IL-3 Inhibits Terminal Granulocytic Differentiation by Inducing the Binding of Stabilizing Proteins to a 300 Base Pair Region within p21cip1/waf1 Messenger RNA.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3152-3152
Author(s):  
Louis Ghanem ◽  
Richard A. Steinman
Keyword(s):  
Progenitor Cells ◽  
Adaptor Protein ◽  
Proteinase 3 ◽  
Granulocytic Differentiation ◽  
Induced Differentiation ◽  
Aberrant Expression ◽  
P21 Protein ◽  
Mrna Stabilization ◽  
Granule Proteins ◽  
Primary Granule

Abstract Elevated levels of the molecular adaptor protein p21cip1/waf1 (p21) and of the IL-3 receptor α chain are correlated with chemoresistance and poor prognosis in acute myeloid leukemia (AML). p21 is a core regulator of many biological functions including cell cycle control, apoptosis and differentiation. Our laboratory has demo−nstrated that p21 undergoes dynamic changes in expression levels and subcellular compartmentalization during cytokine-induced granulocytic differentiation, suggesting that p21 may play an important role in myeloid development. Based on our observation that p21 protein levels decrease during granulocytic differentiation of CD34+ human progenitor cells, we hypothesized that p21 antagonizes granulopoiesis. The proliferative cytokine IL-3 is required to maintain the undifferentiated state in murine 32Dcl3 cells and has been shown to slow the kinetics of differentiation of normal human myeloid progenitors (Hevehan DL, 2000). Given that 32Dcl3 myeloblasts express high basal levels of p21, we also hypothesized that IL-3 inhibition of 32Dcl3 differentiation is mediated in part by p21. Our findings demonstrate that siRNA knockdown of murine p21 is correlated with premature expression of the primary granule proteins myeloperoxidase and proteinase-3 that are normally not abundant in cells maintained as myeloblasts by IL-3. Rescue of p21 knockdown myeloblasts with human p21 suppressed aberrant expression of granule proteins. The upregulation of myeloperoxidase and proteinase-3 occurred at a posttranscriptional level. These findings indicated that p21 prevented premature expression of primary granule proteins and may contribute to maintenance of the myeloblast phenotype. p21 knockdown was also found to accelerate morphologic granulocytic differentiation in 32Dcl3 cells stimulated with G-CSF, indicating that p21 antagonized the entire differentiation process rather than only suppressing primary granule proteins. We then determined how IL-3 maintains p21 expression in myeloblast cells. We demonstrated that IL-3 stabilized p21 mRNA in myeloblasts leading to high levels of p21 protein. This effect mapped to the 3′ untranslated region (UTR) of the p21 transcript. IL-3 also rescued the decrease in p21 mRNA stability noted during G-CSF-induced differentiation. This has been shown to coincide with differentiation blockade. p21 transcript stabilization by IL-3 was independent of PI3-kinase and ERK pathway signaling. In vitro binding assays provided evidence that distinct sets of RNA:protein interactions occur within the proximal 303 nucleotides of the p21 3′ UTR and are regulated by IL-3 and G-CSF signaling. Association of a ~60–65 kDa protein with p21 riboprobes correlated with IL-3 mediated p21 mRNA stabilization, whereas binding by a ~40–42 kDa protein was associated with destabilization of p21 transcripts in 32Dcl3 cells undergoing G-CSF-induced differentiation. These findings provide the first evidence for IL-3-mediated stabilization of mRNA transcripts in myeloid progenitor cells. The finding that p21 antagonized granulopoiesis is also novel. Because high levels of the IL-3 receptor and high p21 expression have separately been linked to poor outcomes in AML, IL-3 mediated p21 mRNA stabilization may contribute to differentiation blockade during AML pathogenesis.

scholarly journals Neutrophils Promote Hematopoiesis Via a Novel Mechanism Involving Leucine Rich α-2 Glycoprotein (LRG)

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2725-2725
Author(s):  
Lawrence J Druhan ◽  
Shimena Li ◽  
Sarah A Baxter ◽  
Amanda Lance ◽  
Andrea E Price ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Progenitor Cells ◽  
Cell Lines ◽  
Myeloid Cell ◽  
Hematopoietic Progenitor Cells ◽  
Human Neutrophils ◽  
Neutrophil Granulocyte ◽  
Granulocytic Differentiation ◽  
Hematopoietic Progenitor ◽  
Granule Proteins

