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1.  G-CSFR Ubiquitination Critically Regulates Myeloid Cell Survival and Proliferation 
PLoS ONE  2008;3(10):e3422.
The granulocyte colony-stimulating factor receptor (G-CSFR) is a critical regulator of granulopoiesis. Mutations in the G-CSFR in patients with severe congenital neutropenia (SCN) transforming to acute myelogenous leukemia (AML) have been shown to induce hypersensitivity and enhanced growth responses to G-CSF. Recent studies have demonstrated the importance of the ubiquitin/proteasome system in the initiation of negative signaling by the G-CSFR. To further investigate the role of ubiquitination in regulating G-CSFR signaling, we generated a mutant form of the G-CSFR (K762R/G-CSFR) which abrogates the attachment of ubiquitin to the lysine residue at position 762 of the G-CSFR that is deleted in the Δ716 G-CSFR form isolated from patients with SCN/AML. In response to G-CSF, mono-/polyubiquitination of the G-CSFR was impaired in cells expressing the mutant K762R/G-CSFR compared to cells transfected with the WT G-CSFR. Cells stably transfected with the K762R/G-CSFR displayed a higher proliferation rate, increased sensitivity to G-CSF, and enhanced survival following cytokine depletion, similar to previously published data with the Δ716 G-CSFR mutant. Activation of the signaling molecules Stat5 and Akt were also increased in K762R/G-CSFR transfected cells in response to G-CSF, and their activation remained prolonged after G-CSF withdrawal. These results indicate that ubiquitination is required for regulation of G-CSFR-mediated proliferation and cell survival. Mutations that disrupt G-CSFR ubiquitination at lysine 762 induce aberrant receptor signaling and hyperproliferative responses to G-CSF, which may contribute to leukemic transformation.
doi:10.1371/journal.pone.0003422
PMCID: PMC2561048  PMID: 18923646
2.  Increased granulocyte colony-stimulating factor responsiveness but normal resting granulopoiesis in mice carrying a targeted granulocyte colony-stimulating factor receptor mutation derived from a patient with severe congenital neutropenia. 
Journal of Clinical Investigation  1998;102(3):483-492.
The role of mutations of the granulocyte colony-stimulating factor receptor (G-CSFR) in the pathogenesis of severe congenital neutropenia (SCN) and the subsequent development of acute myeloid leukemia (AML) is controversial. Mice carrying a targeted mutation of their G-CSFR that reproduces the mutation found in a patient with SCN and AML have been generated. The mutant G-CSFR allele is expressed in a myeloid-specific fashion at levels comparable to the wild-type allele. Mice heterozygous or homozygous for this mutation have normal levels of circulating neutrophils and no evidence for a block in myeloid maturation, indicating that resting granulopoiesis is normal. However, in response to G-CSF treatment, these mice demonstrate a significantly greater fold increase in the level of circulating neutrophils. This effect appears to be due to increased neutrophil production as the absolute number of G-CSF-responsive progenitors in the bone marrow and their proliferation in response to G-CSF is increased. Furthermore, the in vitro survival and G-CSF-dependent suppression of apoptosis of mutant neutrophils are normal. Despite this evidence for a hyperproliferative response to G-CSF, no cases of AML have been detected to date. These data demonstrate that the G-CSFR mutation found in patients with SCN is not sufficient to induce an SCN phenotype or AML in mice.
PMCID: PMC508908  PMID: 9691084
3.  Neutrophil elastase downmodulates native G-CSFR expression and granulocyte-macrophage colony formation 
Background
The granulocyte colony-stimulating factor receptor (G-CSFR) plays a critical role in maintaining homeostatic levels of circulating neutrophils (PMN). The mechanisms modulating G-CSFR surface expression to prevent chronic neutrophilia are poorly understood. Here, we report that neutrophil elastase (NE) proteolytically cleaves the G-CSFR on human PMN and blocks G-CSFR-mediated granulopoiesis in vitro.
Methods
Human peripheral blood PMN isolated from healthy donors were incubated with NE. Expression of the G-CSFR was analyzed by flow cytometry and western blot analyses. Detection of G-CSFR cleavage products from the culture supernatants was also performed. Human bone marrow mononuclear cells were also cultured in the presence or absence of NE to determine its effects on the proliferation of granulocyte-macrophage colony forming units (CFU-GM).
Results
Treatment of PMN with NE induced a time-dependent decrease in G-CSFR expression that correlated with its degradation and the appearance of proteolytic cleavage fragments in conditioned media. Immunoblot analysis confirmed the G-CSFR was cleaved at its amino-terminus. Treatment of progenitor cells with NE prior to culture inhibited the growth of granulocyte-macrophage colony forming units.
Conclusions
These findings indicate that in addition to transcriptional controls and ligand-induced internalization, direct proteolytic cleavage of the G-CSFR by NE also downregulates G-CSFR expression and inhibits G-CSFR-mediated granulopoiesis in vitro. Our results suggest that NE negatively regulates granulopoiesis through a novel negative feedback loop.
doi:10.1186/1476-9255-7-5
PMCID: PMC2824667  PMID: 20205821
4.  Filgrastim XM02 (Tevagrastim®) after autologous stem cell transplantation compared to lenograstim: favourable cost-efficacy analysis 
ecancermedicalscience  2013;7:327.
