Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2013 Jan 1.
Published in final edited form as: Differentiation. 2011 Oct 8;83(1):47–59. doi: 10.1016/j.diff.2011.08.003

The GM-CSF receptor utilizes β-catenin and Tcf4 to specify macrophage lineage differentiation

Anna L Brown a,b, Diana G Salerno a, Teresa Sadras a,b, Grant A Engler a, Chung H Kok a,b, Christopher R Wilkinson c,d, Saumya E Samaraweera b, Timothy J Sadlon c, Michelle Perugini a,b, Ian D Lewis a,e, Thomas J Gonda f, Richard J D’Andrea a,b,e,g,*
PMCID: PMC3394929  NIHMSID: NIHMS331631  PMID: 22099176

Abstract

Granulocyte-macrophage colony stimulating factor (GM-CSF) promotes the growth, survival, differentiation and activation of normal myeloid cells and is essential for fully functional macrophage differentiation in vivo. To better understand the mechanisms by which growth factors control the balance between proliferation and self-renewal versus growth-suppression and differentiation we have used the bi-potent FDB1 myeloid cell line, which proliferates in IL-3 and differentiates to granulocytes and macrophages in response to GM-CSF. This provides a manipulable model in which to dissect the switch between growth and differentiation. We show that, in the context of signaling from an activating mutant of the GM-CSF receptor β subunit, a single intracellular tyrosine residue (Y577) mediates the granulocyte fate decision. Loss of granulocyte differentiation in a Y577F second-site mutant is accompanied by enhanced macrophage differentiation, accumulation of β-catenin together with activation of Tcf4 and other Wnt target genes. These include the known macrophage lineage inducer, Egr1. We show that forced expression of Tcf4 or a stabilised β-catenin mutant is sufficient to promote macrophage differentiation in response to GM-CSF and that GM-CSF can regulate β-catenin stability, most likely via GSK3β. Consistent with this pathway being active in primary cells we show that inhibition of GSK3β activity promotes the formation of macrophage colonies at the expense of granulocyte colonies in response to GM-CSF. This study therefore identifies a novel pathway through which growth factor receptor signalling can interact with transcriptional regulators to influence lineage choice during myeloid differentiation.

Keywords: Myeloid, transcription-factor, β-catenin, Tcf4, signal-transduction

Introduction

In this study we examine the process of granulocyte and macrophage lineage specification mediated via Granulocyte-macrophage colony stimulating factor (GM-CSF) signaling. GM-CSF is a hematopoietic growth factor that can provide both permissive and instructive signals for myeloid differentiation (Kondo et al., 2000) and has been shown to play a critical role in DC function (Conti and Gessani, 2008). In vivo administration of GM-CSF promotes increased production of granulocytes and macrophages demonstrating its key role in vivo in driving coordinated proliferation and differentiation of GM progenitors (Metcalf et al., 1987). In particular, under steady-state conditions, GM-CSF signaling has a non-redundant role in mature macrophage production, with loss of GM-CSF signaling in mouse and humans leading to pulmonary alveolar proteinosis due to defects in alveolar macrophages (Dranoff et al., 1994; Suzuki et al., 2008; Martinez-Moczygemba et al., 2008; Nishinakamura et al., 1995). Null animals also display compromised antigen-specific and LPS-induced T-cell responses and IFNγ production, which may be DC-mediated, have defects in macrophage function and are susceptible to various infectious agents (Enzler et al., 2003; Paine et al., 2000). The high affinity receptors for human GM-CSF (GMR), IL-3 (IL3R) and IL-5 (IL5R) are each comprised of unique ligand-specific α subunits (GMRα, IL3Rα or IL5Rα) and a shared β subunit (hβc) which are all members of the cytokine receptor superfamily (for review see (Miyajima et al., 1993; Lopez et al., 2010). Each ligand binds to its specific α-subunit to form a low affinity intermediate which we, and others have shown to form a signaling complex that is likely to include a dimer of hβc and, at least in the case of the GMR, has been recently shown to form a higher order dodecameric complex for the full range of ligand induced signaling (McClure et al., 2001; McClure et al., 2003; Hansen et al., 2008). hβc is the primary signaling subunit and mutation can result in constitutive activation with a range of mutants now described that display alternative phenotypes and signaling profiles (D'Andrea et al., 1998; McCormack and Gonda, 1999; Jenkins et al., 1995; Brown et al., 2004; Perugini et al.; 2010).

Mutational studies of the GMR have identified intracellular regions and key residues of the GMRα and hβc that are responsible for the signaling required for myeloid differentiation versus growth. In particular, the region of hβc containing Tyr577 is important for mediating GM-CSF induced myeloid differentiation of M1 and WEHI-3B D+ cells, where macrophage differentiation is induced in response to ligand, however particular residues in this region were not linked to the response (Smith et al., 1997). Studies with activated mutants of hβc, showing reduced signaling complexity compared to the wild type receptor, have facilitated dissection of signaling networks downstream of the GM-CSF receptor, and allowed particular signaling events to be assigned to cellular outcomes (Brown et al., 2004; Perugini et al., 2010; Jenkins et al., 1998). In this study we use the well-characterised activated hβc mutant, FIΔ, and a second-site mutant with a tyrosine to phenylalanine substitution at position 577 (Y577F) that selectively abolishes granulocyte differentiation and enhances macrophage differentiation (Brown et al., 2004). This has provided a model system in which to dissect GM differentiation induced through the GM-CSF receptor. The Tyrosine 577 residue of hβc has been previously shown to be a key signaling residue associated with binding of the Shc adapter molecule and is part of a small phosphorylation-dependent motif, which regulates alternative survival and proliferation pathways (Okuda et al., 1997; Powell et al., 2009; Guthridge et al., 2006; Ramshaw et al., 2007). Here we focus on defining downstream events, associated with the Tyr577 residue, and on linking these to the lineage-fate choice between granulocyte and macrophage differentiation. We show that the Y577F mutation is associated with enhanced β-catenin protein accumulation and Tcf4 gene expression and we demonstrate a central role for these factors in promoting macrophage differentiation at the expense of granulocyte differentiation.

Materials and Methods

Cell culture

The culture conditions of FDB1 cells, the construction of FIΔ and FIΔY577F retroviral expression plasmids, and the generation of stable cell lines have been previously described (Brown et al., 2004). Before treatment of cells with inhibitors, cells were washed 3 times and starved of growth factor for 16 hours in medium containing serum. Stimulation was carried out for 5 minutes at 37°C by the use of 500 bone marrow units (BMU)/mL mouse (m)IL-3 or mouse (m)GM-CSF. The GSK-3 Inhibitor IX, BIO, and control, MeBIO, (Merk Chemicals, Nottingham, UK) were dissolved in DMSO and used at a final concentration of 2 µM.

Colony forming assays

Bone marrow cells were plated in methylcellulose medium M3134 (Stem cell technologies, Vancouver, BC, Canada) with 100 ng/ml of rmGM-CSF (Peprotech, Rocky Hill, NJ) and concentrations of Me-BIO or BIO indicated in Fig. 5. Cells were plated at a density of 2×104 cells per plate in triplicate and scored for colony formation 8 days later

Fig. 5. GSK3β inhibition increases macrophage differentiation of primary cells.

Fig. 5

Open in a new tab

A. Bone marrow cells were plated in methylcellulose medium containing GM-CSF and the indicated concentrations of BIO or the control analogue Me-BIO and scored for colony formation 8 days later. CFU=colony forming unit, M=macrophage, G=granulocyte, GM= granulocyte-macrophage. Error bars represent SEM (n=2) and *=p<0.05, **=<0.01, (unpaired, two sided t-test). B. Overlapping TCF4/β-catenin targets and macrophage genes with an FDR p<0.05 (113) were subjected to Ingenuity Pathway Analysis (IPA). The genes were subject to IPA mapping of network interactions and filtered for direct interactions and networks containing genes identified as involved in Wnt/β-catenin signaling. The colour indicates the direction of differential expression in our macrophage regulated gene list with red indicating that gene expression increases between 0 and 72 hours and green indicating that the gene expression decreases with the colour intensity indicating the magnitude of regulation. CTNNB1= β-catenin.