Abstract Leucine-rich α2 glycoprotein (LRG), the founding member of the leucine-rich repeat superfamily of proteins, was initially identified in serum more than 30 years ago, but its biologic function has remained elusive. A role for LRG has been implicated in inflammation and angiogenesis. Our laboratory previously identified cDNA and genomic clones for human and murine LRG, and showed that ectopically expressed LRG localizes to the granule compartment in transfected myeloid cell lines and promotes their granulocytic differentiation. We also demonstrated that expression of LRG is transcriptionally regulated during neutrophil granulocyte differentiation in a manner similar to that reported for genes encoding the different subsets of neutrophil granule proteins. The presence of LRG in primary neutrophils and a role for LRG in hematopoiesis, however, have not been previously described. Based on our prior studies in transfected myeloid cell lines, we considered the tantalizing possibility that LRG is a novel neutrophil granule protein that is secreted extracellularly upon neutrophil activation to modulate hematopoiesis. To investigate this, we examined LRG in primary human neutrophils isolated from healthy volunteers. Immunoblot analysis of whole cell lysates from neutrophils (97% purity) identified a higher molecular weight LRG species in neutrophils (62 kDa) compared to serum (50 kDa); our data demonstrate the difference in apparent molecular weight is due to differential glycosylation. Immunofluorescence microscopy using antibodies to human LRG and antibodies to the neutrophil granule proteins myeloperoxidase (MPO), lactoferrin (LF), and matrix metalloproteinase 9 (MMP-9, also known as gelatinase), along with fluorescently-labeled secondary antibodies, demonstrated the presence of LRG in the cytoplasm of neutrophils in a compartment corresponding to LF. ELISA and immunoblot analyses of subcellular fractions from isolated neutrophils prepared by nitrogen cavitation demonstrated the presence of LRG in LF-containing fractions as well as some MMP-9-containing fractions, consistent with localization of LRG to the secondary/tertiary granule compartment. Neutrophil exocytosis assays using ionomycin, phorbol-12-myristate 13-acetate, and f-Met-Leu-Phe as stimulants also indicated that LRG is co-released with LF and MMP9, but not with MPO. Notably, LRG secreted from activated neutrophils could bind cytochrome c as reported for LRG purified from serum. Recent reports that LRG can also bind to the TGFβR1 receptor on endothelial cells prompted us to investigate the effects of LRG on TGFβ signaling in hematopoietic cells. LRG significantly antagonized the inhibitory effect of TGFβ on HL-60 cell proliferation (n=3; p<0.05) and also on colony growth of human hematopoietic progenitor cells. When LRG was added to hematopoietic progenitor cells cultured in TGFβ-containing Methocult (SF H4436, serum free), a 50% increase in CFU-GMs was observed. Collectively, these data suggest a novel mechanism whereby neutrophils modulate hematopoiesis in the microenvironment via extracellular release of LRG, and invoke an additional role for neutrophils in innate immunity that has not previously been reported Disclosures No relevant conflicts of interest to declare.

scholarly journals Identification of a Novel CEBPE Enhancer Essential for Granulocytic Differentiation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3834-3834
Author(s):  
Pavithra Shyamsunder ◽  
Mahalakshmi Shanmugasundaram ◽  
Anand Mayakonda ◽  
Weoi Woon Teoh ◽  
Lin Han ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Progenitor Cells ◽  
Core Promoter ◽  
Upstream Region ◽  
Open Chromatin ◽  
Granulocytic Differentiation ◽  
Specific Expression ◽  
Granule Formation ◽  
Granule Proteins ◽  
Secondary Granule

Abstract CEBPE is a member of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors essential for granulocytic differentiation. CEBPE is expressed in a stage-specific manner during myeloid differentiation and regulates transition from the promyelocyte to the myelocyte stage. It is essential for secondary and tertiary granule formation in granulocytes. We and others found germline mutations of the CEBPE gene in patients with neutrophil-specific granule deficiency. Their neutrophils display atypical bilobed nuclei, lack expression of granule proteins and these patients often have frequent bacterial infections. Cebpe knock-out mice resemble this clinical phenotype displaying a block in terminal differentiation and absence of secondary granule proteins. Given the tissue specific expression of CEBPE, we were interested in identifying genomic regions and factors that could regulate its lineage specific expression. Our CEBPE ChIP-seq in murine bone marrow cells showed binding of CEBPE to a region 6kb upstream of Cebpe gene. Chromosome conformation capture-on-chip (4C-seq) demonstrated an interaction between this putative regulatory element (6kb upstream region) and the core promoter of Cebpe. Analysis of available DNase-seq data sets revealed that the region bound by CEBPE displayed an open chromatin only in myeloid lineage cells. Further examination revealed binding of a myriad of hematopoietic transcription factors to the +6kb enhancer in HPC-7 (hematopoietic progenitor cells) and in 416B (myeloid progenitor cells), indicating that this region/enhancer might regulate the expression of CEBPE. Targeting of this region using dCas9-KRAB in murine 32D cells caused significant downregulation of RNA and protein levels of CEBPE compared to control cells. These targeted cells also exhibited impaired granulocytic differentiation with lower transcript levels of secondary granule proteins (Ltf and Ngp). To investigate further the role of the +6kb enhancer region in myelopoiesis, mice were generated with deletion of this region using CRISPR/Cas9 technology. Germ line deletion of the +6kb enhancer resulted in reduced levels of CEBPE and its target genes, accompanied by a severe block in granulocytic differentiation and a complete absence of CD11b+/Gr1hi population. This phenotype is nearly identical to our Cebpe KO mice. In summary, we have identified a novel enhancer crucial for regulating Cebpe, and required for normal granulocytic differentiation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Targeting the Calmodulin-Regulated ErbB/Grb7 Signaling Axis in Cancer Therapy

