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HEMATOPOIESIS
From the Ludwig Institute for Cancer Research,
Melbourne Tumour Biology Branch, and the Walter and Eliza Hall
Institute of Medical Research, Parkville, Victoria, Australia.
Granulocyte colony-stimulating factor (G-CSF) is the major
regulator of granulopoiesis and acts through binding to its specific receptor (G-CSF-R) on neutrophilic granulocytes. Previous studies of
signaling from the 4 G-CSF-R cytoplasmic tyrosine residues used model
cell lines that may have idiosyncratic, nonphysiological responses.
This study aimed to identify specific signals transmitted by the
receptor tyrosine residues in primary myeloid cells. To bypass the
presence of endogenous G-CSF-R, a chimeric receptor containing the
extracellular domain of the epidermal growth factor receptor in place
of the entire extracellular domain of the G-CSF-R was used. A series of
chimeric receptors containing tyrosine mutations to phenylalanine,
either individually or collectively, was constructed and expressed in
primary bone marrow cells from G-CSF-deficient mice. Proliferation and
differentiation responses of receptor-expressing bone marrow cells
stimulated by epidermal growth factor were measured. An increased 50%
effective concentration to stimulus of the receptor Ynull
mutant indicated that specific signals from tyrosine residues were
required for cell proliferation, particularly at low concentrations of
stimulus. Impaired responses by mutant receptors implicated G-CSF-R
Y764 in cell proliferation and Y729 in
granulocyte differentiation signaling. In addition, different
sensitivities to ligand stimulation between mutant receptors indicated
that G-CSF-R Y744 and possibly Y729 have an
inhibitory role in cell proliferation. STAT activation was not affected
by tyrosine mutations, whereas ERK activation appeared to depend, at
least in part, on Y764. These observations have suggested
novel roles for the G-CSF-R tyrosine residues in primary cells that
were not observed previously in studies in cell lines.
(Blood. 2002;99:879-887) Granulocyte colony-stimulating factor (G-CSF) plays
a crucial role in proliferation, differentiation, and survival of the granulocytic lineage of myeloid cells exemplified by the neutropenia found in G-CSF-deficient mice.1 G-CSF binding to its
receptor causes receptor homodimerization,2 resulting in
tyrosine (Y) phosphorylation of the receptor itself and in
phosphorylation of associated kinases such as those of the Janus kinase
(JAK) family.3-6 Other important signaling molecules
activated by the G-CSF receptor (G-CSF-R) are signal transducer and
activator of transcription (STAT) proteins STAT1, STAT3, and
STAT5,4,7-9 some members of mitogen-activated protein
kinase (MAPK) pathways,5,10-12 and the tyrosine kinases
Lyn, Syk, and Hck.13,14 Phosphorylated tyrosine residues
of the hG-CSF-R in the cytoplasmic region (Y704, Y729, Y744, and Y764) potentially
serve as docking sites for SH2 (src homology 2) and PTB
(phospho-tyrosine binding) domain-containing proteins to initiate
signaling pathways. Deletion studies have identified the membrane
proximal 56 amino acids of the cytoplasmic domain as essential for cell
proliferation. In contrast, the differentiation response has been
reported to require the C-terminal region of the receptor containing
the tyrosine residues.15-19
Some signaling pathways activated by G-CSF-R tyrosine phosphorylation
have been analyzed by examining the effects of mutating cytoplasmic
tyrosine residues in immortalized cell lines. However, the importance
of each tyrosine residue appeared to differ in the cell lines, so that
the role of tyrosine residues in signal transduction in primary cells
remained unclear. The Y703 and Y728 residues of
the murine G-CSF-R (analogous to human Y704 and
Y729, respectively) transmitted differentiation signals in
murine interleukin-3 (mIL-3)-dependent myeloid LGM-1
cells.20 Exogenous expression of mG-CSF-R in LGM-1 cells
induced cell differentiation into neutrophils in response to G-CSF.
However, transfectants expressing mG-CSF-R lacking Y703
retained their blast cell morphology and showed reduced expression of
the differentiation marker, myeloperoxidase (MPO), in response to
G-CSF. Moreover, cells expressing mG-CSF-R lacking Y728
became macrophagelike in their morphology and failed to express MPO in
response to G-CSF.
In contrast, Nicholson et al8 reported the significance of
Y744 and Y704 of the hG-CSF-R in myeloid cell
line (M1) differentiation. Unlike LGM-1 cells, M1 cells expressing
exogenous wild-type (WT) G-CSF-R differentiated into macrophages,
becoming enlarged and vacuolated in response to G-CSF. Point mutation
of the receptor Y744, and to a lesser degree
Y704, diminished their differentiation responses.
Mutation of Y729 had little effect. In these cells, STAT1, STAT3, and STAT5 were activated by G-CSF, but the activation was
largely tyrosine independent. Multiple receptor tyrosine residues contributed to cell differentiation and survival at low concentrations of G-CSF in transfected myeloid 32D cells, with Y704 having
the greatest effect followed by Y744 and Y729,
respectively. STAT3 activation correlated with differentiation and
survival in these cells.21 Moreover, Y764 of
the receptor supported a strong proliferation response in 32D cells.
Mutants lacking Y764 were unable to proliferate, but they
still differentiated into morphologically mature neutrophils in
response to G-CSF. Thus, mutation of Y764 resulted in fewer differentiated cells in culture.22 In Ba/F3 cells,
however, the Y764 mutation did not affect cell
proliferation.23 In summary, specific signals are
initiated from phosphorylated receptor tyrosine residues, depending on
the type of cultured cells used as a model for neutrophilic granulocyte
development. Receptors Y704, Y729, and
Y744 may activate signaling for cell differentiation,
whereas Y764 may activate signaling for cell proliferation.
