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Departament de Fisiologia (Biologia del Macròfag), Facultat de Biologia and Fundació August Pi i Sunyer, Universitat de Barcelona, Barcelona, Spain
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, and PKC
.
PKC is not inhibited by GF109203X/calphostin C. Of the other two
isoforms, PKC is the best candidate to mediate MKP-1 induction.
Prolonged exposure to PMA slightly inhibits MKP-1 expression in
response to M-CSF. In bone marrow macrophages, this treatment leads to
a complete depletion of PKCßI, but only a partial down-regulation of
PKC. Moreover, no translocation of PKCßI or PKC from the
cytosol to particulate fractions was detected in response to M-CSF,
whereas PKC was constitutively present at the membrane and underwent
significant activation in M-CSF-stimulated macrophages. In conclusion,
we remark the role of PKC, probably isoform , in the negative
control of ERK-1/2 through the induction of their specific
phosphatase. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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M-CSF induces macrophage proliferation, and it is the major and specific growth factor for this cell type (3). M-CSF is recognized by a specific receptor encoded by the protooncogene c-fms (4). The first step in transduction of the M-CSF signal is the activation of the tyrosine kinase domain of the receptor. As a consequence, the receptor becomes autophosphorylated on several tyrosine residues (4, 5). These residues are recognized by distinct tyrosine kinases, including members of the Src family (6, 7), which then phosphorylate a large number of intracellular substrates, and Tyk2, a Janus kinase that phosphorylates and activates transcription factors from the STAT family (8, 9). M-CSF also activates phosphatidylinositol 3-kinase (PI3-K)3 (10), which subsequently generates phosphatidylinositol (3, 4, 5) trisphosphate (PIP3), a molecule that acts as a second messenger that activates p21Ras and some protein kinase C (PKC) isoforms (11, 12).
M-CSF also induces the production of diacylglycerol (DAG) and the
subsequent activation of PKC (13, 14). The PKC family
consists of multiple isoforms that are classified in three main groups
(conventional, novel, and atypical PKCs) depending on their primary
structure and activation requirements (15, 16).
Conventional PKCs, i.e., 
, ßI, ßII, and 
, require both
calcium (Ca2+) and DAG/phorbol esters for
activation and phosphatidylserine as a cofactor. Novel PKCs require
DAG/phorbol esters and phosphatidylserine, but do not depend on
Ca2+ for activation; they include the isozymes
, , 
, 
, and µ. Atypical PKCs, represented by isoforms
and 
/
, cannot be activated by Ca2+ or
DAG/phorbol esters, but are regulated by PIP3,
ceramide, and phosphatidic acid (17, 18, 19, 20).
M-CSF triggers the activation of the Raf/MEK/ERK pathway in macrophages (21, 22). Raf-1, a serine/threonine protein kinase, phosphorylates and activates the threonine/tyrosine protein kinase MEK-1 (23), which, in turn, phosphorylates and activates extracellular signal-regulated protein kinase-1 (ERK-1) and ERK-2 (24). These are proline-directed serine/threonine protein kinases, also known as p44- and p42-mitogen-activated protein kinases (-MAPK), respectively (25). Active ERKs phosphorylate and regulate several cellular proteins, including additional protein kinases, cytoskeletal components, phospholipase A2, and nuclear transcription factors, such as Elk1/TCF and c-Jun, which regulate the expression of immediate early genes (25, 26).
The negative regulation of ERK activity may be mediated by the members of a family of dual specificity tyrosine phosphatases, including MAPK phosphatase-1 (MKP-1) (27, 28). Phosphorylation on both tyrosine and threonine residues is required for ERK activity (29). MKP-1 dephosphorylates both phosphotyrosine and phosphothreonine residues on ERK-1 and -2 both in vitro and in vivo, thus suggesting that this phosphatase is crucial for keeping the balance between ERK phosphorylation and dephosphorylation (27, 28). Recent reports have also shown the capability of MKP-1 to dephosphorylate and inactivate other MAPKs, including JNK/SAPK and p38/RK (30). In fibroblasts, overexpression of MKP-1 inhibits ERK-regulated reporter gene expression, Ras-induced DNA synthesis, and growth factor-induced entry into the S phase (31, 32). The expression of MKP-1 constitutes a mechanism of control and attenuation of mitogenic signaling pathways.
