The Distal Cytoplasmic Domain of the Erythropoietin Receptor Induces Granulocytic Differentiation in 32D Cells

Blood ◽  
1998 ◽  
Vol 92 (4) ◽  
pp. 1219-1224 ◽  
Author(s):  
Kevin W. Harris ◽  
Xian-Jue Hu ◽  
Suzanne Schultz ◽  
Murat O. Arcasoy ◽  
Bernard G. Forget ◽  
...  
Keyword(s):  
Myeloid Cell ◽  
Cytoplasmic Domain ◽  
Receptor Activation ◽  
Erythropoietin Receptor ◽  
Full Length ◽  
Cytokine Receptor ◽  
Myeloid Differentiation ◽  
Hematopoietic Growth Factors ◽  
Granulocytic Differentiation

Abstract The role of hematopoietic growth factors in lineage commitment and differentiation is unclear. We present evidence that heterologous expression of an erythroid specific receptor allows granulocytic differentiation of a myeloid cell line. We have previously characterized a truncation mutant of the erythropoietin receptor (EpoR), which is associated with familial erythrocytosis (Blood89:4628, 1997). This truncated EpoR lacks the distal 70 amino acids of the cytoplasmic domain. To study the functional role of this distal receptor domain, 32D cells, a murine interleukin-3 (IL-3)–dependent myeloid line, were transfected with the wild-type EpoR (32D/EpoR WT) or the truncated EpoR (32D/EpoR FE). 32D cells expressing either the full-length or truncated EpoR display equivalent proliferative rates in saturating concentrations of Epo. There is a dramatic difference in maturational phenotype between the two cell lines, however. The 32D/EpoR FE cells and mock transfected 32D cells have an immature, monoblastic morphology and do not express the primary granule protein myeloperoxidase. The 32D/EpoR WT cells, on the other hand, demonstrate granulocytic differentiation with profuse granulation, mature, clumped chromatin, and myeloperoxidase expression. There is no evidence of erythroid differentiation in 32D cells transfected with either the full-length or truncated EpoR. Treatment of the cells with the specific Jak2 inhibitor tyrphostin AG 490 inhibits myeloid differentiation driven by the distal EpoR. We conclude that: (1) the distal cytoplasmic domain of the EpoR is able to induce a specific myeloid differentiation signal distinct from mitogenic signaling, and (2) these data extend to myelopoiesis the growing body of evidence that the cellular milieu, not the specific cytokine receptor, determines the specificity of differentiation after cytokine receptor activation. © 1998 by The American Society of Hematology.

The Distal Cytoplasmic Domain of the Erythropoietin Receptor Induces Granulocytic Differentiation in 32D Cells

Blood ◽  
1998 ◽  
Vol 92 (4) ◽  
pp. 1219-1224 ◽  
Author(s):  
Kevin W. Harris ◽  
Xian-Jue Hu ◽  
Suzanne Schultz ◽  
Murat O. Arcasoy ◽  
Bernard G. Forget ◽  
...  
Keyword(s):  
Myeloid Cell ◽  
Cytoplasmic Domain ◽  
Receptor Activation ◽  
Erythropoietin Receptor ◽  
Full Length ◽  
Cytokine Receptor ◽  
Myeloid Differentiation ◽  
Hematopoietic Growth Factors ◽  
Granulocytic Differentiation

