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INTRODUCTION |
The immediate-early response gene c-myc encodes one of
the common effectors of mitogen-regulated proliferation and
transformation (1, 2). Myc is a transcription factor that associates in dimeric complexes with its partner Max to activate a series of Myc-responsive genes, at least some of which are necessary for entry
into the DNA synthetic (S) phase of the cell division cycle. The
critical events underlying Myc control of S phase entry are still
unresolved. Myc antagonizes the function of the
cyclin-dependent kinase inhibitor
p27Kip1 by promoting its degradation and/or
sequestration into inactive complexes with other proteins, and this
leads in turn to the activation of cyclin E-CDK2 prior to the
G1/S transition (3-5). Several direct transcriptional
targets of c-Myc have now been identified, which include the dual
specificity phosphatase cdc25A (6), ornithine decarboxylase
(7), cyclins E and A (8), dihydrofolate reductase (9),
-prothymosin
(10), RNA helicase MrDb (11), and nucleolin. The combined
effects of these and other as yet unrecognized target genes are
required to drive quiescent cells into S phase and to maintain
proliferating cells in cycle (2, 12).
Mitogen-induced signaling pathway(s) that mediate the induction of
c-Myc expression have also largely remained an enigma. The ability of
c-Myc to override cell cycle arrest imposed by dominant negative forms
of Src kinases suggests that Src-mediated signaling may be responsible
for c-Myc expression (13). One of the best characterized growth
factor-induced signaling pathways is the
Ras/Raf/MEK1/MAPK pathway,
and several lines of evidence indicate that it can regulate c-Myc
expression. First, the c-myc promoter contains conserved
binding sites for Ets family transcription factors, which can be
activated by Ras/Raf/MEK/ERK signaling (14-17). Second, inhibition of
cell growth caused by overexpression of the carboxyl-terminal catalytic
domain of the Ras GTPase-activating protein can be rescued by enforced
c-Myc expression (18). Third, induction of c-Myc expression by the
oncoprotein v-Abl requires both functional Ras and Raf-1 (19). Finally,
overexpression of a dominant negative and growth-suppressive form of
Raf-1 (Raf-1-C4B) significantly reduces serum-dependent
induction of c-Myc, whereas conditional expression of an inducible
Raf-1 fusion protein is in itself sufficient to induce c-Myc expression
(20). The downstream effectors of activated Raf-1 are MEK1 and MEK2,
but to date neither of these kinases has been linked to regulation of
c-Myc expression.
The colony-stimulating factor-1 receptor (CSF-1R), a growth factor
receptor with intrinsic tyrosine kinase activity, is expressed specifically in macrophages and trophoblasts (21), and bone marrow-derived macrophages require CSF-1 for cell proliferation, survival, and differentiation (22). Ectopic expression of human CSF-1R
in murine fibroblasts endows them with the ability to proliferate in
chemically defined medium containing CSF-1 as the sole growth factor
(23). One of the major autophosphorylation sites within ligand-stimulated CSF-1R is tyrosine 809, located in the activation loop of the kinase domain (24). Substitution of this residue with
phenylalanine (Y809F) leads to reduction but not abolition of receptor
protein kinase activity (25). NIH-3T3 cells expressing CSF-1R (Y809F)
fail to proliferate in response to CSF-1 and instead arrest in the
early G1 phase of the cell cycle (24). These
CSF-1-stimulated cells fail to activate MAP kinases, ERK1 and 2, and to
induce c-Myc and cyclin D1, both of which are required for entry into S
phase (26-28). Enforced expression of c-Myc or D-type cyclins rescues
ligand-induced mitogenesis by CSF-1R (Y809F), enabling the cells to
proliferate continuously and to form colonies in semisolid medium (26,
27).
We have used macrophages as well as NIH-3T3 fibroblasts expressing
different forms of CSF-1R to assess the role of the MEK/ERK pathway in
the induction of c-Myc expression. We report that MEK1 activity is
necessary for c-Myc expression, although maximal c-Myc induction and S
phase entry require the cooperation of additional signaling molecules.
