N-terminal Region of CCAAT/Enhancer-binding Protein ϵ Is Critical for Cell Cycle Arrest, Apoptosis, and Functional Maturation during Myeloid Differentiation

Abstract Background Adenosine is a major signaling nucleoside that activates cellular signaling pathways through a family of four different G protein-coupled adenosine receptors (ARs), A 1, A 2A, A 2B, and A 3. At steady state conditions, extracellular levels of adenosine remain low (10 to 200 nM) either through its rapid cellular uptake by specialized nucleoside transporters, mainly through the equilibrative nucleoside transporter 1 (ENT1), or its degradation by adenosine deaminases. However, the extracellular levels of adenosine can be rapidly elevated up to 100 μM in response to hypoxia, inflammation, or tissue injury. Under pathophysiological conditions, adenosine signaling is involved in modulating inflammation, fibrosis, and ischemic tissue injury. In sickle cell disease (SCD), adenosine signaling is enhanced and contributes to the pathophysiology of the disease. Despite the importance of adenosine signaling in regulating cell proliferation, and stem cell regeneration, as well as in red blood cell functions and adaptation to hypoxia, very little is known about its implication in hematopoiesis, and more specifically during erythropoiesis. Here, we aimed to investigate the effects of high extracellular adenosine on the erythroid commitment and differentiation of hematopoietic progenitors, and to decipher the implication of ARs in these processes. Results To investigate the role of high extracellular adenosine in regulating erythroid commitment and differentiation of hematopoietic progenitors, we performed ex vivo erythropoiesis of healthy CD34 + cells in the presence or absence of increased extracellular adenosine concentrations ranging from 10 to 200 µM. Our results showed that adenosine decreases erythroid proliferation in a dose dependent manner. High adenosine levels (>50μM) inhibited the proliferation of erythroid precursors and increased apoptosis via a cell cycle arrest in G1. Accordingly, western blots revealed the accumulation of p53 and its downstream target p21, a well-known mediator of G1 cell-cycle arrest, in adenosine-treated cells. Moreover, adenosine treatment led to the persistence of a non-erythroid GPA neg subpopulation expressing myeloid markers (CD18, CD11a, CD13, CD33). May-Grünwald Giemsa staining of this subset revealed granular cells at different stages of differentiation. The culture of FACS-sorted CD36 + and CD36 - cells suggested that this adenosine-induced GPA neg population originates from the survival of CD36 - myeloid progenitors even in the presence of erythropoietin. Importantly, these effects were specific to adenosine as neither guanosine, uridine nor cytidine affected the proliferation and differentiation of erythroid precursors. Furthermore, we have recently shown that ENT1-mediated adenosine uptake is essential for optimal erythroid differentiation. Therefore, we suggested that elevated extracellular adenosine perturbs erythropoiesis via its signaling upon ARs activation. To confirm this hypothesis, we assessed the effect of ARs activation during erythropoiesis. Given that A 2B and A 3 are the only known ARs expressed in human hematopoietic progenitors and erythroid precursors, we used BAY60-6583 and CI-IB-MECA, two highly selective agonists for A 2B and A 3 receptors, respectively. Both BAY60-6583 and CI-IB-MECA increased apoptosis and decreased erythroblast maturation and enucleation, while only Cl-IB-MECA led to the upregulation of CD33 and CD11a myeloid markers and promoted the differentiation of a GPA neg myeloid subpopulation. Conclusion Overall, our results place adenosine signaling as a new player in hematopoiesis regulation. Adenosine signaling via A 3 perturbs erythropoiesis and promotes the survival and differentiation of myeloid progenitors even in an erythroid favoring environment. While the activation of A 2B hampers optimal erythropoiesis without impacting the myeloid differentiation. As both ineffective erythropoiesis and increased leucocyte counts are reported in SCD, and given the detrimental role of high adenosine levels in its pathophysiology, further studies are ongoing to address the impact of adenosine signaling on hematopoiesis in this disease. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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CCAAT/Enhancer Binding Protein alpha uses distinct domains to prolong pituitary cells in the Growth 1 and DNA Synthesis phases of the cell cycle