To resolve such discrepancies and to understand more about the function
of receptor tyrosine residues in primary cells, we have attempted to
identify the role of G-CSF-R tyrosine residues in bone marrow cells. To
bypass endogenous G-CSF-R, a chimeric receptor substituting the whole
extracellular domain of G-CSF-R with that of the epidermal growth
factor receptor (EGF-R) was constructed and expressed in murine bone
marrow cells from G-CSF-deficient mice by retroviral infection. In
addition, chimeric receptors carrying cytoplasmic tyrosine mutations,
either individually or collectively, were similarly expressed in
G-CSF-deficient murine bone marrow cells. Infected bone marrow cells
were tested for their ability to proliferate and differentiate in
response to EGF and to signal through the STAT and MAPK pathways.
Cell culture, cytokines, and antibodies
Construction of chimeric EGF-G-CSF receptor and its
tyrosine mutants
Generation of stable virus-producing packaging cells All EG-R constructs were inserted into the XhoI site of the retroviral vector pMSCVpac,26 transfected into GP+E-86 packaging cells by electroporation with subsequent selection in puromycin (2 µg/mL) 36 to 48 hours after transfection. Supernatants of the puromycin-resistant transfectants were filtered (0.2 µm) and stored at 70°C. GP+E-86 cells (4 × 105/plate) were
incubated in growth medium supplemented with tunicamycin (1 µg/mL)
overnight. The medium was aspirated, and the supernatant from the
transfections was added (2.5 mL/plate). Cells were incubated 4 hours
before another aliquot of the supernatant was added. After 2 days, the
original medium was replaced with fresh medium containing puromycin (2 µg/mL). Single clones of the infected cells were analyzed by flow
cytometry for EG-R expression, and those with a high expression level
were selected for these studies.
Identification of EG-R-expressing clones by flow cytometry Receptor-expressing GP+E-86 clones were incubated with mouse antihuman EGF-R monoclonal antibody 528 (10 µg/mL) for 30 minutes, then incubated with fluorescein isothiocyanate-conjugated anti-mouse immunoglobulin antibody (Silenus Labs, Melbourne, Australia) for 30 minutes, followed by propidium iodide (1 µg/mL) staining with washes between each staining. The clones were analyzed using a FACScan flow cytometer (Becton Dickinson, Bedford, MA). At least 3 high EG-R-expressing clones of each construct were expanded and tested for infection efficiency on Ba/F3 cells.Infection of bone marrow cells G-CSF-deficient mice1 (6 to 7 weeks old) were injected intraperitoneally with 5-fluorouracil (5-FU) at 150 mg/kg body weight. On day 4 after injection, bone marrow cells were harvested and cocultured (3 × 106 cells/T25 flask) with adhered virus-producing GP+E-86 packaging clones (5 × 105/flask, irradiated at 30 Gy before seeding) for 5 days in Dulbecco modified Eagle medium supplemented with fetal calf serum (15%), WEHI-3B D cell-conditioned medium (10%), mSCF (10 ng/mL), mIL-6
(10 ng/mL), 2-mercaptoethanol (0.1 mM), and polybrene (5 µg/mL).
After infection, bone marrow cells were transferred to fresh flasks and
incubated at 37°C for 2 hours to remove adherent cells. Nonadherent
cells were then washed extensively and assayed. Infection efficiency of
the bone marrow cells was determined by flow cytometry.
Detection of receptor-expressing bone marrow cells by flow cytometry Bone marrow cells were blocked with mouse serum (1:500) and antimouse Fc III/II receptor antibody (1:250) (Becton Dickinson) for
10 minutes on ice. Cells were then incubated with biotinylated 528 (10 µg/mL), washed, and incubated in streptavidin-phycoerythrin conjugate (PharMingen, San Diego, CA) for 30 minutes. Live cells were
identified by propidium iodide (1 µg/mL) exclusion and were analyzed
by flow cytometry.
Proliferation assays The proliferation assay was performed as described previously,27 except that cell density was 20 000/well and cultures were pulsed with methyl-3H-thymidine for the last 20 hours of the 3-day culture.Agar colony-forming unit assay Infected bone marrow cells were seeded at a density of 30 000/plate in semisolid agar (0.3%) supplemented with a range of mEGF concentrations (0-60 ng/mL).28 As controls, cells were seeded in the presence of G-CSF (0-10 ng/mL) (15 000/plate) or without any growth factors (30 000/plate). Cultures were incubated in a humidified atmosphere of 37°C with 10% CO2 in air. On day 4, colonies of 50 or more cells were counted using a dissecting microscope at × 40 magnification.Staining the whole-plate cultures To analyze the type of stimulated colonies, glutaraldehyde (2.5%)-fixed agar cultures were transferred onto slides and stained with Luxol fast blue (1 hour) and hematoxylin (4 minutes). Stained colony counts tended to be higher than unstained colony counts. The mean ratio of stained colony counts to unstained colony counts (± SD) was 1.1 ± 0.6 at 20 ng/mL mEGF and 1.5 ± 0.9 at 0.7 ng/mL mEGF.Myeloperoxidase staining Six agar colonies stimulated by each receptor construct were picked using a 200-µL pipette tip under a dissecting microscope. Cells were washed and stained for MPO as described.29 This experiment was performed twice.Isolation of RNA, reverse transcription-polymerase chain reaction analysis Total RNA was extracted from G-CSF-deficient bone marrow cells or from a pool of 5 EGF-stimulated agar colonies expressing each receptor construct, using TRIzol reagent (Life Technologies-Gibco, Melbourne, Australia) according to the manufacturer's instructions. Total RNA was reverse transcribed using 500 ng random hexadeoxynucleotide primers (Promega, Sydney, Australia) and 200 U M-MuLV Reverse Transcriptase (New England Biolabs, Beverly, MA) in 20 µL total reaction volume. One tenth reaction volume was amplified (1 minute at 95°C, 1 minute at 52°C, 1 minute at 72°C) for 45 cycles followed by 10 minutes at 72°C in a Stratagene Robocycler. The sequence of the specific primers was as follows: -actin,30 5'-ATGCCATCCTGCGTCTGGACCTGGC-3', 5'-AGCATTTGCGGTGCACGATGGAGGG-3'; gelatinase,31
5'-ACGGTTGGTACTGGAAGTTCC-3', 5'-CCAACTTATCCAGACTCCTGG-3'.