Purpose
Granulocyte colony-stimulating factors (G-CSFs), filgrastim and lenograstim, are recognised to be useful in accelerating engraftment after autologous stem cell transplantation. Several forms of biosimilar non-glycosylated G-CSF have been approved by the European Medicines Agency, with limited published data supporting the clinical equivalence in peripheral blood stem cell mobilisation and recovery after autologous stem cell transplantation.
Method
With the aim of comparing cost-effective strategies in the use of G-CSF after autologous stem cell transplantation, we retrospectively evaluated 32 patients consecutively treated with biosimilar filgrastim XM02 (Tevagrastim) and 26 with lenograstim. All patients received G-CSF (biosimilar or lenograstim) at a dosage of 5 mcg/kg/day subcutaneously from day 5 to absolute neutrophil count of 1500/mmc for three days.
Results
The median time to absolute neutrophil count engraftment was 11 days for the filgrastim XM02 group and 12 days for the lenograstim group. As for platelets recovery, the median time was 12 days in both groups. The median number of G-CSF vials used for patients was 9.5 for Tevagrastim and 10.5 for lenograstim, reflecting a mean estimated cost of about 556.1 euros for Tevagrastim versus 932.2 euros for lenograstim (p< 0.001). The median days of febrile neutropenia were 1.5 and 1 for filgrastim XM02 and lenograstim, respectively. No adverse event related to the use of XM02 filgrastim was recorded.
Conclusion
In our experience, filgrastim XM02 and lenograstim showed comparable efficacy in shortening the period of neutropenia after cytoreduction and autologous stem cell transplantation, with a favourable cost effect for filgrastim XM02.
doi:10.3332/ecancer.2013.327
PMCID: PMC3694838  PMID: 23818939
biosimilar G-CSF; autologous bone marrow transplantation; engraftment; filgrastim; lenograstim
5.  G-CSF influences mouse skeletal muscle development and regeneration by stimulating myoblast proliferation 
Granulocyte colony-stimulating factor and its receptor are needed for skeletal muscle development and injury-induced regeneration in mice.
After skeletal muscle injury, neutrophils, monocytes, and macrophages infiltrate the damaged area; this is followed by rapid proliferation of myoblasts derived from muscle stem cells (also called satellite cells). Although it is known that inflammation triggers skeletal muscle regeneration, the underlying molecular mechanisms remain incompletely understood. In this study, we show that granulocyte colony-stimulating factor (G-CSF) receptor (G-CSFR) is expressed in developing somites. G-CSFR and G-CSF were expressed in myoblasts of mouse embryos during the midgestational stage but not in mature myocytes. Furthermore, G-CSFR was specifically but transiently expressed in regenerating myocytes present in injured adult mouse skeletal muscle. Neutralization of endogenous G-CSF with a blocking antibody impaired the regeneration process, whereas exogenous G-CSF supported muscle regeneration by promoting the proliferation of regenerating myoblasts. Furthermore, muscle regeneration was markedly impaired in G-CSFR–knockout mice. These findings indicate that G-CSF is crucial for skeletal myocyte development and regeneration and demonstrate the importance of inflammation-mediated induction of muscle regeneration.
doi:10.1084/jem.20101059
PMCID: PMC3135344  PMID: 21422169
6.  Expression of the G-CSF receptor in monocytic cells is sufficient to mediate hematopoietic progenitor mobilization by G-CSF in mice 
Expression of the G-CSF receptor on bone marrow monocytes is sufficient to trigger HSC mobilization in response to G-CSF, in part via effects on osteoblast lineage cells.
Granulocyte colony-stimulating factor (G-CSF), the prototypical mobilizing cytokine, induces hematopoietic stem and progenitor cell (HSPC) mobilization from the bone marrow in a cell-nonautonomous fashion. This process is mediated, in part, through suppression of osteoblasts and disruption of CXCR4/CXCL12 signaling. The cellular targets of G-CSF that initiate the mobilization cascade have not been identified. We use mixed G-CSF receptor (G-CSFR)–deficient bone marrow chimeras to show that G-CSF–induced mobilization of HSPCs correlates poorly with the number of wild-type neutrophils. We generated transgenic mice in which expression of the G-CSFR is restricted to cells of the monocytic lineage. G-CSF–induced HSPC mobilization, osteoblast suppression, and inhibition of CXCL12 expression in the bone marrow of these transgenic mice are intact, demonstrating that G-CSFR signals in monocytic cells are sufficient to induce HSPC mobilization. Moreover, G-CSF treatment of wild-type mice is associated with marked loss of monocytic cells in the bone marrow. Finally, we show that bone marrow macrophages produce factors that support the growth and/or survival of osteoblasts in vitro. Together, these data suggest a model in which G-CSFR signals in bone marrow monocytic cells inhibit the production of trophic factors required for osteoblast lineage cell maintenance, ultimately leading to HSPC mobilization.
doi:10.1084/jem.20101700
PMCID: PMC3039862  PMID: 21282380
7.  Granulocyte colony-stimulating factor receptor expression on human transitional cell carcinoma of the bladder. 
British Journal of Cancer  1997;75(10):1489-1496.
