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INTRODUCTION |
The differentiation of myeloid progenitors into
monocyte-macrophages and granulocytes is regulated by the combined
action of microenvironmental signals and lineage-specific transcription factors (1). One of the transcription factors involved in granulocytic differentiation is CCAAT/enhancer-binding protein
(C/EBP)1
: the promoters
of G-CSFR and other granulocytic genes contain binding sites for
C/EBP-
(1, 2); C/EBP-
knockout mice have no mature neutrophils or
eosinophils (3); and conditional expression of C/EBP-
alone can
induce granulocytic differentiation in U937, HL-60, and 32D cells (4,
5). The importance of this transcription factor for normal myelopoiesis
is underlined by the recent finding that a subset of acute myeloid
leukemias exhibit heterozygous mutations in the C/EBP-
protein. This
results in truncated C/EBP-
molecules, which act as dominant
negative mutants and block the expression of C/EBP-
target genes
(6). The mechanism of transcriptional activation of C/EBP-
is not well understood, but recent evidence suggests that it involves its
phosphorylation on Ser through a Ras-dependent mechanism
(7). Another transcription factor that has been implicated in
granulocyte development is signal transducer and activator of
transcription (STAT) 3 (8). STAT3 is also activated by phosphorylation.
In response to stimulation by a number of cytokines, STAT3 is
phosphorylated on tyrosine, and several tyrosine kinases including Jak,
Fes, and Src family kinases have been shown to activate STAT3
(9-15).
The c-fps/fes proto-oncogene encodes a 92-kDa
cytoplasmic protein-tyrosine kinase that is primarily expressed in
vascular endothelial cells (16, 17), monocyte-macrophages and
granulocytes (18-20). Expression studies with cloned
fps/fes genes have provided considerable evidence
that this kinase is involved in macrophage differentiation. The
expression of human c-fps/fes in K562
erythroleukemia cells resulted in their differentiation to
macrophage-like cells (21), whereas infection of avian bone marrow
progenitors with v-fps/fes viruses led to
macrophage differentiation without added M-CSF (22). More recently we
reported that a gain-of-function allele of Fes (Fesact)
was capable of inducing terminal macrophage differentiation of U937
cells and other monocyte progenitors. This was accompanied by
activation of PU.1, a transcription factor that is essential for
macrophage development, providing evidence that Fes may regulate macrophage maturation through activation of lineage-specific
transcription factors (23).
Although much less is known about the biological role of Fes in
granulocytes, this kinase is likely to be an important regulatory molecule in this cell type. Antisense inhibition of Fes expression during retinoic acid-induced granulocytic differentiation of HL60 cells
leads to apoptosis (24), suggesting a role of this kinase in the
survival of differentiating granulocytic cells. More recently, it was
reported that both Fes and the Fes-related kinase Fer can be activated
after cross-linking of high affinity Fc receptor (Fc
RI)
in mast cells (25). Thus, Fes and Fer may have overlapping functions in leukocytes.
To further investigate the possible functions of Fes in granulocytes we
have introduced Fesact into 32D, an
IL-3-dependent cell line that undergoes granulocytic differentiation in the presence of G-CSF (26, 27). These cells have a
normal karyotype, are non-tumorigenic, and have characteristics of
normal granulocyte progenitors. Therefore, they have been used extensively as a model to study the mechanisms involved in granulocytic differentiation. Here we report that expression of Fesact
in 32D protected these cells from apoptosis after IL-3 withdrawal, and
that this was followed by terminal granulocyte differentiation in the
absence of G-CSF. We also found that Fesact expression
caused the transcriptional activation of C/EBP-
and STAT3. Our
results suggest that Fes kinase may contribute to the regulation of
myeloid gene expression during granulopoiesis through the activation of
specific transcription factors.
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MATERIALS AND METHODS |
Cells--
32D and WeHi-3B cells (27) were obtained from Weiqun
Li (Georgetown University, Washington, DC). 32D cells were grown in RPMI 1640 medium supplemented with 15% fetal bovine serum, 1% penicillin, 1% streptomycin, and 5% WeHi-3B conditioned medium as a
source of murine IL-3. The amphotropic retroviral packaging cell line
Phoenix ampho was grown as described (23). For differentiation with
G-CSF, 32D cells were incubated with 20 ng/ml recombinant murine G-CSF
for the indicated times in the absence of IL-3.