BMC Cell Biology ◽

10.1186/1471-2121-3-6 ◽

2002 ◽

Vol 3 (1) ◽

Cited By ~ 9

Author(s):

Weiqun Liu ◽

John F Enwright ◽

William Hyun ◽

Richard N Day ◽

Fred Schaufele

Keyword(s):

Cell Cycle ◽

Dna Synthesis ◽

Binding Protein ◽

Pituitary Cells ◽

Enhancer Binding Protein ◽

Ccaat Enhancer Binding Protein

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Granulocyte colony-stimulating factor regulates myeloid differentiation through CCAAT/enhancer-binding protein ε

Blood ◽

10.1182/blood.v98.4.897.h8000897_897_905 ◽

2001 ◽

Vol 98 (4) ◽

pp. 897-905 ◽

Cited By ~ 2

Author(s):

Hideaki Nakajima ◽

James N. Ihle

Keyword(s):

Molecular Mechanisms ◽

Binding Protein ◽

Hematopoietic Cells ◽

Myeloid Differentiation ◽

Granulocyte Colony Stimulating Factor ◽

Colony Stimulating Factor ◽

Granulocytic Differentiation ◽

Enhancer Binding Protein ◽

Ccaat Enhancer Binding Protein ◽

Stimulating Factor

Granulocyte colony-stimulating factor (G-CSF) is a major cytokine that regulates proliferation and differentiation of myeloid cells, although the underlying mechanisms by which G-CSF controls myeloid differentiation are largely unknown. Differentiation of hematopoietic cells is regulated by lineage-specific transcription factors, and gene-targeting studies previously revealed the critical roles of CCAAT/enhancer-binding protein (C/EBP) α and C/EBPε, respectively, in the early and mid-late stages of granulocyte differentiation. The expression of C/EBPε in 32Dcl3 cells and FDCP1 cells expressing mutant G-CSF receptors was examined and it was found that G-CSF up-regulates C/EBPε. The signal for this expression required the region containing the first tyrosine residue of G-CSF receptor. Dominant-negative signal transducers and activators of transcription 3 blocked G-CSF–induced granulocytic differentiation in 32D cells but did not block induction of C/EBPε, indicating that these proteins work in different pathways. It was also found that overexpression of C/EBPε greatly facilitated granulocytic differentiation by G-CSF and, surprisingly, that expression of C/EBPε alone was sufficient to make cells differentiate into morphologically and functionally mature granulocytes. Overexpression of c-myc inhibits differentiation of hematopoietic cells, but the molecular mechanisms of this inhibition are not fully understood. In 32Dcl3 cells overexpressing c-myc that do not differentiate by means of G-CSF, induction of C/EBPε is completely abrogated. Ectopic expression of C/EBPε in these cells induced features of differentiation, including changes in nuclear morphologic characteristics and the appearance of granules. These data show that C/EBPε constitutes a rate-limiting step in G-CSF–regulated granulocyte differentiation and that c-myc antagonizes G-CSF–induced myeloid differentiation, at least partly by suppressing induction of C/EBPε.

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Granulocyte colony-stimulating factor regulates myeloid differentiation through CCAAT/enhancer-binding protein epsilon

Blood ◽

10.1182/blood.v98.4.897 ◽

2001 ◽

Vol 98 (4) ◽

pp. 897-905 ◽

Cited By ~ 53

Author(s):

H. Nakajima

Keyword(s):

Binding Protein ◽

Myeloid Differentiation ◽

Granulocyte Colony Stimulating Factor ◽

Colony Stimulating Factor ◽

Enhancer Binding Protein ◽

Ccaat Enhancer Binding Protein ◽

Stimulating Factor

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Post-Translational Modification and Differential Protein Binding Partners of CCAAT Enhancer Binding Protein Alpha (C/EBPα) p42 Versus p30 May Contribute to Aberrant C/EBPα Activity in Acute Leukemia

Blood ◽

10.1182/blood.v112.11.3577.3577 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3577-3577

Author(s):

Matthew Silver ◽

Nirmalee Abayasekara ◽

Dylan Perry ◽

Hong Sun ◽

Nancy Berliner ◽

...