Receptors for granulocyte colony-stimulating factor (G-CSFRs) have been confirmed on the cell surfaces of several non-haematopoietic cell types, including bladder cancer cells. This observation has naturally led to the hypothesis that the expression of G-CSFR on these cells may enhance their growth by G-CSF. In this study, the expression of G-CSFR was determined in both established human bladder cancer cell lines and primary bladder cancers. We studied five different human bladder cancer cell lines (KU-1, KU-7, T-24, NBT-2 and KK) and 26 newly diagnosed bladder tumours. G-CSFR mRNA expressions on cultured cell lines were determined using the reverse transcriptase polymerase chain reaction (RT-PCR) method. Furthermore, the G-CSFR binding experiments on the cultured cell lines were conducted using the Na(125)I-labelled G-CSF ligand-binding assay method. Moreover, the G-CSFR mRNA expressions on primary bladder tumour specimens were assessed using the in situ RT-PCR method. Three out of the five cultured cell lines (KU-1, NBT-2 and KK) exhibited G-CSFR mRNA signals when the RT-PCR method was used. The G-CSFR binding experiments showed an equilibrium dissociation constant (K[d]) of 490 pM for KU-1, 340 pM for NBT-2 and 103 pM for KK cells. With in situ RT-PCR, the tumour cells of 6 out of 26 primary bladder tumour specimens (23.1%) presented positive G-CSFR mRNA signals. Thus, in this study, G-CSFR expression was frequently observed on bladder cancer cells. Therefore, the clinical use of G-CSF for patients with bladder cancer should be selected with great care.
Images
PMCID: PMC2223497  PMID: 9166942
8.  Granulocyte Colony-Stimulating Factor Receptor Mutations in Myeloid Malignancy 
Granulocyte colony-stimulating factor is a cytokine able to stimulate both myelopoiesis and hematopoietic stem cell mobilization, which has seen it used extensively in the clinic to aid hematopoietic recovery. It acts specifically via the homodimeric granulocyte colony-stimulating factor receptor (G-CSFR), which is principally expressed on the surface of myeloid and hematopoietic progenitor cells. A number of pathogenic mutations have now been identified in CSF3R, the gene encoding G-CSFR. These fall into distinct classes, each of which is associated with a particular spectrum of myeloid disorders, including malignancy. This review details the various CSF3R mutations, their mechanisms of action, and contribution to disease, as well as discussing the clinical implications of such mutations.
doi:10.3389/fonc.2014.00093
PMCID: PMC4013473  PMID: 24822171
G-CSF; G-CSFR; CSF3R; AML; SCN; CNL; MDS
9.  Enforced Granulocyte/Macrophage Colony-stimulating Factor Signals Do Not Support Lymphopoiesis, but Instruct Lymphoid to Myelomonocytic Lineage Conversion 
The Journal of Experimental Medicine  2003;197(10):1311-1322.
We evaluated the effects of ectopic granulocyte/macrophage colony-stimulating factor (GM-CSF) signals on hematopoietic commitment and differentiation. Lineage-restricted progenitors purified from mice with the ubiquitous transgenic human GM-CSF receptor (hGM-CSFR) were used for the analysis. In cultures with hGM-CSF alone, hGM-CSFR–expressing (hGM-CSFR+) granulocyte/monocyte progenitors (GMPs) and megakaryocyte/erythrocyte progenitors (MEPs) exclusively gave rise to granulocyte/monocyte (GM) and megakaryocyte/erythroid (MegE) colonies, respectively, providing formal proof that GM-CSF signals support the GM and MegE lineage differentiation without affecting the physiological myeloid fate. hGM-CSFR transgenic mice were crossed with mice deficient in interleukin (IL)-7, an essential cytokine for T and B cell development. Administration of hGM-CSF in these mice could not restore T or B lymphopoiesis, indicating that enforced GM-CSF signals cannot substitute for IL-7 to promote lymphopoiesis. Strikingly, >50% hGM-CSFR+ common lymphoid progenitors (CLPs) and >20% hGM-CSFR+ pro-T cells gave rise to granulocyte, monocyte, and/or myeloid dendritic cells, but not MegE lineage cells in the presence of hGM-CSF. Injection of hGM-CSF into mice transplanted with hGM-CSFR+ CLPs blocked their lymphoid differentiation, but induced development of GM cells in vivo. Thus, hGM-CSF transduces permissive signals for myeloerythroid differentiation, whereas it transmits potent instructive signals for the GM differentiation to CLPs and early T cell progenitors. These data suggest that a majority of CLPs and a fraction of pro-T cells possess plasticity for myelomonocytic differentiation that can be activated by ectopic GM-CSF signals, supporting the hypothesis that the down-regulation of GM-CSFR is a critical event in producing cells with a lymphoid-restricted lineage potential.
doi:10.1084/jem.20021843
PMCID: PMC2193786  PMID: 12756267
commitment; lineage conversion; cytokine; plasticity
10.  Granulocyte colony-stimulating factor and leukemogenesis. 
Mediators of Inflammation  2004;13(3):145-150.