DNAs--
Human c-Fes and Fesact cDNAs, the
IRES-GFP-containing retroviral vector MigR1, MigR1-c-Fes,
MigR1-Fesact, and the pCEFL vector were previously
described (23). Details of construction of pCEFL-Fesact are
available upon request. Luciferase reporters containing a 4× tandemly
repeated C/EBP-
element, a wild type G-CSFR promoter that spans bp
1324 to +67, and a mutant C/EBP-
G-CSFR promoter that has a
mutation from bp
49 to
45, corresponding to the C/EBP-
binding
site, were obtained from Daniel Tenen (Harvard Medical School, Boston,
MA) (2). cDNA probes to lactoferrin, lysozyme, and myeloperoxidase
were obtained from Alan Friedman (Johns Hopkins University, Baltimore,
MD) (28). Northern blot analysis was carried out as we described
(29).
Transfection and Infection--
The transfection of Phoenix
ampho cells with retroviral constructs, virus production, and infection
of 32D cells were carried out as described previously (23). 32D cells
infected with MigR1-Fesact virus were selected by
incubation without IL-3 for 2 days. After this time dead cells were
removed by centrifugation in HistopaqueTM (Sigma). The live
cells were then expanded by incubation in IL-3 medium for 2-5 days. To
assess the biological properties of Fesact, the cells were
again resuspended in medium without IL-3 and analyzed as described in
the text.
For transient transfection analysis, 32D cells were transfected with 15 µg of total plasmid DNA by electroporation. Transfection efficiency
was normalized using a cotransfected promoter-less renilla vector pRL-0
(Promega, Madison, WI). Transactivation of the luciferase-linked
C/EBP-
element and G-CSFR promoter were assayed 16 h after
transfection using a dual luciferase kit from Promega.
Flow Cytometry--
Flow cytometric analysis was carried out as
previously described (23). For surface marker staining, we used
Phycoerythrin-labeled antibodies against murine CD11b and Gr-1
(BD Pharmingen), and Phycoerythrin-labeled antibodies against F4/80
(Caltag Laboratories, Burlingame, CA).
Immunoprecipitation and Western Blot Analysis--
The
preparation of cell lysates, immunoprecipitation, SDS-PAGE, and
immunoblotting with specific antibodies were carried out as previously
described (23). Monoclonal anti-Fes antibodies have been previously
described (30). The antibodies to phosphotyrosine (PY99), C/EBP-
,
STAT3, and IL-3R
were purchased from Santa Cruz (Santa Cruz, CA).
Antibodies to phospho-STAT3 (Y705) were purchased from Cell Signaling
Technology (Beverly, MA).
Preparation of Nuclear Extract and Electrophoretic Gel Mobility
Shift Assay (EMSA)--
The preparation of nuclear protein extracts
and gel shift assays (EMSA) were carried out as previously described
(23). The oligonucleotide sequences used were: C/EBP consensus (31),
sense 5'-TGCAGATTGCGCAATCTGCA-3, antisense
5'-TGCAGATTGCGCAATCTGCA-3'; STAT3-SIE (sis-inducible
element) (14), sense 5'-GTGCATTTCCCGTAAATCTTGTC-3', antisense,
5'-GACAAGATTTACGGGAAATGCAC-3'; PU.1-CD11b (32), sense 5'-CTTCTGCCTCCTACTTCTCCTTTTCTGGCCT-3', antisense,
5'-AGGCCAGAAAAGGAGAAGTAGGAGGCAGAAG-3'.
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RESULTS |
Constitutively Active Fes Protects 32D Cells from Apoptosis Induced
by IL-3 Withdrawal--
To gain insight into the possible role of Fes
in granulocyte development we expressed Fesact, a
constitutively active form of Fes in 32D cells. The recently described
Fesact is a chimera between human and avian
fps/fes cognate genes in which part of the kinase
domain of human c-fes was replaced with the corresponding
region from avian v-fps (Fig.