The role of hematopoietic growth factors in lineage commitment and differentiation is unclear. We present evidence that heterologous expression of an erythroid specific receptor allows granulocytic differentiation of a myeloid cell line. We have previously characterized a truncation mutant of the erythropoietin receptor (EpoR), which is associated with familial erythrocytosis (Blood89:4628, 1997). This truncated EpoR lacks the distal 70 amino acids of the cytoplasmic domain. To study the functional role of this distal receptor domain, 32D cells, a murine interleukin-3 (IL-3)–dependent myeloid line, were transfected with the wild-type EpoR (32D/EpoR WT) or the truncated EpoR (32D/EpoR FE). 32D cells expressing either the full-length or truncated EpoR display equivalent proliferative rates in saturating concentrations of Epo. There is a dramatic difference in maturational phenotype between the two cell lines, however. The 32D/EpoR FE cells and mock transfected 32D cells have an immature, monoblastic morphology and do not express the primary granule protein myeloperoxidase. The 32D/EpoR WT cells, on the other hand, demonstrate granulocytic differentiation with profuse granulation, mature, clumped chromatin, and myeloperoxidase expression. There is no evidence of erythroid differentiation in 32D cells transfected with either the full-length or truncated EpoR. Treatment of the cells with the specific Jak2 inhibitor tyrphostin AG 490 inhibits myeloid differentiation driven by the distal EpoR. We conclude that: (1) the distal cytoplasmic domain of the EpoR is able to induce a specific myeloid differentiation signal distinct from mitogenic signaling, and (2) these data extend to myelopoiesis the growing body of evidence that the cellular milieu, not the specific cytokine receptor, determines the specificity of differentiation after cytokine receptor activation. © 1998 by The American Society of Hematology.

Re-Expression of the AML1/ETO Target Gene LAT2/NTAL/LAB Results In Direct Interference with Myeloid Differentiation In AML1/ETO-Positive Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2474-2474
Author(s):  
Jesus Duque-Afonso ◽  
Aitomi Essig ◽  
Leticia M Solari ◽  
Tobias Berg ◽  
Heike L. Pahl ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Lines ◽  
Functional Role ◽  
Target Gene ◽  
Conflicts Of Interest ◽  
Myeloid Cell ◽  
Leukemia Cells ◽  
Myeloid Differentiation ◽  
Granulocytic Differentiation

Abstract Abstract 2474 Background: The leukemia-specific oncofusion protein AML1/ETO regulates different target genes, including the LAT2 gene (encoding the adaptor molecule LAT2/NTAL/LAB), which is epigenetically repressed by AML1/ETO as we have previously described. LAT2 is phosphorylated by c-kit and has a role in mast cell and B cell activation. To address the functional role of LAT2 during myeloid differentiation, expression studies were performed in myeloid cell lines, and LAT2 was overexpressed by retroviral gene transfer in AML1/ETO-positive Kasumi-1 cells and AML1/ETO-negative U-937 cells. Methods: To induce monocytic and granulocytic differentiation, the myeloid cell lines U-937, HL-60 and NB4 were treated with PMA and ATRA, respectively, and LAT2 expression measured by both Northern and Western blot. LAT2 was overexpressed in Kasumi-1 and U-937 cells by use of the retroviral vector pMYSiG-IRES-GFP. Virus was produced in 293T cells and titrated in TE671 cells. Following transduction, GFP-positive cells were sorted by fluorescence-activated cell sorting (FACS). Transduced cells were treated with PMA (2 and 10 nM for 24 and 48 hours) and ATRA (0.1 μM and 0.5 μM for 48 and 96 hours), respectively. Results: The AML1/ETO-negative myeloid cell lines HL-60, NB4 and U-937 readily expressed LAT2, which was further upregulated by PMA, and transiently downregulated with ATRA. In the AML1/ETO-positive Kasumi-1 and SKNO-1 cells, LAT2 expression was absent. To address the functional role of this repression, forced expression of LAT2 was achieved in Kasumi-1 and U-937 cells and resulted in effective processing of LAT2 protein (confirmed by Western blot), and a decrease in the expression of the differentiation markers CD11b and CD11c (FACS analysis) in Kasumi-1 but not U-937, with only minor effects of LAT2 overexpression upon apoptosis and cell growth arrest. Notably, during both PMA- and ATRA-induced differentiation, a striking maturation block occurred in Kasumi-1 (measured by CD11b/CD11c expression, observed at different doses and time points of these treatments), while differentiation of U-937 cells was unimpaired by overexpression of LAT2. Conclusions: In AML1/ETO-negative leukemia cells, LAT2 expression is differentially regulated during monocytic and granulocytic differentiation. In AML1/ETO-positive leukemia cells, in which LAT2 is repressed, LAT2 re-expression imposes a striking maturation block. Graded expression of this novel AML1/ETO target gene may therefore play an important role in maintaining the phenotypic characteristics of this leukemia subtype. Disclosures: No relevant conflicts of interest to declare.