2013 ◽  
Vol 16 (2) ◽  
pp. 177 ◽  
Author(s):  
Antonio Villalobo ◽  
Irene García-Palmero ◽  
Silviya R. Stateva ◽  
Karim Jellali
Keyword(s):  
Cancer Therapy ◽  
Growth Factor Receptor ◽  
Adaptor Protein ◽  
Short Review ◽  
Receptor Protein ◽  
Pharmacological Intervention ◽  
Erbb Receptors ◽  
Aberrant Expression ◽  
Signaling Mechanism ◽  
Downstream Signaling

Signal transduction pathways essential for the survival and viability of the cell and that frequently present aberrant expression or function in tumors are attractive targets for pharmacological intervention in human cancers. In this short review we will describe the regulation exerted by the calcium-receptor protein calmodulin (CaM) on signaling routes involving the family of ErbB receptors - highlighting the epidermal growth factor receptor (EGFR/ErbB1) and ErbB2 - and the adaptor protein Grb7, a downstream signaling component of these receptors. The signaling mechanism of the ErbB/Grb7 axis and the regulation exerted by CaM on this pathway will be described. We will present a brief overview of the current efforts to inhibit the hyperactivity of ErbB receptors and Grb7 in tumors. The currently available information on targeting the CaM-binding site of these signaling proteins will be analyzed, and the pros and cons of directly targeting CaM versus the CaM-binding domain of the ErbB receptors and Grb7 as potential anti-cancer therapy will be discussed. This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.

Specific Signals Generated by the Cytoplasmic Domain of the Granulocyte Colony-Stimulating Factor (G-CSF) Receptor Are Not Required for G-CSF–Dependent Granulocytic Differentiation

Blood ◽  
1998 ◽  
Vol 92 (2) ◽  
pp. 353-361 ◽  
Author(s):  
Jason Jacob ◽  
Jeffery S. Haug ◽  
Sofia Raptis ◽  
Daniel C. Link
Keyword(s):  
Progenitor Cells ◽  
Ectopic Expression ◽  
Cytoplasmic Domain ◽  
Hematopoietic Progenitor Cells ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Granulocytic Differentiation ◽  
Hematopoietic Progenitor ◽  
Stimulating Factor ◽  
Factor G

Abstract Granulocyte colony-stimulating factor (G-CSF) is the principal growth factor regulating the production of neutrophils, yet its role in lineage commitment and terminal differentiation of hematopoietic progenitor cells is controversial. In this study, we describe a system to study the role of G-CSF receptor (G-CSFR) signals in granulocytic differentiation using retroviral transduction of G-CSFR–deficient, primary hematopoietic progenitor cells. We show that ectopic expression of wild-type G-CSFR in hematopoietic progenitor cells supports G-CSF–dependent differentiation of these cells into mature granulocytes, macrophages, megakaryocytes, and erythroid cells. Furthermore, we show that two mutant G-CSFR proteins, a truncation mutant that deletes the carboxy-terminal 96 amino acids and a chimeric receptor containing the extracellular and transmembrane domains of the G-CSFR fused to the cytoplasmic domain of the erythropoietin receptor, are able to support the production of morphologically mature, chloroacetate esterase-positive, Gr-1/Mac-1–positive neutrophils in response to G-CSF. These results demonstrate that ectopic expression of the G-CSFR in hematopoietic progenitor cells allows for multilineage differentiation and suggest that unique signals generated by the cytoplasmic domain of the G-CSFR are not required for G-CSF–dependent granulocytic differentiation.