1A). Fesact is
isogenic and colinear with both human c-Fes and avian v-Fps (23).
Fesact was introduced into 32D cells by retrovirus-mediated
gene transfer using the IRES-GFP vector MigR1 (33). 32D cells were
infected with MigR1, MigR1-c-Fes, or MigR1-Fesact viruses
(Fig. 1A) in the presence of IL-3 and 2 days later, IL-3 was
removed from the culture medium. The uninfected, vector-, and
c-Fes-infected 32D cells underwent apoptosis within 2 days of IL-3
withdrawal, but about 30% of the Fesact-infected 32D cells
survived without IL-3. All of the surviving cells were green
fluorescent, indicating that only Fesact-expressing cells
were protected from apoptosis (Fig. 1B). This enabled us to
rapidly isolate a population where 100% of the cells expressed
Fesact. In the absence of IL-3, Fesact-infected
cells remained viable for several days but their cell numbers did not
appreciably increase (Fig. 2,
A and B). This indicated that Fesact
promoted the survival of 32D cells deprived of IL-3 but had no significant mitogenic effect. In the presence of IL-3, uninfected and
Fes-infected cells proliferated at the same rate (Fig. 2B). As shown in Fig. 3, Fesact
had no significant effect on the levels of expression of IL-3 receptor,
as determined by immunoblot analysis using antibodies directed against
the
subunit of IL-3 receptor.

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Fig. 1.
Schematic representation of
Fesact and constructs used for retroviral-mediated gene
transfer. A, Fesact was constructed
by replacing a portion of the kinase domain of human c-Fes with the
corresponding v-Fps sequence from avian Fujinami sarcoma virus. The
details of construction of this chimera have been described (23). In
the switched kinase region, avian c-Fps and FSV-v-Fps differ in six
point mutations, and the percentage of amino acid homology between
human c-Fes and avian v-Fps in this region is 88% (Ref. 23, and
references therein). c-Fes and Fesact were subcloned into
the MigR1 retroviral vector, which contains a GFP cassette downstream
of IRES. These constructions have been described (23). B,
32D cells were infected with MigR1 or MigR1-Fesact viruses
as described under "Materials and Methods." Two days later cells
were resuspended in medium without IL-3 and cultured for another 2 days. Dead cells were removed by centrifugation in
HistopaqueTM and the remaining
MigR1-Fesact-infected live cells were observed under a
fluorescence microscope. After 2 days without IL-3, no surviving
uninfected or vector-infected cells were recovered. Magnification:
×40.
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Fig. 2.
Growth curve and viability of
Fesact-infected 32D cells. 32D cells infected with
Fesact virus were isolated and expanded as described under
"Materials and Methods." After expansion,
Fesact-infected cells were cultured in the presence or
absence of IL-3 for the indicated times. Viability was determined by
trypan blue exclusion (A), and cell numbers were determined
using a hemocytometer (B). Cell numbers and viability were
also followed in 32D cultures grown in the presence or absence of IL-3,
and in 32D cultures treated with G-CSF in the absence of IL-3.
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Fig. 3.
Activated Fes does not affect the expression
of IL-3 receptor. 32D cells were infected with
MigR1-Fesact viruses and the cells were allowed to
differentiate in the absence of IL-3 for 4 days after which time, the
cells were lysed. Total cell lysates (25 µg) from control 32D cells
(lane 1) and from Fesact-infected 32D cultures
(lane 2) were analyzed by SDS-PAGE (8.5% gel) followed by
immunoblotting (WB, Western blot) with specific antibodies
to the subunit of IL-3 receptor. Numbers at the
left indicate the position of Mr
markers. The position of IL-3R is indicated by an
arrow.
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We conclude from these results that activated Fes protected 32D cells
from apoptosis caused by IL-3 withdrawal. This was not secondary to a
mitogenic effect because Fesact-expressing cells remained
factor-dependent for proliferation.