Although several cell types require ERK activation to proliferate in response to a number of growth factors (reviewed in Ref. 33), only a correlation between ERK activation and M-CSF signal transduction has been found in macrophages, and the exact role of ERK-1/2 in the proliferation of these cells is still unclear. In fact, in the macrophagic cell line BAC1.2F5, v-Raf conferred M-CSF-independent growth without ERK activation (34). On the other hand, the extent of ERK activation appeared to be similar in both proliferating and poorly proliferating macrophages, thus suggesting that ERK activation was not sufficient to induce macrophage proliferation (22). In the present report we show with certainty that blockage of the activation of ERK-1/2 inhibits M-CSF-induced proliferation of bone marrow macrophages, leading to a growth arrest of these cells at the G1 phase of the cell cycle. This blockage was not accompanied by programmed cell death.
The time course of ERK activation determines the fate of several cell
responses, including cell proliferation (35, 36, 37). Our
studies show that in bone marrow macrophages, M-CSF induces the
transient expression of MKP-1, which correlates with most of the
dephosphorylation and inactivation of ERK-1/2. Thus, the induction of
this phosphatase seems to be an important mechanism for the negative
control of ERK activation in this system. Although the induction of
MKP-1 has been also described in M-CSF-stimulated BAC1.2F5 cells
(38), the signaling mechanisms that control the expression
of this phosphatase in response to M-CSF are unknown. In this report we
show that activation of ERK-1/2 is not required for the induction of
MKP-1. Instead, the expression of this phosphatase is induced by M-CSF
through a PKC-dependent pathway. Of all the PKC isoforms detected in
macrophages, the main candidate to mediate MKP-1 expression is PKC.
This suggests an important role for PKC, putatively isoform , in the
negative control of ERK activity through the induction of its specific
phosphatase.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Bone marrow macrophages were obtained from 6- to 10-wk-old BALB/c mice (Charles River Laboratories, Wilmington, MA) as previously described (39). The cells were cultured in 150-mm plates in DMEM (Sigma, St. Louis, MO) supplemented with 20% FBS (Sigma) and 30% L cell-conditioned medium as a source of M-CSF. Once macrophages were 60–80% confluent, they were deprived of L cell-conditioned medium for 14–16 h to render the cells quiescent and then were subjected to different treatments.
Reagents
Recombinant M-CSF was a gift from DNAX (Palo Alto, CA). In some
experiments we used L cell-conditioned medium as the source
of this growth factor. We used selective inhibitors/activators to
either block or activate specific signal transduction pathways.
Bisindolylmaleimyde I (GF109203X), PMA, and calphostin C were purchased
from Calbiochem (San Diego, CA). Before its use, calphostin C was light
activated during 3 min, as recommended by its manufacturer. Wortmannin,
L--phosphatidylserine, and
1,2-sn-dioleoylglycerol were obtained from Sigma. PD98059
was purchased from New England Biolabs (Beverly, MA).
Proliferation assay
Cell proliferation was measured as previously described (40, 41) with minor modifications. Quiescent cells (105) were incubated for 24 h in 24-well plates (3424 MARK II, Costar, Cambridge, MA) in 1 ml of medium with different concentrations of M-CSF. The medium was aspirated and replaced with 0.5 ml of medium containing [3H]thymidine (1 µCi/ml; ICN, Costa Mesa, CA). After 4–6 h of incubation at 37°C, the medium was removed, and the cells were fixed in ice-cold 70% methanol. After three washes in ice-cold 10% TCA, the cells were solubilized in 1% SDS and 0.3 M NaOH at room temperature. Radioactivity was counted by liquid scintillation using a 1400 Tri-Carb Packard scintillation counter (Packard, Downers Grove, IL). Each point was performed in triplicate, and the results were expressed as the mean ± SD.
Analysis of DNA content with 4',6'-diamidino-2-phenylindole (DAPI)
Cells (106) were resuspended and fixed in ice-cold 70% ethanol. The cells were then washed in PBS; resuspended in 0.2 ml of a solution containing 150 mM NaCl, 80 mM HCl, and 0.1% Triton X-100; and incubated at 0–4°C for 10 min. Afterward, 1 ml of a solution containing 180 mM Na2HPO4, 90 mM citric acid, and 2 µg/ml DAPI (pH 7.4), was added to each sample. After incubating the cells at 4°C for 24 h, their fluorescence was measured with an EPICS Elite flow cytometer (Coulter, Miami, FL). For this analysis, we used a UV laser with an excitation beam of 25 mW at 333–364 nm, and fluorescence was collected with a 525-nm band-pass filter. Cell doublets were gated out by comparing the pulse area vs the pulse width. Cells (12,000) were counted for each histogram, and cell cycle distributions were analyzed with the Multicycle program (Phoenix Flow Systems, San Diego, CA).