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EXPERIMENTAL PROCEDURES |
Cells and Culture Conditions--
A murine macrophage cell line,
Bac1.2F5 (29), was grown in Dulbecco's modified Eagle medium
supplemented with 15% fetal bovine serum (FBS), 2 mM
L-glutamine, 100 units each of penicillin and streptomycin
per ml, and 25% L cell conditioned medium (LCM) as a source of murine
CSF-1. For CSF-1 stimulation, Bac1.2F5 cells were growth arrested in
medium lacking LCM for 18 h, and quiescent cells were restimulated
with medium containing LCM for the indicated times. For treatment with
the MEK inhibitor PD98059 (kindly provided by Stuart J. Decker,
Warner-Lambert Co., Ann Arbor, MI), quiescent cells were pretreated
with 60 µM PD98059 or the vehicle control dimethyl
sulfoxide (Me2SO) (0.3%) for 30 min before the addition of
medium containing LCM with PD98059 or Me2SO.
NIH-3T3 cells stably expressing human CSF-1R or mutant CSF-1R (Y809F)
were maintained in Dulbecco's modified Eagle's medium supplemented
with 10% FBS, glutamine, and antibiotics (23). For CSF-1 stimulation,
cells were growth arrested in serum-free medium (Dulbecco's modified
Eagle's medium with 0.4 mg/ml bovine serum albumin, 0.1% FBS,
glutamine, penicillin, and streptomycin) for 36 h. Quiescent cells
were restimulated to enter the cell cycle with serum-free medium
containing purified recombinant human CSF-1 at 10 ng/ml (kindly
provided by Steven C. Clark, Genetics Institute, Cambridge, MA).
S phase entry was monitored by incorporation of bromodeoxyuridine
(BrdUrd) into newly synthesized DNA (30). To measure cell growth in
NIH-3T3 fibroblasts coexpressing CSF-1R (Y809F) and zinc-inducible
activated MEK1, cells were growth arrested in serum-free medium, and
quiescent cells were switched to medium containing either 25 µM ZnSO4, CSF-1, or both, harvested daily,
and counted.
Expression Vectors--
The plasmid pMTCB6-MEK1* contains a
mutant, constitutively active murine MEK1 gene under the
control of a zinc-inducible metallothionein promoter (31). The plasmid
pSR
MSV-CSF-1R-tkneo was constructed by inserting human CSF-1R
cDNA downstream of the MSV promoter into the EcoRI site
of the pSR
MSV(EcoRI)-tkneo vector, provided by Dr.
Charles Sawyers, UCLA. The plasmid pRK5-Ras N17 was constructed by
inserting the murine Ha-Ras N17 cDNA into the NheI and
XhoI sites of pRK5 (32). The plasmids pCEP4-HA and
pCEP4-HA-ERK1 (K71R), which encode a dominant negative form of murine
ERK1 (K71R) (33), were provided by Dr. Melanie H. Cobb (University of
Texas Southwestern Medical Center, Dallas).
Transfection and Selection of Cell Lines--
The calcium
phosphate precipitation method (34) was used for transfection. The
plasmid pMTCB6-MEK1* (20 µg) was cotransfected with pJ6
-puro (5 µg), encoding puromycin resistance (35), into NIH-3T3 cells
expressing the mutant CSF-1R (Y809F). After selection for 3 weeks in
complete medium containing 7.5 µg/ml puromycin, MEK1*-positive
subclones were isolated by limiting dilution in 96-well microtiter plates.
For transient transfection, NIH-3T3 cells were cotransfected with
pSR
MSV-CSF-1R-tkneo together with either a control vector (pCEP4-HA), a vector encoding dominant negative Ras N17 (pRK5-Ras N17),
or a vector encoding dominant negative ERK1 (pCEP4-ERK1 (K71R)).
30 h after transfection, cells were growth arrested for 18 h
in serum-free medium, and quiescent cells were restimulated with
serum-free medium containing 10 ng/ml CSF-1 for 8 h or 14 h
as indicated. The transfection efficiency of NIH-3T3 cells was about
10% as determined by transfecting the cells with
-galactosidase expression plasmid and subsequent cellular staining of
-galactosidase (data not shown).