The granulocyte colony-stimulating factor (G-CSF) plays an important role in normal granulopoiesis. Its functions are mediated by specific receptors on the surface of responsive cells and, upon ligand binding, several cytoplasmic tyrosine kinases are activated. The cytoplasmic region proximal to the membrane of the G-CSF receptor (G-CSF-R) transduces proliferative and survival signals, whereas the distal carboxy-terminal region transduces maturation signals and suppresses the receptor's proliferative signals. Mutations in the G-CSF-R gene resulting in truncation of the carboxy-terminal region have been detected in a subset of patients with severe congenital neutropenia who developed acute myelogenous leukemia (AML). In addition, the AML1-ETO fusion protein, expressed in leukemic cells harboring the t(8;21), disrupt the physiological function of transcription factors such as C/EBPalpha and C/EBPepsilon, which in turn deregulate G-CSF-R expression. The resulting high levels of G-CSF-R and G-CSF-dependent cell proliferation may be associated with pathogenesis of AML with t(8;21). Moreover, in vitro and in vivo studies demonstrated that G-CSF may act as a co-stimulus augmenting the response of PML-RARalpha acute promyelocytic leukemia cells to all-trans-retinoic acid treatment. Finally, in the PLZF-RARalpha acute promyelocytic leukemia transgenic model, G-CSF deficiency suppressed leukemia development. Altogether, these data suggest that the G-CSF signaling pathway may play a role in leukemogenesis.
doi:10.1080/09511920410001713574
PMCID: PMC1781560  PMID: 15223604
11.  G-CSF does not influence C2C12 myogenesis despite receptor expression in healthy and dystrophic skeletal muscle 
Granulocyte-colony stimulating factor (G-CSF) increases recovery of rodent skeletal muscles after injury, and increases muscle function in rodent models of neuromuscular disease. However, the mechanisms by which G-CSF mediates these effects are poorly understood. G-CSF acts by binding to the membrane spanning G-CSFR and activating multiple intracellular signaling pathways. Expression of the G-CSFR within the haematopoietic system is well known, but more recently it has been demonstrated to be expressed in other tissues. However, comprehensive characterization of G-CSFR expression in healthy and diseased skeletal muscle, imperative before implementing G-CSF as a therapeutic agent for skeletal muscle conditions, has been lacking. Here we show that the G-CSFR is expressed in proliferating C2C12 myoblasts, differentiated C2C12 myotubes, human primary skeletal muscle cell cultures and in mouse and human skeletal muscle. In mdx mice, a model of human Duchenne muscular dystrophy (DMD), G-CSF mRNA and protein was down-regulated in limb and diaphragm muscle, but circulating G-CSF ligand levels were elevated. G-CSFR mRNA in the muscles of mdx mice was up-regulated however steady-state levels of the protein were down-regulated. We show that G-CSF does not influence C2C12 myoblast proliferation, differentiation or phosphorylation of Akt, STAT3, and Erk1/2. Media change alone was sufficient to elicit increases in Akt, STAT3, and Erk1/2 phosphorylation in C2C12 muscle cells and suggest previous observations showing a G-CSF increase in phosphoprotein signaling be viewed with caution. These results suggest that the actions of G-CSF may require the interaction with other cytokines and growth factors in vivo, however these data provides preliminary evidence supporting the investigation of G-CSF for the management of muscular dystrophy.
doi:10.3389/fphys.2014.00170
PMCID: PMC4013466  PMID: 24822049
G-CSF; cytokine receptor; skeletal muscle; duchenne muscular dystrophy; mdx; C2C12; proliferation; differentiation
12.  A functional granulocyte colony-stimulating factor receptor is required for normal chemoattractant-induced neutrophil activation 
Journal of Clinical Investigation  1999;103(6):825-832.
Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor that is widely used to treat neutropenia. In addition to stimulating polymorphonuclear neutrophil (PMN) production, G-CSF may have significant effects on PMN function. Because G-CSF receptor (G-CSFR)–deficient mice do not have the expected neutrophilia after administration of human interleukin-8 (IL-8), we examined the effect of the loss of G-CSFR on IL-8–stimulated PMN function. Compared with wild-type PMNs, PMNs isolated from G-CSFR–deficient mice demonstrated markedly decreased chemotaxis to IL-8. PMN emigration into the skin of G-CSFR–deficient mice in response to IL-8 was also impaired. Significant chemotaxis defects were also seen in response to N-formyl-methionyl-leucyl-phenylalanine, zymosan-activated serum, or macrophage inflammatory protein-2. The defective chemotactic response to IL-8 does not appear to be due to impaired chemoattractant receptor function, as the number of IL-8 receptors and chemoattractant-induced calcium influx, actin polymerization, and release of gelatinase B were comparable to those of wild-type PMNs. Chemoattractant-induced adhesion of G-CSFR–deficient PMNs was significantly impaired, suggesting a defect in β2-integrin activation. Collectively, these data demonstrate that selective defects in PMN activation are present in G-CSFR–deficient mice and indicate that G-CSF plays an important role in regulating PMN chemokine responsiveness.
PMCID: PMC408143  PMID: 10079103
13.  Granulocyte colony-stimulating factors for febrile neutropenia prophylaxis following chemotherapy: systematic review and meta-analysis 
BMC Cancer  2011;11:404.
Background
Febrile neutropenia (FN) occurs following myelosuppressive chemotherapy and is associated with morbidity, mortality, costs, and chemotherapy reductions and delays. Granulocyte colony-stimulating factors (G-CSFs) stimulate neutrophil production and may reduce FN incidence when given prophylactically following chemotherapy.