Microarray hybridisation and analysis

FDB1 cells expressing either FIΔ and FIΔY577F were washed 3 times to remove growth factor and a proportion of cells were harvested at time zero. Cells were then cultured without factor for 72 hours for the second time point. Two biological replicates were prepared for each cell line (supplemental Fig. 1). Comparisons were performed for each cell line over time and direct comparisons between cell lines done at 0 and 72 hours. Additional dye swaps were done with the FIΔ/FIΔY577F 0 hour direct comparison and the FIΔ 0–72 hour comparison (supplemental Fig. 2). RNA preparation and microarray analysis and hybridisation were performed as described previously (Brown et al., 2006). Lineage specific gene lists were defined as follows. Neutrophil associated genes were defined as genes with a significant change over time in FIΔ expressing cells (p<0.001) but not FIΔY577F cells. Macrophage associated genes were defined as those with a significantly larger fold change over time in FIΔY577F cells compared to FIΔ cells (p<0.001, supplemental Fig.2). We performed gene-set enrichment analysis as previously described (Brown et al., 2006). Briefly, to test for association of published TF target gene sets based on either differential expression (C/EBP) (Gery et al., 2005) or ChIP (TCF and β-catenin) (Hatzis et al., 2008; Yochum et al., 2007) with our data sets, we used the non-parametric Wilcoxon rank sum test as implemented in the R statistical program (http://www.r-project.org/). We constructed our reference set of genes based on ranking the false discovery rate (FDR) p-value for comparisons of reference-set and test-set. For the C/EBP comparison we generated a reference set by ranking all of the genes on our array using the FDR p-values for the change in expression over time in FIΔ expressing cells and examined the distribution of C/EBP target genes in our reference list. For the β-catenin and TCF4 comparisons we generated a reference set by ranking all of the genes on our array using FDR p-values for the change in expression over time in FIΔY577F expressing cells and examined the distribution of β-catenin and TCF4 target genes in our reference list. A significant result from the Wilcoxon rank sum test indicated that the gene-set of interest displays an association with the indicated reference-set. Unsupervised clustering and heatmaps of data were generated with MeV (http://www.tm4.org/mev/). The raw microarray data is available in the Gene Expression Omnibus (GSE25857).

QRT-PCR

FDB1 cells expressing either FIΔ and FIΔY577F were washed 3 times to remove growth factor and a proportion of cells were harvested at time zero. Cells were then cultured without factor and cells harvested at 24 hours and 72 hours. Parental FDB1 cells were also cultured in GM-CSF and harvested at 24, 72 and 120 hours. Two biological replicates of this experiment were performed. RNA was prepared using Trizol reagent (Invitrogen, Carlsbad, CA, USA) as per the manufacturers instructions. cDNA was prepared using the QuantiTect Reverse Transcription Kit (Qiagen, Valencia, CA, USA). The sequences of the oligonucleotides used for PCR are listed in supplemental Table 1. Gene-specific PCR reactions were performed for 40 cycles using FastStart Taq (Roche diagnostics, Indianapolis, IN, USA) and recommended conditions. SYBR green (Invitrogen, Carlsbad, CA, USA) was added to a final concentration of 0.6× per reaction and PCR was performed on the Rotor-Gene 6000 and related software used for data collection and to determine mean expression values relative to beta-actin (Corbett Research, Version 5.0, Qiagen, Valencia, CA, USA). Amplification products were analyzed by melt curve and sequenced to confirm specificity.

Construction of retroviral expression plasmids and transduction of FDB1 cells

Constructs for FLAG-tagged full length TCF4 and ΔN-TCF4 in pcDNA3.1/Zeo were obtained from Hans Clever (van de et al., 2002). The cDNAs were amplified from the constructs using the oligos indicated in supplemental Table 1. The products were digested with SfiI and ligated into SfiI digested murine stem cell virus (MSCV)-internal ribosome entry site (IRES)-green fluorescence protein (GFP) retroviral vector (modified to contain SfiI sites in the MCS). The construct for stabilised β-catenin (pCS2MMBCS33A) was obtained from Rolf Kemler (Rottbauer et al., 2002). The β-catenin cDNA contains 4 amino acid substitution mutations (Ser33Ala, Ser37Ala, Thr41Ala and Ser45Ala). β-catenin was amplified using the oligos indicated in supplemental materials and methods and cloned into MSCV-IRES-GFP as described above.

Virus production and FDB1 transduction was performed as previously described (Perugini et al., 2009). For the TCF constructs, cells expressing GFP were sorted using flow cytometry and allowed to recover overnight before using in assays. For the βcatS33A construct, cells expressing GFP were sorted to generate a line with stable βcatS33A expression. Cell sorting was performed on Beckman-Coulter ALTRA (Beckman-Coulter, Brea, CA, USA) or Becton-Dickinson ARIA cell sorters (Becton-Dickinson, Franklin Lakes, NJ, USA).

Proliferation and differentiation assays

Growth was assessed using either CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA), or trypan blue exclusion. The proportion of viable cells was determined by trypan blue exclusion. To assess differentiation, cytospin preparations were made, and slides stained with May-Grünwald Giemsa. 200 cells were scored microscopically for morphology. In addition, cell surface staining was performed using a rat anti-mouse cfms-PE conjugate and isotype control, IgG2a-PE, from eBiosciences (San Diego, CA, USA) and analyzed by flow cytometry using a FC500 or XL-MS analyser (Beckman-Coulter, Brea, CA, USA).

Western immunoblot analysis and antibodies

Cells were lysed in modified RIPA lysis buffer, separated by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis, and transferred to nitrocellulose. Proteins of interest were detected by use of the appropriate antibodies and SuperSignal West Pico, West Dura or West Femto substrates (Pierce Biotechnology, Thermo Scientific, Rockford, IL, USA). Mouse monoclonal anti-β-catenin was purchased from BD Biosciences (Becton-Dickinson, Franklin Lakes, NJ, USA). Rabbit polyclonal p38MAP Kinase and rabbit monoclonal GSK3β antibody, were purchased from Cell Signaling Technology (Beverly, MA, USA). Secondary immunoglobulin G (IgG) antibodies were obtained from Pierce Biotechnology (Thermo Scientific, Rockford, IL, USA).

Online Supplemental Material

Supplemental Table 1 includes sequences for oligonucleotides used for PCR and cloning. Supplemental Table 2 includes full lists of genes differentially regulated in macrophage and granulocyte lineages by Y577. Supplemental Table 3 includes the full list of β-catenin/Tcf4 target genes differentially regulated by Y577. Supplemental Fig. 1 contains mutant hβc expression and differentiation information for the cells used in the microarray analysis. Supplemental Fig. 2 contains microarray experimental design and volcano plots of genes differentially regulated by Y577. Supplemental Fig. 3 shows the gene set enrichment plot for β-catenin/Tcf4 target genes in FIΔ-Y577F regulated genes.

Results

A single tyrosine residue in GMR is required for granulocyte differentiation: a model of GM-CSF-induced lineage specification

When cultured in GM-CSF, FDB1 cells form granulocytes and macrophages in approximately equal proportions and this can be mimicked in the absence of growth factor by ectopic expression of the hβc FIΔ activated mutant (McCormack and Gonda, 2000). To characterise the signaling pathways and downstream genes supporting granulocyte and macrophage differentiation induced by GMR we performed comparative gene expression profiling of the differentiation response for FDB1 cells expressing the FIΔ mutant and its derivative FIΔY577F, which supports differentiation predominantly to macrophages (Brown et al., 2004). Cultures of FDB1 cells expressing the parental FIΔ mutant or the Y577F derivative were cultured in the absence of growth factor for 72 hours and assessed for the expected morphological differentiation prior to RNA extraction (supplemental Fig. 1). Microarray hybridisation and processing was carried out on the Compugen Mouse Oligo Library (v2, 21,997 65mers comprising 21,587 unique genes) as previously described (Brown et al., 2006). The differential gene expression in each population was determined over this time period and gene expression changes associated with mutation of Tyr577 were determined by linear modelling (supplemental Fig. 2A) (Brown et al., 2006). Gene expression changes were defined as Tyr577-associated and involved in promoting granulocyte differentiation if the change in expression was selectively lost in cells responding to the FIΔY577 mutant. This was determined by analysing the gene expression data to include only those genes with a significant change over time in FIΔ expressing cells but not FIΔY577F cells (supplemental Fig. 2B).