Electrophoretic mobility shift assay Bone marrow cells were starved of growth factors for 6 hours, followed by 10 minutes stimulation in the presence of EGF (100 ng/mL), G-CSF (25 ng/mL), or no factors at a density of 1 × 106/mL. Electrophoretic mobility shift assay was performed essentially as described.32 The oligonucleotides used were-cas,33 derived from the 5' region of the
-casein gene, which binds STAT5 and STAT1 and a
high-affinity mutant of the sis-inducible element (SIE)34
of the human c-fos gene, which binds STAT1 and STAT3.
Western blot analysis Starved bone marrow cells (1 × 106/mL) were stimulated with EGF (100 ng/mL) for 10 minutes, followed by the addition of 10 vol ice-cold phosphate-buffered saline supplemented with 10 µM Na3VO4. Cells were pelleted and lysed in 50 mM Tris/HCl, 150 mM NaCl, 1% Triton-X-100, 1 mM EDTA, 0.1 mM Na3VO4, 1 mM dithiothreitol, 1 mM Pefablock, 50 µg/mL aprotinin, 50 µg/mL leupeptin, and 50 µg/mL bacitracin, followed by centrifugation at 13 000g for 15 minutes. Soluble proteins were mixed with sample buffer and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, using a 10% gel under nonreducing conditions. Proteins were electrophoretically transferred to a polyvinylidene fluoride membrane (Millipore, Sydney, Australia) and were blocked with TBST (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% (vol/vol) Tween 20) containing 3% bovine serum albumin for 1 hour. Membranes were incubated with antiphospho-p44/42 MAPK (ERK 1 and 2) antibody diluted in TBST containing 1% bovine serum albumin for 2 hours. After washing, the activated ERK 1 and 2 were detected with horseradish peroxidase-conjugated anti-rabbit immunoglobulin (Bio-Rad Laboratories, Sydney, Australia) and enhanced chemiluminescence (Amersham, Sydney, Australia). Membranes were stripped in 62.5 mM Tris-HCl, pH 6.7, 2% SDS, and 100 mM-mercaptoethanol at 50°C for 30 minutes, washed, reblocked, and
probed with anti-ERK 1 antibody as a loading control.
Statistics All statistical analyses were performed using SPSS version 6.1 (SPSS, Sydney, Australia). EGF titration curves were analyzed using multiple regression analysis. Because there was far greater variability at high EGF concentrations, data transformation was necessary. Logarithmic transformation of agar colony numbers was not applicable because of a large number of zero values. Instead, square root transformation was performed. In methyl-3H-thymidine uptake assays, however, data were transformed logarithmically. In colony composition analysis, raw data were analyzed except for macrophage colony numbers, which were transformed to square root because of their low value. Differences were considered significant if P < .01.
Construction of EG-R chimeric mutants To bypass endogenous G-CSF-R, this study used chimeric receptors in which the extracellular domain of EGF-R replaced that of G-CSF-R (Figure 1). All experiments were performed on bone marrow cells isolated from G-CSF-deficient mice to eliminate any effect of endogenous G-CSF in the functional assays. To confirm the integrity of the chimeric construct, so-called WT EG-R was expressed from pEF-BOS in murine myeloid leukemic (M1) cells, and the differentiation of the cells in response to EGF was examined. This mimicked the effect of G-CSF on G-CSF-R-transfected M1 cells, in which the cells became enlarged and vacuolated (data not shown). Similarly, proliferation of Ba/F3 cells expressing EG-R WT in response to EGF mimicked the proliferation of the cells expressing G-CSF-R in response to G-CSF (data not shown). In addition to WT EG-R, chimeric receptors containing Y F mutations individually and collectively were
constructed (Figure 1). A receptor with 2 PS substitutions in the
Box1 region, conserved in cytokine receptors, was also constructed for
use as a negative control. This receptor is unable to activate JAK and,
therefore, does not activate downstream signaling
pathways.8,17
Selection of high-titer packaging clones Receptor constructs were expressed from the retroviral vector pMSCVpac in GP+E-86 packaging cells. At least 3 individual packaging clones were selected on the basis of high receptor expression, and their infection efficiency was measured on Ba/F3 cells. These were cocultured with the virus-producing packaging clones and subsequently were used in an agar colony assay in which infection efficiency was measured as the proportion of Ba/F3 colonies formed in response to EGF to that in response to WEHI-3B D
cell-conditioned medium (data not shown). Single GP+E-86
packaging clones expressing each receptor construct at
similar levels (Figure 2) with comparable
infection efficiencies were selected for bone marrow cell
infection.
Expression of EG-R in murine bone marrow cells To understand the role of receptor tyrosine residues in signal transduction in bone marrow cells, all the receptor constructs were expressed by retroviral infection of bone marrow progenitor cells from 5-FU-treated mice. No evidence indicates that any EGF-R family member is expressed in mouse bone marrow cells35; therefore, chimeric receptors are expected to signal through homodimerization. Indeed, no colony growth or proliferation was observed in the presence of EGF. To prevent signaling from endogenous G-CSF production, G-CSF-deficient mice were used as a source of bone marrow cells. During the infection period, bone marrow cells were grown in a cocktail of growth factors that favored myeloid lineage development.36 Approximately 5% to 11% of the bone marrow cells expressed each receptor construct in each experiment (Table 1). Morphologic analysis of the infected bone marrow cells showed 93% of the receptor-expressing cells were of blast cell or myeloid lineage (data not shown). Infection efficiency was taken into account when calculating the EGF response in agar colony formation and in methyl-3H-thymidine uptake assays.