Fesact Induces Granulocytic Differentiation in the
Absence of G-CSF--
We then examined whether Fesact had
any other biological activity in 32D cells. To this end, 32D cells were
infected with Fesact followed by selection in the absence
of IL-3 for 2 days. The surviving Fesact-infected
population was then allowed to expand for 3 days by supplementing the
culture medium with IL-3. No phenotypic changes were detectable at the
end of this period. Because IL-3 suppresses the biological activity of
G-CSF and may also interfere with that of Fesact, IL-3 was
removed from the Fesact-infected population once again, and
the cells were monitored for phenotypic changes. Remarkably, the
Fesact-expressing cells differentiated into granulocytes
(Fig. 4A, panels D1
and D2). Differentiation by Fes required IL-3
deprivation for a total of 4-8 days, including the initial selection
step. These cells acquired a characteristic mature granulocyte
morphology, namely smaller cell size and multilobulated nuclei. Control
uninfected 32D cells treated with G-CSF displayed granulocyte
morphology within 5 days, and at day 9 more cells showed typical
nuclear fragmentation (Fig. 4A, panels B and
C). The highly fragmented nuclei seen after 9 days in G-CSF
were not readily observed in Fesact-expressing 32D cells,
indicating that Fesact-induced differentiation was not as
complete as that obtained with G-CSF. Nevertheless, the mature
granulocyte type was found in about 20% of this
Fesact-expressing population, whereas the rest exhibited
intermediate or normal blast morphologies. By comparison, after 5 days
in G-CSF-containing medium, about 30% of the treated 32D cells
underwent terminal differentiation. IL-3 greatly suppressed both G-CSF-
and Fesact-dependent granulocytic
differentiation (data not shown). Essentially the same results were
obtained whether the source of IL-3 used to propagate uninfected or
Fesact-infected 32D cells was recombinant IL-3- or
WeHi-conditioned medium. In the experiments described above,
Fesact-infected cells were reincubated with IL-3 after the
initial IL-3 deprivation step, before removing it again to allow
completion of differentiation. The expansion phase in IL-3 was done to
obtain enough cells for our biochemical analysis, but it was not
necessary to observe Fes-dependent differentiation. If the
IL-3 expansion step was omitted, the Fesact-expressing
cells differentiated to the same extent as those that were expanded in
IL-3 (Fig. 4A, panel D3).

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Fig. 4.
Fesact induces granulocyte
differentiation of 32D cells. A, uninfected 32D cells kept
in IL-3 (panel A), 32D cells treated with G-CSF for 5 (panel B) or 9 (panel C) days, and
Fesact-infected 32D cells that were deprived of IL-3 for 4 days (panel D) were stained with May-Grünwald Giemsa.
Three different microscopic fields for each group are presented
(1-3). In panels D1 and D2
Fesact-expressing cells were expanded in IL-3 as indicated
in the text, whereas panel D3 corresponds to
Fesact-expressing cells that were differentiated without an
IL-3 expansion phase. B, a portion of the cultures described
in A, above, were analyzed for surface expression of CD11b,
Gr-1, and F4/80 by flow cytometry using specific (solid
bars) or isotype-matched antibodies (striped bars) as
described under "Materials and Methods." The plots represent the
mean fluorescence intensity for isotype-matched and specific
antibodies.
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Coordinate Up-regulation and Down-regulation of Granulocyte and
Macrophage Markers by Fesact--
To further characterize
the biological activity of Fesact, we analyzed the
expression of differentiation markers in Fesact-infected
versus control 32D cells. As shown in Fig. 4B,
Fesact up-regulated the expression of the common myeloid
marker CD11b and the granulocyte-specific marker Gr-1 by severalfold,
whereas expression of the macrophage-specific marker F4/80 was
down-regulated by this kinase (Fig. 4B). Treatment of
control 32D cells with G-CSF for 5 days was more efficient than
Fesact in inducing CD11b and Gr-1 expression, but G-CSF was
slower than Fesact in down-regulating F4/80. Like G-CSF,
Fesact induced the expression of lysozyme and lactoferrin,
two markers of late granulocytic differentiation, whereas
expression of the early marker myeloperoxidase was down-regulated by
Fes (Fig. 5). Although the time kinetics
of up- and down-regulation of lineage-specific markers by
Fesact and G-CSF was slightly different, their
overall biological activities on granulocytic differentiation were
comparable.

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Fig. 5.
Up-regulation of late granulocytic marker
expression by Fesact. 32D cells infected with
Fesact virus were deprived of IL-3 for the indicated times.