ERK Kinase Assay--
The ERK immune complex kinase assay was
performed as described previously (36) with minor modifications. Cells
were lysed in ERK lysis buffer (50 mM HEPES, pH 7.4, 1%
Triton X-100, 150 mM NaCl, 1 mM
phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 4 µg/ml aprotinin, 4 µg/ml pepstatin, 10 mM
p-nitrophenyl phosphate, 0.5 mM sodium
orthovanadate, 1 mM sodium fluoride, and 10 mM
-glycerophosphate). Lysates were clarified by centrifugation in an
Eppendorf microcentrifuge at 10,000 × g for 10 min,
and the protein concentration of the supernatant was measured with a
BCA protein assay kit (Pierce Chemical Co.). Cell lysates (400 µg of
protein) were mixed with 2 µg of either ERK1 (K-23) or ERK2 (C-14)
antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and 20 µl of
protein A-Sepharose beads 1:1 in lysis buffer (v/v) and incubated at
4 °C for 2 h. Beads were pelleted by centrifugation, washed
four times with ERK lysis buffer, two times with kinase reaction buffer
(50 mM HEPES, pH 7.4, containing 10 mM
MgSO4), and resuspended in 30 µl of kinase reaction
buffer. For the kinase reaction, the beads were incubated with 5 µg
of myelin basic protein (Sigma) and 50 µM ATP containing
10 µCi of [32P]ATP (Amersham Pharmacia Biotech) in 30 µl of kinase reaction buffer at 30 °C for 30 min. Reactions were
stopped by adding 1/3 volume of sample buffer (50 mM Tris,
pH 6.8, 2% SDS, 10% glycerol, 100 mM dithiothreitol,
0.1% bromphenol blue) and heating at 85 °C for 5 min. Labeled
proteins were resolved on denaturing polyacrylamide gels, which were
dried and subjected to autoradiography.
Northern Blotting--
Total cellular RNA (20 µg/sample) was
separated by formaldehyde-agarose gel electrophoresis and transferred
to nitrocellulose membranes as described previously (26). Membranes
were baked at 85 °C, blocked with Denhardt's solution (37),
hybridized with a 32P-labeled mouse c-myc
cDNA probe prepared by random priming (Boehringer Mannheim), and
washed membranes were subjected to autoradiography (26).
Immunoblotting--
To detect the c-Myc protein, 2 × 106 cells were lysed in 100 µl of gel sample buffer, and
cell lysates were boiled for 10 min. After brief sonication, the
proteins were resolved by SDS-polyacrylamide gel electrophoresis and
transferred to nitrocellulose membrane (Micron Separations,
Westborough, MA). Nonspecific binding sites were blocked by incubation
for 1 h at room temperature with Tris-buffered saline (25 mM Tris, pH 8.0, 150 mM NaCl, 2 mM
KCl) containing 3% nonfat dry milk. Filters were incubated with the
c-Myc antibody (06-340, Upstate Biotechnology, Lake Placid, NY) in
Tris-buffered saline and milk for 2 h at room temperature, and
sites of antibody binding were identified using horseradish
peroxidase-conjugated donkey anti-rabbit IgG followed by enhanced
chemiluminescence detection (ECL kit; Amersham Pharmacia Biotech).
Detection of cyclin D1 (72-13G-11), CDK4 (RY), human CSF-1R
(RA), Myc-tagged active MEK1 (MEK1*) (9E10), and actin
(C-11, Santa Cruz Biotechnology) with the indicated antibodies was
described previously (31, 38-40).
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RESULTS |
Induction of c-Myc by CSF-1 Requires Ras/MEK/ERK
Activation--
To address the role of MEK1/2 in CSF-1R-induced c-Myc
expression, Bac1.2F5, a macrophage-derived cell line that requires
CSF-1 for proliferation, was treated with the specific MEK1/2 inhibitor PD98059 (41, 42). Cells made quiescent by deprivation of CSF-1 for
16 h were pretreated with PD98059 or with Me2SO
solvent for 30 min and then restimulated with medium containing CSF-1
together with PD98059 or Me2SO. Treatment with 60 µM PD98059 blocked CSF-1R-induced activation of ERK1 and
ERK2 as measured by in vitro kinase assays (Fig.