Receptor Y764 supports cell proliferation Bone marrow cell proliferation was measured by agar colony formation and methyl-3H-thymidine uptake in response to EGF. In agar, the receptor-expressing bone marrow cells were stimulated by increasing concentrations of EGF, and the total number of colonies formed on day 4 was counted (Figure 3A). Colony formation stimulated by the WT receptor was characterized by a sigmoidal curve, and the effect of tyrosine mutations on variation from this curve was analyzed statistically (see "Materials and methods"). Colony formation by the receptor Y764 mutant was significantly reduced compared with that stimulated by the WT receptor (P < .01), although cells bearing the receptor Y744 mutant formed more colonies than bone marrow cells bearing the WT receptor (P < .01). Receptor Y704 and Y729 mutants stimulated similar numbers of colonies to the WT receptor. Receptor Ynull supported maximal colony formation in response to EGF (the curve maximal response occurs at 60 ng/mL EGF; data at higher concentrations not shown), but it showed reduced sensitivity to EGF the 50%
effective concentration (EC50) of EGF was increased by more
than 10-fold (9.0 vs 0.4 ng/mL) in cells expressing receptor
Ynull compared with WT cells. Dose responsiveness was not
altered in any of the single tyrosine mutants
(EC50 = 0.8, 0.4, 0.3 and 0.4 ng/mL for Y704,
Y729, Y744 , and Y764 mutants,
respectively). As expected, the P640, 642 mutant did not
form any colonies in response to EGF.
The requirement for the receptor Y764 for maximal bone marrow cell proliferation was evident in the methyl-3H-thymidine uptake assay, where the bone marrow cells stimulated by the receptor Y764 mutant showed significantly reduced proliferation compared with those stimulated by the WT receptor (Figure 3B) (P < .01). Other tyrosine mutations also showed some effect on cell proliferation. Receptor Y704 mutant supported a reduced proliferation of bone marrow cells, particularly at lower concentrations of EGF (P < .01). In contrast, receptor Y729 and Y744 mutants caused hyperproliferation of the bone marrow cells in response to EGF (P < .01). The P640, 642 mutant did not support bone marrow cell proliferation, and the receptor Ynull mutant caused an increase in EC50 compared with the WT receptor (0.8 vs 0.03). Overall, the results of the methyl-3H-thymidine uptake assay were similar to those of the colony assay. As an additional control, the G-CSF response of the bone marrow cells, expressing the WT receptor and mutants, was also measured. Their titration curves were similar to each other (Figure 3C), indicating that the different bone marrow infection cultures gave rise to similar progenitor populations. Receptor Y729 supports granulocytic cell differentiation The WT receptor stimulated 3 types of bone marrow colonies in agar: granulocyte (G), macrophage (M), and granulocyte-macrophage (GM) colonies (Figure 4). Granulocyte colonies appeared morphologically normal, whereas macrophages were activated (with several projections). Receptor mutants stimulated the same types of colonies, but in different proportions. Although the total number of colonies stimulated by the receptor Y729 mutant was similar to that stimulated by the WT receptor, the number of granulocyte colonies stimulated by this mutant was significantly reduced (P < .01) (Figure 5A,B). Instead, this mutant supported more macrophage colony formation than the WT receptor (P < .01). Reduction in granulocyte colony formation was also apparent, but less marked, in the bone marrow cells stimulated by the receptor Ynull (P < .01). Receptor Y704 mutant stimulated similar numbers of G, GM, and M colonies compared with the WT receptor. Although the receptor Y744 mutant stimulated a higher number of colonies than the WT receptor, the increase was not significant for any individual colony type.
Proportions of G, GM, and M colonies were also compared between the WT
and mutant receptors to determine whether the reduced colony number
observed with the Y764 mutant resulted in the loss of a
particular colony type (Table 2).
Proportions of G, GM, and M colonies stimulated by the receptor
Y704, Y744, or Y764 mutants were
not significantly altered compared with the WT receptor at either
stimulus concentration. As we saw in the absolute colony type counts,
the receptor Y729 mutant stimulated a lower proportion of
granulocyte colonies and a higher proportion of macrophage colonies
than the WT receptor at either stimulus concentration. The increased
proportion of stimulated macrophage colonies was also evident through
the receptor Ynull. A higher proportion of granulocyte
colonies and a lower proportion of macrophage colonies at lower
concentrations of EGF were generated by all the receptor constructs
(except for Y764 mutant, probably because of the small macrophage colony numbers), suggesting that granulocyte progenitors have higher sensitivity to stimuli than macrophage progenitors.
Activation of STAT and MAPK To gain further insight into the signaling mechanisms used by the G-CSF-R to mediate the proliferation and differentiation responses observed, we examined activation of the STAT and MAPK pathways. First, we tested the ability of the WT receptor to activate STAT. This showed that the same complexes were activated as those we reported for +/+ bone marrow stimulated with G-CSF37 (Figure 6A,B). Next, we examined the various mutants: all activated STAT1, STAT3, and STAT5 with only minor differences that appeared to be related to the relative infection efficiencies. Similar results were observed at 0.7 ng/mL EGF stimulation. Strong ERK 1/2 phosphorylation was observed through receptor WT, Y704, and Y729 mutants, whereas a modest activation was observed through receptor Y744 mutant. In contrast, receptor Y764 and Ynull mutants failed to phosphorylate ERK1/2 (Figure 6C).
Expression of myeloperoxidase and gelatinase do not require tyrosine-mediated signaling To measure the differentiation status of bone marrow granulocyte agar colonies stimulated by each receptor construct, granulocytes were isolated from agar colonies and stained for MPO. The WT receptor and all single- and multiple-tyrosine mutants supported MPO expression in granulocytes in a similar manner (Figure 7A). Expression of neutrophil gelatinase (NG) in agar colonies was examined by reverse transcription-polymerase chain reaction (RT-PCR) (Figure 7B). Bone marrow granulocyte colonies expressing the WT receptor or its tyrosine mutants all expressed NG.