Total RNA was extracted from Fesact-infected (lanes
3-4, 7-8, and 11-12), control
32D (lanes 1, 5, and 9), and 32D cells
allowed to differentiate in the presence of G-CSF for 5 days
(lanes 2, 6, and 10) as described
under "Materials and Methods." The RNA was subjected to Northern
blot analysis (upper panel) using probes specific for
lactoferrin (lanes 1-4), lysozyme (lanes 5-8),
and myeloperoxidase (lanes 9-12) as described under
"Materials and Methods." The lower panel corresponds to
an ethidium bromide stain of the loaded RNA, showing the position of 18 S rRNA.
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We conclude from these results that Fesact is capable of
regulating myeloid gene expression in a lineage-specific manner. The extent of differentiation by Fes was not as complete as that induced by
G-CSF. This suggests that under physiological conditions, additional cooperating signals are required to complete the granulocyte
differentiation program. Nevertheless, the remarkable biological
activity of Fesact we observed suggests that Fes kinase may
play a key role in granulopoiesis.
Fesact Activates STAT3 and C/EBP-
but Not
PU.1--
Since Fesact was able to induce granulocyte
differentiation, we examined whether this kinase activated
transcription factors that are important for myeloid development (1).
To this end, we carried out EMSA of nuclear extracts from
Fesact-infected 32D cells. As shown in Fig.
6A, lanes 3-5,
both G-CSF and Fesact induced DNA-binding activity of
STAT3. This activity was competed out by excess unlabeled specific
oligonucleotide, but not by a nonspecific oligonucleotide (Fig.
6A, lanes 6 and 7). The addition of
anti-STAT3 antibodies to the EMSA reaction resulted in a band supershift (Fig. 6A, lane 8), confirming the
presence of STAT3 in the DNA-binding complexes induced by Fes.

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Fig. 6.
Fesact induces tyrosine
phosphorylation and DNA-binding activity of Stat3. A, 32D
cells infected with MigR1-Fesact viruses (lanes
4-8) were allowed to differentiate in the absence of IL-3 for the
indicated times. Nuclear extracts were prepared from uninfected
(lane 2), G-CSF-treated (lane 3), and infected
32D cultures (lanes 4-8), and STAT3 DNA-binding activity
was assayed by EMSA as described under "Materials and Methods."
Unlabeled, specific (S) STAT3-SIE (lane 6), or
nonspecific (NS) PU.1-CD11b (lane 7)
oligonucleotides were included in the EMSA for competition. Anti-STAT3
antibody was used for supershift (lane 8). Arrows
and asterisk at the right indicate the positions
of STAT3 and supershifted STAT3 complexes. B, total cell
lysates prepared from a portion of the cultures described in
A, above were analyzed by SDS-PAGE followed by
immunoblotting (WB, Western blot) with anti-phospho-STAT3
(Y705) antibodies (upper panel). The membrane was stripped
and reblotted with antibodies to STAT3 (lower panel).
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Transcriptional activation of STAT3 requires its phosphorylation on
Tyr-705 (15). Consistent with the results of our EMSA, Fesact induced the phosphorylation of STAT3 on Tyr-705, as
determined by immunoblot analysis using phosphospecific antibodies
(Fig. 6B, lanes 3 and 4). The
phosphorylated species migrated as a doublet, which probably
corresponds to STAT3-
and its spliced form STAT3-
. Neither G-CSF
treatment or Fesact expression had any detectable effect on
the expression levels of STAT3 (Fig. 6B).
Fesact also induced binding of nuclear extracts to an
oligonucleotide that is specific for C/EBP family members (Fig.
7). This band was competed out by excess
unlabeled specific oligonucleotide, but not by a nonspecific
oligonucleotide (Fig. 7A, lanes 6 and 7). G-CSF also induced DNA binding to this oligonucleotide
(Fig. 7A, lane 3). Although the G-CSF- and
Fes-induced complexes had slightly different electrophoretic
mobilities, anti-C/EBP-
antibodies caused a supershift of both
transcriptional complexes indicating that they contained C/EBP-
(Fig. 7A, lanes 8 and 9). Neither G-CSF treatment nor Fesact expression modified the
expression levels of C/EBP-
(data not shown). Whether other C/EBP
family members are also activated by Fes will require further analysis.