Methods
A systematic review and meta-analysis assessed the effectiveness of G-CSFs (pegfilgrastim, filgrastim or lenograstim) in reducing FN incidence in adults undergoing chemotherapy for solid tumours or lymphoma. G-CSFs were compared with no primary G-CSF prophylaxis and with one another. Nine databases were searched in December 2009. Meta-analysis used a random effects model due to heterogeneity.
Results
Twenty studies compared primary G-CSF prophylaxis with no primary G-CSF prophylaxis: five studies of pegfilgrastim; ten of filgrastim; and five of lenograstim. All three G-CSFs significantly reduced FN incidence, with relative risks of 0.30 (95% CI: 0.14 to 0.65) for pegfilgrastim, 0.57 (95% CI: 0.48 to 0.69) for filgrastim, and 0.62 (95% CI: 0.44 to 0.88) for lenograstim. Overall, the relative risk of FN for any primary G-CSF prophylaxis versus no primary G-CSF prophylaxis was 0.51 (95% CI: 0.41 to 0.62). In terms of comparisons between different G-CSFs, five studies compared pegfilgrastim with filgrastim. FN incidence was significantly lower for pegfilgrastim than filgrastim, with a relative risk of 0.66 (95% CI: 0.44 to 0.98).
Conclusions
Primary prophylaxis with G-CSFs significantly reduces FN incidence in adults undergoing chemotherapy for solid tumours or lymphoma. Pegfilgrastim reduces FN incidence to a significantly greater extent than filgrastim.
doi:10.1186/1471-2407-11-404
PMCID: PMC3203098  PMID: 21943360
14.  Reconstituted human granulocyte-macrophage colony-stimulating factor receptor transduces growth-promoting signals in mouse NIH 3T3 cells: comparison with signalling in BA/F3 pro-B cells. 
Molecular and Cellular Biology  1993;13(3):1440-1448.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) plays a critical role in growth and differentiation of myeloid cells. We previously reconstituted high-affinity human GM-CSF receptor (hGM-CSFR) in a pro-B cell line, BA/F3, by cotransfecting alpha- and beta-chain cDNA clones and showed that the reconstituted receptor could transduce growth-promoting signals. The high-affinity hGM-CSFR was also reconstituted in mouse NIH 3T3 cells, but its ability to transduce signals in fibroblasts remained undetermined. In the present study, we further characterized signal transduction by the reconstituted hGM-CSFR in both NIH 3T3 cells and BA/F3 cells. We found that the reconstituted hGM-CSFR transduces signals in NIH 3T3 fibroblasts and BA/F3 cells in response to hGM-CSF to activate transcription of the c-fos, c-jun, and c-myc proto-oncogenes. hGM-CSF also induces protein tyrosine phosphorylation and DNA synthesis in both cell types. These results indicated that hGM-CSFR is functional in fibroblasts, that signal transduction via hGM-CSFR in fibroblasts involves tyrosine kinase(s), and that association of hGM-CSFR with a factor(s) specific to hematopoietic cell lineage is not essential to transduce growth-promoting signals.
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PMCID: PMC359454  PMID: 8441389
15.  ROLE OF THE PROTEASOME IN MODULATING NATIVE G-CSFR EXPRESSION 
Cytokine  2008;43(2):114-123.
The granulocyte colony-stimulating factor receptor (G-CSFR) is a critical regulator of granulopoiesis, but the mechanisms controlling its surface expression are poorly understood. Recent studies using transfected cell lines have suggested the activated G-CSFR is routed to the lysosome and not the proteasome. Here, we examined the role of the ubiquitin/proteasome system in regulating G-CSFR surface expression in both ts20 cells that have a temperature-sensitive E1 ubiquitin-activating enzyme and in primary human neutrophils. We show that the G-CSFR is constitutively ubiquitinated, which increases following ligand binding. In the absence of a functional E1 enzyme, ligand-induced internalization of the receptor is inhibited. Pre-treatment of ts20 transfectants with either chloroquine or MG132 inhibited ligand-induced G-CSFR degradation, suggesting a role for both lysosomes and proteasomes in regulating G-CSFR surface expression in this cell line. In neutrophils, inhibition of the proteasome but not the lysosome was found to inhibit internalization/degradation of the activated G-CSFR. Collectively, these data demonstrate the requirement for a functional ubiquitin/proteasome system in G-CSFR internalization and degradation. Our results suggest a prominent role for the proteasome in physiologic modulation of the G-CSFR, and provide further evidence for the importance of the ubiquitin/proteasome system in the initiation of negative signaling by cytokine receptors.
doi:10.1016/j.cyto.2008.04.015
PMCID: PMC2556513  PMID: 18554923
G-CSFR; ubiquitination; proteasome; internalization; neutrophil
16.  Distinct regions of the human granulocyte-colony-stimulating factor receptor cytoplasmic domain are required for proliferation and gene induction. 
Molecular and Cellular Biology  1993;13(4):2384-2390.
Using two different cell systems, we show that the cytoplasmic domain of the granulocyte-colony-stimulating factor receptor (G-CSFR) may be composed of at least two functional regions. The first, within the membrane-proximal 57 amino acids, is absolutely required to deliver a proliferative signal. This region contains two sequence motifs conserved between members of the hematopoietin receptor family. The second functional region resides between amino acids 57 and 96. This region is required for the induction of acute-phase plasma protein gene expression when the G-CSFR is transfected into human hepatoma cell lines. The G-CSFR-transfected hepatoma cells respond to G-CSF by increasing the production of the same set of plasma proteins as stimulated by interleukin-6, suggesting that the two cytokines share a common signal transduction pathway.