1A). Analysis of total RNA by
Northern blotting demonstrated that the addition of 60 µM
PD98059 significantly impaired CSF-1-mediated induction of
c-myc expression, reducing c-myc
mRNA by 80% by 6 h of treatment (Fig. 1B,
lane 5 versus lane 3). In contrast,
c-myc mRNA levels were not significantly altered by
treatment with Me2SO (Fig. 1B, lanes
6 and 7). PD98059 also inhibited the induction of c-Myc
protein in response to CSF-1, whereas Me2SO had no
significant effect (Fig. 1C). In Bac1.2F5 macrophages, the
expression of CDK4 is dependent on the addition of CSF-1 to the culture
medium (39). CDK4 expression was unaffected by PD98059 (Fig.
1C), indicating that this inhibitor did not indiscriminately
block CSF-1 signaling.

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Fig. 1.
MEK1/2 activity is required for
CSF-1R-mediated c-Myc expression and S phase entry. Panel
A, quiescent Bac1.2F5 macrophages were left untreated ( ) or were
pretreated with 20, 40, or 60 µM PD98059 (PD)
or 0.3% Me2SO (DMSO) for 30 min. Cells were
restimulated with medium containing CSF-1 with or without the inhibitor
at the same concentration. ERK1 kinase activity was measured with
myelin basic protein as a substrate. 32P-Labeled proteins
were resolved on denaturing polyacrylamide gels, which were dried and
subjected to autoradiography. Panel B, quiescent Bac1.2F5
macrophages were left untreated ( ) or were pretreated with 60 µM PD98059 or 0.3% Me2SO for 30 min and were
restimulated with medium containing CSF-1 with or without the
inhibitor. Total RNA extracted at the indicated times after stimulation
was hybridized sequentially with 32P-labeled probes
specific for c-Myc and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) used for a loading control. Panel C,
c-Myc, CDK4, and actin protein levels were measured by immunoblotting
with c-Myc antibody (06-340), CDK4 antiserum (RY), or actin
antibody (C-11), and the sites of binding were visualized with ECL
reagent. Panel D, DNA synthesis was measured in cells
pretreated with or without the inhibitor and restimulated with CSF-1
for 18 h. BrdUrd was added to the culture medium between 12 and
18 h of CSF-1 treatment. The cells were fixed and immunostained
with BrdUrd antibody, and the percentage of BrdUrd-positive cells was
determined by fluorescence microscopy.
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Because c-Myc is essential for CSF-1-mediated S phase entry (26, 43),
the effect of these inhibitors on entry into the cell cycle was
determined. As expected, PD98059-treated quiescent macrophages failed
to enter S phase after CSF-1 stimulation, whereas Me2SO-treated cells were not inhibited (Fig.
1D). Similar results were obtained after PD98059 treatment
of NIH-3T3 fibroblasts engineered to express CSF-1R (data not shown).
Although PD98059 is a specific inhibitor of MEK1/2, it remained
possible that some of its inhibitory effects might be caused by
inhibition of other as yet unidentified MEKs. Given that Ras signals
via MEKs to activate ERKs, we assessed the effects of dominant negative
forms of Ras (N17) and ERK1 (K71R) on c-Myc induction in response to
CSF-1. NIH-3T3 cells were transiently transfected with expression
plasmids encoding wild-type human CSF-1R alone or together with
plasmids encoding Ras (N17) or ERK1 (K71R). Transfected cells growth
arrested by serum depletion were stimulated with serum-free medium
containing human CSF-1 as the only mitogen (Fig.
2A). Those transfected with
CSF-1R alone expressed c-Myc (Fig. 2A, lane 2)
and began to enter S phase by 14 h after CSF-1 stimulation (Fig.
2B, lane 2). In contrast, coexpression of CSF-1R
with the dominant negative Ras (N17) or ERK1 (K71R) significantly
impaired CSF-1R-mediated induction of c-Myc (Fig. 2A,
lanes 3-6), and most cells failed to enter S phase (Fig.
2B, lanes 3-6), even though comparable CSF-1R
levels were observed in all transfectants (Fig. 2A).
Together, these results support the view that signaling through the
Ras/MEK/ERK pathway is required for CSF-1-dependent
induction of c-Myc and S phase entry.

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Fig. 2.