As shown in Fig. 7B, the expression of Fesact in
32D cells had no detectable effect on the DNA binding activity of
PU.1. We conclude from these results that in 32D cells,
Fesact induced the formation of C/EBP-
and STAT3
DNA-binding complexes.

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Fig. 7.
Fesact expression results in
activation of C/EBP- . A,
nuclear lysates were prepared from sister cultures of the control
(lane 2), G-CSF-treated (lanes 3 and
9), and Fesact-infected (lanes 4-8)
32D cells described in the legend to Fig. 6A. C/EBP-
DNA-binding activity was assayed by EMSA as described under
"Materials and Methods." C/EBP (lane 6) and STAT3-SIE
(lane 7) were used as specific and nonspecific
oligonucleotides, respectively, for competition. Anti-C/EBP-
antibody was used for supershift (lanes 8 and 9).
The arrow and asterisk at the right
indicates the positions of C/EBP- and supershifted C/EBP-
complexes. B, the nuclear lysates described in A
were assayed for PU.1 DNA-binding activity. PU.1 (lane 6)
and STAT3-SIE (lane 7) were used as specific and nonspecific
oligonucleotides, respectively, for competition. Anti-PU.1 antibody was
used for supershift (lane 8). The arrow and
asterisk at the right indicates the positions of
PU.1 and supershifted PU.1 complexes.
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Fesact Transactivates C/EBP-
and G-CSFR Reporter
Genes--
To further examine the ability of Fes to induce
transcriptional activation of C/EBP-
, we analyzed whether
Fesact was able to transactivate a minimal
luciferase-linked promoter containing 4 tandem repeats of the
C/EBP-
-binding site. As shown in Fig.
8, Fesact was able to
transactivate this C/EBP-
element. Since C/EBP-
is essential for
transcription of the G-CSFR gene (2, 34), we also examined whether
Fesact would be able to transactivate the G-CSFR promoter.
Fesact transactivated the G-CSFR promoter, but not a
promoter with a mutation in the C/EBP-
site (Fig. 8). We conclude
from these results that Fesact can induce terminal
granulocytic differentiation and the transcriptional activation of
C/EBP-
and STAT3.

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Fig. 8.
Fesact transactivates a
C/EBP- element and the G-CSF receptor
promoter. A, 32D cells were transfected with a 4× tandemly
repeated C/EBP- element linked to a luciferase reporter together
with pCEFL vector or pCEFL-Fesact, by electroporation.
Cells were lysed 16 h after transfection, and luciferase activity
was measured as described under "Materials and Methods."
B, 32D cells were transfected with a luciferase-linked G-CSF
receptor promoter or a mutant from which the C/EBP- site was
mutated, along with pCEFL vector or pCEFL-Fesact.
Luciferase activity was measured as described in A.
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DISCUSSION |
In this paper we present evidence that Fes kinase can regulate
lineage-specific gene expression during granulocytic differentiation. We also identified the transcription factors C/EBP-
and STAT3 as
potential Fes targets, which may mediate the biological activity of
this kinase.
Although 32D cells behave as granulocytic progenitors, these cells are
still capable of differentiating along the monocyte/macrophage lineage.
Introduction of another tyrosine kinase, M-CSF receptor, into 32D cells
allowed them to undergo partial monocytic differentiation in the
presence of M-CSF (35). Also, ectopic expression of the transcription
factor EGR-1 enabled these cells to undergo macrophage differentiation
by GM-CSF treatment (36). Since Fesact was an inducer of
macrophage differentiation in monocyte progenitors (23), it was of
interest to determine whether in 32D cells, Fesact would
induce macrophage or granulocytic differentiation, or both. By contrast
with M-CSFR, Fesact was not able to change the lineage
commitment of 32D cells but completed their granulocyte differentiation
program. The results of these and our previous studies in monocytic
cells (23) suggest that Fes may be involved in regulating the
completion of terminal granulocyte and macrophage differentiation
rather than a lineage decision step.