Chromatin fragmentation assay
Fragmentation of DNA due to internucleosomal cleavage was determined using a commercial ELISA kit (Cell Death Detection ELISA Kit plus, Boehringer Mannheim, Indianapolis, IN). Briefly, the cells were harvested and washed in ice-cold PBS. The cells were then lysed in 0.5 ml of lysis buffer (50 mM Tris-HCl, 10 mM EDTA, and 1% SDS, pH 8.0) for 16 h at 4°C, and the lysates were centrifuged (15,000 x g) to separate high m.w. DNA (pellet) from cleaved low m.w. DNA (supernatant). The DNA present in the supernatants was analyzed in ELISA plates following the commercial kit instructions. Each point was performed in triplicate, and the results were expressed as the mean ± SD.
RNA extraction and Northern blot analysis
The cells were washed twice in PBS, and total RNA was extracted
as previously described (42). Total RNA samples (20 µg)
were separated on 1.2% agarose gels containing formaldehyde and
transferred to nylon membranes (GeneScreen, DuPont-New England Nuclear,
Boston, MA). For MKP-1 mRNA detection, we obtained a probe
corresponding to the full-length cDNA of MKP-1 by digesting pBS/MKP-1
(provided by Dr. R. Bravo, Bristol-Myers Squibb, Princeton, NJ) with
HindIII. To detect the L32 transcript, we used the
EcoRI/HindIII fragment of pGEM1/L32 as a probe
(43). The probes were labeled with
[-32P]dCTP (ICN). The membranes were
hybridized in a solution containing 20% formamide, 5x Denhart’s
solution, 5x SSC, 10 mM EDTA, 1% SDS, 25 mM
Na2HPO4, 25 mM
NaH2PO4, and 0.2 mg/ml
salmon sperm DNA for 18 h at 65°C. Afterward, the membranes were
subjected to three washes of 5 min each at room temperature in 2x
SSC/0.1% SDS, and one more wash at 65°C for 20 min in 0.1x
SSC/0.1% SDS. The membranes were finally exposed to Kodak X-AR films
(Eastman Kodak, Rochester, NY). The bands of interest were quantified
with a Molecular Analyst (Bio-Rad, Richmond, CA).
Protein extraction and Western blot analysis
The cells were washed twice in cold PBS and lysed on ice with a lysis solution containing 1% Triton X-100, 10% glycerol, 50 mM HEPES (pH 7.5), 150 mM NaCl, and protease inhibitors (1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml iodoacetamide, and 1 mM PMSF). Sodium orthovanadate (1 mM) was added when inhibition of the activity of tyrosine phosphatases was required. For PKC detection experiments, 1 mM EGTA and 2 mM EDTA were incorporated in the lysis solution. Insoluble material was removed by centrifugation at 13,000 x g for 8 min at 4°C. The protein concentration of the samples was determined by the Bio-Rad protein assay. Proteins from cell lysates (50–100 µg) were heated at 95°C in Laemmli SDS loading buffer, separated by 10% SDS-PAGE, unless stated otherwise, and electrophoretically transferred to nitrocellulose membranes (Hybond-ECL, Amersham, Arlington Heights, IL). The membranes were blocked in 2% BSA in Tris buffer saline-0.5% Tween-20 (TBS-T) for 3 h at room temperature and then incubated with the primary Ab in 2% BSA in TBS-T. For MKP-1 immunoblotting, we used a primary Ab purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The incubation was performed for 1 h at room temperature. To recognize PKC isozymes, the incubation with primary Abs (provided by Dr. P. J. Parker, Imperial Cancer Research Fund, London, U.K.) was performed overnight at 4°C. After three washes of 15 min each in TBS-T, the membranes were incubated for 1 h with peroxidase-conjugated anti-rabbit IgG Ab (Cappel-Organon Teknik., Durham, NC). After three washes with TBS-T, enhanced chemiluminescence detection was performed (Amersham), and the membranes were exposed to x-ray films (Amersham). The bands of interest were quantified by densitometric analysis.