Conversely, macrophage associated genes were defined as those suppressed by Tyr577 signaling, and these were identified by selecting only those genes with a significantly larger change over time in FIΔY577F cells compared to FIΔ cells (supplemental Fig. 2B). Lists of differentially-expressed genes were generated using an FDR p-value of 0.001 and a fold change cut-off of 1.4. Using this approach we identified 153 up-regulated and 83 down-regulated genes associated specifically with intact Tyr577 signaling and differentiation of the granulocyte lineage and 166 up-regulated and 55 down-regulated genes associated with enhanced differentiation of the macrophage lineage in the absence of Tyr577 signaling (supplemental Table 2). The top 25 up and down regulated genes associated specifically with the granulocyte and macrophage lineages are shown in Fig. 1A and C respectively.

Fig. 1. Y577 regulated genes.

Fig. 1

Open in a new tab

A. Heatmap showing the expression of the top 25 up and down regulated genes associated FIΔ induced factor independent granulocyte differentiation over 72 hours (see materials and methods for details of gene-set derivations). B. IPA defined canonical pathways significantly associated with the neutrophil gene set Lysine= lysine degradation, LES= leukocyte extravasation signaling, CES= caveolar-mediated endocytosis signaling. C. Heatmap showing the expression of the top 25 up and down regulated genes associated with FIΔ-Y577F induced factor independent macrophage differentiation over 72 hours, * marks Tcf4. D. IPA defined canonical pathways associated with the macrophage gene set, AP= acute phase response signaling, RA= rheumatoid arthritis. The M value (log2 fold change) for both FIΔ or FIΔY577F expressing cells was converted to a colour scale with red indicating that gene expression increases between 0 and 72 hours and green indicating that the gene expression decreases with the colour intensity indicating the magnitude of regulation (range log2, −3 to +3). Black boxes indicate individual genes involved in the indicated pathway.

Consistent with the loss of neutrophil differentiation associated with the Y577 second-site mutant we identified differential expression of many known neutrophil genes amongst the Tyr577-regulated genes. These included granule proteins such as Ltf, Ngp, Mmp8, Itgb2l, Camp, Prtn3, Lcn2, Ceacam1, S100a8. Many of these genes are also targets of the C/EBP family of transcription factors several members of which, including C/EBPα and C/EBPε, are important regulators of this lineage. Indeed, using gene-set enrichment analysis we identified a significant association of these genes with the C/EBP target gene-set generated by Gery et al (Gery et al., 2005) (p=0.00023). Ingenuity pathway analysis (IPA) of the full neutrophil associated gene list identified significantly associated canonical pathways. These revealed a network of down-regulated genes involved in IL-4 signaling (and T and B cell differentiation and function) consistent with suppression of some alternative lineages, and up-regulation of genes associated with leukocyte function (Fig. 1B).

As predicted, the genes that show enhanced up-regulation over time in the presence of the Y577F mutation were strongly associated with macrophage differentiation. These included genes involved in macrophage function, such as proteins normally found in the lysosomal compartment of macrophages (e.g. Ctss, Psap, Mmp12, Asahl) or cell surface proteins involved in the innate immune response (e.g. Clecsf12, Clecsf10, Trem2, Ccr2, Itgax). Of particular interest are known key regulators of macrophage differentiation such as CSF1 receptor and ligand, PKCδ and the transcription factors Egr1, Fos and C/EBPβ all of which displayed large increases in expression selectively in the Y577F mutant over the 72 hour time course (Fig. 1C–D). IPA canonical pathway analysis identified pathways associated with rheumatoid arthritis, a disease with known GM-CSF induced macrophage involvement (Hamilton, 2008) (Fig. 1D). In addition, the macrophage associated gene list also contained genes significantly associated with pathways of PPAR, IL-6, p38MAPK, GM-CSF and IL-3 signaling, that were not identified with the neutrophil associated gene list (Fig. 1B). Thus gene expression analysis using this FDB1 differentiation model is consistent with signaling from Tyr577 promoting a granulocyte differentiation program while suppressing key inducers of macrophage differentiation.

Tcf4 is a repressed target of Tyr577 signaling and a promoter of macrophage differentiation

Given that key regulators of macrophage differentiation are significantly up-regulated over time in cells expressing the FIΔY577 mutant we next used this characteristic pattern to identify additional potential regulators of macrophage differentiation. By microarray the transcription factor Tcf4 showed significantly enhanced up-regulation in cells expressing the Y577F second-site mutant of FIΔ (FDR p=1.8×10−11, Fig. 1C). A comparison of the change in expression for Tcf4 and other macrophage lineage transcription factors (TFs) (Egr1, Fos and C/EBPβ) in FIΔ and FIΔY577F expressing cells was determined by quantitative RT-PCR (Q-PCR) and is shown in Fig. 2A. The expression changes of Tcf4, Egr1, Fos and Cebpb were consistent with the results obtained by microarray (Fig. 2A). Expression of these genes also increases during GM-CSF-directed bi-lineage terminal differentiation of FDB1 cells (which proceeds more slowly than FIΔ-induced GM differentiation) over 120 hours (Fig. 2B). By QPCR Tcf4 mRNA expression increased from 1.8 fold at 72 hours in FIΔ cells to 7 fold in FIΔY577F cells and by 3.8 fold in GM-CSF-treated parental FDB1 cells at 72 hours (Fig. 2 A,B). This is consistent with Tcf4 being a repressed target of the Y577 signaling pathway with a potential role in promoting macrophage differentiation and/or suppressing granulocyte differentiation. Tcf4 is itself a target gene activated by the Tcf4/β-catenin complex (Hatzis et al., 2008) and up-regulation of Tcf4 expression is likely to be an indication of Wnt pathway activation. Further support for this was provided from IPA pathway analysis which identified an association between the genes associated with Tyr577 signaling and the Wnt/β-catenin pathway, in particular up-regulation of Tcf4, as well as down-regulation of the Wnt antagonist Dkkl1 (Fig. 1D).

Fig. 2. Tcf4 is regulated by GM-CSF and promotes macrophage differentiation.

Fig. 2

Open in a new tab

Quantitative real-time PCR was used to determine the expression of the indicated genes. Expression was normalised to β-actin and expression is shown relative to 0 hours. Results shown are the average of two experiments A. FDB1 cells expressing FIΔ and FIΔY577F were cultured without factor and RNA extracted at 0, 24 and 72 hours. B. Parental FDB1 cells were cultured in GM-CSF and RNA extracted at 0, 24, 72 and 120 hours. C. Full length TCF4 and the engineered form lacking the N-terminal β-catenin interaction domain are depicted. The β-catenin interaction domain is indicated by a black box and the DNA binding domain is indicated by a grey box. D. FDB1 parental cells were transduced with a MSCV-IRES-GFP (MIG) retrovirus encoding TCF4, ΔN-TCF4 or a control retrovirus and selected for GFP expression by FACS. Cells were washed and placed in the indicated growth conditions. Expansion of GFP positive cells was determined by assessing viable cell number using trypan blue exclusion and flow cytometry to determine the proportion of GFP positive cells at the indicated time points. E. At day 5, cells were cytocentrifuged, Wright-Giemsa stained and 200 cells for each condition were scored microscopically for morphology. F. At day 5 cells were stained with PE-conjugated anti-c-fms and the percentage of c-fms positive cells in the GFP positive population was determined for each cell line. A representative flow histogram is shown. Error bars represent SEM (n=4) and *=p<0.05, **=<0.01, ***=p<0.001 (unpaired, two sided t-test).

