Hematopoietic-Specific CSNK2B Loss in Mice Causes Impaired Erythropoiesis

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.

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Mel Aggravates Pyroptosis Induced by Temozolomide Through Inhibiting NRF2-are Pathway in Glioblastoma

10.21203/rs.3.rs-35576/v1 ◽

2020 ◽

Author(s):

Hao Pan ◽

Handong wang ◽

Qiang Wang ◽

Wenhao Niu ◽

Qi Wu ◽

...

Keyword(s):

Western Blot ◽

Cell Viability ◽

Combined Treatment ◽

Flow Cytometric Analysis ◽

Glioma Cells ◽

Mtor Pathway ◽

Tumor Growth Inhibition ◽

Dependent Manner ◽

Related Protein

Abstract Background:Glioblastoma(GBM) is a common malignant tumor of the brain. It has been verified that melatonin(MEL) can inhibit glioma proliferation. But its mechanism has not been fully elucidated. We intend to examine the mechanism of MEL on glioma cells from the perspective of pyroptosis and Nrf2. Methods:Expression of MEL receptor in glioma was detected by western blot. GBM cell viability treated with temozolomide(TMZ) plus MEL was detected by CCK-8. Pyroptosis rate was determinate by flow cytometric analysis. Western blot was used to detect the Nrf2 and pyroptosis related protein level after MEL treatment. Orthotopic tumor growth inhibition study was performed to further investigate the tumor inhibition effect of TMZ plus MEL.Results:We first confirmed MEL receptor was abundant in glioma tissue and cell lines. After combined treatment of TMZ and MEL, cell viability decreased significantly as compared to those of TMZ treatment alone. Also, the ratio of pyroptosis and ROS level increased, followed by elevated expression of pyroptosis related protein. Furthermore, MEL can induce a diminution of Nrf2 expression in glioma in dose- and time-dependent manner. TMZ can increase Nrf2-ARE pathway expression, which also can be deprived by MEL. Its inhibition of Nrf2 depends on dephosphorylation of IGF-1/AKT/mTOR pathway. More importantly, after overexpression of Nrf2 in glioma cells, the level of pyroptosis-related protein elevated by MEL decreased, suggesting that the effect of MEL on promoting pyroptosis is dependent on its inhibition of Nrf2. In vivo results further confirmed that MEL plus TMZ induced significantly decreased tumor size and increased pyroptosis rate, but had no significant effect on mouse body weight, ALT, AST.Conclusion:MEL can inhibit the phosphorylation of IGF-1/AKT/mTOR pathway at millimol level, which further reduces the expression of Nrf2 and promotes pyroptosis of glioma cells. Considering the modest efficacy of TMZ chemotherapy, MEL can be considered as a potential chemotherapy sensitizer to improve the chemotherapy effect of glioma.

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Cardiac deletion of α-E-catenin leads to redused expression of the main component of desmosomes – γ-catenin

Faktori eksperimental'noi evolucii organizmiv ◽

10.7124/feeo.v21.854 ◽

1970 ◽

Vol 21 ◽

pp. 293-296

Author(s):

V. V. Balatskyi ◽

L. L. Matsevych ◽

O. O. Piven

Keyword(s):

Heart Failure ◽

Transgenic Mice ◽

Western Blot ◽

Real Time ◽

Knockout Mice ◽

Protein Level ◽

Conditional Knockout ◽

Protein Levels ◽

Main Component ◽

Real Time Qpcr

Aim. In our present work, we have addressed to the γ-catenin, known main component of desmosomes, expression in hearts with heterozygous and homozygous knockout of α-E-catenin gene. Methods. Alpha-E-catenin conditional knockout mice were bred with α-MHC-Cre transgenic mice. We analyze expression of γ-catenin with real time qPCR and Western blot. Results. Cardiac α-E-catenin deletion leads to downregulation of γ-catenin mRNA and protein levels only in homozygous mice, while we not observed any perturbation of γ-catenin expression in heterozygous mice. Conclusions. We have shown that homozygous knockout of α-E-catenin gene in embryonic heart occur reduction of the main component of desmosomes – γ-catenin mRNA and protein level of expression, which can lead to disruption of the desmosomes structure in adult myocardium. Keywords: α-E-catenin, heart failure, γ-catenin.