Determination of the ERK phosphorylation state by mobility shift assay
This assay was performed as described for the Western blot analysis with slight modifications (44). Proteins from cell lysates (50–100 µg) were subjected to 7.5% SDS-PAGE to allow efficient separation of phosphorylated and dephosphorylated forms of ERKs. The blocking of the membrane was conducted in 5% nonfat dry milk in TBS-T for at least 1 h at room temperature. Incubations with anti-ERK-1/2 primary Ab (1/10,000; provided by Dr. M. J. Weber, University of Virginia School of Medicine, Charlottesville, VA) and with peroxidase-conjugated anti-mouse IgG Ab (1/5000; Cappel) were performed in TBS-T for 1 h each at room temperature.
Determination of ERK activity by in-gel kinase assay
This assay was performed as previously described
(45). Briefly, 50 µg of total protein was separated by
12.5% SDS-PAGE in the presence of 0.1 mg/ml of myelin basic protein
(MBP; Sigma) copolymerized in the gel. After electrophoresis, SDS was
removed by washing the gel with two changes of 20% 2-propanol in 50 mM
Tris-HCl (pH 8.0) for 1 h at room temperature. The gel was then
incubated with 50 mM Tris-HCl (pH 8.0) containing 5 mM 2-ME (buffer A)
for 1 h at room temperature. The proteins were denatured by
incubating the gel with two changes of 6 M guanidine-HCl for 1 h
at room temperature and then renatured by incubating with five changes
of buffer A containing 0.04% Tween-20 for 16 h at 4°C. To
perform the phosphorylation assay, the gel was first equilibrated in 40
mM HEPES-NaOH (pH 7.4) containing 2 mM DTT, 0.1 mM EGTA, 15 mM
MgCl2, and 300 µM sodium orthovanadate for 30
min at 25°C and then incubated for 1 h in the same solution
containing 50 µM ATP and 100 µCi
[-32P]ATP (ICN). The reaction was stopped by
washing the gel with 5% TCA containing 10 mM sodium pyrophosphate to
inhibit phosphatase activity. The gel was dried, exposed to x-ray films
(Kodak), and quantified with a Bio-Rad Molecular Analyst.
Determination of PKC translocation
PKC translocation was determined as previously described (46) with some modifications. The cells were lysed by scraping in cold hypotonic buffer T10 (10 mM Tris-HCl (pH 7.5), 1 mM EGTA, and 10 mM NaCl) containing protease inhibitors and 100 µM sodium orthovanadate. The cell lysates were centrifuged (100,000 x g) for 30 min at 4°C, and the supernatants were collected (cytosolic fraction). The pellets were resuspended in cold T10 buffer containing 1% Triton X-100 and homogenized with a Dounce homogenizer on ice (15–20 strokes; Kontes, Vineland, NJ). To allow PKC extraction from the cell membrane, the samples were incubated for 1 h at 4°C and then centrifuged (100,000 x g) for 30 min at 4°C. The supernatants were collected (plasma membrane fraction), and the pellets were resuspended in cold T10 containing 1% SDS, repeatedly passed through a 19-gauge needle, and heated at 100°C for 5 min. Insoluble material was removed by centrifugation (13,000 x g) for 10 min, and the supernatants were collected (cytoskeleton fraction). Samples from each fraction (25 µg of protein) were boiled at 95°C in loading buffer and separated by 8% SDS-PAGE. The proteins were electrophoretically transferred to Hybond-ECL membranes (Amersham) and immunoblotted with Abs against each PKC isoform.
Measurement of PKC activity
This assay was performed as previously described
(47) with modifications. Specific Abs against PKC (Life
Technologies, Grand Island, NY) were used to immunoprecipitate this
isoform from subcellular fractions (2 µg of Ab/150 µg of total
protein in a total volume of 300 µl). Incubation was conducted for
2 h at 4°C. Immunocomplexes were separated by addition of 75
µl of 20% protein A-Sepharose slurry, incubated 2 h at 4°C,
and pelleted. The pellets were washed twice with RIPA buffer (50 mM
Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 2
mM EDTA, and 1 mM EGTA) supplemented with protease inhibitors and 1 mM
sodium orthovanadate and once with prereaction buffer (50 mM
ß-glycerophosphate, 10 mM MgCl2, 20 mM Tris-HCl
(pH 7.5), 50 mM NaCl, 1 mM DTT, protease inhibitors, 1 mM sodium
orthovanadate) and then resuspended in reaction buffer (prereaction
buffer supplemented with 100 µM ATP, 33 µM
1,2-sn-dioleoylglycerol, 40 µg/ml
L--phosphatidylserine, 1 µM
[25Ser]PKC, and 5 µCi
[-32P]ATP).