Images
PMCID: PMC359559  PMID: 7681146
17.  Cytokine signals through STAT3 promote expression of granulocyte secondary granule proteins in 32D cells 
Experimental hematology  2005;33(3):308-317.
Objective
In a previous study, we showed that activation of a transfected human erythropoietin receptor (EPOR) in the murine myeloid cell line 32D resulted in the development of morphologic features of granulocytic differentiation and expression of the neutrophil primary granule protein myeloperoxidase. We now studied if EPOR signaling could also mediate secondary granule protein gene expression and investigated the signal transduction requirements for induction of secondary granule gene expression in 32D cells.
Materials and Methods
Wild-type and variant 32D cells expressing normal or chimeric EPORs or receptors for granulocyte colony-stimulating factor (G-CSFRs) were stimulated with EPO or G-CSF and the expression of granulocyte-specific genes was analyzed by Northern blot analysis. To determine the signaling mechanisms required for secondary granule protein gene induction, the activation of STAT pathways following growth factor stimulation was studied by Western blot analysis.
Results
We found that EPO treatment of 32D cells engineered to express EPOR did not result in induction of the secondary granule protein genes encoding lactoferrin and 24p3 lipocalin, the mouse homolog of human N-Gal, or the myeloid transcription factor C/EBPε. Replacement of the intracellular domain of EPOR with the intracellular domain of G-CSFR in a chimeric receptor was associated with EPO-mediated induction of lactoferrin, 24p3 lipocalin, and C/EBPε genes. We found that STAT3 phosphorylation was mediated by the intracellular domain of G-CSFR, but not EPOR. Replacement of one or two of the STAT5 binding sites in the intracytoplasmic domain of the EPOR with STAT3 binding sites resulted in EPO-mediated STAT3 activation and a marked increase in the expression of the 24p3 lipocalin gene. Knockdown of STAT3 protein levels with siRNA caused significant decrease in 24p3 lipocalin gene induction.
Conclusion
These results indicate that EPOR signaling cannot substitute for G-CSFR signaling to stimulate secondary granule protein gene expression in 32D cells. In addition, STAT3 is a critical mediator of 24p3 lipocalin gene expression in these cells.
doi:10.1016/j.exphem.2004.11.014
PMCID: PMC2388245  PMID: 15730854
18.  Granulocyte-Colony-Stimulating Factor Stimulation of Bone Marrow Mesenchymal Stromal Cells Promotes CD34+ Cell Migration Via a Matrix Metalloproteinase-2-Dependent Mechanism 
Stem Cells and Development  2012;21(17):3162-3172.
Human hematopoietic stem/progenitor cells (HSPCs) can be mobilized into the circulation using granulocyte-colony stimulating factor (G-CSF), for graft collection in view of hematopoietic transplantation. This process has been related to bone marrow (BM) release of serine proteases and of the matrix metalloproteinase-9 (MMP-9). Yet, the role of these mediators in HSC egress from their niches remains questionable, because they are produced by nonstromal cells (mainly neutrophils and monocytes/macrophages) that are not a part of the niche. We show here that the G-CSF receptor (G-CSFR) is expressed by human BM mesenchymal stromal/stem cells (MSCs), and that G-CSF prestimulation of MSCs enhances the in vitro trans-stromal migration of CD34+ cells. Zymography analysis indicates that pro-MMP-2 (but not pro-MMP-9) is expressed in MSCs, and that G-CSF treatment increases its expression and induces its activation at the cell membrane. We further demonstrate that G-CSF-stimulated migration depends on G-CSFR expression and is mediated by a mechanism that involves MMPs. These results suggest a molecular model whereby G-CSF infusion may drive, by the direct action on MSCs, HSPC egress from BM niches via synthesis and activation of MMPs. In this model, MMP-2 instead of MMP-9 is implicated, which constitutes a major difference with mouse mobilization models.
doi:10.1089/scd.2012.0048
PMCID: PMC3495113  PMID: 22651889
19.  Ribosomal p70S6K Basal Activity Increases upon Induction of Differentiation of Myelomonocytic Leukemic Cell Lines HL60, AML14 and MPD 
Leukemia research  2004;28(7):755-762.
The role of ribosomal p70S6K in the cell cycle has been studied extensively, and it is known that this enzyme is crucial for cell advancement through G1. Conversely, the participation of p70S6K in cell differentiation is not well understood. We have studied the response of p70S6K to the cytokine granulocyte-macrophage colony stimulating factor (GM-CSF) in three differentiation-capable leukemic cell lines (MPD, AML-14 and HL-60) and in normal mature neutrophils. Immature leukemic cells starved for 16 hours showed a robust (~3.5-fold over controls) p70S6K phosphorylation on T421/S424 residues in response to an acute (5 min) 10 nM GM-CSF stimulation. On the other hand, cells that had been induced to differentiate and express granulocytic phenotypes, showed an increased (~6-fold) basal level of p70S6K T421/S424 phosphorylation over immature cells, as well as an increased baseline tyrosyl phosphorylation of the GM-CSF receptor β subunit (GM-CSF•Rβ). However, these cells displayed a weak (~1.4-fold over controls) response to GM-CSF even at prolonged incubation times (20 min). In vitro p70S6K enzymatic activity paralleled p70S6K T421/S424 phosphorylation in both high basal, unstimulated, levels in immature cells and a low degree of response to GM-CSF. Lastly, peripheral blood mature neutrophils had low basal GM-CSF•Rβ and p70S6K activity, with both parameters being robustly stimulated following addition of GM-CSF, a situation in contrast with the cell lines, indicative perhaps of their incomplete terminal differentiation. In summary, these findings show the increase in basal phosphorylation of p70S6K upon granulocytic differentiation of myeloid leukemic cells and their responses to GM-CSF that are closely paralleled with tyrosyl phosphorylation of its receptor, and help in pointing to specific cell signaling molecules that are different in leukemic blasts from normal leukocytes.