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Histone Methyltransferase Setd8 Represses Gata2 Expression and Regulates Erythroid Maturation

Molecular and Cellular Biology ◽

10.1128/mcb.01413-14 ◽

2015 ◽

Vol 35 (12) ◽

pp. 2059-2072 ◽

Cited By ~ 18

Author(s):

Jeffrey Malik ◽

Michael Getman ◽

Laurie A. Steiner

Keyword(s):

Global Gene Expression ◽

Regulatory Elements ◽

Histone Methyltransferase ◽

Tissue Type ◽

Erythroid Cell ◽

Erythroid Cells ◽

Histone H4 ◽

Nuclear Condensation ◽

Erythroid Maturation ◽

In Erythroid Cells

Setd8 is the sole histone methyltransferase in mammals capable of monomethylating histone H4 lysine 20 (H4K20me1). Setd8 is expressed at significantly higher levels in erythroid cells than any other cell or tissue type, suggesting that Setd8 has an erythroid-cell-specific function. To test this hypothesis, stable Setd8 knockdown was established in extensively self-renewing erythroblasts (ESREs), a well-characterized, nontransformed model of erythroid maturation. Knockdown of Setd8 resulted in impaired erythroid maturation characterized by a delay in hemoglobin accumulation, larger mean cell area, persistent ckit expression, incomplete nuclear condensation, and lower rates of enucleation. Setd8 knockdown did not alter ESRE proliferation or viability or result in accumulation of DNA damage. Global gene expression analyses following Setd8 knockdown demonstrated that in erythroid cells, Setd8 functions primarily as a repressor. Most notably, Gata2 expression was significantly higher in knockdown cells than in control cells and Gata2 knockdown rescued some of the maturation impairments associated with Setd8 disruption. Setd8 occupies critical regulatory elements in the Gata2 locus, and knockdown of Setd8 resulted in loss of H4K20me1 and gain of H4 acetylation at the Gata2 1S promoter. These results suggest that Setd8 is an important regulator of erythroid maturation that works in part through repression of Gata2 expression.

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Silencing of RhoA nucleotide exchange factor, ARHGEF3, reveals its unexpected role in iron uptake

Blood ◽

10.1182/blood-2011-02-337295 ◽

2011 ◽

Vol 118 (18) ◽

pp. 4967-4976 ◽

Cited By ~ 23

Author(s):

Jovana Serbanovic-Canic ◽

Ana Cvejic ◽

Nicole Soranzo ◽

Derek L. Stemple ◽

Willem H. Ouwehand ◽

...

Keyword(s):

Blood Cell ◽

Cell Biology ◽

Iron Uptake ◽

Meta Analysis ◽

Erythroid Cell ◽

Erythroid Cells ◽

Nucleotide Exchange ◽

Exchange Factor ◽

Nucleotide Exchange Factor ◽

Genomewide Association

Abstract Genomewide association meta-analysis studies have identified > 100 independent genetic loci associated with blood cell indices, including volume and count of platelets and erythrocytes. Although several of these loci encode known regulators of hematopoiesis, the mechanism by which most sequence variants exert their effect on blood cell formation remains elusive. An example is the Rho guanine nucleotide exchange factor, ARHGEF3, which was previously implicated by genomewide association meta-analysis studies in bone cell biology. Here, we report on the unexpected role of ARHGEF3 in regulation of iron uptake and erythroid cell maturation. Although early erythroid differentiation progressed normally, silencing of arhgef3 in Danio rerio resulted in microcytic and hypochromic anemia. This was rescued by intracellular supplementation of iron, showing that arhgef3-depleted erythroid cells are fully capable of hemoglobinization. Disruption of the arhgef3 target, RhoA, also produced severe anemia, which was, again, corrected by iron injection. Moreover, silencing of ARHGEF3 in erythromyeloblastoid cells K562 showed that the uptake of transferrin was severely impaired. Taken together, this is the first study to provide evidence for ARHGEF3 being a regulator of transferrin uptake in erythroid cells, through activation of RHOA.

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Csnk2β Knockout during Hematopoiesis Results in Lethality at Mid/Late Gestation Mostly Due to Impaired Fetal Erythropoiesis

Blood ◽

10.1182/blood.v124.21.4329.4329 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4329-4329

Author(s):

Laura Quotti Tubi ◽

Sara Canovas Nunes ◽

Alessandro Casellato ◽

Elisa Mandato ◽

Fortunato Zaffino ◽

...