Ser25-substituted peptide obtained from the
pseudosubstrate region of PKC (Calbiochem, La Jolla, CA) was used as
the substrate for the phosphorylation assay, because it represents an
appropriate substrate for measuring PKC activity (48).
The reaction was conducted for 10 min at 30°C. Each sample was
spotted on a phosphocellulose filter (Whatman 3 MM, Clifton, NJ) and
subjected to five washes of 30 min each in 5% TCA and 10 mM sodium
pyrophosphate. Radioactivity was counted by liquid scintillation using
a 1400 Tri-Carb Packard scintillation counter.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A). In the
presence of M-CSF, bone marrow macrophages proliferated in a
dose-dependent manner, as measured by thymidine incorporation (Fig. 1B). This method gives an efficient indication of macrophage
proliferation as previously determined (41). Macrophages
preincubated with PD98059 were unable to proliferate in response to
M-CSF, thus showing that activation of the MEK/ERK pathway is necessary
for M-CSF-dependent macrophage proliferation. We also analyzed the cell
cycle distribution of M-CSF-stimulated macrophages in the presence or
the absence of PD98059. To do this, we stained the cells with DAPI, a
fluorescent dye that specifically binds to DNA, and we measured the
cellular DNA content by flow cytometric analysis. Cultures of bone
marrow macrophages growing in M-CSF-containing medium (control cells)
showed a random distribution among the different phases of the cell
cycle, with 51% of cells in
Go/G1, 30% in S, and 17%
in G2/M (Fig. 1C). When these cells
were deprived of M-CSF for 24 h (starving cells), the tetraploid
peak corresponding to the G2/M phase was
drastically reduced, whereas a subdiploid peak representing apoptotic
cells (Sub-G1) appeared. When macrophages growing in the
presence of M-CSF were also incubated with PD98059 over a period of
24 h, they turned out to be distributed homogeneously (74% of
total cells) in the G0/G1
phase of the cell cycle, with no peaks of apoptotic cells. This
suggests that the blockage of M-CSF-induced ERK activation causes a
growth arrest of macrophages at the
G0/G1 phase of the cell
cycle and that this growth arrest is not due to induction of apoptosis.
To support this observation, the induction of apoptosis was further
analyzed using a cell death detection kit, which measures DNA
fragmentation caused by internucleosomal cleavage. Apoptosis was
observed in cells deprived of M-CSF for 24 h (positive control),
but not in cells growing in the presence of M-CSF or treated with
PD98059 (Fig. 1D). These results show that blockage of ERK
activation inhibits M-CSF-induced proliferation by inducing a growth
arrest of macrophages at the G1 phase of the cell
cycle without causing apoptosis.
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A), which decreased
progressively after 30 min of stimulation. Moreover, the activity of
ERK-1/2 was analyzed by an in-gel kinase assay, using myelin basic
protein as the substrate. ERK activation was detected as soon as 2–3
min after M-CSF treatment and, again, decreased progressively after 20
min of stimulation (Fig. 2B). Some residual activity was
observed at prolonged times of M-CSF treatment.
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C). The expression of the protein was detected after 20
min, peaked at 60–75 min, and returned to basal levels within 2 h
of stimulation with M-CSF (Fig. 2D). MKP-1 expression
correlated with the inactivation by dephosphorylation of ERK-1/2 during
the macrophage response to M-CSF (Fig. 2E), thus suggesting
that synthesis of this phosphatase is an important mechanism induced by
M-CSF to control the time course of activation of the ERK pathway.
We next analyzed the mechanisms that mediate the induction of MKP-1 in
response to M-CSF. In fibroblasts, transcription of the MKP-1 gene is
under the control of the ERK pathway (51). For this
reason, we studied whether the activation of ERK-1/2 was required for
the induction of this phosphatase by M-CSF. Fig. 3 shows that blockage of the ERK pathway
with PD98059 did not alter MKP-1 expression at the level of either mRNA
or protein synthesis, thus indicating that activation of ERK-1/2 is not
strictly required for MKP-1 induction in M-CSF-stimulated macrophages.
Instead, M-CSF must activate other signaling pathways that ensure MKP-1
expression.
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, B and
C). This assay was also performed with subsaturant
concentrations of the growth factor without obtaining any inhibitory
effect of wortmannin (data not shown). Taken together, these results
indicate that activation of PI3-K is not required for the induction of
MKP-1 by M-CSF.