doi:10.1016/j.leukres.2003.11.012
PMCID: PMC3082395  PMID: 15158097
ribosomal p70S6K; leukemic cell lines; AML; MPD; GM-CSF
20.  Expression of the macrophage colony-stimulating factor and c-fms genes in human acute myeloblastic leukemia cells. 
Journal of Clinical Investigation  1988;81(4):1030-1035.
Macrophage colony-stimulating factor (CSF-1; M-CSF) is a growth factor required for growth and differentiation of mononuclear phagocytes. The effects of CSF-1 are mediated through binding to specific, high-affinity surface receptors encoded by the c-fms gene. CSF-1 and c-fms gene expression was investigated in fresh human acute myeloblastic leukemic cells by Northern blot hybridization using cDNA probes. 4.0-kb CSF-1 transcripts were detected in 10 of 17 cases of acute myeloblastic leukemia (AML), while c-fms transcripts were detected in 7 of 15. Coexpression of CSF-1 and c-fms was observed in five cases, and in five other cases neither gene was expressed. In situ hybridization demonstrated that transcripts for CSF-1 were present in 70-90% of cells in each of three cases studied while c-fms mRNA was detected in 40-70% of cells. The constitutive expression of CSF-1 transcripts was associated with production of CSF-1 protein, although detectable amounts of CSF-1 were not secreted unless the cells were exposed to phorbol ester. These results demonstrate that leukemic myeloblasts from a subset of patients with AML express transcripts for both the CSF-1 and CSF-1 receptor genes, often in the same leukemic cells in vitro.
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PMCID: PMC329627  PMID: 2832442
21.  Induction of the granulocyte-macrophage colony-stimulating factor (CSF) receptor by granulocyte CSF increases the differentiative options of a murine hematopoietic progenitor cell. 
Molecular and Cellular Biology  1990;10(9):4846-4853.
32DC13(G) is an interleukin-3-dependent murine hematopoietic precursor cell line which differentiates into neutrophilic granulocytes upon exposure to granulocyte colony-stimulating factor (G-CSF) but ceases to proliferate and dies when exposed to granulocyte-macrophage (GM)-CSF. Surface receptors for GM-CSF are undetectable on 32DC13(G) cells but can be induced by priming the cells with G-CSF. Exposure of the G-CSF-primed cells to GM-CSF then results in the generation of monocytes as well as granulocytes. The acquired competence to respond to GM-CSF remains irreversibly encoded in the primed cells, although the GM-CSF receptor can be down regulated by interleukin-3. This phenomenon suggests a mechanism by which hematopoietic precursors may obtain additional receptors, thereby increasing their differentiative potential.
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PMCID: PMC361095  PMID: 1697033
22.  Human Immunodeficiency Virus Type 1 Infection Inhibits Granulocyte-Macrophage Colony-Stimulating Factor-Induced Activation of STAT5A in Human Monocyte-Derived Macrophages 
Journal of Virology  2003;77(23):12630-12638.
Human immunodeficiency virus type 1 (HIV-1) infects cells of the monocyte/macrophage lineage. While infection of macrophages by HIV-1 is generally not cytopathic, it does impair macrophage function. In this study, we examined the effect of HIV-1 infection on intracellular signaling in human monocyte-derived macrophages (MDM) stimulated with the growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF is an important growth factor for cells of both the macrophage and granulocyte lineages and enhances effector functions of these cells via the heterodimeric GM-CSF receptor (GM-CSFR). A major pathway which mediates the effects of GM-CSF on macrophages involves activation of the latent transcription factor STAT5A via a Janus kinase 2 (JAK2)-dependent pathway. We demonstrate that GM-CSF-induced activation of STAT5A is inhibited in MDM after infection in vitro with the laboratory-adapted R5 strain of HIV-1, HIV-1Ba-L, but not after infection with adenovirus. HIV-1 infection of MDM did not decrease the STAT5A or JAK2 mRNA level or STAT5A protein level or result in increased constitutive activation of STAT5A. Surface expression of either the α-chain or common βc-chain of GM-CSFR was also unaffected. We conclude that HIV-1 inhibits GM-CSF activation of STAT5A without affecting expression of the known components of the signaling pathway. These data provide further evidence of disruption of cellular signaling pathways after HIV-1 infection, which may contribute to immune dysfunction and HIV-1 pathogenesis.
doi:10.1128/JVI.77.23.12630-12638.2003
PMCID: PMC262552  PMID: 14610185
23.  Stoichiometric Structure-Function Analysis of the Prolactin Receptor Signaling Domain by Receptor Chimeras 
Molecular and Cellular Biology  1998;18(2):896-905.
