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Blood, 1 February 2002, Vol. 99, No. 3, pp. 879-887
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
From the Ludwig Institute for Cancer Research,
Melbourne Tumour Biology Branch, and the Walter and Eliza Hall
Institute of Medical Research, Parkville, Victoria, Australia.
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Abstract |
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)
© 2002 by The American Society of Hematology.
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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) 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.
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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 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 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.
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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 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
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| Figure 1.
Schematic diagram of the retroviral vector pMSCVpac and
a series of chimeric EG-R mutants.
EG-R (Y704F), (Y729F),
(Y744F), (Y764F), and (Ynull)
are mutant receptors with tyrosine substitution(s) as indicated; EG-R
(P640, 642S) 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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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.
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| 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).
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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.
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| 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, ( );
Y704F, ( ); Y729F, ( );
Y744F, ( ); Y764F,
( ); Ynull, ( ); P640,
642S, ( ); and vector alone ( ).
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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.
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| 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).
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| 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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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).
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| 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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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.
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| 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)
Y704F, (Aiii) Y729F, (Aiv)
Y744F, (Av) Y764F, 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.
|
|
| |
Discussion |
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.
| |
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.
| |
Footnotes |
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.
| |
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Abstract
Introduction