The enzyme PKC has been also involved in the signal transduction of
M-CSF (13, 14). PKC may mediate the activation of the
transcriptional complex AP-1 (53, 54, 55), which, in turn,
recognizes and activates transcription from gene promoters containing
o-tetradecanoylphorbol 13-acetate (TPA) response elements
(TREs) (56, 57). Because a TRE site has been described at
position -450 bp in the promoter of the MKP-1 gene (58),
we studied the involvement of PKC in the induction of MKP-1 in
M-CSF-stimulated macrophages. The cells were preincubated with the PKC
inhibitor GF109203X (59) before adding M-CSF. GF109203X
inhibited the expression of MKP-1 in a dose-dependent manner (Fig. 4, A and B). Eighty
percent inhibition of MKP-1 expression was observed when macrophages
were pretreated with 5 µM GF109203X. Recently, it has been shown that
GF109203X can also inhibit the activation of two other molecules, Rsk-2
and p70 S6 kinase (60). However, it is unlikely that
blockage of MKP-1 expression is due to the inhibition of any of these
proteins for two reasons. First, Rsk-2 is an enzyme that lies
immediately downstream of ERK-1/2 and is activated by the
same agonists that activate this pathway (61). As shown
above, specific blockage of this cascade with PD98059 does not inhibit
MKP-1 induction by M-CSF (Fig. 3A). Second, rapamycin, a
selective inhibitor for p70 S6 kinase (62) does not modify
the levels of MKP-1 mRNA in response to M-CSF (data not shown). These
results suggest that inhibition of MKP-1 expression by GF109203X is
due to selective blockage of PKC. To confirm this, we also used
calphostin C (63), a PKC inhibitor not related to
GF109203X. As shown in Fig. 4C, the induction of MKP-1 by
M-CSF was also inhibited by calphostin C. Taken together,
these results indicate that a PKC-dependent pathway mediates
MKP-1 induction in M-CSF-stimulated macrophages.
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D), thus
indicating that PKC activity is not required for ERK activation by
M-CSF. However, the extent of ERK activation was significantly more
prolonged in macrophages in which MKP-1 expression had been inhibited
by preincubation with GF109203X. This observation further supports the
involvement of PKC in the negative regulation of ERK activity by
inducing the expression of the phosphatase MKP-1 in M-CSF-stimulated
macrophages.
Atypical isotypes of PKC are not sensitive to inhibition by GF109203X
(64). Therefore, our results suggest that either a
classical or a novel isoform of PKC mediates the induction of MKP-1 by
M-CSF. By using polyclonal Abs raised against isoform-specific
peptides, we studied the expression of isoenzymes , ßI, , ,
, , , /, and in bone marrow macrophages. We detected
the expression of PKCßI, PKC, and PKC (Fig. 5A). None of the other
isoforms mentioned above was detected under our experimental
conditions. We did not assay the presence of PKCßII. However, ßI
and ßII result from differential splicing of the same transcript, and
in most cases, one of the two isoforms is mainly expressed in a certain
tissue (reviewed in 15). In support of the absence of PKC,
rottlerin (1–20 µM), a specific inhibitor of this isoform, had no
effect on the induction of MKP-1 by M-CSF (data not shown). Of the
three isoforms detected in our system, only PKCßI and PKC are
sensitive to inhibition by GF109203X. To assess the involvement of
these two isoforms, the cells were exposed to prolonged treatment with
PMA (12 h). This treatment resulted in a complete down-regulation of
PKCßI (Fig. 5B). However, in bone marrow macrophages,
expression of PKC was highly resistant to down-regulation by this
treatment. We found that the induction of MKP-1 by M-CSF was only
slightly affected (20% inhibition) by prolonged exposure to PMA (Fig. 5C), thus suggesting that the induction of MKP-1 does not
depend on the presence of PKCßI and that the main candidate to
mediate MKP-1 expression is PKC.
