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INTRODUCTION |
In addition to being necessary for proliferation, colony
stimulating factor-1 (CSF-1)1
or macrophage CSF is absolutely required for the continued survival of
certain populations of cells of the monocyte/macrophage lineage (1).
Several lines of evidence suggest that the maintenance of survival and
the stimulation of proliferation are mediated by either different
signaling pathways activated by the CSF-1 receptor or require different
thresholds of activation of similar pathways (2-4). Advances have been
made in describing the signal transduction molecules activated in
response to CSF-1 stimulation of macrophages, although the details of
the pathways leading to their activation are still in many cases
unresolved (5). The role of the CSF-1 receptor in controlling passage
through the G1 phase of the cell cycle has been relatively
well studied, and it has been shown, for example, for cell cycle
progression that continued stimulation by CSF-1 is required for the
synthesis of D-type cyclins as is the up-regulation of
c-myc (6-8). Relatively little, however, is known about the
signal transduction pathways activated by CSF-1 involved in maintaining
cell survival.
Recent research has suggested roles for different members of the MAP
kinase family (ERKs, p38/(CSBP), JNK (stress-activated protein kinase))
in promoting either cell survival and/or proliferation, in response to
growth factor stimulation, or apoptosis, in response to various stress
stimuli. There are many examples where enhanced ERK activity has been
implicated in growth factor-mediated cell survival and/or proliferation
(for example, see Ref. 9 for review); significant downstream responses
of ERK-dependent signal transduction pathways that have
been proposed to be important for cell cycle progression are cyclin D1
and c-myc transcription, including in CSF-1-stimulated
NIH-3T3 cells expressing the CSF-1 receptor (c-Fms) (Ref. 10 and
references therein). The relationship between ERK activation and other
responses in macrophages is, however, much more complicated than the
above discussion suggests. In this cell type, activation of ERKs has
not been found to correlate strongly with the level of subsequent DNA
synthesis (11, 12), and there is some evidence to suggest that there
may be Ras-independent control over ERK activity (5). Also, in
macrophages, elevated intracellular cAMP enhances CSF-1-stimulated ERK
activity while suppressing the corresponding rise in cyclin D1
mRNA, suggesting a possible dissociation (12).
The p38 and JNK cascades are primarily activated by various
environmental stresses: proinflammatory cytokines (TNF
and
interleukin-1), osmotic shock, ultraviolet radiation, heat shock, x-ray
irradiation, hydrogen peroxide, and protein synthesis inhibitors (for
review, see Ref. 13). They have also been proposed to be involved in the regulation of apoptosis, because, for example, overexpression of
kinases that activate them resulted in the induction of apoptosis (14,
15), withdrawal of a growth factor (nerve growth factor) from PC-12
cultures can result in a transient activation of the p38 and JNK
families (14), and inhibition of p38 MAP kinase activity can suppress
apoptosis (16). Contrary to its role in cellular stress responses, it
has recently been reported that CSF-1 activates p38 activity in a
myeloid cell line, and a role for this enzyme in hemopoietic cell
proliferation was postulated (17). p38 also has a well established role
in cytokine production by lipopolysaccharide (LPS)-activated
macrophages (18). However, there has been controversy as to whether
CSF-1 is a strong stimulator of cytokine production in macrophages
relative to stimuli such as LPS (for review, see Ref. 19). Reports from
our laboratory and those of others have shown that CSF-1 treatment of
monocytes and macrophages does not lead to high levels of secreted
proinflammatory cytokine production (19, 20). It is therefore of
interest to know whether CSF-1 is a potent stimulator of p38 activity
in cells of the macrophage lineage and what the significance of such stimulation may be.
From the above background information, it is clear that the role of the
MAP kinase family in cell survival and/or proliferation is cell type-
and/or stimulus-specific. It has been maintained before (5) that the
relevance of signal transduction pathways to the cell biology of a
growth factor such as CSF-1 must be analyzed in each individual case
and is best studied in cells where the receptor (c-Fms) is normally
expressed and preferably in primary cultures of such cells rather than
in immortalized cell lines, where proliferation and survival pathways
are, ipso facto, altered. We therefore have examined the
roles of the MAP kinase family members, including potentially dependent
pathways, in CSF-1-treated murine bone marrow-derived macrophages
(BMM); because BMM depend upon CSF-1 for survival (1), we also examined
the activities of the MAP kinase family members upon CSF-1 withdrawal.
We report evidence for some involvement of ERK activity in
CSF-1-stimulated BMM DNA synthesis but, interestingly, and in contrast
to reports in fibroblasts ectoptically expressing c-fms, no
evidence for its involvement in the control of cyclin D1 or
c-myc mRNA expression. p38 activity was weakly induced
by CSF-1. No data were obtained to support a role for p38 or JNK-1 in
BMM apoptosis resulting from CSF-1 withdrawal.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Bone marrow-derived macrophages (BMM) were
obtained by culturing bone marrow cells from femurs of CBA mice in the
presence of CSF-1, as described previously (21). Cells were cultured in
RPMI, 15% fetal bovine serum (FBS), 30% L-cell-conditioned medium (a
source of CSF-1), routinely growth-arrested by washing three times in
phosphate-buffered saline, and then cultured in RPMI, 10% FBS for
18 h or for times indicated.
