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HEMATOPOIESIS
Tyrosine residues of the granulocyte colony-stimulating factor receptor transmit
proliferation and differentiation signals in murine bone marrow cells
Shiva Akbarzadeh, Alister C. Ward, Dora O. M. McPhee, Warren S. Alexander, Graham J. Lieschke, and Judith E. Layton
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 impli-
cated 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 GCSF-R tyrosine residues in primary cells
that were not observed previously in studies in cell lines. (Blood. 2002;99:879-887)
© 2002 by The American Society of Hematology
Introduction
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) domaincontaining 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
From the Ludwig Institute for Cancer Research, Melbourne Tumour Biology
Branch, and the Walter and Eliza Hall Institute of Medical Research, Parkville,
Victoria, Australia.
Sciences in Australia (G.J.L.).
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
BLOOD, 1 FEBRUARY 2002 䡠 VOLUME 99, NUMBER 3
Reprints: Judith E. Layton, Ludwig Institute for Cancer Research, Royal
Melbourne Hospital, PO Box 2008, Victoria 3050, Australia; e-mail:
[email protected].
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.
© 2002 by The American Society of Hematology
879
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880
AKBARZADEH et al
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.
Materials and methods
Cell culture, cytokines, and antibodies
GP⫹E-86 cells were grown under conditions described before.24 Murine
stem cell factor (mSCF) was provided by N. Nicola (Walter and Eliza Hall
Institute of Medical Research, Parkville, Australia). Murine interleukin-6
(mIL-6) and murine EGF (mEGF) were provided by R. Simpson and E.
Nice (Ludwig Institute for Cancer Research, Parkville, Australia), respectively. Recombinant hG-CSF was a gift from L. Souza (Amgen, Thousand
Oaks, CA). WEHI-3B D⫺ cell-conditioned medium was used as a source of
mIL-3. Anti-EGF-R monoclonal antibody 52825 was provided by F. Walker
(Ludwig Institute for Cancer Research). Antiphospho-p44/42 MAPK
antibody was purchased from New England Biolabs (Beverly, MA), and the
anti-ERK 1 antibody was purchased from Santa Cruz Biotechnology (Santa
Cruz, CA).
Construction of chimeric EGF–G-CSF receptor
and its tyrosine mutants
To replace the extracellular domain of the G-CSF-R with that of the EGF-R,
a KpnI site was introduced in G-CSF-R cDNA in pBluescript by substituting C2071CT with T2071AC, using a site-directed mutagenesis kit (QuickChange kit; Stratagene, La Jolla, CA). Similarly, a KpnI site was introduced
in the EGF-R cDNA by replacing G2142GC with T2142AC, which resulted in
the substitution of Ala629 to Thr629 in the encoded EGF-R protein. The 2
cDNAs were subcloned into pEF-BOS and were digested with KpnI and
AatII. The 2 KpnI/AatII fragments were swapped and ligated, resulting in a
cDNA coding for a chimeric protein (Thr629 of EGF-R was joined to Leu613
of G-CSF-R in the transmembrane region). In addition to the so-called WT
EGF–G-CSF receptor (EG-R), a series of chimeric receptors containing
tyrosine residues Y704, Y729, Y744, and Y764 mutated to phenylalanine (F),
either individually or collectively, and a receptor containing 2 prolines (P640
and P642) mutated to serine (S) were constructed in pEF-BOS from the
BLOOD, 1 FEBRUARY 2002 䡠 VOLUME 99, NUMBER 3
corresponding G-CSF-R mutants.8 This was achieved by subcloning the
cytoplasmic regions of each G-CSF-R mutant into the WT EG-R by a MscI
digest. Junctions were confirmed by sequencing.
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 ␮g/mL) 36 to 48 hours after
transfection. Supernatants of the puromycin-resistant transfectants were
filtered (0.2 ␮m) and stored at ⫺70°C. GP⫹E-86 cells (4 ⫻ 105/plate) were
incubated in growth medium supplemented with tunicamycin (1 ␮g/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 ␮g/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 ␮g/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 ␮g/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 ␮g/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 ␮g/mL), washed,
and incubated in streptavidin–phycoerythrin conjugate (PharMingen, San
Diego, CA) for 30 minutes. Live cells were identified by propidium iodide
(1 ␮g/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–3Hthymidine 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
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BLOOD, 1 FEBRUARY 2002 䡠 VOLUME 99, NUMBER 3
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.