The aim of this study was to improve our understanding of the role of G-CSF-R tyrosine residues in signal transduction in primary cells. Previous studies have been undertaken in cell lines that can show idiosyncratic responses that may not reflect the situation in normal, nonimmortalized cell types. We found that in primary bone marrow, receptor tyrosine residues are required for cell proliferation at lower concentrations of stimulus. Receptor Ynull showed less sensitivity to stimulation than the WT receptor in colony formation and methyl-3H-thymidine uptake assays. Multiple tyrosine residues contribute to signal transduction from the receptor. Y729 initiates granulocyte differentiation pathway(s), whereas Y764 transmits strong signals for cell proliferation in bone marrow cells, evident in agar colony formation and methyl-3H-thymidine uptake assays. This study confirms some of the findings in related cell lines and provides additional information about G-CSF-R signaling in primary cells. Lack of receptor Y729 caused a defect in granulocyte differentiation in bone marrow cells. This finding was different from the results in M1 and 32D cells, where Y729 mutation had little effect on cell differentiation.8,22 However, mutation of Y728 of the mG-CSF-R caused a defect in granulocyte differentiation of LGM-1 cells, resulting in macrophage differentiation in these cells.20 It is likely that receptor Y729 initiates differentiation pathway(s) through recruitment of an as yet unidentified molecule(s) in bone marrow cells. Although the reduction of agar granulocyte colonies by the receptor Y729 mutant suggested that this tyrosine initiated neutrophil granulocyte differentiation pathways, we cannot rule out the possibility that the observed reduction in granulocyte colonies was caused by perturbed progenitor cell proliferation. If receptor Y729 sent granulocyte proliferation signals, in its absence there should have been fewer stimulated colonies; we did not observe this. The reduction in granulocyte colony formation was compensated for by increased macrophage colony formation by this mutant, suggesting no loss of total progenitors. Some evidence indicates that the differentiation of granulocytic and monocytic cells is inversely controlled by transcription factors.38 It is possible that the expression of specific transcription factor(s) is altered as a result of receptor Y729 mutation, resulting in a switch of the differentiation program. Another possibility is that in the absence of signals from receptor Y729, the sequence of neutrophil differentiation signals would be incomplete, allowing the cells to differentiate into macrophages instead as a default pathway. Receptor Y729 may have a dual function of promoting differentiation and suppressing cell proliferation as cell maturation coincides with cell cycle arrest in mature neutrophilic granulocytes. In contrast to other studies, receptors Y704 and Y744 were found not to have major roles in bone marrow cell proliferation or differentiation. Unlike the effect in LGM-1 cells, mutation of Y704 or Y729 did not abrogate MPO expression in primary bone marrow cells. This was perhaps not surprising considering that MPO expression is detectable in CD34+, HLA-DR+ progenitor cells,39 probably before G-CSF signaling is required. Although not apparently required for MPO expression, G-CSF signaling does enhance MPO expression in cell lines17 and in bone marrow cells.40 We found that granulocyte colonies, stimulated by WT EG-R or its tyrosine mutants, were terminally differentiated as measured by the expression of NG.41 Little is known about the role of G-CSF signaling in NG expression. However, its expression in G-CSF-deficient mice (this study) and its lack of expression in a G-CSF-R transduced myeloid cell line derived from PU.1 null mice42 suggests G-CSF signaling is redundant or is not involved in NG expression. Strong proliferation signals were initiated from receptor Y764, and we have evidence from methyl-3H-thymidine uptake data that Y704 may also contribute to proliferation. These results are overall in agreement with studies in 32D cells.21,22 However, our results suggest that receptor Y744 and perhaps receptor Y729 engage inhibitory pathways for cell proliferation because bone marrow cells expressing receptors lacking these tyrosines were hyper-proliferative in terms of colony formation and 3H incorporation. Such inhibitory effects have not been detected previously in studies of cell lines. In this study, simultaneous mutation of all 4 receptor tyrosine residues had a different effect from individual mutations of the tyrosine residues. For example, the observed reduction of cell proliferation caused by the mutation of receptor Y764 was not found in the receptor Ynull mutant. Clearly, the mutation of other tyrosine residues in receptor Ynull was compensated for by the mutation of Y764. It is possible that negative regulatory factors interact with tyrosine residues, so that when individual tyrosine residues are mutated, positive and negative signals are eliminated. Y764 binds to SHIP (SH2-containing inositol phosphatase) in Ba/F3 cells.43 SHIP may down-regulate the proliferation signals initiated from this tyrosine. Similarly, other factors may down-regulate signals from other tyrosine residues. The balance of signals transmitted from the whole receptor appears to determine the net outcome. At high concentrations of EGF, signals from receptor Ynull were sufficient for cell proliferation and differentiation, suggesting that there are receptor Y-independent mechanisms for activating cell proliferation and differentiation at optimal stimulus concentration. At lower concentrations of stimulus, however, tyrosine residues were required for signal transduction. Ward et al32 have shown that STAT3 activation is dependent on receptor tyrosine residues at low stimulus concentrations and that additional tyrosine-independent STAT3 activation signals occurred at high stimulus concentration. Together, these observations are consistent with the hypothesis that receptor tyrosine residues may signal under steady state conditions and that additional Y-independent pathways are activated in an emergency state.32 This hypothesis could also explain the decreased sensitivity to EGF stimulation of receptor Ynull that we observed in this study. By corollary, the sensitivity to erythropoietin (EPO) stimulation was reduced in Ba/F3 and DA-3 cells expressing EPO-R (Ynull) compared with cells expressing the WT receptor,44 suggesting that this may represent a more general phenomenon. Because STAT and MAPK family members have been implicated in the control of G-CSF-mediated differentiation and proliferation responses,5,7-12,21 we sought to investigate whether they might play a role in this primary cell system. All receptor mutants were able to activate STAT1, STAT3, and STAT5 in a manner equivalent to that of the WT receptor (and indeed WT bone marrow), even at low ligand concentrations. This suggests that these signaling components are activated in a tyrosine-independent manner in these cells, and it equally suggests that they are not responsible for the differences observed among the various receptor forms. It does not, however, necessarily imply that they are not important for proliferation or differentiation. Given that all mutants do show some proliferation and differentiation, activation of STAT might be required. In contrast, ERK activation was predominantly dependent on Y764; it was absent from cells expressing Y764 and Ynull mutants. This is in agreement with other studies showing Y764-mediated activation of SHP-2, Grb2, and Shc (elements of MAPK pathway) in 32D cells45 and JNK-SAPK (a form of MAPK) in Ba/F3 cells.46 Chimeric growth factor receptors using the EGF-R extracellular domain have been shown to be functional.47-49 Here we analyzed the behavior of the EGF-G-CSF chimeric receptor in comparison with the intact G-CSF-R in cell lines. Although we cannot rule out the possibility that subtle differences in signaling occur from the chimeric receptor in comparison with the WT G-CSF-R, the chimeric receptor stimulated proliferation in Ba/F3 cells and differentiation in M1 cells in the same manner as the intact G-CSF-R. Furthermore, the chimeric receptor formed STAT complexes similar to those of the intact G-CSF-R in bone marrow cells. Therefore, we used chimeric receptors carrying tyrosine mutations to study the tyrosine-specific signals in bone marrow cells. Specific signals from the G-CSF-R were required to stimulate maximal granulocytic cell differentiation, supporting an instructive role for G-CSF-R in granulopoiesis. Further evidence for this was found in transgenic mice expressing G-CSF-EPO chimeric receptor, which did not undergo normal granulopoiesis.50 Although myeloid cell lineage commitment was not altered, neutrophil terminal differentiation and function were impaired in these mice. These observations suggest that general signals are common to cytokine receptors and that, if they are placed in the correct cellular environment, they can mimic each other. However, certain signals specific to each cytokine receptor are essential for its full spectrum of functions.