Tcf4 is a DNA-binding cofactor of β-catenin and a role for Tcf4 has not previously been reported in promoting myeloid differentiation. To further investigate the possibility that β-catenin/TCF4 is involved in promoting macrophage differentiation in FDB1 cells we ectopically expressed full length Tcf4 and Tcf4 lacking the amino terminal β-catenin interaction domain (ΔN-Tcf4; Fig. 2C) using the MSCV-IRES-GFP (MIG) vector (Fig. 2D–F). FDB1 cells were transduced with MIG-Tcf4, MIG-ΔN-Tcf4 or MIG vector control and infected cells (GFP+) enriched by flow cytometry. After sorting, cells were seeded into medium containing IL-3 (for growth) or GM-CSF (GM differentiation) and growth, viability and differentiation were measured over a 5 day period. Expression of Tcf4 resulted in significantly reduced growth over 5 days in both IL-3 and GM-CSF (Fig. 2D) which was also associated with significantly reduced viability (as measured by trypan blue exclusion) at day 5 in IL-3, but not GM-CSF (data not shown). Interestingly, while ΔN-Tcf4 was also able to affect the growth of cells in IL-3, there was no effect on cell expansion in response to GM-CSF (Fig. 2D) indicating that the latter effect is dependent on the β-catenin interaction domain. The Tcf4 and ΔN-Tcf4 growth suppression in IL-3 was associated with a significantly decreased number of blasts and an increase in the number of granulocytes, although the majority of cells in IL-3 in all cases were still of undifferentiated morphology (Fig. 2E). Importantly, under conditions of GM-CSF-induced differentiation, we found that ectopic Tcf4 expression significantly increased the number of cells differentiating to macrophages, a response that was not associated with expression of the ΔN-Tcf4 mutant (Fig. 2E). This enhanced macrophage differentiation was also associated with increased cell surface expression of the macrophage marker, c-fms (Fig. 2F). These results show that Tcf4 has the capacity, most likely via an interaction with β-catenin, to promote differentiation of the macrophage lineage.

GM-CSF receptor activation induces β-catenin stabilization that is negatively regulated by the Tyr577 pathway

We next determined the levels of β-catenin protein present in FDB1 cells expressing the FIΔ mutant or the Y577F derivative, or responding to GM-CSF over 72 hours. In cells responding to GM-CSF or expressing FIΔ we observed accumulation of β-catenin at 24 hours following IL-3 withdrawal, which was absent at 72 hours (Fig. 3A). In contrast, in cells expressing FIΔY577F, which differentiate predominantly to macrophages, β-catenin was constitutively present (Fig. 3A). This is consistent with mutation of Tyr577 disrupting a pathway that normally regulates the stability of β-catenin. To further examine the pathway regulating β-catenin stabilization we treated FDB1 cells with the Glycogen synthase kinase 3β (GSK3β) inhibitor BIO. GSK3β is a well-recognised negative regulator of β-catenin stability and one of the characterised effects of inhibition of GSK3β activity is constitutive β-catenin protein stabilisation (Liu et al., 2002; Holmes et al., 2008b). Parental FDB1 cells were treated with 2 µM BIO or the control N-methylated analogue (Me-BIO). As predicted, inhibition of GSK3β with BIO led to increased β-catenin protein levels measured at 16 hours (Fig. 3B). BIO treatment did not significantly affect viability of the cells, as measured by trypan blue exclusion (data not shown) but resulted in a significant suppression of growth in response to both IL-3 and GM-CSF (Fig. 3C). In response to GM-CSF, there was a significant increase in the number of macrophages with concomitant decreases in both blasts and granulocytes (Fig. 3D). This was also associated with a clear increase in the number of cells expressing c-fms (Fig. 3E). We also observed increased levels of macrophages in response to IL-3 in the presence of BIO although this increase was not significant.

Fig. 3. β-catenin stabilization is associated with increased macrophage differentiation.

Fig. 3

Open in a new tab

A. FDB1 cells expressing FIΔ and FIΔY577F were cultured without factor and parental cells in GM-CSF and cell lysates were made at 0, 24 and 72 hours. Lysates were western blotted with the indicated antibodies. Representative photo-micrographs were taken at the 72 hour timepoint for FDB1 FIΔ and FIΔY577F cells and at 120 hours for parental cells in GM-CSF. B. FDB1 cells growing in response to IL-3 or undergoing GM differentiation in response to GM-CSF were incubated with 2 µM of the GSK3β inhibitor BIO ((2’Z,3’E)-6-Bromoindirubin-3’-oxime) or N-methylated control analogue (Me-BIO). Activity of BIO at 16 hours was assessed by detection of β-catenin protein by western analysis. C. Cell growth was determined at days 2 and 5 of GSK3β inhibition by trypan blue exclusion. D. At day 5 cells were cytocentrifuged, Wright-Giemsa stained and 200 cells for each condition were scored microscopically for morphology. E. At day 5 cells were stained with PE-conjugated anti-c-fms and the percentage of c-fms positive cells was determined for each cell population. Error bars represent SEM (n=3) and *=p<0.05, **=<0.01, ***=p<0.001 (unpaired, two sided t-test).

β-catenin stabilization is sufficient to specify macrophage differentiation in FDB1 cells

We next tested the ability of β-catenin to directly mediate macrophage differentiation by ectopically expressing a stabilized form of β-catenin. For this we used the MIG vector to express an engineered version of β-catenin containing point mutations at the GSK3β and casein kinase phosphorylation sites (Ser33Ala, Ser37Ala, Thr41Ala, Ser45Ala) (Liu et al., 2002; Rottbauer et al., 2002). Expression of this β-catenin mutant (herein referred to as βcatS33A) has previously been shown to render the β-catenin protein resistant to degradation and consequently mimic a constitutive Wnt signal (Aoki et al., 2002; Rottbauer et al., 2002). FDB1 cells were transduced with MIG-βcatS33A or MIG vector control and infected cells (GFP+) enriched by flow cytometry. After sorting, cells were seeded into medium containing IL-3 or GM-CSF and growth, viability and differentiation were measured over a 5 day period. Western immunoblot analysis at day 5 confirmed the increase of β-catenin protein in cells transduced with MIG-βcatS33A, in both IL-3 and GM-CSF (Fig 4A). Over-expression of βcatS33A did not affect growth of FDB1 cells in IL-3, however in the presence of GM-CSF we observed significantly enhanced growth of the MIG-βcatS33A FDB1 cell population (Fig. 4B). There was no difference in the viability of the cell populations (as measured by trypan blue exclusion) over the 5 day period (data not shown). Importantly, after 5 days in GM-CSF, differentiation of the MIG-βcatS33A cell population was associated with a significantly increased proportion of macrophages (p=0.027) compared to vector-control cells (Fig. 4C–D). While having a significant effect on differentiation in GM-CSF, βcatS33A was not sufficient to induce differentiation to macrophages in the presence of IL-3 (Fig. 4C–D). Taken together with the BIO-induction of differentiation this demonstrates that, in the context of GM-CSF signaling, stabilization of β-catenin promotes specification of macrophage differentiation in this FDB1 model system.

Fig. 4. β-catenin directs macrophage differentiation in response to GM-CSF receptor signaling.

Fig. 4

Open in a new tab

FDB1 cells were transduced with MIG encoding β-catenin with mutations S33A, S37A, T41A, S45A (βcatS33A) or vector alone. A. GFP positive cells were placed in IL-3 or GM-CSF and after 5 days were assessed for expression of stabilised β-catenin by western analysis. B. Growth was assessed over 4 days using AQueous one solution proliferation assay. C. Photomicrographs of cytocentrifuged, Wright-Giemsa stained cells at day 5. D. At day 5 200 cells for each condition were scored by morphology. Error bars represent SEM (n=2) and *=p<0.05 (unpaired, two sided t-test).