Immunoprecipitations and Kinase Assays--
Cells were cultured
and stimulated as described in the text, then lysates were prepared
using a Triton-based lysis buffer supplemented with protease and
phosphatase inhibitors (11). Extracts were precleared with 20 µl of
protein A-Sepharose, and samples containing equal protein were
immunoprecipitated at 4 °C overnight with 0.8 µg/ml anti-ERK-1 or
anti-JNK-1 antibody or with 17 µg/ml anti-p38 antibody. Immune
complexes were collected using 20 µl of protein A-Sepharose and
washed with 4 × 1 ml of Triton-based lysis buffer (plus
phosphatase inhibitors) and 1 × 1 ml of kinase assay buffer.
ERK-1 kinase assays were performed as described elsewhere (11). p38
kinase assays were performed in 10 µl of 25 mM Hepes, pH
7.4, 10 mM MgC12, 1 mM vanadate, 20 mM NaF, 0.5 mg/ml myelin basic protein,
[
-32P]ATP (adjusted to a specific activity of 3.7 × 108 MBq/mol). JNK-1 assays were performed after the
addition of 10 µl of assay mix (without myelin basic protein) and 10 µl of c-Jun (1-169)-GST-agarose conjugate. Reactions were for 30 min
at 30 °C and terminated by the addition of SDS sample buffer.
Reaction mixtures were resolved by SDS-polyacrylamide gel
electrophoresis, and the relative extent of substrate phosphorylation
was determined using a Fujix BAS1000 phosphorimager.
The above antibodies were tested for specificity in our system by
carrying out immunoprecipitations using BMM lysates and immunoblotting
with the other MAP kinase antibodies. By this criterion, none of the
antibodies cross-reacted with members of the other MAP kinase families
studied. The measured ERK-1 and p38 activities were proportional to
lysate protein concentration and linear over 30 min. The JNK-1 activity
immunoprecipitated under the conditions used was dependent on, but not
strictly proportional to, lysate concentration and was completely
abolished by 0.4 µg/ml peptide used as epitope to raise the antibody.
Western Analysis--
Cell extracts in Triton-based lysis buffer
containing equal amounts of protein were resolved by SDS-polyacrylamide
gel electrophoresis. Proteins were transferred to nitrocellulose in 25 mM Tris, 192 mM glycine, 20% (v/v) methanol,
and membranes were blocked for 1 h with 5% nonfat milk in
Tris-buffered saline containing 0.5% Triton X-100 (TBST). Membranes
were probed with relevant antibodies at 1:1000 in 0.5% nonfat milk,
TBST, washed extensively with TBST, and probed with swine-anti-rabbit
horseradish peroxidase (1:10000) in 0.5% milk, TBST. Bound secondary
antibody was detected by enhanced chemiluminescence.
Protein Determinations--
Protein content of cell extracts was
determined by the method of Lowry following coprecipitation of protein
with 0.5% sodium deoxycholate using defatted bovine serum albumin as standard.
Apoptosis in BMM Cultures--
3 × 106
cells/time point were lysed in 360 µl of 0.6% SDS, 0.1% EDTA (pH
8.0) and incubated at room temperature for 10 min, 21 µl of 5 M NaC1 was added, and the lysate was incubated at 4 °C
overnight. The low molecular weight DNA was extracted (22) and analyzed
by 2% agarose gel electrophoresis. Any apoptosis indicated by DNA
laddering was confirmed by the terminal
deoxynucleotidyltranserase-mediated dUTP nick end labeling (TUNEL)
assay and characteristic apoptotic morphology as described previously
(23).
DNA Synthesis and Cell Viability--
Tritiated thymidine
([3H]TdR) incorporation was used as a measure of DNA
synthesis as before (24). To measure cell viability, BMM were cultured
in 24-well plates in RPMI, 10% FBS or RPMI, 15% FBS, 30%
L-cell-conditioned medium. Inhibitors were added at indicated
concentrations using Me2SO as a vehicle at a final concentration of 0.5% (v/v). No effect on survival or DNA synthesis was noted with Me2SO at this concentration. Viable cell
numbers were then determined by incubation of cultures for 2 h in
the presence of MTT (0.8 mg/ml). Reduced MTT dye was solubilized
overnight by the addition of 0.5 volumes of acidified 15% Triton
X-100, and the amount was assessed at 595 nm using a Bio-Rad model 450 microplate reader.
mRNA Levels--
Approximately 1 × 107
cells/extraction were used. Cells were lysed in 600 µl of RNA
extraction buffer (5 M guanidine thiocyanate, 10 mM Tris pH 7.6, 10 mM EDTA), and RNA was
extracted and probed as described before (24). cDNA probes to
either c-myc, v-fos, or cyclin D1 were
radiolabeled with [
-32P]dATP (Bresatec) using nick
translation and used to probe the blots. Control hybridization with a
2-microglobulin probe was used to determine RNA loading and quality.
