c-Kit+ bone marrow cells of IDTR+/- mice with or without PF4Cre were transduced with retroviruses carrying MPLW515L. Injection of diphtheria toxin (DT) was initiated on day 28 post-transplant. Depletion of megakaryocytes by DT reduced platelet and white count (A, B), decreased spleen weight (C) and reduced megakaryocyte and erythroid cell infiltration in the spleen (D). *, p<0.05, **, p<0.01. Disclosures Levine: Novartis: Consultancy; Qiagen: Membership on an entity's Board of Directors or advisory committees.

Download Full-text

A Differentiation Block in Erythroid Cells Lacking Erythroid Krupple-Like Factor (EKLF).

Blood ◽

10.1182/blood.v106.11.526.526 ◽

2005 ◽

Vol 106 (11) ◽

pp. 526-526

Author(s):

Patrick G. Gallagher ◽

Murat O. Arcasoy ◽

Serena E. Vayda ◽

Holly K. Dressman ◽

James J. Bieker ◽

...

Keyword(s):

Cell Cycle ◽

Flow Cytometry ◽

Fetal Liver ◽

Erythroid Differentiation ◽

Erythroid Cells ◽

Wild Type ◽

Fetal Liver Cells ◽

Differentiation Block ◽

Microarray Analyses ◽

Deficient Mice

Abstract Mice deficient in the erythroid specific zinc-finger transcription factor EKLF die ~d14-15 of gestation of severe anemia, attributed to decreased expression of β-globin. The morphology of fetal-liver derived erythroid cells in EKLF-deficient mice does not mimic that seen in thalassemia, but instead shows hemolysis with uniform, nucleated erythroid progenitor cells. This has led to the hypothesis that a block in erythroid differentiation contributes to the anemia in EKLF-deficient mice. To address this, we performed microarray analyses with Affymetrix GeneChip Mouse Genome 430 2.0 arrays and RNA from d13.5 fetal livers of wild type (WT) and EKLF-deficient mice. Three independent EKLF +/+ and −/− RNA samples were analyzed. Numerous genes were down regulated including AHSP, pyruvate kinase, ankyrin, β spectrin and band 3. Verification of reduced expression of selected genes demonstrated that expression levels of many genes identified as down regulated via microarray analyses were minimally reduced in EKLF −/− RNA (<20%) compared to normal (Rh 30, protein 4.2, protein 4.9, p55, AQP1, and ALAS-E). Flow cytometry of WT d14.5 fetal liver cells using TER 119 and CD71 was performed. In WT fetal livers, this identifies 5 populations, designated R1-R5, with R1/R2 composed of primitive progenitors and proerythroblasts and R3, R4, and R5 composed of more mature erythroblasts (Blood102:3938, 2003). In EKLF −/− fetal livers, R3, R4, and R5, populations involved in terminal erythroid differentiation, were completely absent, suggesting many of the genes identified by microarray analyses were differentially expressed because of a bias introduced by a differentiation block to more mature erythroid cells. Confirming this hypothesis, we demonstrated that genes with <20% difference in expression between WT and EKLF-deficient fetal liver mRNA had 4-fold or higher levels in wild type R3+R4+R5 RNA compared to R1+R2 RNA. To better understand how differentially expressed genes were integrated into specific regulatory and signaling pathway networks, we used Ingenuity Pathway Analysis. A subset of focus genes incorporated into a biological network with highly a significant scores (>40) was generated containing 35 focus genes. The biological function of this network involved cell cycle and DNA replication. At the central nodes of this network were E2F1 and E2F2, transcription factors involved in cell cycle control. Cell cycle analysis demonstrated that EKLF-deficient R1 cells exhibited a significant delay exiting G0+G1 and entering S phase and both R1 and R2 cells exhibited a defect in exiting S and entering G2+M. Colony assays with R1 and R2 cells revealed that EKLF-deficient fetal liver cells had decreased frequency of CFU-E, but similar absolute numbers of CFU-E as WT. As predicted by the cell cycle defect, EKLF−/− FL cells were severely (~10 fold) deficient in their ability to generate BFU-E. Flow cytometry with annexin V revealed no difference between WT and EKLF-deficient cells indicated that apoptosis was not contributing to the differentiation block. These results support the hypothesis that the failure of definitive erythropoiesis in EKLF deficient mice is due to decreased expression of many erythroid genes involved in erythroid differentiation, stabilization of α-globin protein, membrane stability, and glycolysis, not simply decreased transcription of the β-globin gene.

Download Full-text

K-Ras is essential for normal fetal liver erythropoiesis

Blood ◽

10.1182/blood-2004-05-2021 ◽

2005 ◽

Vol 105 (9) ◽

pp. 3538-3541 ◽

Cited By ~ 23

Author(s):

Waleed F. Khalaf ◽

Hilary White ◽

Mary Jo Wenning ◽

Attilio Orazi ◽

Reuben Kapur ◽

...