The GSK3β/β-catenin signaling axis promotes macrophage differentiation of primary cells in response to GM-CSF

Having shown that Y577 of GMR is able to regulate granulocyte-macrophage differentiation ratios and β-catenin stability, most likely through modulation of GSK3β, we next determined whether this pathway operates in primary hematopoietic cells. To do this we freshly isolated mouse bone marrow cells and plated them in colony assays containing GM-CSF with increasing concentrations of BIO representing ranges used previously on primary hematopoietic cells and including the 2 µM concentration used on FDB1 cells in Fig. 3 (Holmes et al., 2008a). While overall colony numbers did not increase significantly when cells were plated in increasing concentrations of BIO (data not shown), we observed a significant dose dependent increase in the proportion of macrophage colonies formed (41% in 2 µm Me-BIO versus 68% in 2 µm BIO, p<0.01) with a corresponding decrease in the proportion of granulocyte colonies formed (52% in 2 µm Me-BIO versus 26% in 2 µm BIO, p<0.01; Fig. 5A). Mixed GM colonies were less frequent in all conditions and were not affected by treatment with BIO (Fig. 5A). This data recapitulates the results obtained in FDB1 cells (Fig. 3) and shows that this pathway operates in primary hematopoietic cells to influence macrophage differentiation.

Tcf4/β-catenin target genes are enriched in Tyr577-regulated genes

To further investigate the link between Tyr577 signaling, Tcf4/β-catenin activity and macrophage differentiation we investigated whether other known β-catenin/Tcf4 target genes, are regulated in response to Tyr577 signaling. For this we used gene-set enrichment analysis to compare the full set of Y577-differentially regulated genes with a combined gene-set of direct TCF4 and β-catenin target genes (347 genes) derived from two chromatin immunoprecipitation (ChIP) studies in colorectal cancer cell lines (Hatzis et al., 2008; Yochum et al., 2007). This analysis identified a significant association between the 347 β-catenin/Tcf4 target genes and the genes significantly differentially regulated over time by FIΔY577F (p=1.7×10−11, Wilcoxon rank-sum test, supplemental Fig. 3). The full list of significantly differentially expressed β-catenin/TCF4 targets (113 genes) is shown in supplemental Table 2. This analysis showed a clear association of the Tyr577-regulated genes with β-catenin and Tcf4 direct target genes. Egr1 is the β-catenin/Tcf4 direct-target gene with the largest-fold differential expression in FIΔY577F expressing cells over time suggesting a key macrophage promoting role for this lineage-specific TF downstream of the Tyr577 signaling pathway. Differential regulation of Egr1 in the FIΔY577F mutant was also validated by QPCR (Fig. 2A). As shown in Fig. 5 Egr1, although not specified by IPA as a Wnt/β-catenin signaling-related gene, is an identified ChIP target of the β-catenin/TCF4 complex (Yochum et al., 2007) and is independently connected to multiple β-catenin/Tcf4 targets, consistent with a model of macrophage differentiation that involves Wnt/β-catenin modulation of Egr1 activity (Fig. 6).

Fig. 6. Novel molecular interactions in monopoiesis regulated by GM-CSF signaling.

Fig. 6

Open in a new tab

Signaling emanating from Tyr577 of the GMR leads to stabilisation of the β-catenin protein (possibly though GSK3β inactivation) which, in combination with Tcf4, leads to differentiation of the macrophage lineage at the expense of neutrophil lineage.

Discussion

While genetic and functional studies provide clear evidence for the importance of GM-CSF signaling in macrophage differentiation, the pathways that contribute to this process are still incompletely defined. Here we define a novel signaling pathway from the GM-CSF receptor that we propose contributes to the lineage fate of bi-potential GM progenitors. The key intracellular Tyr577 residue in the common β subunit of the GM-CSF receptor has been shown previously to be a determinant of the extent of granulocyte and macrophage differentiation in the FDB1 cell line model (Brown et al., 2004). An in vivo mutant model of Tyr577 signaling also showed expansion of progenitor cells in response to GM-CSF consistent with a defect in GM progenitor differentiation (Ramshaw et al., 2007). Our analysis comparing gene expression in the activated (FIΔ) mutant of GMR, that mediates factor-independent macrophage and granulocyte differentiation, with that of the second-site mutant FIΔY577F, is consistent with the observed reduction in morphological granulocyte differentiation upon mutation of Tyr577 (Brown et al., 2004). Here we focused on Tcf4 which displayed similar expression to other regulators of macrophage differentiation in the FDB1 system (Fig. 2). Tcf4 is a member of the Tcf/Lef family that combines with β-catenin to form transcriptional complexes that regulate target gene transcription. The Tyr577 mutation results in increased Tcf4 expression and is associated with reduced granulocyte differentiation and enhanced differentiation to the macrophage lineage. Our data are most consistent with Tcf4 functioning as a promoter of differentiation and tumour suppressor in the GM lineage. Forced expression of Tcf4 in FDB1 cells responding to GM-CSF demonstrated a functional role in promoting macrophage differentiation. This effect was dependent on the β-catenin interaction domain, consistent with the known interaction and cooperation between Tcf4 and β-catenin.

Activation of Tcf4 transcription downstream of GMR is most likely a result of β-catenin accumulation as Tcf4 is itself a target gene of the Tcf4/β-catenin transcriptional complex (Hatzis et al., 2008). We found that β-catenin accumulated in the presence of the FIΔY577F mutant. We also observed transient accumulation of β-catenin at the 24 hour timepoint following GM-CSF stimulation of FDB1 cells (Fig. 3A) and expression of a stabilised β-catenin or inhibition of GSK3β with pharmacological inhibitors mimicked Tcf4 over-expression and increased the number of cells differentiating to macrophages. Thus three independent approaches demonstrate that β-catenin/Tcf4 activity promotes macrophage differentiation in the FDB1 system. The ability of BIO to stabilize β-catenin downstream of GM-CSF signalling suggests that, as in other systems, GSK3β is key to regulating β-catenin protein levels and therefore activation of this pathway in response to GM-CSF. Modification of GSK3β signalling by the GMR has not been previously investigated and although not directly shown here, the accumulation of β-catenin in cells expressing the FIΔY577F mutant suggests that this residue may normally have a role in regulating GSK3β activity in response to GM-CSF. Previous studies have linked Tyr577 to binding of Shc, and activation and regulation of Gab2, SHP2, Akt/PI3K and SHIP related pathways (Gu et al., 2000; Dijkers et al., 1999; Ramshaw et al., 2007). Interestingly, mutation of this residue in different contexts has been associated with both reduced and enhanced activation of Akt (Dijkers et al., 1999; Ramshaw et al., 2007). Preliminary analysis of signalling from FIΔ and FIΔY577F in FDB1 cells suggests alterations in activation of both Akt and p38MAPK (data not shown) both of which are known regulators of the GSK3β/β-catenin axis (Liu et al., 2002; Cross et al., 1995; Thornton et al., 2008; Bikkavilli et al., 2008). Recent data has also shed light on the role of the protein phosphatase, SHP2, in myeloid differentiation. Loss of SHP2 activation leads to a reduction in granulocyte colonies and an increase in macrophage colonies in response to a myeloid differention cytokine cocktail, in part due to reduced cebpa expression (Zhang and Friedman, 2011). As Y577 of GMR mediates activation of SHP2 (Gu et al., 2000) this may represent a mechanism for our observed loss of granulocyte differentiation in the FIΔY577F mutant cells. Interestingly, loss of SHP2 has been reported to increase tyrosine phosphorylation of β-catenin (Grinnell et al., 2010) which in some contexts leads to its nuclear localisation and subsequent activation of Wnt target genes (Kajiguchi et al., 2007). Therefore modulation of SHP2 activation via recruitment to Y577 of the GMR hβc may determine the balance of macrophage and granulocyte differentiation through down-stream regulation of cebpa and β-catenin. Further mapping of this pathway downstream of the GM-CSF receptor in this cell line model will reveal the signalling events important for macrophage differentiation and modulation of β-catenin activity, and shed light on the interplay between growth factor receptor signalling and transcriptional regulation in myeloid differentiation.