Keyword(s):

Protein Binding ◽

Transcriptional Activity ◽

Binding Protein ◽

Myeloid Cells ◽

Leukemic Cells ◽

Myeloid Differentiation ◽

Differential Protein ◽

Binding Partners ◽

Enhancer Binding Protein ◽

Ccaat Enhancer Binding Protein

Abstract CCAAT enhancer binding protein alpha (C/EBPα) is the founding member of a family of basic region/leucine zipper (bzip) transcription factors and has been shown to be a master regulator of granulopoiesis It is expressed at high levels throughout myeloid differentiation and has been shown to bind to the promoters of multiple myeloid- specific gene promoters at different stages of myeloid maturation. Profound hematopoietic abnormalities have been reported for mice nullizygous for including a selective early block in the differentiation of C/EBPα, granulocytes. Mutations in C/EBPα have been demonstrated in a subset of patients with AML presenting with a normal karyotype. These mutations can result in the expression of a 30kD dominant negative C/EBPα isoform which contributes to loss of C/EBPα function. We have sought to understand the molecular basis for this observation. We and others have demonstrated that C/EBPα is post-translationally modified by small ubiquitin-related modifier (SUMO) at a lysine residue (K159) that lies within a region of the C/EBPα protein that can negatively affect transcriptional activity. We have demonstrated that the levels of sumoylated p42C/EBPα decrease upon normal neutrophil maturation and that transactivation of the myeloid-specific lactoferrin (LF) promoter reporter is significantly enhanced by a p42 sumoylation mutant of C/EBPα (K159A). Additionally, in oligonucleotide pull down assays, we show that sumoylated p42C/EBPα binds to the C/EBP site in the LF promoter in immature myeloid cells (which do not express LF) while loss binding and LF of sumoylation correlates with loss of p42C/EBPα expression in more mature cells. Based on these observations we is associated with the negative conclude that sumoylated p42C/EBPα regulation of LF in early myeloid cells. We further demonstrate that sumoylated p42C/EBPα remains bound to the LF promoter following ATRA induction of the leukemic NB4 cells, which do not express LF despite induction of morphologic maturation. Based on these observations we conclude that during normal myeloid differentiation, sumoylated p42C/EBPα is associated with the negative regulation of LF in early myeloid cells, and that LF expression upon maturation is associated with loss of binding of sumoylated p42 C/EBPα In leukemic cells induced toward mature neutrophils, sumoylated p42C/EBPα remains bound to the LF promoter, contributing to the lack of expression of LF in these cells. We show in addition, that p30 C/EBPα can also be sumoylated. In transactivation assays, however, sumoylated p42C/EBPα suppresses LF promoter activity more efficiently than p30C/EBPα in 293 cells. In order to identify differential protein binding partners of p30 and p42C/EBPα that could account for the differential transcriptional activity of the two isoforms, we have used a one step purification method that allows isolation of biotinylated C/EBPα p30 and p42- containing complexes using magnetic-streptavidin beads. The K562 myelomonocytic cell line stably expressing a biotin ligase (BirA) plasmid was transfected with p30C/EBPα or p42C/EBPα each containing a 23 amino acid tag at the N-terminus that allows for in vivo biotinylation. Proteins complexed with the two C/EBP isofoms have been isolated and are currently being identified by LC- MS MS analysis. Their differential association with the two isofoms of C/EBPα will be confimed by coimmunoprecipitation assays in normal myeloid and in leukemic cells. The identification of differentially bound proteins to p30 and p42 C/EBPα may identify molecular targets for future drug development.

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Elevated EGR2 Expression Provides a Potential Link Between Cell Cycle Arrest and Induced Differentiation in Myeloid Progenitor Cells

Blood ◽

10.1182/blood.v124.21.2733.2733 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2733-2733

Author(s):

Joshua B. Bland ◽

Jose R. Peralta ◽

William T. Tse

Keyword(s):