scholarly journals PML-RAR Binds to the +7kb Enhancer of CEBPE and Inhibits Its Expression

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 43-43
Author(s):  
Pavithra Shyamsunder ◽  
Shree Pooja Sridharan ◽  
Pushkar Dakle ◽  
Zeya Cao ◽  
Vikas Madan ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Regulatory Element ◽  
Promyelocytic Leukemia ◽  
Luciferase Reporter ◽  
Myeloid Differentiation ◽  
Atra Treatment ◽  
Granulocytic Differentiation ◽  
Nb4 Cells ◽  
Acute Myeloid

Acute promyelocytic leukemia (APL) is a unique subtype of acute myeloid leukemia (AML). The disease is identified by distinctive morphology and is distinguished by a balanced reciprocal translocation between chromosomes 15 and 17. This aberration leads to the fusion between promyelocytic leukemia (PML) gene located on chromosome 15q21, and retinoic acid receptor α (RARA) gene from chromosome 17q21, leading to the resultant chimeric onco-fusion protein PML-RARA, which is detectable in more than 95% patients and disturbs proper promyelocytic differentiation. All-trans retinoic acid (ATRA) can induce granulocytic differentiation in APL and is used to treat APL patients. Genes containing PML-RARA-targeted promoters are transcriptionally suppressed in APL and most likely constitute a major mechanism of transcriptional repression occurring in APL. A growing body of evidence points to the role of distal regulatory elements, including enhancers, in the control of gene expression. In order to understand the unique sets of enhancers that might be under the control of PML-RAR and crucial for granulocytic differentiation of NB4 cells, we analysed the enhancer landscape of control and ATRA treated NB4 cells. H3K9Ac mapping identified a repertoire of enhancers that were gained in NB4 cells treated with ATRA. Closer investigation of these enhancer elements revealed enrichment of H3K9Ac signals around major drivers of myeloid differentiation. Of note, we identified a gain in enhancer signature for a region about 7kb downstream of the CEBPE gene. Our previous studies identified a novel enhancer for CEBPE in murine hematopoietic cells, which was 6 downstream of CEBPE core promoter. It appears that the +7kb region we identified in human APL cells may be analogous to the murine enhancer. We also observed that PML-RAR binds this +7kb region and ATRA treatment of NB4 cells displaced binding of PML-RAR from the + 7kb region, suggestive of a transcriptional repressive effect of PML-RAR at such enhancer elements. To test the transcription regulating potential of this +7kb region, we used catalytically inactive Cas9 fused to Krüppel associated box (KRAB) domain (dCas9-KRAB). We designed three guide RNAs covering this regulatory region. The sgRNAs effectively repressed expression of CEBPE accompanied by lowered granulocytic differentiation of these guide RNA targeted NB4 cells after ATRA treatment. To explore transcription factor (TF) occupancy at this +7 kb region, we analysed public available ChIP-seq datasets for hematopoiesis-specific factors. Analysis revealed that the +7kb region was marked by an open chromatin signature, accompanied by binding of a majority of hematopoietic TFs around this putative regulatory element with concurrent binding of EP300. Strikingly we noticed binding of CEBPA, CEBPB and CEBPE at this regulatory element. To assess whether binding of these members of the CEBP family of TFs is functionally relevant, luciferase reporter and electrophoretic mobility shift assays (EMSA) were performed. Co expression of the CEBP TFs led to significant induction of luciferase expression, and this data was further confirmed using EMSA assays. Based on these observations, we propose that PML-RAR blocks granulocytic differentiation by occupying this +7kb enhancer of CEBPE, hinders binding of other cell type/lineage specific TFs, and blocks CEBPE expression. When cells are stimulated with ATRA, PML-RAR is displaced from the CEBPE enhancer, allowing for efficient binding of myeloid-specific TFs. This results in increased CEBPE expression, which in turn promotes efficient granulocytic differentiation. The findings from our study expands our current understanding of the mechanism of differentiation therapy, the role of onco-fusion proteins in inhibiting myeloid differentiation, and may provide new therapeutic approaches to many acute myeloid leukemias. Disclosures Ong: National University of Singapore: Other: Royalties.