The intracellular domain of the prolactin (PRL) receptor (PRLr) is required for PRL-induced signaling and proliferation. To identify and test the functional stoichiometry of those PRLr motifs required for transduction and growth, chimeras consisting of the extracellular domain of either the α or β subunit of human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GM-CSFr) and the intracellular domain of the rat PRLr were synthesized. Because the high-affinity binding of GM-CSF results from the specific pairing of one α- and one β-GM-CSFr, use of GM-CSFr/PRLr chimera enabled targeted dimerization of the PRLr intracellular domain. To that end, the extracellular domains of the α- and β-GM-CSFr were conjugated to one of the following mutations: (i) PRLr C-terminal truncations, termed α278, α294, α300, α322, or β322; (ii) PRLr tyrosine replacements, termed Y309F, Y382F, or Y309+382F; or, (iii) PRLr wild-type short, intermediate, or long isoforms. These chimeras were cotransfected into the cytokine-responsive Ba/F3 line, and their expression was confirmed by ligand binding and Northern and Western blot analyses. Data from these studies revealed that heterodimeric complexes of the wild type with C-terminal truncation mutants of the PRLr intracellular domain were incapable of ligand-induced signaling or proliferation. Replacement of any single tyrosine residue (Y309F or Y382F) in the dimerized PRLr complex resulted in a moderate reduction of receptor-associated Jak2 activation and proliferation. In contrast, trans replacement of these residues (i.e., αY309F and βY382F) markedly reduced ligand-driven Jak2 activation and proliferation, while cis replacement of both tyrosine residues in a single intracellular domain (i.e., αY309+382F) produced an inactive signaling complex. Analysis of these GM-CSFr–PRLr complexes revealed equivalent levels of Jak2 in association with the mutant receptor chains, suggesting that the tyrosine residues at 309 and 382 do not contribute to Jak association, but instead to its activation. Heterodimeric pairings of the intracellular domains from the known PRLr receptor isoforms (short-intermediate, short-long, and intermediate-long) also yielded inactive receptor complexes. These data demonstrate that the tyrosine residues at 309 and 382, as well as additional residues within the C terminus of the dimerized PRLr complex, contribute to PRL-driven signaling and proliferation. Furthermore, these findings indicate a functional requirement for the pairing of Y309 and Y382 in trans within the dimerized receptor complex.
PMCID: PMC108801  PMID: 9447986
24.  Constitutive expression of the granulocyte-macrophage colony-stimulating factor gene in acute myeloblastic leukemia. 
Journal of Clinical Investigation  1987;79(1):100-106.
Expression of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene was studied by Northern blot analysis in normal human hematopoietic cells and a series of leukemias. GM-CSF messenger (m)RNA was detected in activated T cells, but not in normal bone marrow cells, monocytes, or nonactivated T cells. In contrast, leukemic cells from 11 of 22 cases of acute myeloblastic leukemia expressed GM-CSF transcripts. Biologically active CSF was detected in supernatant conditioned by 6 of these 11 leukemias. Expression of the GM-CSF gene was not detected in "common" (pre-B cell) acute lymphoblastic leukemia (11 cases tested) or chronic myeloid leukemia (4 cases tested). These results show that the GM-CSF gene is constitutively expressed in a subset of patients with AML, and further suggest that expression of this gene could contribute to the abnormal growth properties characteristic of AML.
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PMCID: PMC423997  PMID: 3491836
25.  Expression of granulocyte colony stimulating factor receptor in human colorectal cancer 
Yang, X | Liu, F | Xu, Z | Chen, C | Wu, X | Li, G | Li, J
Postgraduate Medical Journal  2005;81(955):333-337.
Aims: To discover if human colorectal cancer expresses granulocyte colony stimulating factor receptor (G-CSFR) and if granulocyte colony stimulating factor (G-CSF) plays an important part in the development and progression of human colorectal cancer.
Methods: Forty two specimens of colorectal cancer and normal colorectal mucosa were investigated, taken from the colon or rectum in group of colorectal cancer patients. Immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) technique were used to show G-CSFR expression. The relation between expression of G-CSFR and clinical or pathological factors was analysed.
Results: Immucohistochemical analyses showed that G-CSFR was expressed in the human colorectal cancer (25 of 42, 59.52%) and seemed to be up-regulated compared with the normal mucosa (14 of 42, 33.33%, p<0.001). In pronounced contrast with mostly strongly positive tumours, corresponding normal colorectal mucosa was negative or weakly positive. A significant correlation was found between G-CSFR expression and tumour stage (p = 0.001), tumour differentiation (p<0.001), but there was no significant relation between the expression of G-CSFR and the age, sex, and tumour size (p = 0.346, p = 0.686, p = 0.459). In RT-PCR, 21 of all 42 tumours had G-CSFR mRNA expression, while only 11 of 42 normal colorectal mucosa had such expression.
Conclusions: These data show that G-CSFR is commonly expressed in human colorectal cancers, thus supporting a possible role for G-CSF in colorectal cancer physiology.
doi:10.1136/pgmj.2004.024646
PMCID: PMC1743273  PMID: 15879049

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