We thank Prof Donald Metcalf and Dr George Hodgson for their assistance with agar colony typing, Dr Nicholas Wilson for his kind gift of antiphospho-ERK antibody, Dr Brendan Jenkins for the gift of anti-ERK antibody, and George Rennie for undertaking the statistical analysis. We also thank Fiona Connell for technical assistance.
Submitted November 1, 2000; accepted September 20, 2001.
Supported in part by a Research Scholarship of The University of Melbourne (S.A.), a grant from the National Health and Medical Research Council (no. 981133) (J.E.L.), a Sylvia and Charles Viertel Senior Medical Research Fellowship (A.C.W.), and a Wellcome Senior Research Fellowship in Medical Sciences in Australia (G.J.L.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Judith E. Layton, Ludwig Institute for Cancer Research, Royal Melbourne Hospital, PO Box 2008, Victoria 3050, Australia; e-mail: judy.layton{at}ludwig.edu.au.
1.
Lieschke GJ, Grail D, Hodgson G, et al.
Mice lacking granulocyte colony-stimulating factor have chronic neutropenia, granulocyte and macrophage progenitor cell deficiency, and impaired neutrophil mobilization.
Blood.
1994;84:1737-1746
2.
Fukunaga R, Ishizaka-Ikeda E, Nagata S.
Purification and characterization of the receptor for murine granulocyte colony-stimulating factor.
J Biol Chem.
1990;265:14008-14015
3.
Nicholson SE, Oates AC, Harpur AG, et al.
Tyrosine kinase JAK1 is associated with the granulocyte colony-stimulating factor receptor and both become tyrosine phosphorylated after receptor activation.
Proc Natl Acad Sci U S A.
1994;91:2985-2988
4.
Tian SS, Lamb P, Seidel HM, Stein RB, Rosen J.
Rapid activation of the STAT3 transcription factor by granulocyte colony-stimulating factor.
Blood.
1994;84:1760-1764
5.
Nicholson SE, Novak U, Ziegler SF, Layton JE.
Distinct regions of the granulocyte colony-stimulating factor receptor are required for tyrosine phosphorylation of the signaling molecules JAK2, Stat3, and p42, p44MAPK.
Blood.
1995;86:3698-3704
6.
Shimoda K, Feng J, Murakami H, et al.
Jak1 plays an essential role for receptor phosphorylation and Stat activation in response to granulocyte colony-stimulating factor.
Blood.
1997;90:597-604
7.
Tweardy DJ, Wright TM, Ziegler SF, et al.
Granulocyte colony-stimulating factor rapidly activates a distinct STAT-like protein in normal myeloid cells.
Blood.
1995;86:4409-4416
8.
Nicholson SE, Starr R, Novak U, Hilton DJ, Layton JE.
Tyrosine residues in the granulocyte colony-stimulating factor (G-CSF) receptor mediate G-CSF-induced differentiation of murine myeloid leukemic (M1) cells.
J Biol Chem.
1996;271:26947-26953
9.
Tian SS, Tapley P, Sincich C, et al.
Multiple signaling pathways induced by granulocyte colony-stimulating factor involving activation of JAKs, STAT5, and/or STAT3 are required for regulation of three distinct classes of immediate early genes.
Blood.
1996;88:4435-4444
10.
Bashey A, Healy L, Marshall CJ.
Proliferative but not nonproliferative responses to granulocyte colony-stimulating factor are associated with rapid activation of the p21ras/MAP kinase signaling pathway.
Blood.
1994;83:949-957
11.
Rausch O, Marshall CJ.
Cooperation of p38 and extracellular signal-regulated kinase mitogen-activated protein kinase pathways during granulocyte colony-stimulating factor-induced hemopoietic cell proliferation.
J Biol Chem.
1999;274:4096-4105
12.
Suzuki K, Hino M, Hato F, Tatsumi N, Kitagawa S.
Cytokine-specific activation of distinct mitogen-activated protein kinase subtype cascades in human neutrophils stimulated by granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-alpha.
Blood.
1999;93:341-349
13.
Corey SJ, Burkhardt AL, Bolen AL, et al.
Granulocyte colony-stimulating factor signaling involves the formation of a three-component complex with Lyn and Syk protein-tyrosine kinases.
Proc Natl Acad Sci U S A.
1994;91:4683-4687 14. Ward AC, Monkhouse JL, Csar XF, Touw IP, Bello PA. The Src-like tyrosine kinase Hck is activated by granulocyte colony-stimulating factor (G-CSF) and docks to the activated G-CSF receptor. Biochem Biophys Res Commun. 1998;251:117-123[CrossRef][Medline] [Order article via Infotrieve]. 15. Fukunaga R, Ishizaka-Ikeda E, Pan C-X, Seto Y, Nagata S. Functional domains of the granulocyte colony-stimulating factor receptor. EMBO J. 1991;10:2855-2865[Medline] [Order article via Infotrieve].