Keyword(s):

Transcription Factors ◽

High Throughput ◽

Fetal Liver ◽

Cell Development ◽

Conditional Knockout ◽

Late Gestation ◽

Cell Maturation ◽

Erythroid Cell ◽

Erythroid Cells ◽

Fetal Erythropoiesis

Abstract Background. CK2, a serine-threonine kinase composed of two catalytic (α) and two regulatory (β) subunits, has been clearly involved in several hematologic malignancies. This kinase regulates the PTEN/PI3K/AKT, Wnt/βcatenin, Hedgehog, JAK-STAT, cMyc and NF-κB signalling cascades, all of which are known to be of critical importance for hematopoietic stem cell (HSC) biology and normal hematopoiesis. However, the role played by CK2 during blood cell development has remained as yet unexplored. Aims and methods. CK2 function in hematopoiesis was investigated generating conditional knockout mice for CK2β by crossing Csnk2β-Flox/Flox mice with Vav1-CRE transgenic mice. Inactivation of Csnk2β started from 9.5 dpc during embryonic development. Histo-cytological methods, FACS analysis, colony-forming assays (CFA), signal transduction studies by western blotting and RT-PCR were employed to characterize the cellular and molecular phenotype. High throughput RNAseq analysis was also performed on purified Ter119-positive erythroid cells from Csnk2β knockout and Csnk2β control mice to identify differentially expressed CK2-dependent transcriptional targets. Results. Csnk2β knockout in hematopoiesis resulted lethal at mid-late gestation. Rarely some pups were found dead at birth. Macroscopic and phenotypic analysis during gestation revealed the appearance of pale and hydropic fetuses after 12.5 dpc. The majority of pups showed teleangiectasic vessels and haemorrhages. Fetal livers appeared smaller and paler. Cytological analysis and CFA studies unveiled a great depletion of hematopoietic elements belonging to both the erythroid, megakaryocytic and granulocytic-monocytic precursors. A more thorough analysis of the erythroid phenotype revealed that Csnk2β loss caused impairment/loss of red cell maturation at two developmental stages: the earlier stages of Megakaryocyte-Erythroid Precursors (MEP) and pro-erythroblasts and the later stages of terminal maturation (orthocromatic erythroblasts towards reticulocytes). Expression analysis of proteins/genes belonging to known hematopoietic and erythroid-regulating pathways showed perturbations in cell cycle regulatory molecules, cellular apoptosis, a marked reduction of total and phosphorylated Akt in Ser473 and Ser129, a decrease of GATA1 protein levels and a decrease of Hedgehog/Wnt target genes such as Gli-1 and Cyclin D1. Erythropoietin-dependent AKT activation and GATA1 phosphorylation was impaired by Csnk2β loss. Moreover, starting at 14.5 dpc, blood cells displayed a massive p53-dependent response, marked by high levels of p21 and a progressive clear apopototic phenotype. At 17.5 dpc residual hematopoietic cells in the fetal liver were represented by dying erythroid cells, immature myelo-monocytic precursors (expressing high CD11b and low Gr1 levels on the surface) and B-cells displaying an aberrant phenotype with low intensity of expression of B220 and CD19 on the surface. High throughput RNAseq analysis of Ter119-expressing fetal liver cells (erythroid lineage) obtained from 14.5 dpc pups revealed the upregulation of 145 transcripts and the downregulation of 68 transcripts. Among the most increased transcripts were the transcription factors Jun/AP1 and stress-related intermediaries and embryonal globin ε and ζ chains. Among the most decreased transcripts were sugar transporters, glycoproteins CD36 and CD59a, Duffy Blood Group Atypical Chemokine Receptor and component members. Conclusions. We found that Csnk2β plays a critical role in mouse blood development by regulating definitive hematopoiesis of all the hematopoietic cell lineages; however, Csnk2β was needed for the early and late erythropoiesis whilst its loss could be compatible with a certain extent of immature/altered myelo-monocytic and B cell development. Among the pathways found targeted by Csnk2β loss were the PI3K/Akt and the p53-p21 cascades. Our data also suggest that Csnk2β might have a role in the proper activation of the erythroid master regulator GATA1. Moreover, RNAseq analysis revealed that this kinase might have a broader impact during erythroid cell maturation by regulating the activity of critical stress related transcription factors, of molecules regulating energy-managing cellular processes and of mechanisms controlling the switch from embryonal to fetal erythropoiesis. Disclosures No relevant conflicts of interest to declare.