Cytokines and Other Reagents--
Human recombinant CSF-1 was a
gift from Chiron Corp. (Emeryville, CA). Other reagents used were from
the following commercial sources: LPS, purified from Escherichia
coli strain 0111:B4, Difco; myelin basic protein, Sigma;
[methyl-3H]thymidine (3Mbq/mmol), Hybond C
nitrocellulose, Enhanced Chemiluminescence (ECL) Western blotting
detection reagents and Hyperfilm-ECL, Amersham Pharmacia Biotech;
stabilized [
-32P]ATP (148 TBq/mmol), Bresatec
(Adelaide, Australia.); protease inhibitors, Roche Molecular
Biochemicals. c-Jun-GST fusion protein was expressed in E. coli, strain MC1061, and purified by glutathione agarose
chromatography. cDNA probes were as before (24, 25).
Antibodies and Inhibitors--
The following antibodies were
used for immunoprecipitations and in the kinase assays: anti-ERK-1
(C-16, Santa Cruz Biotechnology), anti-p38/CSBP (obtained from J. C. Lee; recognizing both CSBP1 and CSBP2). The following antibodies
were used for Western blotting: anti-ERK (K23 (Santa Cruz), which
recognizes both ERK-1 and -2), anti-JNK-1 (C17; Santa Cruz), and
anti-phosphorylated ERK (New England Biolabs, which recognizes both
pERK-1 and pERK-2). SB203580 (lot AJK-19080-74) and SKB202190 (lot
10148-25A) were gifts from Smith Kline Beecham (King of Prussia, PA)
and Amgen Inc. (Boulder, CO), respectively. Secondary
antibody-horseradish peroxidase conjugates were from DAKO (Glostrup,
Denmark). PD98059 was from New England Biolabs Inc, Beverly, MA).
LPS Contamination of Culture Media and Reagents--
All
practical precautions were taken for minimizing endotoxin
contamination. Media and cytokines were made using pyrogen-free water
(Delta West Pty Ltd., Bentley, Western Australia), and endotoxin levels
in media and cytokines were routinely monitored using the Limulus
lysate assay (Commonwealth Serum Laboratories, Parkville, Australia).
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RESULTS |
ERK Activity in Cycling and Noncycling BMM--
We have previously
shown that addition of CSF-1 to noncycling BMM causes an increase in
ERK-1 and ERK-2 activities with a peak activity at around 5 min (11).
We now show in Table I that cycling BMM,
i.e. BMM maintained in the presence of saturating concentrations of CSF-1, have an approximately 2-fold higher ERK-1 activity than factor-deprived BMM, suggesting that the large and transient increase measured when growth-arrested cells are stimulated by CSF-1 ( Ref. 11 and Table I) is paralleled by an elevated level of
activity in cycling versus growth-arrested BMM. LPS also stimulates ERK-1 activity in BMM via a kinetically distinguishable pathway (11). The activity of ERK-1 15 min after stimulation of
growth-arrested cells with LPS is also shown for comparison. The
observation that CSF-1-deprived BMM have a lower ERK-1 activity than
cycling cells was verified by measuring the kinetics of loss of ERK-1
activity after CSF-1 withdrawal (Fig.
1a). In other experiments, we
found that the level of ERK activity per unit cell protein was
maintained between 24 and 36 h after withdrawal of CSF-1 from the
culture medium (data not shown). It should be noted that in these
experiments the ERK-1 activities were measured in lysate samples
containing equal amounts of protein; furthermore, we verified that the
ERK-1 activity was not affected by cell density in these experiments
because a similar activity was measured in cycling cultures harvested
at the beginning and at the end of the 18-h period (data not
shown).
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Table I
Relative ERK-1 activities in BMM
Lysates were prepared from exponentially growing BMM cultures
(Cycling), from cultures that were growth-arrested by 18 h of
incubation in RPMI, 10% FBS (growth-arrested), and from the
growth-arrested population stimulated with CSF-1 (5000 units/ml) or LPS
(100 ng/ml) for 5 min and 15 min, respectively. 50 µg of precleared
lysates were immunoprecipitated and assayed for ERK-1 activity
("Experimental Procedures"). Kinase activities are expressed
relative to that found in cycling cells, and the data are mean
values (±S.E.) for triplicate determinations.
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Fig. 1.