Slug Plays an Essential Role in the Radioprotection of Hematopoietic Progenitors In Vivo by Antagonizing p53-Mediated Apoptotic Pathways.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 31-31
Author(s):  
Wen-Shu Wu ◽  
Dong Xu ◽  
Stefan Heinrichs ◽  
A. Thomas Look
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Leukemia Cells ◽  
Dependent Manner ◽  
Wild Type ◽  
Γ Irradiation ◽  
Aberrant Expression ◽  
Hematopoietic Stem ◽  
Myeloid Progenitors

Abstract An antiapoptotic role for Slug/Snail in mammals was suggested by studies in C. elegans, where CES-1/Scratch, a member of the Slug/Snail superfamily, was found to control the apoptotic death of NSM sister neurons by acting as a transcriptional repressor of EGL-1, a BH3-only proapoptotic protein. Identification of Slug as the target gene of the E2A-HLF oncoprotein in human pro-B leukemia cells led us to demonstrate its antiapoptotic function in IL-3-dependent murine pro-B cells. In contrast to its aberrant expression in pro-B leukemia cells, endogenous Slug is normally expressed in both LT-HSC and ST-HSC, as well as committed progenitors of the myeloid series, but not in pro-B and pro-T cells, implying its function in myelopoiesis. Using Slug−/− mice produced in our laboratory, we showed that these knockouts are much more radiosensitive than Slug+/− and wild-type mice, and that apoptotic cells increase significantly in the hematopoietic progenitor cells of Slug−/− mice as compared to wild-type mice following γ-irradiation, indicating a radioprotective function in vivo. We showed here that although the development of myeloid progenitors is not impaired under steady-state conditions, their repopulation is incomplete γ-irradiated in in Slug−/− mice. We demonstrate further the radiation-induced death of Slug−/− mice is exclusively a result of bone marrow failure with no apparent contribution from systemic injures to other tissues. By two-way bone marrow transplantation, we provide firm evidence that Slug protects mice from γ-irradiation-induced death in a cell-autonomous manner. Interestingly, regenerative capacity of hematopoietic stem cells (HSC) was retained in irradiated Slug−/− mice, which could be rescued by wild-type bone marrow cells after irradiation, indicating that Slug exerts its radioprotective function in myeloid progenitors rather than HSCs. Furthermore, we establish that Slug radioprotects mice by antagonizing downstream of the p53-mediated apoptotic signaling through inhibition of the p53-resposive proapoptotic gene Puma, leading in turn to inhibition of the mitochondria-dependent apoptotic pathway activated by γ-irradiation in myeloid progenitors. More interestingly, we observed that Slug is inducible by γ-irradiation in a p53-dependent manner. Together, our findings implicate a novel Slug-mediated feedback mechanism by which p53 control programmed cell death in myeloid progenitor cells in vivo in response to γ-irradiation.

Inhibition of Elastase or SDF-1/CXCR4 Axis Promotes Differentiation of Human AML Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1891-1891
Author(s):  
Sigal Tavor ◽  
Jasmine Jacob-Hirsch ◽  
Manny Eisenbach ◽  
Sigi Kay ◽  
Shoshana Baron ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Critical Role ◽  
Marker Genes ◽  
Granulocytic Differentiation ◽  
Elastase Inhibitor ◽  
Granule Proteins ◽  
Cxcr4 Axis

Abstract Elastase, along with other azurophil granule proteins like proteinase 3 regulates normal and leukemic granulopoiesis in an un-defined mechanism. We have recently showed that human acute myeloid leukemic (AML) cells constitutively express and secrete stromal derived factor 1 (SDF-1) dependent cell surface elastase, which regulates their migration and proliferation. To elucidate the molecular events and genes regulated by elastase and SDF-1/CXCR4 axis in AML cells, we examined gene expression of U937 AML cell line treated with neutralizing anti-CXCR4 Abs or elastase inhibitor (EI) compared to untreated cells, using DNA microarray technology. Unsupervised hierarchical clustering analysis showed very similar gene expression profiles of EI and anti CXCR4 Abs treated cells as compared to control. 230 of 8400 genes interrogated were repressed, and 164 were induced after culturing AML cells in the presence of EI or anti CXCR4 Abs at different time points as compared to untreated cells. Inhibition of elastase or CXCR4 was accompanied by down regulation of the transcripts of primary granule proteins. Functional classification of elastase or SDF-1/CXCR4 axis regulated genes revealed downregulation of HOXA9, HOXA10, ETS2, as well as other transcription factors that are over expressed in AML and are important for the development of leukemia. Whereas, transcriptional factors and regulators known to be induced during myeloid differentiation like C/EBPε, ID1, RUNX3 and HHEX were up-regulated in treated cells. Expression patterns of apoptosis genes indicated decline in death control by the p53 dependent pathway and a more prominent control by mitochondrial mediated apoptotic pathway like bcl2 related genes. In addition, receptors for interleukins, growth factors (G-CSFR and GM-CSF), complement component (C1QR1) were upregulated in the treated cells. In contrast, FLT-3, a growth factor receptor stimulating growth of early progenitor cells and AML blasts, was down regulated in AML cell treated with EI or anti CXCR4 Abs. These data were confirmed by real time PCR for selected marker genes of granulocytic differentiation. Interestingly, many of the differentially expressed genes were common to the transcriptional program of normal terminal granulocytic differentiation (Theilgaard-Monch & Borregarrd 2005. Blood 105:1785) suggesting that inhibition of elastase may induce differentiation in AML cells. Thus we further analyzed the effect of elastase inhibition on AML cell differentiation and growth. Treatment of HL60 AML cell line with EI triggered a proliferative arrest, apoptosis and mimicked terminal granulocytic differentiation, including morphologic changes, increased CD11b expression, and the ability to produce oxidative bursts. In summary, our study showed that inhibition of elastase or SDF-1/CXCR4 axis in AML cells affects similar pathways related to differentiation and malignant transformation, implying a critical role for those molecules in regulating leukemic development. Repression of elastase decreases proliferation and induces differentiation of AML cells, suggesting a potential new therapeutic approach for AML.