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were located in the
cytoplasm of unstimulated cells and did not translocate to the plasma
membrane or the cytoskeleton in response to M-CSF (Fig. 6A). As a positive control, we
show the translocation of PKCßI in response to PMA (Fig. 6B). In contrast to the other two isoforms, PKC was
constitutively present at the plasma membrane. We further studied the
capability of membrane-bound PKC to be activated by M-CSF. To do
this, PKC was immunoprecipitated from the Triton-soluble fraction of
cells stimulated or not with M-CSF, and its activity was measured as
the capability of the immunoprecipitates to phosphorylate the specific
substrate 25Ser-substituted PKC peptide. The
levels of PKC activity in the Triton-soluble fraction of
nonstimulated macrophages were comparable to the background signal
detected in the cytosolic fraction of untreated macrophages (data not
shown), thus indicating that PKC was not constitutively activated in
macrophage membranes. However, after 15 min of M-CSF stimulation, there
was a significant activation of membrane-bound PKC (3.7-fold
induction; Fig. 6C). These results further correlate PKC
activation with the induction of MKP-1 in bone marrow macrophages.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The time course of ERK activation is a critical aspect for determining some cellular responses, including cell proliferation (35, 36, 37, 49, 50). We have determined the time course of ERK-1/2 activation in M-CSF-stimulated bone marrow macrophages by studying both the state of phosphorylation of these kinases and their ability to phosphorylate MBP in an in-gel kinase assay. Our results confirm previous observations about the transient pattern of ERK activity in M-CSF-stimulated macrophages (22). However, by using a totally distinct substrate of phosphorylation, a recombinant Ets-2 protein, it has been recently described that M-CSF induces persistent activation of ERK-1/2 in bone marrow macrophages (71). In our experiments we detected some residual ERK activity at prolonged times of M-CSF stimulation, which may be sufficient to mediate Ets-2 phosphorylation.
During the macrophage response to M-CSF, the major part of ERK inactivation correlated with the synthesis of the phosphatase MKP-1, thus suggesting that this phosphatase is involved in the negative control of ERK activity in our system. The induction of MKP-1 in bone marrow macrophages confirms a previous report that described accumulation of the mRNA coding for this phosphatase after stimulation of the macrophagic cell line BAC1.2F5 with M-CSF (38). However, we have further explored the mechanisms involved in the negative control of ERK activity by studying the signaling pathway that mediates MKP-1 induction in M-CSF-stimulated macrophages.
In contrast to what has been described in serum-stimulated fibroblasts (51), induction of MKP-1 by M-CSF was not dependent on activation of the MEK/ERK cascade. Thus, MKP-1 is not expressed in M-CSF-stimulated macrophages as a consequence of the ERK pathway being able to induce its own attenuation in a direct negative feedback loop. Instead, M-CSF ensures the expression of this phosphatase by an alternative mechanism.
In this report we show that the induction of MKP-1 is dependent on PKC activation. In parallel studies we found that LPS also induces the expression of MKP-1 in bone marrow macrophages through a PKC-dependent pathway.4 In that report we also showed that, in contrast to what has been described in other systems, an increase in the intracellular levels of cAMP did not induce the expression of this phosphatase. This indicates that important differences exist regarding transcription of the MKP-1 gene in different cell types. Our observations suggest a major role for PKC-dependent events in the control of MKP-1 expression in macrophages.
The time course of ERK activity was significantly extended in macrophages in which MKP-1 expression was inhibited in response to a PKC inhibitor. Although we cannot discard the involvement of phosphatases other than MKP-1, especially since some dephosphorylation of ERK-1/2 still occurred in the absence of normal MKP-1 induction, our results allow us to conclude that PKC plays an important role in the control of the time course of ERK activity by inducing the expression of MKP-1.
There is increasing evidence that individual PKC isoforms mediate
specific events in signal transduction (16). We have shown
that bone marrow macrophages express PKC isoforms ßI, , and .
In contrast to the observations recently reported by Pingel et al.
(72), we did not detect the expression of PKC and
PKC in this cell type, although the experiment was conducted several
times. The fact that MKP-1 induction was not altered by rottlerin, a
specific inhibitor of PKC, further supports our results. Differences
in the pattern of expression of PKC isoforms in a certain cell type may
be caused by the state of maturation of the cells (73, 74, 75, 76)
or by the specific culture conditions. In fact, there are significant
variations in the expression of PKC isozymes when comparing different
macrophagic cell lines (21, 77, 78, 79) or even different
primary monocytic/macrophagic populations (80, 81, 82, 83),
perhaps as a consequence of their specific state of
differentiation/maturation. To study the response of bone marrow
macrophages to M-CSF, we need to use nonconfluent cultures of
macrophages that maintain a high rate of proliferation, because
prolonged culture of these cells in the presence of M-CSF correlates
with a loss of their capability to further proliferate in response to
M-CSF or other growth factors. Besides, before the stimulation with
M-CSF, we need to render the cells quiescent by incubating them in the
absence of this growth factor for 16 h. The use of macrophages at
a different stage of terminal differentiation may explain the
discrepancies between our results and the data reported by Pingel et
al. (72).