ERK-1 activity and apoptosis following CSF-1
removal from BMM. a, ERK-1 activity. Cycling BMM
cultured in 10-cm dishes were washed three times to remove CSF-1 and
then transferred to RPMI, 10% FBS for the indicated times. ERK-1
activity was measured in cell lysates as in Table I. Kinase activity is
expressed relative to that found in cycling cells; the data are
expressed as mean values (±S.E.) for triplicate determinations and are
representative of three independent experiments. b,
apoptosis. Cycling BMM in 10-cm dishes were washed three times to
remove CSF-1 and then transferred to RPMI, 10% FBS for 24 h. DNA
was extracted from the cycling (t = 0) and
CSF-1-deprived populations and analyzed by agarose gel electrophoresis
("Experimental Procedures"). Gels were stained with ethidium
bromide and photographed under UV light.
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It is generally assumed that removal of growth factor from hemopoietic
cells leads to cell death via apoptosis (26). We found that removal of
CSF-1 from cycling BMM led to cell death accompanied by the DNA
laddering that is often characteristic of apoptosis (Fig.
1b). We also observed apoptotic macrophages within
phagolysosomes of neighboring cells, which is to be expected given the
known phagocytic activity of macrophages toward cells undergoing
apoptosis. This removal makes quantitation of apoptotic BMM in such
cultures difficult; however, quantitation of cells possessing the
morphologic and histological characteristics of late stage apoptosis
both by bisbenzimide or TUNEL staining ("Experimental Procedures")
indicated that there were approximately 0.3% apoptotic BMM in cycling
BMM cultures with this proportion increasing to 8-10% after 24 h
of growth factor removal (data not shown). These data indicate that at
least a proportion of the CSF-1-deprived BMM are dying via an apoptotic pathway.
It has previously been shown in nerve growth factor-treated PC-12
cells, for example, that growth factor withdrawal results in loss of
ERK activity and cell death by apoptosis, leading to the suggestion
that ERK activity is important for growth factor-mediated cell survival
(14, 27). A specific inhibitor of MEK activation, PD98059, is widely
used to determine the relevance of a MEK/ERK pathway to cellular
function (28); we have found previously that a 30-min preincubation of
BMM with this compound before stimulation with CSF-1 inhibited the
activation of both ERK-1 and ERK-2 with an IC50 of 5-10
µM (Ref. 12 and data not shown). We therefore determined
whether PD98059 would reverse the ability of CSF-1 to suppress
apoptosis in BMM. However, it can be seen in Fig. 2 that the inhibitor of MEK activation
did not do this, suggesting that ERK activity per se is not
critical for the maintenance of BMM viability by CSF-1. In support of
this we also found that incubation of BMM with 50 µM
PD98059 did not accelerate the loss of viability (measured using MTT
dye reduction) following CSF-1 withdrawal (data not shown).

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Fig. 2.
Effect of PD98059 and 8BrcAMP on the reversal
of BMM apoptosis by CSF-1. Low molecular weight DNA was isolated
from growth-arrested BMM restimulated for 24 h with CSF-1 (5000 units/ml) in the absence or presence of PD98059 (50 µM),
8BrcAMP (1 mM), or their combination, as indicated. DNA was
separated by electrophoresis on a 2% agarose gel then visualized by UV
light and photographed. The figure is a representative of four separate
experiments.
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We also examined the effect of 8BrcAMP, an agent that can suppress a
number of CSF-1-induced responses in BMM while activating the ERK
activity (12, 24). We see in Fig. 2 that 8BrcAMP by itself cannot
prevent the reversal of apoptosis by CSF-1 but interestingly, when
added with PD98059, can do so. This finding suggests that multiple
pathways may be governing the maintenance of BMM viability by CSF-1 and
that at least one of these pathways is ERK-dependent (see below).
We have found previously that expression of ERK activity in BMM in
response to a particular stimulus did not always correlate with the
subsequent appearance of a DNA synthesis response (11). This
observation does not necessarily mean that ERK activity is not required
for DNA synthesis to occur in BMM, especially because we have shown
above (Table I; Fig. 1a) that ERK-1 activity in cycling BMM
was higher than that in CSF-1-deprived cells. To test this possibility
directly we examined the effect of PD98059 on DNA synthesis in
CSF-1-treated BMM. We see in Table II
that PD98059 inhibited CSF-1-stimulated DNA synthesis in BMM measured
20 h after the addition of both CSF-1 and the inhibitor to
growth-arrested BMM, suggesting that a MEK/ERK pathway may play a role
in DNA synthesis control in this system.
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Table II
Effect of PD98059 on CSF-1-stimulated BMM DNA synthesis
Growth-arrested BMM (control) were treated with CSF-1 (5000 units/ml)
in the absence or presence of PD98059 (50 µM). DNA
synthesis was measured by pulsing cells with [3H]TdR at
20 h and harvesting at 22 h ("Experimental Procedures").
Values are means of quadruplicate cultures (±S.D.) and are from a
representative experiment that was repeated four times.