Keyword(s):

Fetal Liver ◽

Erythroid Cell ◽

Wild Type ◽

Erythroid Progenitors ◽

Kit Ligand ◽

Ras Activation ◽

Ras Isoforms ◽

Fetal Erythropoiesis

AbstractIn vitro studies suggest that Ras activation is necessary for erythroid cell development. However, genetic inactivation of the Ras isoforms H-Ras, N-Ras, and K-Ras in mice reportedly did not affect adult or fetal erythropoiesis, though K-Ras-/- embryos were anemic. Given these discrepancies, we performed a more detailed analysis of fetal erythropoiesis in K-Ras-/- embryos. Day-13.5 K-Ras-/- embryos were pale with a marked reduction of mature erythrocytes in their fetal livers. The frequency and number of both early (erythroid burst-forming unit [BFU-E]) and late erythroid progenitors (erythroid colony-forming unit [CFU-E]) were reduced in K-Ras-/- fetal livers compared with wild-type controls and displayed a delay in terminal erythroid cell maturation. Further, K-Ras-/- hematopoietic progenitors had reduced proliferation in response to erythropoietin and Kit ligand compared with control cells. Thus, these studies identify K-Ras as a unique Ras isoform that is essential for regulating fetal erythropoiesis in vivo.

Download Full-text

Elevated P21 (CDKN1a) Mediates Apoptosis of Beta-Thalassemic Erythroid Cells in Mice but Its Ablation Doesn't Improve Erythroid Maturation

Blood ◽

10.1182/blood-2020-143384 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 19-19

Author(s):

Miao Lin ◽

Vijay Menon ◽

Raymond Liang ◽

Tasleem Arif ◽

Laura Breda ◽

...

Keyword(s):

Bone Marrow ◽

Double Mutant ◽

Complete Blood Count ◽

Globin Gene ◽

Mutant Mice ◽

Cell Maturation ◽

Erythroid Cell ◽

Beta Thalassemia ◽

Wild Type ◽

The Status

Beta-thalassemias are caused by mutations in the β-globin gene leading to anemia. In β-thalassemia, excessive accumulation of unpaired α globin chains in erythroblasts, triggers redox-mediated reactions, which is associated with increased production of immature erythroid precursors that fail to mature. This impaired maturation is in part due to increased apoptosis of late maturing erythroblasts in β-thalassemic patients that aggravates anemia despite enhanced erythropoiesis leading to what is called ineffective erythropoiesis and ultimately resulting in extramedullary expansion of hematopoiesis. The mechanism of apoptosis in beta-thalassemia remains poorly understood. To investigate this, we examined the status of mediators of stress response during erythroid cell maturation ofHbbth3/+ (th3/+) mice, a model that mimics the beta-thalassemia intermedia phenotype in humans. We found that both Foxo3 and p53 were prematurely activated in th3/+ beta-thalassemic erythroblasts as compared to wild type controls. We crossed Hbbth3/+ (th3/+) and Foxo3-/- mice and found that red blood cell (RBC) count and hemoglobin content were improved (by 1g/L, n=10), and erythroblast apoptosis was decreased to similar levels as in the WT during erythroblast maturation of double mutant mice. However, loss of Foxo3 did not ameliorate the splenomegaly of th3/+mice. We also found that p53 direct target, p21 the cyclin-dependent kinase inhibitor was greatly upregulated in th3/+erythroblasts as well as in beta-thalassemic patients' erythroblasts. To address the contribution of p21, we crossed p21-/- and Th3/+. It showed a significant decrease of apoptosis in CD45- TER119+ erythroblasts both in the bone marrow and spleen of double mutant mice (30% and 23% reduction respectively, n=6 mice each genotype). Although, as in beta-thalassemic patients, serum erythropoietin (Epo) was elevated in the peripheral blood of th3/+mice, the double mutant mice had significantly lower level of Epo than th3/+ (45% reduction, n=3 mice per genotype).In p21-/-th3/+, CD45- TER119+ cells also showed lesser ROS accumulation(12% less, n=3 per genotypes). However, to our surprise, the deletion of p21 on beta-thalassemic background did not have any effect on splenomegaly (n=6 mice each genotype), complete blood count, hemoglobin, RBC production or bone marrow erythroid cell maturation (n=12 mice each genotype). To further examine the underlying mechanism, we analyzed cell cycle in double mutant p21-/-th3/+ erythroblast at distinct stages of maturation identified by CD45, TER119, CD44 and cell size (n=3 mice per genotype) using ki67 staining at distinct stages of maturation. We found p21-/-th3/+erythroblasts proliferate much less than their th3/+ counterparts (basophilic erythroblasts G2 14% less, polychromatic erythroblasts 20% less, p<0.05 n=3 mice per genotype). This may partially explain lack of improvement of RBC production and anemia despite enhanced erythroblast survival. ROS levels were also reduced in double mutant p21-/-th3/+ erythroblasts as compared to controls. Next we investigated the status of p53 and Foxo3 in double mutant p21-/-th3/+ erythroblasts as compared to controls. We confirmed as we had observed earlier that nuclear p53 and Foxo3 expression were greater in th3/+ primitive erythroid (TER119-/low, c-KIT+, CD71Hi) cells than in wild type (n=3 mice per genotype) controls. Strikingly, the double mutant p21-/-th3/+ erythroblasts exhibited the greatest nuclear Foxo3 in all four groups, while nuclear p53 was dramatically reduced by over 80% (n=2 mice each genotype. Each mouse taking >=30 cells to calculate nuclear MFI) even as compared to wild type. These combined studies suggest that ameliorating apoptosis may not improve anemia in beta-thalassemia. Disclosures Liang: Hemogenyx Pharmaceuticals LLC: Current Employment.

Download Full-text