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Zfp281 (ZBP-99) plays a functionally redundant role with Zfp148 (ZBP-89) during erythroid development

Blood Advances ◽

10.1182/bloodadvances.2018030551 ◽

2019 ◽

Vol 3 (16) ◽

pp. 2499-2511

Author(s):

Andrew J. Woo ◽

Chelsea-Ann A. Patry ◽

Alireza Ghamari ◽

Gabriela Pregernig ◽

Daniel Yuan ◽

...

Keyword(s):

Transcription Factor ◽

Hypochromic Anemia ◽

Conditional Knockout ◽

Erythroid Cell ◽

Core Set ◽

Perinatal Lethality ◽

Delayed Recovery ◽

Erythroid Maturation ◽

Erythroid Cell Differentiation ◽

Erythroid Development

Abstract Erythroid maturation requires the concerted action of a core set of transcription factors. We previously identified the Krüppel-type zinc finger transcription factor Zfp148 (also called ZBP-89) as an interacting partner of the master erythroid transcription factor GATA1. Here we report the conditional knockout of Zfp148 in mice. Global loss of Zfp148 results in perinatal lethality from nonhematologic causes. Selective Zfp148 loss within the hematopoietic system results in a mild microcytic and hypochromic anemia, mildly impaired erythroid maturation, and delayed recovery from phenylhydrazine-induced hemolysis. Based on the mild erythroid phenotype of these mice compared with GATA1-deficient mice, we hypothesized that additional factor(s) may complement Zfp148 function during erythropoiesis. We show that Zfp281 (also called ZBP-99), another member of the Zfp148 transcription factor family, is highly expressed in murine and human erythroid cells. Zfp281 knockdown by itself results in partial erythroid defects. However, combined deficiency of Zfp148 and Zfp281 causes a marked erythroid maturation block. Zfp281 physically associates with GATA1, occupies many common chromatin sites with GATA1 and Zfp148, and regulates a common set of genes required for erythroid cell differentiation. These findings uncover a previously unknown role for Zfp281 in erythroid development and suggest that it functionally overlaps with that of Zfp148 during erythropoiesis.

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

Blood ◽

10.1182/blood.v124.21.4356.4356 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4356-4356

Author(s):

Kelsey Swartz ◽

Gangjian Qin ◽

Alexander C Minella

Keyword(s):

Flow Cytometry ◽

Knockout Mice ◽

Transcriptional Activity ◽

Fetal Liver ◽

Cyclin E ◽

Cell Maturation ◽

Erythroid Cell ◽

Wild Type ◽

Independent Functions ◽

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.

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SGK1 Inhibition Induces Fetal Hemoglobin in Erythroid Cells

Blood ◽

10.1182/blood-2021-146071 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 963-963

Author(s):

Yannis Hara ◽

Mark Stottlemyer ◽

Kim Alving ◽

Nis Halland ◽

Alexandra Hicks ◽

...

Keyword(s):