scholarly journals Essential role of spi-1–like (spi-1l) in zebrafish myeloid cell differentiation

Blood ◽  
2009 ◽  
Vol 113 (9) ◽  
pp. 2038-2046 ◽  
Author(s):  
Alex Bukrinsky ◽  
Kevin J. P. Griffin ◽  
Yan Zhao ◽  
Shuo Lin ◽  
Utpal Banerjee
Keyword(s):  
Functional Analysis ◽  
Transcription Factor ◽  
Cell Differentiation ◽  
Essential Role ◽  
Critical Role ◽  
Myeloid Cell ◽  
Myeloid Differentiation ◽  
Adult Kidney ◽  
Necessary And Sufficient

The ETS protein Spi-1/Pu.1 plays a pivotal and widespread role throughout hematopoiesis in many species. This study describes the identification, characterization, and functional analysis of a new zebrafish spi transcription factor spi-1–like (spi-1l) that is expressed in primitive myeloid cells, erythro-myelo progenitor cells, and in the adult kidney. Spi-1l functions genetically downstream of etsrp, scl, and spi-1/pu.1 in myeloid differentiation. Spi-1l is coexpressed in a subset of spi-1/pu.1 cells and its function is necessary and sufficient for macrophage and granulocyte differentiation. These results establish a critical role for spi-1l in zebrafish myeloid cell differentiation.

scholarly journals Styryl Quinazolinones as Potential Inducers of Myeloid Differentiation via Upregulation of C/EBPα

Molecules ◽  
2018 ◽  
Vol 23 (8) ◽  
pp. 1938 ◽  
Author(s):  
Radhakrishnan Sridhar ◽  
Hisashi Takei ◽  
Riyaz Syed ◽  
Ikei Kobayashi ◽  
Liu Hui ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Myeloid Cell ◽  
Leukemic Cells ◽  
Myeloid Differentiation ◽  
Granulocytic Differentiation ◽  
Leukemia Cell Lines ◽  
Expression Activity ◽  
Differentiation Marker ◽  
Neutrophil Differentiation

The CCAAT enhancer-binding protein α (C/EBPα) plays an important role in myeloid cell differentiation and in the enhancement of C/EBPα expression/activity, which can lead to granulocytic differentiation in acute myeloid leukemia (AML) cells. We found that styryl quinazolinones induce upregulation of C/EBPα expression, and thereby induce myeloid differentiation in human myeloid leukemia cell lines. We screened a series of active styryl quinazolinones and evaluated the structure–activity relationship (SAR) of these small molecules in inducing C/EBPα expression—thereby prompting the leukemic cells to differentiate. We observed that compound 78 causes differentiation at 3 μM concentration, while 1 induces differentiation at 10 μM concentration. We also observed an increase in the expression of neutrophil differentiation marker CD11b upon treatment with 78. Both the C/EBPα and C/EBPε levels were found to be upregulated by treatment with 78. These SAR findings are inspiration to develop further modified styryl quinazolinones, in the path of this novel differentiation therapy, which can contribute to the care of patients with AML.

Role of Different C/EBPα Mutations in AML Transformation.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1343-1343
Author(s):  
Oscar Quintana-Bustamante ◽  
S. Lan-Lan Smith ◽  
Jude Fitzgibbon ◽  
Dominique Bonnet
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Cell Cycle Arrest ◽  
Myeloid Differentiation ◽  
Granulocytic Differentiation ◽  
Self Renewal ◽  
Cycle Arrest