While a role for β-catenin in stem cell function and progenitor commitment has been reported previously (Zhao et al., 2007), our observation of a differentiation-promoting and lineage modulation capacity represents a novel function for Tcf4/β-catenin in the hematopoietic system. Activation of β-catenin in response to M-CSF signaling supports the growth and survival of macrophages, consistent with our findings, although lineage specification was not examined in this context (Otero et al., 2009). Interestingly we also note that conditional activation of β-catenin in the hematopoietic system acts to reduce the number of GM progenitors, and colonies formed in response to GM-CSF overall, and blocks granulocyte colony formation completely, while maintaining permissive conditions for some macrophage colony formation (Scheller et al., 2006). This is also consistent with our manipulation of the β-catenin/GSK3β axis by treating primary myeloid progenitors with BIO. Inhibition of GSK3β shifted the differentiation of myeloid progenitors from granulocytes to macrophages, an observation that has not been made in the context of other in vitro studies using BIO as these focused on the stem cell compartment (Huang et al., 2009; Holmes et al., 2008a). These data support our hypothesis that the β-catenin/GSK3β signalling axis may have independent roles at different stages of haemopoiesis, including a role in committed progenitors for determining macrophage versus granulocyte differentiation decisions.

To shed light on the possible mechanism of β-catenin/Tcf4 mediated macrophage differentiation we analysed the Tyr577-regulated genes and showed a significant enrichment of β-catenin/TCF4 target genes. Of particular note was the known transcriptional mediator of macrophage differentiation, Egr1 which displayed an 8-fold increase in expression over 72 hours during FIΔY577F-induced macrophage differentiation and was the most significant differentially regulated macrophage-associated gene. In primary myeloid progenitors Egr1 is controlled by the macrophage master regulator PU.1 and induction of Egr1 promotes the transcription of macrophage genes, while repressing the alternative granulocyte lineage associated genes (Laslo et al., 2006). This provides a likely mechanism by which β-catenin/Tcf4 could influence macrophage differentiation. Recent reports have shown that Egr1 directly regulates Tcf4 in endometrial carcinoma cells and link Egr1 expression to progression of colorectal cancer, a process which also involves β-catenin/Tcf4 (Saegusa et al., 2008; Ernst et al.), suggesting the possibility of a positive feedback loop. Interestingly like β-catenin, Egr1 is also important for hematopoietic stem cell homeostasis (Min et al., 2008; Zhao et al., 2007) suggesting that this regulatory loop may operate with different outputs at the level of the HSC and the committed myeloid progenitor. Further dissection of these roles will require stage or lineage specific deletion approaches to separate stem cell effects from those important for later cell fate decisions and will shed light on their role in GM lineage choice.

Supplementary Material

01
02

Acknowledgements

The authors would like to acknowledge the Adelaide Microarray Centre for their technical assistance with the microarray and Mrs Silvia Nobbs, Mr Sandy McIntyre and Ms Kate Pilkington for assistance with flow cytometry. This work was supported by the U.S. National Institutes of Health, National Health and Medical Research Council of Australia and the Cancer Council of South Australia. A.L.B was a fellow of the Cancer Council of South Australia.

Abbreviations

GM

granulocyte macrophage

GMR

GM-CSF receptor

DC

dendritic cell

IPA

ingenuity pathway analysis

BIO

(2’Z,3’E)-6-Bromoindirubin-3’-oxime

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Author Contributions

A.L.B. and R.J.D. wrote the manuscript. A.L.B. designed the experiments, performed the experiments and analyzed the data; D.G.S., T.S., G.E. and M.P. performed experiments and analysed the data; C.W. and C.H.K. analysed microarray data; S.S. and T.S. provided technical assistance and advice; I.L. and T.J.G were involved in data interpretation and critical review of the manuscript.

Conflicts of Interest

The authors declare no competing financial interests.