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MEK/ERK-dependent Pathways in CSF-1-treated
BMM--
Previous studies in CSF-1-treated macrophages have examined
the pathways governing CSF-1-dependent activation of ERK
activity (29, 30); pathways downstream of the enhanced ERK activity that may be involved in, for example, the control of BMM DNA synthesis by CSF-1 have not been widely examined. Much evidence has implicated both cyclin D1 and c-Myc as important regulators of the mammalian cell
cycle (31), including in CSF-1-treated macrophages (25, 32-34); cyclin
D1 and c-myc transcription have been shown to be dependent
upon upstream ERK activity in a number of cell systems, including
CSF-1-stimulated NIH3T3 cells ectopically expressing c-Fms (see Ref. 10
and references therein). We therefore examined whether PD98059
suppresses cyclin D1 and c-myc mRNA expression as part
of its inhibitory action in controlling CSF-1-stimulated BMM DNA
synthesis. Our data, however, show that PD98059, even at concentrations
as high as 100 µM, failed to inhibit the increase in
cyclin D1 and c-myc mRNA expression resulting from
stimulation of BMM with CSF-1 (Fig.
3a); in contrast, 8BrcAMP did,
as we and others have previously reported (33-35). These results
contrast with our earlier findings that CSF-1-stimulated
c-fos mRNA expression was inhibited by PD98059 (Fig.
3b (12)). These data suggest that a MEK/ERK pathway does not
govern CSF-1 stimulated cyclin D1 mRNA and c-myc
mRNA expression in macrophages and that regulation of the levels of
these mRNA species is different from that found in CSF-1-stimulated
NIH 3T3 cells (10) (see "Discussion"). The above data indicate
that, unlike 8BrcAMP, PD98059 does not suppress cyclin D1 or
c-myc mRNA expression as part of its mechanism in inhibiting DNA synthesis in CSF-1 treated BMM.

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Fig. 3.
Effect of inhibitors of ERK and p38 pathways
on CSF-1-stimulated mRNA expression. a, total RNA was
isolated from growth-arrested BMM or from BMM restimulated for 6 h
with CSF-1 (5000 units/ml) in the absence or presence of PD98059 (100 µM) or 8BrcAMP (1 mM), as indicated. The
three autoradiograms represent the same Northern blot hybridized
successively with radiolabeled cDNA fragments of cyclin D1,
c-myc, and (as a control for RNA loading)
2-microglobulin. b, total RNA was isolated from
CSF-1-deprived BMM or from BMM restimulated for 30 min with CSF-1 (5000 units/ml) in the absence or presence of PD98059 (100 µM)
or SB202190 (10 µM), as indicated. The two autoradiograms
represent the same Northern blot hybridized successively with
radiolabeled cDNA fragments of c-fos and
2-microglobulin, the latter as a control for RNA loading.
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p38 Activity in Cycling and Noncycling BMM--
p38 activity has
been implicated in the control of cytokine biosynthesis in
LPS-stimulated monocytes/macrophages (18). Activation of p38 by CSF-1,
measured by its tyrosine phosphorylation status on a Western blot, has
recently been reported in a murine myeloid cell line (17). Given the
evidence that CSF-1 is a poor stimulator of monocyte/macrophage
inflammatory cytokine production when compared with LPS (Ref. 20 and
references cited in Ref. 19), we decided to compare p38 activity
directly in BMM stimulated with CSF-1 and, as a comparison, LPS. For
this purpose we used a quantitative immunoprecipitation kinase assay
("Experimental Procedures"). Our data (Fig.
4a) show that cycling BMM have
about a 2-fold higher p38 activity than CSF-1-deprived cells and that
when added back, CSF-1 is a relatively weak activator of p38 compared
with LPS. This contrasts with the activation of ERK-1 (and ERK-2) in
macrophages, where we have shown that CSF-1 and LPS activate this
enzyme to similar extents (Table I and Ref. 11). The kinetics of
activation of p38 by LPS and CSF-1 are different, with LPS activation
being maximal at 15 min whereas the small amount of activation observed with CSF-1 peaks earlier at around 5 min (data not shown). These time
courses are identical to those that we have observed previously for ERK
activation by these stimuli (11).

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Fig. 4.
p38 activity in BMM and its lack of
involvement in apoptosis. a, lysates were prepared from
exponentially growing cultures, from cultures that were growth
factor-deprived by an 18-h incubation in RPMI, 10% FBS, and from the
CSF-1-deprived cells stimulated again with CSF-1 (5000 units/ml) or
with LPS (100 ng/ml) for 5 min and 15 min, respectively. 100 µg of
precleared lysates were immunoprecipitated and assayed for p38 activity
("Experimental Procedures"). The degrees of substrate (myelin basic
protein (MBP)) phosphorylation in single lanes of acrylamide
gels are shown. Kinase activities are expressed relative to those found
in cycling cells (arbitrarily given a value of 1), and the values shown
are means (±S.E.) from triplicate determinations. b,
cycling BMM were washed three times to remove CSF-1 and then
transferred to RPMI, 10% FBS for the indicated times. Lysate aliquots
containing 100 µg of protein were assayed for p38 activity as in
a, and kinase activities are expressed relative to those
found in cycling cells; the data are expressed as mean values (±S.E.)
from triplicate determinations and are representative of three
independent experiments. c, CSF-1 was withdrawn from cycling
BMM, which were then either untreated ( ) or treated ( ) with 10 µM SKB202190 in 2% v/v Me2SO at the same
time as CSF-1 withdrawal. Viable cell numbers were determined using a
MTT reduction assay ("Experimental Procedures"). Data are mean
values (±S.E.) from triplicate cultures and are representative of two
experiments.