CD15 (Lewis x) Expression in Human Myeloid Cell Differentiation Is Regulated by Sialidase Activity.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1931-1931
Author(s):  
Samah Z. Gadhoum ◽  
Robert Sackstein
Keyword(s):  
De Novo ◽  
Morphological Changes ◽  
Critical Role ◽  
Myeloid Cells ◽  
Granulocytic Differentiation ◽  
Induced Differentiation ◽  
Lewis X ◽  
Sialidase Activity ◽  
The Core ◽  
Sialyl Lex

Abstract The cell surface carbohydrate Lewis x (Lex, CD15) is now well established as an important marker for immunophenotyping leukocytes and for immunoregulatory functions. Lex is related to sialyl Lex (sLex) by the addition of a sialic acid in the core Lex trisaccharide. This sialylation has profound implications as it confers novel biological function(s) to the core trisaccharide. In particular, expression of sLex correlates with E-selectin ligand activity. Though the expression of Lex in myeloid differentiation is now widely recognized, no studies to date have addressed the molecular mechanism regulating its expression. To examine this issue, we employed a leukemic model of differentiation based on the capacity of anti-CD44 mAbs to induce maturation and Lex expression. We found that anti-CD44 mAb-induced expression of Lex (> 30% increase in Mean Fluorescence Intensity (MFI) by flow cytometry) was accompanied by decreased expression (> 20% decrease in MFI) of its related structure sialyl Lex (sLex). Two non-mutually exclusive processes could account for the increase in Lex expression during myeloid differentiation:neo-synthesis of the Lex epitope and/orde-sialylation of the existing sLex. To test whether increased expression of Lex during CD44-induced differentiation resulted from augmented synthesis, Acute Myeloid Leukemia (AML) cells were treated with anti-CD44 mAb in the presence of 4F-GlcNAc (2-acetamido-1,3,6-tri-O-acetyl-4-fluoro-D-glucopyranose), an inhibitor of poly-N-acetyl-lactosamine synthesis. 4-F-GlcNAc did not significantly alter the increase of Lex induced by the anti-CD44 mAb indicating that the CD44-mediated increase of Lex does not require de-novo polylactosamine synthesis. In presence of the sialidase inhibitor DANA (2,3-dehydro-2-deoxy-N-Acetyl-neuraminic acid) the anti-CD44-induced increase of Lex and decrease of sLex were abrogated indicating that sialidase plays an important role in the modulation of Lex expression during CD44 mAb-induced granulocytic differentiation. Moreover, using the exogenous substrate 4MU-NANA to measure endogenous sialidase activity in AML cells we found that the anti-CD44 mAb Hermes-1 induced a significant increase in the sialidase activity of AML cells, strongly suggesting that CD44-induced differentiation causes the elaboration of endogenous sialidases that are responsible for the modulation of Lex level by converting sLex into Lex. Furthermore, treatment of immature myeloid cells with G-CSF induced a 2-fold increase in sialidase activity, coincident with increased morphological changes of differentiation suggesting that the induction of surface sialidase activity is associated dynamically with the conversion of immature to mature phenotypes among native myeloid cells. Our results unveil a new paradigm, demonstrating a critical role for glycosidase activity in the “biosynthesis” of a key glycan determinant expressed on human cells.

Sign in / Sign up

Export Citation Format

Share Document