G-CSF-R Tyr RESIDUE SIGNALING IN PRIMARY CELLS
881
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.
Myeloperoxidase staining
Six agar colonies stimulated by each receptor construct were picked using a
200-␮L 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 ␮L 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.
Results
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
Y3F mutations individually and collectively were constructed
(Figure 1). A receptor with 2 P3S 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
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 ␮M 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
␮g/mL aprotinin, 50 ␮g/mL leupeptin, and 50 ␮g/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.
Figure 1. Schematic diagram of the retroviral vector pMSCVpac and a series of
chimeric EG-R mutants. EG-R (Y7043F), (Y7293F), (Y7443F), (Y7643F), and (Ynull)
are mutant receptors with tyrosine substitution(s) as indicated; EG-R (P640, 6423S) is
a nonfunctional receptor with 2 amino acid substitutions as indicated. LTR, long
terminal repeat; pgk, phosphoglycerate kinase; pac, puromycin N-acetyltransferase;
TM, transmembrane.
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882
AKBARZADEH et al
BLOOD, 1 FEBRUARY 2002 䡠 VOLUME 99, NUMBER 3
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
Figure 2. Expression of EG-R mutants. Expression of receptor constructs in
GP⫹E-86 packaging clones detected by flow cytometry. Cells were incubated with
mouse antihuman EGF-R antibody 528 or IgG2b (control), followed by fluorescein
isothiocyanate–conjugated anti–mouse immunoglobulin. Receptor expression (unfilled histograms) is shown against the control (filled histograms).
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
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–3Hthymidine 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.
Table 1. Infection efficiency in bone marrow cells
Receptor
Infection efficiency (%)
EG-R WT
6.9 ⫾ 1.0
EG-R (Y704 3 F)
6.2 ⫾ 1.0
EG-R (Y729 3 F)
6.2 ⫾ 1.0
EG-R (Y744 3 F)
6.1 ⫾ 1.7
EG-R (Y764 3 F)
9.2 ⫾ 1.3
EG-R (Ynull)
8.0 ⫾ 1.6
EG-R (P640, 642 3 S)
6.1
Infection efficiency was determined by flow cytometry. Data represent average
infection efficiency ⫾ SE in 3 independent experiments, shown in Figures 3 and 5.
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BLOOD, 1 FEBRUARY 2002 䡠 VOLUME 99, NUMBER 3
G-CSF-R Tyr RESIDUE SIGNALING IN PRIMARY CELLS
883
Figure 4. Typical examples of bone marrow colony types in agar in response to
EGF. EG-R WT receptor-stimulated (A) granulocyte, (B) granulocyte-macrophage,
and (C) macrophage colonies. A macrophage and a granulocyte are indicated with an
open and a closed arrow, respectively, in panel B (⫻ 400).
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,
Figure 3. Proliferation and differentiation response of EG-R–expressing bone
marrow cells. (A) Total number of colonies per plate stimulated in response to
increasing concentrations of EGF (0-60 ng/mL) in agar was counted on day 4, as
described in “Materials and methods.” Colony numbers were corrected for infection
efficiency. Data represent the average of 3 independent experiments. (B) Bone
marrow cells were incubated in increasing concentrations of EGF (0-60 ng/mL) for 3
days, containing methyl–3H-thymidine for the last 20 hours of culture. Proliferation
response was measured in counts per minute (cpm) and was corrected for infection
efficiency. This figure is a representative of 2 independent experiments. (C)
Proliferation response of bone marrow cells to G-CSF (0-30 ng/mL) as a control was
also measured. This figure is representative of 2 independent experiments. WT, (E);
Y7043F, (F); Y7293F, (Œ); Y7443F, (); Y7643F, (⽧); Ynull, (f); P640, 6423S, (‚); and
vector alone (ƒ).