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In PC-12 cells, a 2-3-fold elevation in p38 activity preceded the
onset of apoptosis after nerve growth factor withdrawal (14). We
therefore followed p38 activity in macrophages deprived of CSF-1 over
an 18-h period, a time frame in which, given the time of onset of
apoptosis in BMM (Fig. 1b), we might expect by analogy (14)
to see increases in p38 activity. We show, however, that there is no
significant elevation of p38 activity following CSF-1 withdrawal from
cycling BMM (Fig. 4b). Similar results were obtained using
the CSF-1-dependent macrophage cell line, BAC1.2F5 (data
not shown). In BMM, no elevation in the p38 activity was noted if its
activity was monitored between 30 min and 3 h after CSF-1 removal
(data not shown). What we found instead is a loss of p38 activity
compared with the value for the cycling cells. It should be noted that
the p38 activities are expressed relative to cell protein in each
lysate and that they were not affected by cell density in these
experiments because similar activities were measured in cycling
cultures harvested at the beginning and at the end of the 18-h period
(data not shown). Therefore, for both primary cultures of macrophages
and a CSF-1-dependent macrophage cell line, withdrawal of
CSF-1 results in a decrease in p38 activity with no indication of the
involvement of p38 activation in the cell death that occurs when the
CSF-1 is removed.
To further test the above conclusion, we examined the effect of
specific inhibitors of this kinase on BMM survival. The phenolic triaryl imidazole derivatives SB203580 and SKB202190 have been shown to
inhibit cytokine production in human monocytes by binding selectively
to and inhibiting the enzymatic activity of p38 (18, 36); they have
been widely used in the investigation of the biological functions of
p38 (28). As a prelude to studying their effects on macrophage
survival, we tested the inhibitory activity and selectivity of these
compounds against p38 versus ERK-1 isolated from murine
macrophages. SKB202190 strongly inhibited p38 activity immunoprecipitated from BMM with an IC50 of about 45 nM (data not shown). By contrast, there was no effect on
ERK-1 activity at concentrations as high as 0.5 µM.
Although the IC50 for the inhibition of murine p38 was
slightly higher than the 10 nM reported for human p38,
there is still a strong selectivity with respect to the murine p38
member(s) of the MAP kinase family. This result also confirms the
specificity of the kinase assays used above ("Experimental
Procedures"). The effect of SKB202190 on BMM survival was assessed
using an MTT dye reduction assay. No significant effect on loss of
viability induced by CSF-1 withdrawal, assessed by MTT dye reduction,
was observed when the macrophages were cultured for up to 2 days in the
presence of 10 µM SKB202190 (Fig. 4c). Similar
results were obtained using SB203580 where we measured in
vitro an IC50 for murine p38 of about 20 nM (data not shown). These data reinforce the conclusion
arrived at above that p38 activity is not required for macrophage cell
death under conditions of growth factor deprivation. When the BMM were
cultured in the presence of SKB202190, administered under the same
conditions as the viability experiments, the cells became highly
vacuolated after 24 h, consistent with a role for p38 in
osmoregulation and indicating that, although viability was not
significantly affected, the drug is still active throughout the time
frame of the experiment.
Because cycling BMM have approximately 2-fold higher p38 enzymatic
activity per unit cell protein than growth-arrested BMM (Fig.
4a), we measured the effect of SKB202190 on BMM DNA
synthesis. The data presented in Fig. 5
show that there was an inhibitory effect of this compound on the
incorporation of [3H]TdR into DNA. This effect was
consistently observed at relatively high concentrations, and in five
independent experiments, inhibition of [3H]TdR
incorporation occurred with an IC50 of 4-5
µM. This high IC50 is comparable with what
has been reported for DNA synthesis inhibition in other murine cell
lines (37). We found that SKB202190 (5 µM) did not
prevent the CSF-1-induced increase in c-fos mRNA expression measured 30 min after CSF-1 addition to growth-arrested BMM
(Fig. 3b); in fact there appeared to be a slight increase in
the c-fos mRNA levels. The suppressive effect of PD98059
on c-fos mRNA induction, found previously (12), is shown
for comparison. However, in preliminary experiments, we found that the
addition of 5 µM SB202190 resulted in a decrease in
CSF-1-dependent induction of c-myc mRNA,
measured at 6 h, with no effect noted on cyclin D1 mRNA levels
measured at the same time (not shown).