Abstract Acute Myeloid Leukemia (AML) is characterized by an abnormal hematopoietic differentiation and uncontrolled cell proliferation. Mutations in several transcription factors (TFs) have been implicated in the development of leukemia. One of these TFs is CCAAT/enhancer-binding protein-α (C/EBPα). In normal hematopoiesis, C/EBPα plays a central role to coordinate myeloid differentiation and growth arrest. C/EBPα is mutated in approximately 9% of AML; these mutations take place either in C or N terminal domains of the protein, although there are several familial cases of AML where both types of mutations have been found. We use C and/or N terminal C/EBPα mutations from one case of sporadic AML to investigate the role of each mutation in leukemic transformation (Smith et al., 2004, N Engl J Med 351, 2403–2407). Human lineage negative (Lin-) umbilical cord blood were transduced with lentiviral vectors carrying the wild type C/EBPα (WT), N terminal mutated C/EBPα (N-ter) or N and C terminal mutated (NC-ter) C/EBPα cloned from this sporadic case of AML. We observed differences in proliferation of transduced Lin- in vitro: WT C/EBPα expression resulted in G0 cell cycle arrest causing a progressive extinction of the transduced cells overtime; N-ter cells showed a higher proliferative advantage over untransduced cells. The NC-ter CEBPα cells like untransduced cells kept their levels throughout culture. Furthermore, when induced into myeloid differentiation in vitro, WT C/EBPα cells were mainly inducing fully mature granulocytes whereas N-ter C/EBPα was not able to induce terminal granulocytic differentiation; in contrast NC-ter C/EBPα did not increase myeloid differentiation. Additionally, their ability to form Colony Forming Units (CFUs) in primary, secondary and tertiary replating was also tested: WT transduced cells gave rise to few primary CFUs; contrary, N and NC-ter could generate both primary and secondary CFUs, but only NC-ter cells were able to produce CFUs in tertiary replating, indicating its ability to maintain undifferentiated hematopoietic progenitors in vitro. These results were confirmed using Long-Term Culture Initiating Cells (LTC-IC) where the NC-ter mutated cells showed the highest LTC-IC after 5 weeks. Finally, in vivo transplantation in NOD/SCID/β2mnull indicated that NC-ter mutated cells engraft better than WT and N-ter 8 week post- transplant. Serial transplantation experiments are underway to evaluate their self-renewal capacity. Our results confirmed some known functions of WT C/EBPα in human hematopoiesis, such as inducing myeloid differentiation and cell cycle arrest. On the other hand, we showed new functions for the C/EBPα mutants. The N-ter C/EBPα mutation caused an increase in cell proliferation and blockage of terminal granulocytic differentiation, whereas the NC-ter C/EBPα mutation increased the self-renewal capacity of progenitor/stem cells without having an influence on myeloid differentiation. This work provides further insight into the mechanisms by which different C/EBPα mutations induce AML.

Phospho- AKT Is Not Sufficient To Delay Neutrophil Apoptosis.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3366-3366
Author(s):  
Liliana R. Souza ◽  
Carlos Cabrera ◽  
Morgan L. McLemore
Keyword(s):  
Bone Marrow ◽  
Expression System ◽  
Active Form ◽  
Mtor Pathway ◽  
Myeloid Differentiation ◽  
Neutrophil Apoptosis ◽  
Granulocytic Differentiation ◽  
Early Stages ◽  
Conditional Expression