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
Figure 5. Absolute number of agar colony types stimulated with EGF. Colony
composition stimulated with (A) 20 ng/mL and (B) 0.7 ng/mL EGF by each receptor
construct is shown. Differentially shaded bars represent the number of each colony
type. Numbers of G (*), GM (**), or M (***) colonies stimulated by the receptor mutant
are significantly different from those stimulated by the WT receptor. Data are
presented as the mean ⫾ SE of 3 independent experiments.
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884
BLOOD, 1 FEBRUARY 2002 䡠 VOLUME 99, NUMBER 3
AKBARZADEH et al
Table 2. Proportion of bone marrow agar colony types stimulated
by receptor constructs
Discussion
Colony formation (%)*
G
Receptor
GM
M
High
Low
High
Low
EG-R WT
23
37
69
59
9
EG-R (Y704 3 F)
27
40
55
50
19
11
EG-R (Y729 3 F)
3†
High
Low
4.2
15†
67
64
30†
21†
EG-R (Y744 3 F)
15
36
72
59
13
6
EG-R (Y764 3 F)
19
33
80
62
2
6
EG-R (Ynull)
14
34
58
47
28†
19†
*SE ⬍ 25% in 90% of the colony numbers at high (20 ng/mL) and in 80% of the
colony numbers at low (0.75 ng/mL) EGF concentrations.
†Proportion of colonies stimulated by the receptor mutant is significantly different
from those stimulated by the WT receptor at each particular EGF concentration.
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.
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 mGCSF-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
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.
Figure 6. STAT and MAPK activation by receptor tyrosine mutants. Nuclear
extracts of 3 ⫻ 106 bone marrow cells were stimulated with or without EGF (100
ng/mL) and were incubated with 0.2 ng 32P-labeled double-stranded (A) ␤-cas
(derived from the 5⬘ region of the ␤-casein gene) or (B) a high-affinity mutant of SIE.
As a control, bone marrow cells of a normal mouse were stimulated with G-CSF (25
ng/mL), and STAT complexes were identified as described.37 S1, STAT1; S3, STAT3;
S5, STAT5. These figures represent 1 of 3 independent experiments. (C) Bone
marrow cells (3 ⫻ 106) expressing each receptor construct were stimulated with or
without EGF (100 ng/mL), lysed, and electrophoresed on a 10% polyacrylamide gel.
Proteins were transferred to a polyvinylidene difluoride membrane and were
incubated with antiphospho-p44/42 MAPK antibody, followed by horseradish peroxidase-conjugated anti–rabbit immunoglobulin and enhanced chemiluminescence.
The membrane was stripped and reprobed with anti-ERK 1 antibody. The main panel
represents 1 of 2 independent experiments. The additional panel represents the
repeated results for the Y744 mutant. Infection efficiency (Inf Eff) of bone marrow cells
expressing each receptor construct for all experiments is also indicated.
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BLOOD, 1 FEBRUARY 2002 䡠 VOLUME 99, NUMBER 3
Figure 7. Expression of differentiation markers MPO and NG in bone marrow
granulocyte colonies. (A) Expression of MPO (black staining) is shown in the
cytoplasm of granulocytes from agar colonies stimulated by EG-R (Ai) WT, (Aii)
Y7043F, (Aiii) Y7293F, (Aiv) Y7443F, (Av) Y7643F, and (Avi) Ynull (⫻ 1000). (B)
Expression of NG mRNA (207 base pair [bp]) in granulocytes was detected by
RT-PCR. As a control, bone marrow cells isolated from G-CSF–deficient mice (⫺/⫺)
were also analyzed. (C) Negative control with no RNA. Expression of ␤-actin (605 bp)
was shown as a control. This figure is representative of 2 independent experiments.
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
G-CSF-R Tyr RESIDUE SIGNALING IN PRIMARY CELLS
885
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 hyperproliferative 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
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886
BLOOD, 1 FEBRUARY 2002 䡠 VOLUME 99, NUMBER 3
AKBARZADEH et al
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.
Acknowledgments
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.
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2002 99: 879-887
doi:10.1182/blood.V99.3.879
Tyrosine residues of the granulocyte colony-stimulating factor receptor
transmit proliferation and differentiation signals in murine bone marrow
cells
Shiva Akbarzadeh, Alister C. Ward, Dora O. M. McPhee, Warren S. Alexander, Graham J. Lieschke and
Judith E. Layton
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