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Fig. 5.
Effect of SKB202190 on BMM DNA
synthesis. CSF-1-deprived (growth-arrested) BMM cultures in
96-well plates were stimulated with CSF-1 (5000 units/ml) (solid
bars) or left without CSF-1 (open bars) in the absence
or presence of the indicated concentrations of SKB202190. DNA synthesis
was measured by pulsing cells with [3H]TdR at 20 h
and harvesting the cells at 22 h. Data represent means ±S.E. of
quadruple determinations in a typical experiment that was repeated a
further four times.
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JNK-1 Activity in BMM--
Enhanced JNK-1 activity has also been
implicated in apoptosis; for example, in that found in PC-12 cells
following nerve growth factor removal (14). JNK-1 activity was not
detected in BMM from which the CSF-1 had been removed nor after its
readdition for 5 min (data not shown), although robust activation by
100 ng/ml LPS and TNF
(used as positive controls) was detected. No statistically significant activity was measurable in cycling BMM, and
as for the p38 activity upon CSF-1 removal, no increase was detectable
over a 30-min-24 h period (n = 3; data not shown).
 |
DISCUSSION |
In the above studies we tested the effects on BMM functions of
PD98059, a highly specific inhibitor of MEK activation widely used to
determine the involvement of a MEK/ERK pathway in particular cell
functions (28). In general, an inhibitor of an intracellular target,
even though it may not be exquisitely specific, can provide evidence
that is consistent with a role for its target in a particular cellular
response (for example, to a growth factor); if its action is very
effective on its target but does not affect a particular cellular
response, then it is likely that the target of the inhibitor is not
required for that response. We have demonstrated above that ERK-1
activity is maintained at a higher level in cycling BMM than in cells
from which the CSF-1 was removed (Table I; Fig. 1a) but,
using PD98059, no evidence could be found for its involvement in the
CSF-1-stimulated enhancement of BMM survival (i.e. reversal
of apoptosis) (Fig. 2). Even though enhanced BMM ERK activity does not
correlate with enhanced DNA synthesis (11, 12) because, for example, a
nonmitogenic stimulus such as LPS can enhance ERK activity, PD98059
suppressed somewhat the CSF-1-stimulated BMM DNA synthesis (Table II);
assuming that PD98059 is specific for the ERK pathway under the
conditions used (28), this suppression suggests MEK/ERK involvement in
a pathway(s) critical for the progression into S-phase in this system.
The elevated ERK activity in cycling BMM is consistent with this
conclusion and suggests that ERK activation may be necessary but not
sufficient for macrophage proliferation.
In a number of other cell systems, it is widely concluded that enhanced
ERK activity leads to transcription of cyclin D1 and c-myc
genes with subsequent progression into S phase. However, in
CSF-1-stimulated BMM we did not find that PD98059 inhibited cyclin D1
mRNA or c-myc mRNA expression (Fig. 3a),
observations at odds with other systems including CSF-1-stimulated NIH
3T3 cells, which had been transfected with the CSF-1R, c-fms
(Ref. 10 and references therein). Interestingly, when the induction of
cyclin D1 protein was monitored by Western blotting, we found in
preliminary experiments that PD98059 (
50 µM) did
suppress it (data not shown). Because PD98059 blocked CSF-1-induced
c-fos mRNA expression (Ref. 12; Fig. 3b), it
is possible that a MEK/ERK/c-Fos-dependent pathway is
relevant for DNA synthesis control in CSF-1-treated BMM; of possible
relevance also is the recent report that persistent ERK activation by
CSF-1 is necessary for both expression and post-translational activation of ets-2 in macrophages (30).
The actions of PD98059 differ from those of another inhibitor of
CSF-1-stimulated BMM DNA synthesis, albeit a more potent one, namely
8BrcAMP (12). This cAMP analogue blocked the CSF-1-stimulated cyclin D1
and c-myc mRNA expression (Refs. 25 and 34; Fig. 3a) while raising c-fos mRNA expression and
ERK activity (12, 24). However, 8BrcAMP, like PD98059, could not
prevent the reversal of BMM apoptosis by CSF-1 (Fig. 2); interestingly,
the combination of the two agents did so, suggesting perhaps they
control complementary pathways emanating from the stimulated CSF-1
receptor, which are each important for cell survival and possibly for
other cellular responses. We have found before that 8BrcAMP inhibited
CSF-1-induced Ras activity (12), raising once again the possibility of
the presence of a Ras-independent pathway controlling ERK activity in
macrophages (5); perhaps enhanced Ras activity but not ERK activity is
relevant for subsequent cyclin D1 mRNA or c-myc mRNA expression in CSF-1-stimulated macrophages.