Western Blot ◽

Blood Donors ◽

Fetal Hemoglobin ◽

Erythroid Cell ◽

Erythroid Cells ◽

Target Engagement ◽

Western Blots ◽

Cd34 Cells ◽

Hbf Induction ◽

In Erythroid Cells

Abstract Introduction: Novel and safe therapeutic targets to increase expression of fetal hemoglobin (HbF) have potential to treat b-hemoglobinopathies (Platt, Brambilla et al. 1994, Steinberg 2020), including sickle cell disease (SCD) in which red blood cell (RBC) hemoglobin S resulting from a mutation in the hemoglobin β-globin subunit causes RBC sickling and hemolysis triggering vascular inflammation (Piel, Steinberg et al. 2017, Kato, Piel et al. 2018). Serum- and glucocorticoid-regulated kinase 1 (SGK1) is a serine/threonine kinase in the AGK kinase family that controls physiological processes such as cell growth, proliferation, migration, and apoptosis (Hayashi, Tapping et al. 2001, Sang, Kong et al. 2020). SGK1 is regulated by multiple ligands (insulin, cAMP, IGF-1, steroids, IL-2 and TGF-β) and phosphorylation by SGK1 modulates the activity of downstream effectors including ion channels (ENaC), Na-Cl cotransporters (NCC), membrane transporters, cellular enzymes (GSK3B) and transcription factors (FOXO3a, β-catenin, NF-κB and SP1) (Brunet, Park et al. 2001, Snyder, Olson et al. 2002, Loffing, Flores et al. 2006, Bruhn, Pearson et al. 2010, Boccitto and Kalb 2011, Wang, Hu et al. 2017). Previous studies show that SGK1 mediates survival signals in HEK cells by inhibiting FOXO3a through phosphorylation at Ser-315 (Brunet, Park et al. 2001). Recently, metformin was shown to induce HbF in erythroid cells through FOXO3a activation and metformin prevents RBC sickling in SCD (Zhang, Paikari et al. 2018). Thus, we hypothesized that inhibition of SGK1 and subsequent alleviation of SGK1-induced FOXO3a inhibition, may induce expression of erythroid cell HbF. Methods: We studied the ability of SGK1 to inhibit HbF induction in erythroid cells by culturing CD34+ hematopoietic progenitor stem cells from both healthy and SCD blood donors using a 21-day differentiation protocol. After confirming expression of SGK1 in CD34+ cells by Western blot, SGK1 activity was inhibited using the selective and potent SGK1 inhibitor RA04075215A (Halland, Schmidt et al. 2015). SGK1 is activated by phosphorylation at Thr256 and we confirmed target engagement through measurement of Thr256 phosphorylation on Western blots. To decipher the effect of SGK1 inhibition on the SGK1 downstream pathway, we assessed the inhibition of FOXO3a triggered by SGK1 through evaluation of FOXO3a phosphorylation Ser315. In parallel, we quantified HbF gene transcripts by qPCR, determined the level of HbF protein by Western blot, and quantified F-cells by flow cytometry. Finally, to evaluate the effect of SGK1 inhibition on RBC sickling, we performed a cell sickling assay upon completion of erythroid differentiation in culture. Fully differentiated CD34+ cells from SCD blood donors were incubated under in hypoxia (2% O 2) for 4 hours and then abnormal shaped cells were analyzed using the Amnis® ImageStream® flow cytometer. Results: By day 21 of differentiation, HbF protein expression in CD34+ cells increases significantly in RA04075215A-treated cells versus untreated controls. In addition, a combination of SGK1 inhibition and hydroxyurea treatment reveals a potential synergistic induction of HbF. Western blot analysis shows a decrease in phospho-SGK1 phosphorylated at Thr-256 with SGK1 inhibition, confirming target engagement and loss of SGK1 activity. Downstream of SGK1, phospho-FOXO3a phosphorylated at Ser-315 was also decreased significantly following SGK1 inhibition, demonstrating alleviation of FOXO3a inhibition. Finally, in the RBC sickling assay, RA04075215A-treated cells were significantly protected from sickling under hypoxia compared to controls. Conclusion: In summary, this study establishes SGK1 as a potential new therapeutic target in SCD. We demonstrate that SGK1 inhibition induces HbF in CD34+ cells through FOXO3a transcription factor activation and prevents CD34+ cells from sickling. In the future, in vivo studies are necessary to confirm the role of SGK1 in HbF induction and to assess the efficacy of SGK1 inhibition in improving markers of SCD. Disclosures No relevant conflicts of interest to declare.

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EKLF Directly Activates the p21WAF1/CIP1 Gene by Proximal Promoter and Novel Intronic Regulatory Regions during Erythroid Differentiation

Molecular and Cellular Biology ◽

10.1128/mcb.01016-09 ◽

2010 ◽

Vol 30 (11) ◽

pp. 2811-2822 ◽

Cited By ~ 30

Author(s):

Miroslawa Siatecka ◽

Felix Lohmann ◽

Sujin Bao ◽

James J. Bieker

Keyword(s):

Cell Cycle ◽

Transcription Factor ◽

Erythroid Differentiation ◽

Proximal Promoter ◽

Erythroid Cell ◽

Cell Phenotype ◽

Cdk Inhibitors ◽

Red Cell ◽

Rich Region ◽

Erythroid Maturation

ABSTRACT The switch from proliferation to differentiation during the terminal stages of erythropoiesis is a tightly controlled process that relies in part on transcription factor-mediated activation of cell cycle components. EKLF is a key transcription factor that is necessary for the initial establishment of the red cell phenotype. Here, we find that EKLF also plays a role during the subsequent differentiation process, as it induces p21WAF1/CIP1 expression independent of p53 to regulate the changes in the cell cycle underlying erythroid maturation. EKLF activates p21 not only by directly binding to an EKLF site within a previously characterized GC-rich region in the p21 proximal promoter but also by occupancy at a novel, phylogenetically conserved region that contains consensus CACCC core motifs located downstream from the p21 TATA box. Our findings demonstrate that EKLF, likely in coordination with other transcription factors, directly contributes to the complex set of events that occur at the final erythroid cell divisions and accentuates terminal differentiation directly by activation of CDK inhibitors such as p21.