When we analyzed the selective implication of PKC isoforms, we found no
evidence that PKC could mediate the expression of MKP-1 in response
to M-CSF. First, the expression of this phosphatase was inhibited by
GF109203X, which blocks the activation of conventional and novel PKCs,
but does not effectively inhibit atypical isoforms at the doses used in
our experiments (64). Second, the expression of MKP-1
induced by M-CSF did not decrease by pretreating the macrophages with
wortmannin, a specific inhibitor of PI3-K. This enzyme mediates the
generation of PIP3 production, a second messenger
that activates PKC (17). Taken together, these results
suggest that PKC is not involved in the M-CSF-induced signaling
pathway that leads to MKP-1 expression.
Our results indicate that of the other two PKC isoforms expressed in
macrophages, the most likely candidate to mediate the induction of
MKP-1 by M-CSF is PKC. First, calphostin C has been shown to inhibit
the novel isoforms of PKC, including PKC, more efficiently than the
conventional ones (63). Second, in bone marrow
macrophages, prolonged treatment with PMA causes the complete depletion
of PCKßI, but leads to only a slight down-regulation of PKC.
Similarly, PKC has been also shown to be resistant to prolonged PMA
treatment in three macrophagic cell lines and in several other cell
systems (84, 85, 86, 87, 88). The fact that this treatment only
reduces slightly MKP-1 induction by M-CSF supports the involvement of
PKC in this event. Third, PKC is associated with the plasma
membrane in both untreated and M-CSF-stimulated macrophages. The
constitutive presence of this isoform in the plasma membrane has also
been described in some other cell types and in the macrophagic cell
line U937 (15, 89). In bone marrow macrophages we have
detected significant activation of PKC within 15 min of M-CSF
stimulation. Our results allow us to conclude that the only presence of
PKC in the macrophage membrane fraction is not sufficient for the
activation of this isoform. In our system stimulation with M-CSF is
required for PKC to become active, perhaps as a consequence of both
the generation of DAG (14) and the induction of
conformational or phosphorylation-based modifications of the enzyme.
Moreover, DAG generated in response to M-CSF mainly derives from the
hydrolysis of phosphatidylcholine mediated by a phospholipase C isoform
specific for this phospholipid (90). Production of DAG
without generation of inositol trisphosphate, a second messenger that
triggers the mobilization of intracellular calcium, may explain the
lack of translocation of PKCßI in our system. In conclusion, MKP-1
expression in response to M-CSF correlates with the activation of
PKC, and the absence of this activation in nonstimulated macrophages
allows us to explain why MKP-1 is not expressed under nonstimulated
conditions.
However, although PKC is the best candidate to mediate the induction
of MKP-1 by M-CSF, we cannot discard the involvement of other signaling
molecules that have not been studied in this report. In fact, we did
not detect a total inhibition of MKP-1 expression in macrophages
treated with specific PKC inhibitors. This suggests that PKC is
required for this process, but some other mechanism may participate in
the induction of MKP-1 by M-CSF.
In this report we have shown that ERK-1/2 are required for macrophage
proliferation, but not survival, in response to M-CSF. Our results also
yield relevant insights into the mechanisms that control the duration
of ERK activity in M-CSF-stimulated macrophages. M-CSF induces the
activation of ERK-1/2 and the expression of MKP-1 through two distinct
pathways. Induction of MKP-1 is mediated by the activation of a
GF109203X/calphostin C-sensitive isoform of PKC, putatively
isoform .
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Antonio Celada, Departament de Fisiologia, Facultat de Biologia, Avenue Diagonal 645, 08028 Barcelona, Spain. E-mail address:
3 Abbreviations used in this paper: DAG, diacylglycerol; ERK, extracellular signal-regulated kinase; JNK/SAPK, c-Jun N-terminal kinase/stress-activated protein kinase; MAPK, mitogen-activated protein kinase; MBP, myelin basic protein; MEK, MAPK/ERK kinase; MKP-1, MAPK phosphatase-1; PI3-K, phosphatidylinositol-3-kinase; PIP3, phosphatidylinositol (3,4,5)-trisphosphate; PKC, protein kinase C; PLC, phospholipase C; RK, reactivating kinase; TRE, o-tetradecanoylphorbol 13-acetate (TPA) response element; DAPI, 4',6'-diamidino-2-phenylindole.<./>
4 A. F. Valledor, J. Xaus, and A. Celada. 1999. Protein kinase C is required for the induction of MAP kinase phosphatase-1 in LPS-stimulated macrophages. Submitted for publication.
Received for publication January 25, 1999. Accepted for publication June 10, 1999.
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