References

  1. Aoki M, Sobek V, Maslyar DJ, Hecht A, Vogt PK. Oncogenic transformation by beta-catenin: deletion analysis and characterization of selected target genes. Oncogene. 2002;21:6983–6991. doi: 10.1038/sj.onc.1205796. [DOI] [PubMed] [Google Scholar]
  2. Bikkavilli RK, Feigin ME, Malbon CC. p38 mitogen-activated protein kinase regulates canonical Wnt-beta-catenin signaling by inactivation of GSK3beta. J. Cell Sci. 2008;121:3598–3607. doi: 10.1242/jcs.032854. [DOI] [PubMed] [Google Scholar]
  3. Brown AL, Peters M, D'Andrea RJ, Gonda TJ. Constitutive mutants of the GM-CSF receptor reveal multiple pathways leading to myeloid cell survival, proliferation, and granulocyte-macrophage differentiation. Blood. 2004;103:507–516. doi: 10.1182/blood-2003-05-1435. [DOI] [PubMed] [Google Scholar]
  4. Brown AL, Wilkinson CR, Waterman SR, Kok CH, Salerno DG, Diakiw SM, Reynolds B, Scott HS, Tsykin A, Glonek GF, Goodall GJ, Solomon PJ, Gonda TJ, D'Andrea RJ. Genetic regulators of myelopoiesis and leukemic signaling identified by gene profiling and linear modeling. J.Leukoc.Biol. 2006;80:433–447. doi: 10.1189/jlb.0206112. [DOI] [PubMed] [Google Scholar]
  5. Conti L, Gessani S. GM-CSF in the generation of dendritic cells from human blood monocyte precursors: recent advances. Immunobiology. 2008;213:859–870. doi: 10.1016/j.imbio.2008.07.017. [DOI] [PubMed] [Google Scholar]
  6. Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature. 1995;378:785–789. doi: 10.1038/378785a0. [DOI] [PubMed] [Google Scholar]
  7. D'Andrea RJ, Harrison-Findik D, Butcher CM, Finnie J, Blumbergs P, Bartley P, McCormack M, Jones K, Rowland R, Gonda TJ, Vadas MA. Dysregulated hematopoiesis and a progressive neurological disorder induced by expression of an activated form of the human common beta chain in transgenic mice. J.Clin.Invest. 1998;102:1951–1960. doi: 10.1172/JCI3729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dijkers PF, van Dijk TB, de Groot RP, Raaijmakers JA, Lammers JW, Koenderman L, Coffer PJ. Regulation and function of protein kinase B and MAP kinase activation by the IL-5/GM-CSF/IL-3 receptor. Oncogene. 1999;18:3334–3342. doi: 10.1038/sj.onc.1202678. [DOI] [PubMed] [Google Scholar]
  9. Dranoff G, Crawford AD, Sadelain M, Ream B, Rashid A, Bronson RT, Dickersin GR, Bachurski CJ, Mark EL, Whitsett JA. Involvement of granulocyte-macrophage colony-stimulating factor in pulmonary homeostasis. Science. 1994;264:713–716. doi: 10.1126/science.8171324. [DOI] [PubMed] [Google Scholar]
  10. Enzler T, Gillessen S, Manis JP, Ferguson D, Fleming J, Alt FW, Mihm M, Dranoff G. Deficiencies of GM-CSF and interferon gamma link inflammation and cancer. J. Exp. Med. 2003;197:1213–1219. doi: 10.1084/jem.20021258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ernst A, Aigner M, Nakata S, Engel F, Schlotter M, Kloor M, Brand K, Schmitt S, Steinert G, Rahbari N, Koch M, Radlwimmer B, Weitz J, Lichter P. A gene signature distinguishing CD133hi from CD133- colorectal cancer cells: essential role for EGR1 and downstream factors. Pathology (Phila) 2011;43:220–227. doi: 10.1097/PAT.0b013e328344e391. [DOI] [PubMed] [Google Scholar]
  12. Gery S, Gombart AF, Yi WS, Koeffler C, Hofmann WK, Koeffler HP. Transcription profiling of C/EBP targets identifies Per2 as a gene implicated in myeloid leukemia. Blood. 2005;106:2827–2836. doi: 10.1182/blood-2005-01-0358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Grinnell KL, Casserly B, Harrington EO. Role of protein tyrosine phosphatase SHP2 in barrier function of pulmonary endothelium. Am J Physiol Lung Cell Mol Physiol. 2010;298:L361–L370. doi: 10.1152/ajplung.00374.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gu H, Maeda H, Moon JJ, Lord JD, Yoakim M, Nelson BH, Neel BG. New role for Shc in activation of the phosphatidylinositol 3-kinase/Akt pathway. Mol. Cell. Biol. 2000;20:7109–7120. doi: 10.1128/mcb.20.19.7109-7120.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Guthridge MA, Powell JA, Barry EF, Stomski FC, McClure BJ, Ramshaw H, Felquer FA, Dottore M, Thomas DT, To B, Begley CG, Lopez AF. Growth factor pleiotropy is controlled by a receptor Tyr/Ser motif that acts as a binary switch. EMBO J. 2006;25:479–489. doi: 10.1038/sj.emboj.7600948. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hamilton JA. Colony-stimulating factors in inflammation and autoimmunity. Nat Rev Immunol. 2008;8:533–544. doi: 10.1038/nri2356. [DOI] [PubMed] [Google Scholar]
  17. Hansen G, Hercus TR, McClure BJ, Stomski FC, Dottore M, Powell J, Ramshaw H, Woodcock JM, Xu Y, Guthridge M, McKinstry WJ, Lopez AF, Parker MW. The structure of the GM-CSF receptor complex reveals a distinct mode of cytokine receptor activation. Cell. 2008;134:496–507. doi: 10.1016/j.cell.2008.05.053. [DOI] [PubMed] [Google Scholar]
  18. Hatzis P, van der Flier LG, van Driel MA, Guryev V, Nielsen F, Denissov S, Nijman IJ, Koster J, Santo EE, Welboren W, Versteeg R, Cuppen E, van de Wetering M, Clevers H, Stunnenberg HG. Genome-wide pattern of TCF7L2/TCF4 chromatin occupancy in colorectal cancer cells. Mol. Cell. Biol. 2008;28:2732–2744. doi: 10.1128/MCB.02175-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Holmes T, O'Brien TA, Knight R, Lindeman R, Shen S, Song E, Symonds G, Dolnikov A. Glycogen synthase kinase-3beta inhibition preserves hematopoietic stem cell activity and inhibits leukemic cell growth. Stem Cells. 2008a;26:1288–1297. doi: 10.1634/stemcells.2007-0600. [DOI] [PubMed] [Google Scholar]
  20. Holmes T, O'Brien TA, Knight R, Lindeman R, Symonds G, Dolnikov A. The role of glycogen synthase kinase-3beta in normal haematopoiesis, angiogenesis and leukaemia. Curr. Med. Chem. 2008b;15:1493–1499. doi: 10.2174/092986708784638834. [DOI] [PubMed] [Google Scholar]
  21. Huang J, Zhang Y, Bersenev A, O'Brien WT, Tong W, Emerson SG, Klein PS. Pivotal role for glycogen synthase kinase-3 in hematopoietic stem cell homeostasis in mice. J. Clin. Invest. 2009;119:3519–3529. doi: 10.1172/JCI40572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jenkins BJ, Blake TJ, Gonda TJ. Saturation mutagenesis of the beta subunit of the human granulocyte-macrophage colony-stimulating factor receptor shows clustering of constitutive mutations, activation of ERK MAP kinase and STAT pathways, and differential beta subunit tyrosine phosphorylation. Blood. 1998;92:1989–2002. [PubMed] [Google Scholar]
  23. Jenkins BJ, D'Andrea R, Gonda TJ. Activating point mutations in the common beta subunit of the human GM-CSF, IL-3 and IL-5 receptors suggest the involvement of beta subunit dimerization and cell type-specific molecules in signalling. EMBO J. 1995;14:4276–4287. doi: 10.1002/j.1460-2075.1995.tb00102.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kajiguchi T, Chung EJ, Lee S, Stine A, Kiyoi H, Naoe T, Levis MJ, Neckers L, Trepel JB. FLT3 regulates beta-catenin tyrosine phosphorylation, nuclear localization, and transcriptional activity in acute myeloid leukemia cells. Leukemia. 2007;21:2476–2484. doi: 10.1038/sj.leu.2404923. [DOI] [PubMed] [Google Scholar]
  25. Kondo M, Scherer DC, Miyamoto T, King AG, Akashi K, Sugamura K, Weissman IL. Cell-fate conversion of lymphoid-committed progenitors by instructive actions of cytokines. Nature. 2000;407:383–386. doi: 10.1038/35030112. [DOI] [PubMed] [Google Scholar]
  26. Laslo P, Spooner CJ, Warmflash A, Lancki DW, Lee HJ, Sciammas R, Gantner BN, Dinner AR, Singh H. Multilineage transcriptional priming and determination of alternate hematopoietic cell fates. Cell. 2006;126:755–766. doi: 10.1016/j.cell.2006.06.052. [DOI] [PubMed] [Google Scholar]
  27. Liu C, Li Y, Semenov M, Han C, Baeg GH, Tan Y, Zhang Z, Lin X, He X. Control of beta-catenin phosphorylation/degradation by a dual-kinase mechanism. Cell. 2002;108:837–847. doi: 10.1016/s0092-8674(02)00685-2. [DOI] [PubMed] [Google Scholar]
  28. Lopez AF, Hercus TR, Ekert P, Littler DR, Guthridge M, Thomas D, Ramshaw HS, Stomski F, Perugini M, D'Andrea R, Grimbaldeston M, Parker MW. Molecular basis of cytokine receptor activation. IUBMB Life. 2010;62:509–518. doi: 10.1002/iub.350. [DOI] [PubMed] [Google Scholar]