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Embryonic to Fetal Globin Switch Is Associated with Erythroid Cell Maturation in Primitive Hematopoiesis Derived from hESCs.

Blood ◽

10.1182/blood.v106.11.3625.3625 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3625-3625

Author(s):

Caihong Qiu ◽

Emmanuel N. Olivier ◽

Eric E. Bouhassira

Keyword(s):

Liquid Culture ◽

Fetal Liver ◽

Yolk Sac ◽

Erythroid Cell ◽

Globin Genes ◽

Erythroid Cells ◽

Hemoglobin Content ◽

Large Numbers ◽

Erythroid Maturation ◽

High Level

Abstract In humans, the first erythroblasts appear 14–19 days post-fertilization. At week 5, yolk sac erythroblasts synthesize primarily ζ and ε globins, but at weeks 6 and 7, these primitive erythroblasts also synthesize large amounts of α-globin and moderate amounts of γ-globin. Therefore yolk-sac-derived primitive erythrocytes undergo a partial hemoglobin switch. Cells derived from the yolk sac do not synthesize β-globin. At 6 weeks of gestation, erythropoiesis also takes place in the fetal liver, and express ζ, α, ε, γ and small amounts of β-globin. In this organ, the ζ and ε-globin genes are rapidly silenced while the γ genes remain expressed at high level until birth. Little is known about early globin switches in human because experimental material is difficult to obtain. Here we took advantage of a recently developed method (see accompanying abstract) of production of large numbers of human primitive erythroid cells in liquid culture to characterized globin switching in human primitive cells. This method is a 5 steps procedure that leads from hESCs to virtually pure populations of orthochromatic erythroblasts. At step 3, 90% pure populations of hemoglobinized immature polychromatic erythroblasts (hemoglobin content 6pg/106 cells) were analyzed by HPLC for globin expression. Results revealed that these cells express mostly ζ and ε globins and only traces of α and γ globins with α/ζ<0.05. At the end of step 5 the cells had matured to orthochromatic erythroblasts (hemoglobin content 20 pg/106 cells) and expressed much larger amounts of α and γ-globin with α/ζ and γ/ε ratios of 3.74±1.59 and 0.52±0.08 demonstrating a switch in globin expression during the culture. Since differentiation of these erythroid cells is not completely synchronized, the globin switches could happen either gradually during the erythroid maturation process or could be due to a late developing population of progenitors remaining in the third step culture. To discriminate these possibilities we sorted 3rd step cells with α/ζ and γ/ε < 0.05 according to their CD45 and CD235a expression into immature erythroblasts (CD45−/235alow) and polychromatophilic erythroblasts (CD45−/CD235high) and place them back in culture. After 7 days of culture the sorted populations of CD45−/CD235a+ cells had switched and respectively exhibited α/ζ ratios of 1.40 and 1.71, and γ/ε ratios of 0.59 and 0.53. These results suggested that these globin switches are associated with terminal erythroblastic maturation rather than with the production of successive waves of progenitors expressing different globin expression programs. To determine if the switches could be observed in clonally derived population of cells, we developed an assay in which individual CD34+ cells derived from hESCs (step 1 of our procedure) were expanded in liquid culture and analyzed by real-time RT-PCR for globin expression. These experiments revealed that all 8 clonally derived populations of cells switched from producing ζ to α globin with mean α/ζ ratio to 0.11±0.10 at step 2 and 159.80±219.71 at step 5. Only traces of β-globin expression could be detected in all the cells tested by HPLC or PCR. We conclude that the switches in globin expression that occur in the yolk sac during early human development are very different from the switch that occur later in life since they are caused by differential expression of the ζ, α, ε, and γ globin genes during late erythroid maturation rather than to production of progenitors with different expression programs.

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