16.
Dong F, van Buitenen C, Pouwels K, et al.
Distinct cytoplasmic regions of the human granulocyte colony-stimulating factor receptor involved in the induction of proliferation and maturation.
Mol Cell Biol.
1993;13:7774-7781 17. Fukunaga R, Ishizaka-Ikeda E, Nagata S. Growth and differentiation signals mediated by different regions in the cytoplasmic domain of granulocyte colony-stimulating factor receptor. Cell. 1993;74:1-20[CrossRef][Medline] [Order article via Infotrieve].
18.
Ziegler SF, Bird TA, Morella KK, et al.
Distinct regions of the human granulocyte colony-stimulating factor receptor cytoplasmic domain are required for proliferation and gene induction.
Mol Cell Biol.
1993;13:2384-2390
19.
Avalos BR, Hunter MG, Parker JM, et al.
Point mutations in the conserved Box 1 region inactivate the human granulocyte colony-stimulating factor receptor for growth signal transduction and tyrosine phosphorylation of p75c-rel.
Blood.
1995;85:3117-3126 20. Yoshikawa A, Murakami H, Nagata S. Distinct signal transduction through the tyrosine-containing domains of the granulocyte colony-stimulating factor receptor. EMBO J. 1995;14:5288-5296[Medline] [Order article via Infotrieve].
21.
Ward AC, Smith L, de Koning JP, van Aesch Y, Touw IP.
Multiple signals mediate proliferation, differentiation, and survival from the granulocyte colony-stimulating factor receptor in myeloid 32D cells.
J Biol Chem.
1999;274:14956-14962
22.
de Koning JP, Soede BA, Schelen AM, et al.
Proliferation signaling and activation of Shc, p21Ras, and Myc via tyrosine 764 of human granulocyte colony-stimulating factor receptor.
Blood.
1998;91:1924-1933
23.
de Koning JP, Schelen AM, Dong F, et al.
Specific involvement of tyrosine 764 of human granulocyte colony-stimulating factor receptor in signal transduction mediated by p145/Shc/GRB2 or p90/GRB2 complexes.
Blood.
1996;87:132-140
24.
Markowitz D, Goff S, Bank A.
A safe packaging line for gene transfer: separating viral genes on two different plasmids.
J Virol.
1988;62:1120-1124
25.
Gill GN, Kawamoto T, Cochet C, et al.
Monoclonal anti-epidermal growth factor receptor antibodies which are inhibitors of epidermal growth factor binding and antagonists of epidermal growth factor binding and antagonists of epidermal growth factor-stimulated tyrosine protein kinase activity.
J Biol Chem.
1984;259:7755-7760 26. Hawley RG, Lieu FH, Fong AZ, Hawley TS. Versatile retroviral vectors for potential use in gene therapy. Gene Ther. 1994;1:136-138[Medline] [Order article via Infotrieve].
27.
Layton JE, Iaria J, Smith DK, Treutlein HR.
Identification of a ligand-binding site on the granulocyte colony-stimulating factor receptor by molecular modeling and mutagenesis.
J Biol Chem.
1997;272:29735-29741 28. Metcalf D. Clonal Culture of Hemopoietic Cells: Techniques and Applications. Amsterdam, The Netherlands: Elsevier; 1984:107-124. 29. Kaplow LS. Simplified myeloperoxidase stain using benzidine dihydrochloride. Blood. 2000;26:215-219. 30. McKnight AJ, Barclay AN, Mason DW. Molecular cloning of rat interleukin 4 cDNA and analysis of the cytokine repertoire of subsets of CD4+ T cells. Eur J Immunol. 1991;21:1187-1194[Medline] [Order article via Infotrieve].
31.
Anderson KL, Smith KA, Pio F, Torbett BE, Maki RA.
Neutrophils deficient in PU.1 do not terminally differentiate or become functionally competent.
Blood.
1998;92:1576-1585
32.
Ward AC, Hermans MH, Smith L, et al.
Tyrosine-dependent and -independent mechanisms of STAT3 activation by the human granulocyte colony-stimulating factor (G-CSF) receptor are differentially utilized depending on G-CSF concentration.
Blood.
1999;93:113-124 33. Wang D, Stravopodis D, Teglund S, Kitazawa J, Ihle JN. Naturally occurring dominant negative variants of Stat5. Mol Cell Biol. 1996;16:6141-6148[Abstract]. 34. Wagner BJ, Hayes TE, Hoban CJ, Cochran BH. The SIF binding element confers sis/PDGF inducibility onto the c-fos promoter. EMBO J. 1990;9:4477-4484[Medline] [Order article via Infotrieve]. 35. von Ruden T, Wagner EF. Expression of functional human EGF receptor on murine bone marrow cells. EMBO J. 1988;7:2749-2756[Medline] [Order article via Infotrieve]. 36. Dannaeus K, Nilsson K, Jonsson JI. Distinct actions of Flt3 ligand and stem cell factor on myeloid lineage selection and maturation of granulocytes versus macrophages. Exp Hematol. 1998;26:345-352[Medline] [Order article via Infotrieve].
37.
Hermans MH, Antonissen C, Ward AC, et al.
Sustained receptor activation and hyperproliferation in response to granulocyte colony-stimulating factor (G-CSF) in mice with a severe congenital neutropenia/acute myeloid leukemia-derived mutation in the G-CSF receptor gene.
J Exp Med.
1999;189:683-692
38.
Krishnaraju K, Hoffman B, Liebermann DA.
Lineage-specific regulation of hematopoiesis by HOX-B8 (HOX-2.4): inhibition of granulocytic differentiation and potentiation of monocytic differentiation.
Blood.
1997;90:1840-1849
39.
Berliner N, Hsing A, Graubert T, et al.