Dominant negative Ras (N17) or ERK1 (K71R)
inhibits CSF-1R-mediated c-Myc expression. Panel A,
NIH-3T3 cells were transfected with 30 µg of empty vector pSR MSV
( ), with 10 µg of pSR MSV-CSF-1R plus 20 µg of empty vector
(lane 2), with 10 µg of pSR MSV-CSF-1R plus 10 µg
(lane 3) or 20 µg (lane 4) of pRK5-Ras (N17),
or 10 µg (lane 5) or 20 µg (lane 6) of
pCEP4-HA-ERK1 (K71R). 30 h post-transfection, cells were serum
starved and then restimulated with CSF-1 for 8 h. Cell lysates
were assayed for c-Myc, CDK4, and CSF-1R protein levels by
immunoblotting with c-Myc antibody (06-340), CDK4 antiserum
(RY), or CSF-1R antiserum (RA), respectively.
Panel B, DNA synthesis was measured in serum-starved cells
restimulated with CSF-1 for 14 h with BrdUrd. Cells were fixed and
immunostained with BrdUrd antibody, and the percentage of
BrdUrd-positive cells was determined by fluorescence microscopy.
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MEK1 Complements Defective Mitogenic Signaling by Mutant CSF-1R
(Y809F)--
NIH-3T3 cells that stably express a mutant CSF-1R (Y809F)
that is partially defective in its kinase activity fail to induce c-Myc
in response to CSF-1 stimulation (26). After stimulation with CSF-1,
activation of the wild-type receptor induced a rapid transient peak of
ERK1 activation followed by a lower level of activation which persisted
in the presence of CSF-1 (Fig. 3,
A and B). By contrast, much weaker activation of
ERK1 was seen in cells expressing equivalent levels of mutant CSF-1R
(Y809F) (Fig. 3, A and B).

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Fig. 3.
ERK1 activation is impaired in CSF-1R
(Y809F)-mediated signaling. Quiescent NIH-3T3 cells expressing
wild-type (WT) or mutant (Y809F) CSF-1R were stimulated with
serum-free medium containing 10 ng/ml human CSF-1 for the indicated
times, and ERK1 kinase activity was measured with myelin basic protein
(MBP) as a substrate. 32P-Labeled proteins were
resolved on denaturing polyacrylamide gels, which were dried and
subjected to autoradiography.
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To assess whether MEK1 might restore signaling by CSF-1R (Y809F), an
expression plasmid encoding a constitutively active, Myc-tagged form of
MEK1 (designated MEK1*) expressed under the control of a zinc-inducible
metallothionein promoter was introduced into NIH-3T3 cells that stably
express mutant CSF-1R (Y809F). Several individual clones were selected
by limiting dilution (31). In quiescent serum-starved cells, MEK1*
protein was not detected using an antibody to the Myc-epitope tag (Fig.
4A, lane 1). Zinc treatment induced high levels of MEK1* protein expression, which were
readily detectable by 6 h of zinc treatment (Fig. 4A,
lane 3). In response to MEK1* expression, ERK1 activity and
cyclin D1 expression were induced as reported previously (Fig.
4A) (see also Ref. 31). In contrast, MEK1* activation was
insufficient to induce c-Myc expression (Fig. 4A,
lanes 1-5). As expected, high levels of c-Myc were induced
by FBS stimulation (Fig. 4A, lanes 6-10),
indicating that the Myc response was specifically refractory to
signaling via CSF-1R (Y809F).

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Fig. 4.
Ectopic expression of MEK1* restores c-Myc
expression mediated by mutant CSF-1R (Y809F). Panel A,
serum-starved NIH-3T3 cells harboring mutant CSF-1R (Y809F) and
inducible MEK1* were stimulated with ZnSO4 or FBS and
assayed at the indicated times thereafter for MEK1*, c-Myc, cyclin D1,
and CDK4 protein levels by immunoblotting with the respective
antibodies. Panel B, serum-starved NIH-3T3 cells harboring
mutant CSF-1R (Y809F) and inducible MEK1* were treated either with
CSF-1 alone or with both ZnSO4 and CSF-1 for the indicated
times. c-Myc and CDK4 protein levels were determined by immunoblotting
with c-Myc antibody (06-340) and CDK4 antiserum (RY).