Abstract Neutrophils play an important role in the innate immune response and usually are the first cells to defend the organism against infections. The lifetime of neutrophils is tightly regulated and once the mature cells left the bone marrow most of them undergo apoptosis within 48 hours. The rate of neutrophil apoptosis can be modulated by a variety of cytokines, including granulocyte colony-stimulating factor (G-CSF). To investigate the involvement of the G-CSF receptor in the neutrophil susceptibility to apoptosis we utilized mice bearing truncation mutations on the G-CSF receptor (G-CSFR) and also the G:EpoR mutation, where the entire cytoplasmic (signaling) domain of the G-CSFR is replaced with that of the erythropoietin receptor (EpoR). We examined the role of AKT-mTOR pathway in neutrophil apoptosis. Bone marrow neutrophils from wild type (C57BL/6) and mutant mice where cultured in presence or absence of G-CSF and/or Rapamycin (a specific mTor inhibitor); the apoptosis and the level of phosphorylation of AKT and ERK were evaluated. Neutrophils extracted from G:EpoR mice do not respond to G-CSF treatment and undergo apoptosis within 24 hours, although WT neutrophils can be partially protected against apoptosis when treated with G-CSF. Interestingly, G-CSF treatment leads to activation of Akt and Erk kinases, even in G:EpoR neutrophils. Furthermore, the addition of Rapamycin had no effect in the apoptosis of WT neutrophils in presence or absence of G-CSF. To further elucidate the role of the AKT-mTOR pathway, Sca+Lin- WT bone marrow cells were utilized in an in vitro G-CSF dependent granulocytic differentiation assay. The experiments showed that the mTor pathway is important for the early stages of myeloid differentiation but has little influence in the late stages, and also in the life span of neutrophils. Interestingly, over expression of a constitutively active form of AKT (myr-AKT) in Sca+Lin- cells impaired proliferation even though the differentiation was not affected. To overcome this, a tetracycline response element (TRE) conditional expression system was utilized. These experiments showed that myr-AKT expression does not rescue neutrophils from apoptosis. In conclusion, our results indicate that after treatment with G-CSF; AKT and ERK are phosphorylated in WT and G:EpoR neutrophils, despite this, G-CSF does not suppress G:EpoR neutrophil apoptosis while WT neutrophils can have their apoptosis partially delayed. We also found that mTor is involved in the early stages of G-CSF dependent myeloid differentiation and its inhibition can partially arrest differentiation. Conversely, rapamycin does not affect neutrophil apoptosis, in presence or not of G-CSF. In concordance with these results overexpression of a constitutive active form of AKT, myr-AKT does not rescue neutrophils from apoptosis and it is also detrimental to early stages of granulocytic proliferation. These data strongly suggest that the AKT-mTor pathway while important in early myeloid differentiation and proliferation does not play a role in cytokine suppression of neutrophil apoptosis.

Activation of the Jak3 pathway is associated with granulocytic differentiation of myeloid precursor cells

Blood ◽  
2002 ◽  
Vol 100 (8) ◽  
pp. 2753-2762 ◽  
Author(s):  
Sushil G. Rane ◽  
James K. Mangan ◽  
Arshad Amanullah ◽  
Brian C. Wong ◽  
Renu K. Vora ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Differentiation ◽  
Kinase Inhibitor ◽  
Myeloid Cell ◽  
Janus Kinase ◽  
Colony Stimulating Factor ◽  
Granulocytic Differentiation ◽  
Ectopic Overexpression ◽  
Stimulating Factor

Jak3, a member of the Janus kinase family of cytoplasmic tyrosine kinases, is expressed at low levels in immature hematopoietic cells and its expression is dramatically up-regulated during the terminal differentiation of these cells. To better understand the role of Jak3 in myeloid cell development, we have investigated the role of Jak3 in myeloid cell differentiation using the 32Dcl3 cell system. Our studies show that Jak3 is a primary response gene for granulocyte colony-stimulating factor (G-CSF) and the accumulation of tyrosine phosphorylated Jak3 correlated with cell growth inhibition and terminal granulocytic differentiation in response to G-CSF. Ectopic overexpression of Jak3 in 32Dcl3 cells resulted in an acceleration of the G-CSF–induced differentiation program that was preceded by G1 cell cycle arrest, which was associated with the up-regulation of the cyclin-dependent kinase inhibitor p27Kip1 and down-regulation of Cdk2, Cdk4, Cdk6, and Cyclin E. In addition, ectopic overexpression of Jak3 appears to result in the inactivation of PKB/Akt and Stat3-mediated proliferative pathways in the presence of G-CSF. Similarly, overexpression of Jak3 in primary bone marrow cells resulted in an acceleration of granulocytic differentiation in the presence of granulocyte-macrophage colony-stimulating factor, which was associated with their growth arrest in the G1 phase of the cell cycle. Taken together, these results indicate that Jak3-mediated signals play an important role in myeloid cell differentiation.

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