Granulocyte colony-stimulating factor induction of normal human bone marrow progenitors results in neutrophil-specific gene expression.
Blood.
1995;85:799-803 40. Tsuruta T, Tani K, Shimane M, et al. Effects of myeloid cell growth factors on alkaline phosphatase, myeloperoxidase, defensin and granulocyte colony-stimulating factor receptor mRNA expression in haemopoietic cells of normal individuals and myeloid disorders. Br J Haematol. 1996;92:9-22[CrossRef][Medline] [Order article via Infotrieve].
41.
Borregaard N, Sehested M, Nielsen BS, Sengelov H, Kjeldsen L.
Biosynthesis of granule proteins in normal human bone marrow cells: gelatinase is a marker of terminal neutrophil differentiation.
Blood.
1995;85:812-817
42.
Anderson KL, Smith KA, Perkin H, et al.
PU.1 and the granulocyte- and macrophage colony-stimulating factor receptors play distinct roles in late-stage myeloid cell differentiation.
Blood.
1999;94:2310-2318
43.
Hunter MG, Avalos BR.
Phosphatidylinositol 3'-kinase and SH2-containing inositol phosphatase (SHIP) are recruited by distinct positive and negative growth- regulatory domains in the granulocyte colony-stimulating factor receptor.
J Immunol.
1998;160:4979-4987 44. Damen JE, Wakao H, Miyajima A, et al. Tyrosine 343 in the erythropoietin receptor positively regulates erythropoietin-induced cell proliferation and Stat5 activation. EMBO J. 1995;14:5557-5568[Medline] [Order article via Infotrieve]. 45. Ward AC, Monkhouse JL, Hamilton JA, Csar XF. Direct binding of Shc, Grb2, SHP-2 and p40 to the murine granulocyte colony-stimulating factor receptor. Biochim Biophys Acta. 1998;1448:70-76[Medline] [Order article via Infotrieve]. 46. Rausch O, Marshall CJ. Tyrosine 763 of the murine granulocyte colony-stimulating factor receptor mediates Ras-dependent activation of the JNK/SAPK mitogen-activated protein kinase pathway. Mol Cell Biol. 1997;17:1170-1179[Abstract].
47.
Seedorf K, Felder S, Millauer B, Schlessinger J, Ullrich A.
Analysis of platelet-derived growth factor receptor domain function using a novel chimeric receptor approach.
J Biol Chem.
1991;266:12424-12431
48.
Sakamaki K, Wang HM, Miyajima I, et al.
Ligand-dependent activation of chimeric receptors with the cytoplasmic domain of the interleukin-3 receptor beta subunit (beta IL3).
J Biol Chem.
1993;268:15833-15839
49.
Maruyama K, Miyata K, Yoshimura A.
Proliferation and erythroid differentiation through the cytoplasmic domain of the erythropoietin receptor.
J Biol Chem.
1994;269:5976-5980 50. Semerad CL, Poursine-Laurent J, Liu F, Link DC. A role for G-CSF receptor signaling in the regulation of hematopoietic cell function but not lineage commitment or differentiation. Immunity. 1999;11:153-161[CrossRef][Medline] [Order article via Infotrieve].
© 2002 by The American Society of Hematology.
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  P. D. Emanuel Drifting precariously due to lost tyrosines Blood, January 1, 2007; 109(1): 7 - 8. [Full Text] [PDF]
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M. Germeshausen, M. Ballmaier, and K. Welte Incidence of CSF3R mutations in severe congenital neutropenia and relevance for leukemogenesis: results of a long-term survey Blood, January 1, 2007; 109(1): 93 - 99. [Abstract] [Full Text] [PDF]
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 D. Zhuang, Y. Qiu, S. J. Haque, and F. Dong Tyrosine 729 of the G-CSF receptor controls the duration of receptor signaling: involvement of SOCS3 and SOCS1 J. Leukoc. Biol., October 1, 2005; 78(4): 1008 - 1015. [Abstract] [Full Text] [PDF]
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G.-J. M. van de Geijn, J. Gits, L. H. J. Aarts, C. Heijmans-Antonissen, and I. P. Touw G-CSF receptor truncations found in SCN/AML relieve SOCS3-controlled inhibition of STAT5 but leave suppression of STAT3 intact Blood, August 1, 2004; 104(3): 667 - 674. [Abstract] [Full Text] [PDF]
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Q.-S. Zhu, L. J. Robinson, V. Roginskaya, and S. J. Corey G-CSF-induced tyrosine phosphorylation of Gab2 is Lyn kinase dependent and associated with enhanced Akt and differentiative, not proliferative, responses Blood, May 1, 2004; 103(9): 3305 - 3312. [Abstract] [Full Text] [PDF]
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M. A. Guthridge, E. F. Barry, F. A. Felquer, B. J. McClure, F. C. Stomski, H. Ramshaw, and A. F. Lopez The phosphoserine-585-dependent pathway of the GM-CSF/IL-3/IL-5 receptors mediates hematopoietic cell survival through activation of NF-{kappa}B and induction of bcl-2 Blood, February 1, 2004; 103(3): 820 - 827. [Abstract] [Full Text] [PDF]
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 T. S. Kendrick, R. J. Lipscombe, O. Rausch, S. E. Nicholson, J. E. Layton, L. C. Goldie-Cregan, and M. A. Bogoyevitch Contribution of the Membrane-distal Tyrosine in Intracellular Signaling by the Granulocyte Colony-stimulating Factor Receptor J. Biol. Chem., January 2, 2004; 279(1): 326 - 340. [Abstract] [Full Text] [PDF]
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M. H. A. Hermans, G.-J. van de Geijn, C. Antonissen, J. Gits, D. van Leeuwen, A. C. Ward, and I. P. Touw Signaling mechanisms coupled to tyrosines in the granulocyte colony-stimulating factor receptor orchestrate G-CSF-induced expansion of myeloid progenitor cells Blood, April 1, 2003; 101(7): 2584 - 2590. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
);
Y704
); Y729
);
Y744
); Y764
); Ynull, (
); P640,
642
); and vector alone (
).