How does Fes kinase contribute to the execution of alternative programs
of myeloid gene expression? In 32D cells Fes activated STAT3 and
C/EBP-
, two important regulators of granulocytic differentiation (3,
8, 37, 38). On the other hand, PU.1, a transcription factor that is
essential for macrophage development but has a more limited role in
granulopoiesis (5) was activated by Fesact in U937
monoblasts but not in 32D cells. These results suggest that Fes may
select its targets according to the lineage commitment stage of the
cells, and that Fesact may induce granulocyte
differentiation at least in part through transcriptional activation of
STAT3 and C/EBP-
. The mechanism of activation of these factors may
involve direct or indirect phosphorylation by Fes. In
Fesact-expressing 32D cells, STAT3 was phosphorylated on
Tyr-705, an event known to cause its transcriptional activation (15).
Whether Fes activation of C/EBP-
also involves the phosphorylation
of this protein will have to be clarified in future experiments.
Fesact protected 32D cells from apoptosis upon IL-3
withdrawal. This was not due to a conversion of the cells to factor
independence because without IL-3, Fesact-expressing cells
were essentially not dividing. By contrast with Fesact, the
expression of v-Src, v-Abl, or Bcr-Abl in 32D cells does not result in
differentiation but in their conversion to factor-independence and oncogenic transformation (26, 39-41). Thus, among non-receptor tyrosine kinases, Fes has a unique ability to induce granulocytic differentiation in 32D cells.
The fact that in 32D cells, both Fesact and G-CSF activated
C/EBP-
and STAT3 suggests that Fes kinase may have utilized elements of the G-CSF pathway to drive granulocytic differentiation. There were
also other similarities between the biological activities of G-CSF and
Fesact: the time courses of granulocytic differentiation
induced by G-CSF and active Fes were similar; G-CSF and
Fesact induced granulocytic differentiation without IL-3,
but their biological activity was greatly diminished in the presence of this cytokine; and both G-CSF and Fesact protected cells
from apoptosis after IL-3 removal. Future experiments will be aimed at
clarifying the functional relation of Fes to G-CSF and other cytokines
that regulate granulocyte development.
Considering the large body of evidence that Fes plays a role in myeloid
cell development, the phenotype of Fes knockout mice is milder than
expected. Hackenmiller et al. (42) reported that some of the
Fes
/
mice were compromised in their innate immunity,
had low numbers of B cells, and elevated numbers of macrophages and
granulocytes. Fes
/
macrophages exhibited defects in
cell adhesion and GM-CSF signaling, pointing to incomplete maturation
of their myeloid compartment. However, targeting of the Fes locus using
a different strategy did not reveal alterations in the macrophage or
granulocyte populations (43). This low penetrance of the
Fes
/
phenotype may be a result of functional
redundancy. A possible candidate that may compensate for the loss of
Fes is the ubiquitously expressed Fer/NCP94 kinase, which has the same
overall structure as Fes (18, 19, 44, 45). It was recently reported
that in mast cells, Fer and Fes were both activated by
Fc
RI cross-linking (25). Thus, Fes and Fer may have
overlapping functions in mast cells and basophils. These observations
are consistent with the idea that in leukocytes, Fer may partially
compensate for the absence of Fes. In granulocytes, functional
complementation between Src family kinases has masked the phenotypic
consequences of deleting single members of the family. For instance,
defects in adhesion-dependent degranulation of
polymorphonuclear neutrophils were observed only after ablation of both
Hck and Fgr kinases (46). And defects in the respiratory burst response
were only observed in neutrophils from
hck
/
/fgr
/
/lyn
/
triple
knockout mice (47). Since myeloid development can be regulated by
multiple pathways (1, 48), it is likely that in addition to Fer there
may be other mechanisms capable of compensating for the absence of Fes.
For these reasons, we believe that both loss- and gain-of-function
genetic approaches will be required to elucidate the biological
functions and mechanism of action of Fes.
The results presented in this paper suggest that Fes may play an
important role in granulopoiesis. Our observation that C/EBP-
and
STAT3 are activated by Fes during this process suggests a potential
mechanism by which this kinase may contribute to the regulation of
granulocyte-specific gene expression during myeloid development.