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Therefore, whereas CSF-1R-mediated c-Myc expression is dependent on
MEK1/2 activity (Figs. 1 and 2), induction of c-Myc appears to require
CSF-1R-mediated signals in addition to those mediated by the MEK/ERK
pathway. To explore this issue further, NIH-3T3 cells coexpressing
CSF-1R (Y809F) and inducible MEK1* were serum starved and restimulated
with medium containing CSF-1 without or with zinc. Treatment with both
zinc and CSF-1 induced c-Myc expression (Fig. 4B,
lanes 6-10), whereas, as expected, CSF-1 treatment alone
did not (Fig. 4B, lanes 1-5) (24). High levels of c-Myc protein were detected 6 h after stimulation with zinc plus CSF-1 (Fig. 4B, lanes 6-10), correlating
with the onset of MEK1* expression in response to zinc treatment alone
(Fig. 4A, lanes 1-5). However, in the presence
of both CSF-1 and zinc, the levels of Myc achieved were still lower
than those detected in response to FBS stimulation, again implying that
multiple CSF-1R signaling pathways cooperate in Myc induction.
Ectopic expression of either c-Myc or D-type cyclins in NIH-3T3 cells
expressing mutant CSF-1R (Y809F) resensitizes them to the mitogenic
effects of CSF-1 and enables them to proliferate continuously (26, 27).
MEK1/2 activation was shown to be required for
mitogen-dependent c-Myc and cyclin D1 expression (44-48).
Although sufficient to induce cyclin D1 expression (see Fig.
4A), zinc-regulated expression of MEK1* alone does not
induce S phase entry (31). To address whether zinc-inducible MEK1*
could rescue S phase entry and proliferation of cells expressing mutant
CSF-1R (Y809F) in response to CSF-1 stimulation, NIH-3T3 cells stably
transfected with CSF-1R (Y809F) and zinc-inducible MEK1* were serum
starved and restimulated with CSF-1, zinc, or both. As expected,
NIH-3T3 cells expressing the wild-type receptor entered S phase in
response to CSF-1 stimulation, whereas CSF-1-stimulated cells
expressing the mutant CSF-1R (Y809F) failed to enter S phase (Fig.
5A). However, approximately
65% of cells harboring both CSF-1R (Y809F) and inducible MEK1* were
able to enter S phase by 18 h (Fig. 5A) and proliferate (Fig. 5B) in medium containing both zinc and CSF-1.
Consistent with the observations above that FBS was more efficient than
CSF-1 plus zinc in inducing Myc in these cells, their growth rate
(doubling time ~60 h) (Fig. 5B) was significantly slower
than cells grown in FBS (doubling time, ~24 h). NIH-3T3 cells
harboring the wild-type receptor show no such impairment in response to
CSF-1 (23). Therefore, even though MEK1*- and CSF-1R
(Y809F)-dependent signals can cooperate to rescue cell
proliferation in response to CSF-1, other receptor-mediated signals are
required for maximal CSF-1-induced cell proliferation.

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Fig. 5.
Ectopic expression of MEK1* rescues S phase
entry and proliferation of cells expressing mutant CSF-1R (Y809F).
Panel A, serum-starved NIH-3T3 fibroblasts expressing
wild-type (WT) and mutant (Y809F) CSF-1R were either left
untreated ( ) or were stimulated with CSF-1 (+) for 18 h. NIH-3T3
fibroblasts coexpressing CSF-1R (Y809F) and inducible MEK1* were either
untreated ( ) or treated for 18 h with CSF-1 alone,
ZnSO4 alone, or both. Between 12 and 18 h of
treatment, BrdUrd was added to the culture medium. Cells were fixed and
immunostained with BrdUrd antibody, and the percentage of
BrdUrd-positive cells was determined by fluorescence microscopy.
Panel B, NIH-3T3 fibroblasts coexpressing CSF-1R (Y809F) and
inducible MEK1* were seeded on culture plates for 16 h, growth
arrested in serum-free medium, and then treated with serum-free medium
containing ZnSO4, CSF-1, or both. Cells were harvested
daily and counted.