p38 MAP kinase has been shown in a variety of cell systems to be
strongly activated by stress signals such as osmotic shock, UV light,
LPS, and inflammatory cytokines but to be activated only weakly by
growth factors (see Refs. 13 and 27 for reviews). p38 is known to be
involved in the control of cytokine production (e.g. TNF
in LPS-activated macrophages (18)). Some reports but not others have
found that CSF-1 is a relatively poor stimulator of inflammatory
cytokine synthesis (e.g. TNF
, interleukin-1) in
monocytes/macrophages in vitro (19, 20, 38-40). However, it
has been reported that CSF-1 causes an increase in tyrosine phosphorylation in p38 immunoprecipitates in the myeloid cell line,
FD-MACII cells (17). This type of analysis does not measure enzymatic
activation nor give any indication as to the degree of activation of
this enzyme. We therefore decided to measure p38 activity by a specific
and quantitative immunoprecipitation kinase assay in cultures of
primary macrophages and to compare directly the activity induced by
CSF-1 treatment to that resulting from the action of LPS. Our data show
that in BMM the stimulation by CSF-1 of p38 enzymatic activity is very
small compared with that stimulated by LPS (Fig. 4a) and is
kinetically distinguishable from the latter (data not shown). These
findings are entirely consistent with the reports from our laboratory
and those of others that show that CSF-1 is a weak stimulator of
cytokine biosynthesis in monocytes and macrophages compared with LPS
and offer an explanation for these findings. The kinetics of p38
activation by CSF-1 and LPS are similar to those that we, and others,
have previously observed for ERK-1 activation in the same cells and by
the same stimuli (11), although there are clear differences in the
extent of activation. Thus CSF-1 activates both enzymes maximally at 5 min in growth-arrested cells, whereas LPS activates them maximally at
15 min; furthermore, LPS is a strong activator of both MAP kinase
family members, whereas CSF-1 is a weak activator of the p38 enzyme
relative to its effects on the ERK isoforms. Whether the activation of
these enzymes by CSF-1 and LPS represents activation of the same
intracellular pools is not clear, but in this context it is of interest
that a recent report has shown that LPS preferentially activates a
minor microtubule-bound subpopulation of MAP kinase (41).
The p38 pathway has been implicated to play a critical role in
apoptosis, because many apoptotic signals are able to stimulate p38
activity, and p38 activation is correlated with the induction of
apoptosis in several cell types (14-16); however, others have found no
correlation between its activation and apoptosis in other cell types
and even found some activation by growth factors (see for example Refs.
17, 42). Inhibition of p38 activity has been shown to suppress
apoptosis in some cases (see for example Ref. 16) but not in others
(43) and to even induce apoptosis (44). In BMM (Fig. 4b) and
the macrophage cell line BAC.1.2F5 (data not shown), we found a gradual
decrease in p38 activity following CSF-1 withdrawal; furthermore,
SKB202190 failed to inhibit the decrease in BMM viability (Fig.
4c). Our data, therefore, do not support a role for p38
family members, at least those that are susceptible to pyridinyl
imidazole inhibition, in macrophage cell death resulting from growth
factor deprivation and indicate that CSF-1 does not maintain cell
viability by suppressing p38 activity. The significance of the elevated
p38 activity in cycling BMM awaits clarification. p38 activity may play
a permissive role in CSF-1-driven macrophage proliferation because
SKB202190 blocked CSF-1-stimulated BMM DNA synthesis, and our
preliminary data suggest that it inhibits the induction of
c-myc mRNA but not cyclin D1 mRNA; however, it
should be noted that the concentrations required were much higher than
those needed to inhibit the in vitro BMM p38 activity,
suggesting that the drug might have alternative targets in BMM at the
higher concentrations.
JNK activity in various cell types can be enhanced by a similar range
of stress signals as p38 activity and be weakly activated by growth
factors (13). We could find no evidence for an activation of JNK-1
activity in CSF-1-treated BMM above the very low levels found in the
untreated population. In contrast, LPS and TNF
both gave significant
increases; LPS has been shown previously to stimulate JNK activity in
macrophages (45). In BMM we found no detectable increase in JNK
activity as they proceeded to the apoptotic state and, therefore, no
support for its role in the control of apoptosis in these cells.
In summary, the lack of association of MAP kinase family members with
apoptosis and/or survival in the macrophages again highlights the need
to explore the relevance of a signaling pathway to cellular function
for each particular cell type; also, the atypical evidence presented
above that ERK activity may not be controlling cyclin D1 mRNA and
c-myc mRNA expression in CSF-1-stimulated BMM but does
so in CSF-1-treated NIH 3T3 cells (cf. present paper and Ref. 10) again indicates that CSF-1 biology and associated signal transduction pathways are best studied in myeloid cells (5). The
relatively weak stimulation of p38 activity by CSF-1 compared with that
resulting from LPS action contrasts some actions of CSF-1 (a survival
and growth factor) with those of LPS (a stress stimulus) and is
consistent with some reports (19, 20), but not others (38-40), showing
that CSF-1 is a relatively poor stimulator of monocyte/macrophage
inflammatory mediator production.