Cell Cycle ◽

Cell Cycle Arrest ◽

Progenitor Cells ◽

Myeloid Differentiation ◽

Phenotypic Effect ◽

Induced Differentiation ◽

Hl60 Cells ◽

Cycle Arrest ◽

Myeloid Progenitor ◽

Myeloid Progenitor Cells

Abstract Similar to many immature cell types, myeloid progenitor cells need to exit cell cycle to undergo terminal differentiation, but the mechanism linking the two is still unclear. Elucidating this mechanism could lead to the development of new differentiation therapies to treat myeloid leukemia. Recent studies have suggested that the processes regulating myeloid differentiation and cell cycle progression together constitute a positive feedback loop where each process reciprocally affects the other. To study the relationship between these processes, we examined early cellular and molecular events associated with induced differentiation of the HL60 human promyelocytic leukemia cells. We treated HL60 cells with 3 classical inducers of differentiation (vitamin D3 analog EB1089 (EB), all-trans retinoic acid (ATRA), and dimethyl sulfoxide (DMSO)), along with PD0332991 (PD), a selective cyclin D-dependent kinase 4/6 inhibitor that caused G1-phase-specific cell-cycle arrest. We evaluated differentiation of the treated cells by flow cytometric analysis of CD11b (integrin αM) and CD71 (transferrin receptor) expression. In untreated HL60 cells, a baseline subset of 3-5% of cells exhibits a differentiated, CD11b+CD71- phenotype. Exposure to the various inducers revealed a progressive increase in the percentage of CD11b+CD71- cells with time, such that by day 4 of treatment, it has increased to 50-90% in the treated samples, indicating that all 4 agents tested were effective in inducing myeloid differentiation. To understand how differentiation induced by each agent affects cell cycle progression, the cell cycle status of the induced cells were evaluated by a BrdU-incorporation assay after a 30-minute pulse of BrdU labeling. Uninduced cells exhibited a baseline cell cycle phase distribution of 64%-28%-8% (G1-S-G2/M phases). After 1 day of induction, EB-treated sample showed no changes in the distribution (58%-33%-9%), but ATRA, DMSO and PD-treated samples showed significant changes, with an increase of cell numbers in G1 phase and decrease in S phase (74%-18%-8%, 79%-13%-8%, and 93%-4%-3%, respectively). These results reveal that an early induction of G1 arrest was caused by treatment with ATRA, DMSO and PD, but not EB, and that the cell cycle arrest occurred before major changes in the myeloid phenotype were observed. To determine how the cell cycle perturbation relates to changes in the underlying genetic regulatory network, we examined by quantitative RT-PCR analysis the expression of several transcription factors associated with myeloid differentiation. PU.1 and CEBPA were found to be expressed at high levels but these levels did not change upon treatment with the inducing agents. Similarly, the expression levels of GFI1 and EGR1 did not change significantly with induction. In contrast, the expression level of EGR2 (Early Growth Response 2) was found to be low initially but became elevated upon treatment with 3 of the 4 inducers. EGR2 is a zinc finger transcription factor implicated in the control of a switch between pro- and anti-proliferation pathways. EGR2 has been shown to regulate the transition between differentiation states of Schwann cells, induction of anergic and regulatory T cells, growth and survival of osteoclasts, and proliferation and apoptosis of acute myeloid leukemia blasts. We found that EGR2 expression, after 1 day of treatment with ATRA, DMSO or PD, was increased by 5.2 ± 0.9, 7.6 ± 1.9, 5.8 ± 0.9 folds, respectively, whereas treatment with EB led to no significant change (1.5 ± 0.2 fold). We evaluated whether simultaneous treatment of the cells with 2 inducers would result in an additive effect. Treatment of HL60 cells with a combination of ATRA/DMSO, ATRA/PD, or DMSO/PD increased the percentage of CD11b+CD71- cells to 55%, 70% and 25% after just 1 day of treatment. In line with the enhanced phenotypic effect, the expression level of EGR2 was further elevated to 7.7 ± 1.4, 15.4 ± 3.5, and 11.3 ± 3.4 folds, respectively, when the cells were treated with the above inducer combinations, indicating a tight association between EGR2 expression and the phenotypic effect. In summary, our data suggest that elevated expression of EGR2 is an early event in the induction of myeloid differentiation in HL60 cells. Because of its known role in cell cycle regulation, EGR2 could function as a mechanistic link between cell cycle arrest and induced differentiation in myeloid progenitor cells. Disclosures No relevant conflicts of interest to declare.

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