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DISCUSSION |
Our results strongly support the concept that Ras/Raf/MEK/ERK
signaling is an obligate pathway for growth factor receptor-mediated c-Myc induction (20). Experiments utilizing the MEK1/2 inhibitor PD98059 as well as dominant negative forms of Ras (N17) and ERK1 (K71R)
indicate that activation of Ras and MEK/ERK is necessary for c-Myc
expression in response to CSF-1. However, signaling through this
pathway is insufficient to guarantee c-Myc induction because the
ectopic expression of enzymatically active MEK1* in quiescent cells did
not induce c-Myc expression in the absence of other cooperating
signals. When cells coexpressing both CSF-1R (Y809F) and MEK1* were
stimulated with CSF-1, c-Myc was induced, and the cells entered S
phase. Although active MEK1 rescued CSF-1R (Y809F)-mediated c-Myc
expression and mitogenesis, the level of c-Myc induction and rate of
cell proliferation remained below those obtained using cells expressing
wild-type CSF-1R alone. Together, the results imply that several
CSF-1R-mediated signaling pathways cooperate with MEK/ERK to induce
c-Myc maximally and drive entry into S phase.
Our findings are generally consistent with a previous report that
overexpression of a dominant negative form of Raf-1 (Raf-1-C4B) can
reduce serum-mediated c-Myc expression (20). Moreover, c-Myc induction
by v-Abl, an oncogenic tyrosine kinase, requires functional Ras and
Raf-1 (19). However, there are also discrepancies between our results
and those of others. Although regulated expression by tamoxifen of a
fusion protein between the estrogen receptor and Raf-1 (Raf-ER) was
sufficient to induce c-Myc expression and S phase entry in NIH-3T3
fibroblasts (20), our results would suggest that collateral signals in
addition to MEK/ERK activation are required for c-Myc expression. One
possibility is that Raf-ER might activate pathways in addition to
MEK/ERK signaling. For example, cdc25A, a tyrosine phosphatase involved
in the regulation of cell proliferation, has been implicated in Raf-1-
but not MEK1/2-mediated signaling (49). Constitutive expression of
active MEK1 in NIH-3T3 fibroblasts can lead to serum-independent growth
(50), and ectopic expression of active MEK1 under the control of the
estrogen receptor (MEK1-ER) in these same cells resulted in accelerated
S phase entry (51). In contrast, our data indicated that inducible
expression of active MEK1 by zinc in the absence of any other growth
factors in the medium was not sufficient to promote S phase entry (31). The different levels of active MEK1 achieved in the transfected cell
lines might explain the discrepancies between the latter sets of experiments.
Although phosphoinositol 3-kinase is activated quickly in response to
CSF-1, its overall activity is not altered in response to CSF-1R
(Y809F)-mediated signaling (24). Therefore, it is unlikely to be a
candidate for the second signal. Moreover, inhibition of its activation
with wortmannin had no effect on CSF-1R-mediated c-Myc expression (data
not shown). Microinjection of dominant negative Src constructs or
neutralizing antibodies to Src family kinases has shown that they are
required for DNA synthesis in response to CSF-1 or platelet-derived
growth factor (52). Src family kinases associate with and are activated
by growth factor receptors via their Src homology domain-2 and are
substrates for the receptor kinases (53, 54). Src family kinases can
also activate a pathway that leads to the transcription of
c-myc (13). Specifically, constitutively expressed c-Myc
rescues a G1 block elicited by a dominant negative form of
Src, whereas Fos and Jun rescue a G1 block induced by
dominant negative Ras. This has led to the suggestion that Src family
kinases induce c-Myc in a Ras-independent fashion (13).
Even though the downstream targets of Src family kinases have not been
well characterized, the importance of the Ras/Raf/MEK/ERK signal
pathway in transmitting Src-mediated signals has been demonstrated (55,
56). For example, transformation of fibroblasts by oncogenic Src is
blocked by inhibiting Ras function with anti-Ras antibody or by
overexpression of GTPase-activating protein (57, 58). The
Ras/Raf/MEK/ERK pathway is also required for the transcriptional induction of several Src targets including the mitogen-responsive transcription factor Egr-1 (59). Therefore, it is likely that the
induction of c-Myc by the Src family kinases is at least in part
dependent on Ras function.