  29. Martinez-Moczygemba M, Doan ML, Elidemir O, Fan LL, Cheung SW, Lei JT, Moore JP, Tavana G, Lewis LR, Zhu Y, Muzny DM, Gibbs RA, Huston DP. Pulmonary alveolar proteinosis caused by deletion of the GM-CSFRalpha gene in the X chromosome pseudoautosomal region 1. J. Exp. Med. 2008;205:2711–2716. doi: 10.1084/jem.20080759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. McClure BJ, Hercus TR, Cambareri BA, Woodcock JM, Bagley CJ, Howlett GJ, Lopez AF. Molecular assembly of the ternary granulocyte-macrophage colony- stimulating factor receptor complex. Blood. 2003;101:1308–1315. doi: 10.1182/blood-2002-06-1903. [DOI] [PubMed] [Google Scholar]
  31. McClure BJ, Woodcock JM, Harrison-Findik D, Lopez AF, D'Andrea RJ. GM-CSF binding to its receptor induces oligomerisation of the common beta-subunit. Cytokine. 2001;13:240–243. doi: 10.1006/cyto.2000.0826. [DOI] [PubMed] [Google Scholar]
  32. McCormack MP, Gonda TJ. Myeloproliferative disorder and leukaemia induced in mice by different classes of constitutive mutants of the human IL-3/IL-5/GM-CSF receptor common beta subunit. Oncogene. 1999;18:7190–7199. doi: 10.1038/sj.onc.1203226. [DOI] [PubMed] [Google Scholar]
  33. McCormack MP, Gonda TJ. Novel murine myeloid cell lines which exhibit a differentiation switch in response to IL-3 or GM-CSF, or to different constitutively active mutants of the GM-CSF receptor beta subunit. Blood. 2000;95:120–127. [PubMed] [Google Scholar]
  34. Metcalf D, Begley CG, Williamson DJ, Nice EC, De Lamarter J, Mermod JJ, Thatcher D, Schmidt A. Hemopoietic responses in mice injected with purified recombinant murine GM-CSF. Exp. Hematol. 1987;15:1–10. [PubMed] [Google Scholar]
  35. Min IM, Pietramaggiori G, Kim FS, Passegue E, Stevenson KE, Wagers AJ. The transcription factor EGR1 controls both the proliferation and localization of hematopoietic stem cells. Cell Stem Cell. 2008;2:380–391. doi: 10.1016/j.stem.2008.01.015. [DOI] [PubMed] [Google Scholar]
  36. Miyajima A, Mui AL, Ogorochi T, Sakamaki K. Receptors for granulocyte-macrophage colony-stimulating factor, interleukin-3, and interleukin-5. Blood. 1993;82:1960–1974. [PubMed] [Google Scholar]
  37. Nishinakamura R, Nakayama N, Hirabayashi Y, Inoue T, Aud D, McNeil T, Azuma S, Yoshida S, Toyoda Y, Arai K, et al. Mice deficient for the IL-3/GM-CSF/IL-5 beta c receptor exhibit lung pathology and impaired immune response, while beta IL3 receptor-deficient mice are normal. Immunity. 1995;2:211–222. doi: 10.1016/1074-7613(95)90046-2. [DOI] [PubMed] [Google Scholar]
  38. Okuda K, Smith L, Griffin JD, Foster R. Signaling functions of the tyrosine residues in the beta chain of the granulocyte-macrophage colony-stimulating factor receptor. Blood. 1997;90:4759–4766. [PubMed] [Google Scholar]
  39. Otero K, Turnbull IR, Poliani PL, Vermi W, Cerutti E, Aoshi T, Tassi I, Takai T, Stanley SL, Miller M, Shaw AS, Colonna M. Macrophage colony-stimulating factor induces the proliferation and survival of macrophages via a pathway involving DAP12 and beta-catenin. Nat Immunol. 2009;10:734–743. doi: 10.1038/ni.1744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Paine R, III, Preston AM, Wilcoxen S, Jin H, Siu BB, Morris SB, Reed JA, Ross G, Whitsett JA, Beck JM. Granulocyte-macrophage colony-stimulating factor in the innate immune response to Pneumocystis carinii pneumonia in mice. J. Immunol. 2000;164:2602–2609. doi: 10.4049/jimmunol.164.5.2602. [DOI] [PubMed] [Google Scholar]
  41. Perugini M, Brown AL, Salerno DG, Booker GW, Stojkoski C, Hercus TR, Lopez AF, Hibbs ML, Gonda TJ, D'Andrea RJ. Alternative modes of GM-CSF receptor activation revealed using activated mutants of the common beta-subunit. Blood. 115:3346–3353. doi: 10.1182/blood-2009-08-235846. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Perugini M, Kok CH, Brown AL, Wilkinson CR, Salerno DG, Young SM, Diakiw SM, Lewis ID, Gonda TJ, D'Andrea RJ. Repression of Gadd45alpha by activated FLT3 and GM-CSF receptor mutants contributes to growth, survival and blocked differentiation. Leukemia. 2009;23:729–738. doi: 10.1038/leu.2008.349. [DOI] [PubMed] [Google Scholar]
  43. Powell JA, Thomas D, Barry EF, Kok CH, McClure BJ, Tsykin A, To LB, Brown A, Lewis ID, Herbert K, Goodall GJ, Speed TP, Asou N, Jacob B, Osato M, Haylock DN, Nilsson SK, D'Andrea RJ, Lopez AF, Guthridge MA. Expression profiling of a hemopoietic cell survival transcriptome implicates osteopontin as a functional prognostic factor in AML. Blood. 2009;114:4859–4870. doi: 10.1182/blood-2009-02-204818. [DOI] [PubMed] [Google Scholar]
  44. Ramshaw HS, Guthridge MA, Stomski FC, Barry EF, Ooms L, Mitchell CA, Begley CG, Lopez AF. The Shc-binding site of the betac subunit of the GM-CSF/IL-3/IL-5 receptors is a negative regulator of hematopoiesis. Blood. 2007;110:3582–3590. doi: 10.1182/blood-2007-01-070391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Rottbauer W, Saurin AJ, Lickert H, Shen X, Burns CG, Wo ZG, Kemler R, Kingston R, Wu C, Fishman M. Reptin and pontin antagonistically regulate heart growth in zebrafish embryos. Cell. 2002;111:661–672. doi: 10.1016/s0092-8674(02)01112-1. [DOI] [PubMed] [Google Scholar]
  46. Saegusa M, Hashimura M, Kuwata T, Hamano M, Watanabe J, Kawaguchi M, Okayasu I. Transcription factor Egr1 acts as an upstream regulator of beta-catenin signalling through up-regulation of TCF4 and p300 expression during trans-differentiation of endometrial carcinoma cells. J. Pathol. 2008;216:521–532. doi: 10.1002/path.2404. [DOI] [PubMed] [Google Scholar]
  47. Scheller M, Huelsken J, Rosenbauer F, Taketo MM, Birchmeier W, Tenen DG, Leutz A. Hematopoietic stem cell and multilineage defects generated by constitutive beta-catenin activation. Nat Immunol. 2006;7:1037–1047. doi: 10.1038/ni1387. [DOI] [PubMed] [Google Scholar]
  48. Smith A, Metcalf D, Nicola NA. Cytoplasmic domains of the common á-chain of the GM-CSF/IL-3/IL-5 receptors that are required for inducing differentiation or clonal suppression in myeloid leukaemic cell lines. EMBO J. 1997;16:451–464. doi: 10.1093/emboj/16.3.451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Suzuki T, Sakagami T, Rubin BK, Nogee LM, Wood RE, Zimmerman SL, Smolarek T, Dishop MK, Wert SE, Whitsett JA, Grabowski G, Carey BC, Stevens C, van der Loo JC, Trapnell BC. Familial pulmonary alveolar proteinosis caused by mutations in CSF2RA. J. Exp. Med. 2008;205:2703–2710. doi: 10.1084/jem.20080990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Thornton TM, Pedraza-Alva G, Deng B, Wood CD, Aronshtam A, Clements JL, Sabio G, Davis RJ, Matthews DE, Doble B, Rincon M. Phosphorylation by p38 MAPK as an alternative pathway for GSK3beta inactivation. Science. 2008;320:667–670. doi: 10.1126/science.1156037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. van de WM, Sancho E, Verweij C, de Lau W, Oving I, Hurlstone A, van der HK, Batlle E, Coudreuse D, Haramis AP, Tjon-Pon-Fong M, Moerer P, van den BM, Soete G, Pals S, Eilers M, Medema R, Clevers H. The beta-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells. Cell. 2002;111:241–250. doi: 10.1016/s0092-8674(02)01014-0. [DOI] [PubMed] [Google Scholar]
  52. Yochum GS, McWeeney S, Rajaraman V, Cleland R, Peters S, Goodman RH. Serial analysis of chromatin occupancy identifies beta-catenin target genes in colorectal carcinoma cells. Proc. Natl. Acad. Sci. U. S. A. 2007;104:3324–3329. doi: 10.1073/pnas.0611576104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Zhang L, Friedman AD. SHP2 tyrosine phosphatase stimulates CEBPA gene expression to mediate cytokine-dependent granulopoiesis. Blood. 2011 doi: 10.1182/blood-2011-01-331157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Zhao C, Blum J, Chen A, Kwon HY, Jung SH, Cook JM, Lagoo A, Reya T. Loss of beta-catenin impairs the renewal of normal and CML stem cells in vivo. Cancer Cell. 2007;12:528–541. doi: 10.1016/j.ccr.2007.11.003. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01
NIHMS331631-supplement-01.doc (368KB, doc)
02
NIHMS331631-supplement-02.ppt (2.9MB, ppt)

RESOURCES