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Is Required for the Induction of Mitogen-Activated Protein Kinase Phosphatase-1 in Lipopolysaccharide-Stimulated Macrophages1
Departament de Fisiologia (Biologia del Macròfag), Facultat de Biologia and Fundació August Pi i Sunyer, Campus Bellvitge, Universitat de Barcelona, Barcelona, Spain
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, and 
. Of all these, only PKCßI and are inhibited
by GF109203X. The following arguments suggest that PKC is required
selectively for the induction of MKP-1 by LPS. First, in macrophages
exposed to prolonged treatment with PMA, MKP-1 induction by LPS
correlates with the levels of expression of PKC but not with that of
PKCßI. Second, Gö6976, an inhibitor selective for conventional
PKCs, including PKCßI, does not alter MKP-1 induction by LPS. Last,
antisense oligonucleotides that block the expression of PKC, but not
those selective for PKCßI or PKC, inhibit MKP-1 induction and lead
to an increase of extracellular-signal regulated kinase activity during
the macrophage response to LPS. Finally, in macrophages stimulated with
LPS we observed significant activation of PKC. In conclusion, our
results demonstrate an important role for PKC in the induction of
MKP-1 and the subsequent negative control of MAPK activity in
macrophages. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1, and IL-6 (3, 4),
which play important roles in the immune response.
In macrophages, LPS triggers the activation of several signal
transduction pathways involving G proteins, tyrosine kinases,
phospholipase C
(PLC),3 protein kinase
A (PKA), and protein kinase C (PKC) (5, 6, 7, 8). The
serine/threonine kinase PKC family consists of several isoforms that
are distributed in three main groups (conventional, novel, and atypical
PKCs) based on their primary structure and activation requirements
(9, 10). Conventional PKCs, which include , ßI,
ßII, and 
, need both Ca2+ and diacylglycerol
(DAG)/phorbol esters for activation and phosphatidylserine as a
cofactor. Novel PKCs, which include 
, , 
, 
, and µ, need
DAG/phorbol esters and phosphatidylserine, but do not require
Ca2+ for activation. Atypical PKCs, including
and 
v, cannot be activated by Ca2+ or
DAG/phorbol esters, but are regulated by phosphatidylinositol
(3, 4, 5)-trisphosphate, ceramide, and phosphatidic acid
(11, 12, 13).
LPS also activates different mitogen-activated protein kinase (MAPK) cascades, including the extracellular-signal regulated protein kinase (ERK) (14, 15), the c-Jun N-terminal protein kinase, and the p38 MAPK/reactivating kinase pathways (16). For general MAPK activity, phosphorylation in both tyrosine and threonine residues is required (17). 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 (reviewed in Ref. 18).
MAPK phosphatase-1 (MKP-1) is a member of a family of inducible dual-specificity tyrosine phosphatases (19, 20, 21). MKP-1 selectively dephosphorylates tyrosine and threonine residues on ERK-1/2 (22, 23), thus allowing the inactivation of these kinases. Recent reports have also shown the capability of MKP-1 to dephosphorylate and inactivate c-Jun N-terminal kinase/stress-activated protein kinase and p38/reactivating kinase (24).
At the present, little is known about the mechanisms involved in the
induction of MKP-1 by LPS. Because the study of the events that account
for the negative control of MAPK activity is of great interest, in this
report we have characterized the signaling pathway(s) that lead to
MKP-1 expression in response to LPS. We have found that MKP-1
expression was not dependent on the activation of the MAPKs ERK-1/2 in
response to LPS. Instead, the induction of this phosphatase required
the activation of tyrosine kinases and PKC isozyme . This suggests
that, in macrophages, PKC plays a key role in the negative control
of general MAPK activity through the induction of the phosphatase
MKP-1.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Bone marrow-derived macrophages were obtained from 6- to 10-wk-old BALB/c mice (Charles River Laboratories, Wilmington, MA) as described (25). Macrophages were cultured 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 80% confluent, normally after 6 days of culture, they were deprived of L cell-conditioned medium for 16–18 h and treated with LPS (Sigma) in the presence or absence of selective inhibitors/activators. All treatments were not toxic for the cells as determined by flow cytometry analysis.
Reagents
Genistein, bisindolylmaleimyde I (GF109203X), calphostin C, PMA, PLC from Bacillus cereus, KT5720, and KT5823 were purchased from Calbiochem (San Diego, CA). Ionomycin was purchased from ICN Pharmaceuticals (Costa Mesa, CA). 8-Bromo-cAMP (8-Br-cAMP) and 8-bromo-cyclic GMP (8-Br-cGMP) were obtained from Fluka Biochemika (Buchs, Switzerland). Sphingomyelinase from B. cereus and wortmannin were obtained from Sigma. PD98059 was purchased from New England Biolabs (Beverly, MA). Gö6976 was a kind gift from Dr. A. García de Herreros (Institut Municipal d’Investigació Mèdica, Barcelona, Spain). All reagents were used following the manufacturer’s recommendations.
Antisense oligonucleotides
Antisense phosphorothioated-oligonucleotides specific for PKC
isoforms ßI/II, , and were used to block the expression of
specific PKC isoforms. The cells were incubated for 34 h in DMEM
with 5% FBS containing 12 µM thioated-oligonucleotides (Biognostik,
Göttingen, Germany). Oligonucleotides specific for mouse
PKCßI/II corresponded to the reverse complement of a target sequence
as described (C. L. Ashendel, unpublished observations) (accession
no. X53532). Oligonucleotides against mouse PKC were designed as
described (26). Oligonucleotides specific for mouse PKC
corresponded to the sequence: 5'-GCTCACCGCCTCGCAGATTT-3'.
RNA extraction and Northern blot analysis
The cells were washed twice in cold PBS, and extraction of total
RNA was performed as described (27). Total RNA samples (15
µg) were separated on 1.2% agarose gels containing formaldehyde and
transferred to nylon membranes (Genescreen; NEN Life Science Products,
Boston, MA). For MKP-1 mRNA detection, we obtained the full-length cDNA
fragment of MKP-1 following purification from a HindIII
digestion of the plasmid pBluescript KS/MKP-1 (kindly provided by Dr.
R. Bravo, Bristol-Myers Squibb Pharmacology Research Institute,
Princeton, NJ). For TNF- mRNA detection, we used the
EcoRI/HindIII fragment of pSP65/TNF- (kindly
supplied by Dr. M. Nabholz, Institut Suisse de Recherches
Experimentales sur le Cancer, Epalinges, Switzerland). To study the
expression of IL-1ß, we obtained a probe by digesting the construct
pGEM1/IL-1ß (kindly provided by Dr. R. Wilson, Glaxo Research and
Development Limited, Greenford, U.K.) with
EcoRI/PstI. For ß-actin mRNA detection, we
obtained the PstI fragment of pSP65/ß-actin
(28). To detect the L32 transcript, we used the
EcoRI/HindIII fragment of pGEM1/L32 as a probe
(29). All probes were labeled with
[-32P]dCTP (ICN Pharmaceuticals). After
incubating in hybridization solution (20% formamide, 5x Denhardt’s,
5x SSC, 10 mM EDTA, 1% SDS, 25 mM
Na2HPO4, 25 mM
NaH2PO4, and 0.2 mg/ml
salmon sperm DNA) at 65°C, the membranes were washed and exposed to
Kodak X-AR films (Kodak, Rochester, NY). Besides, 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 lysis solution (1% Triton X-100, 10% glycerol, 50 mM HEPES, pH 7.5, 150 mM NaCl, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml iodoacetamide, 1 mM PMSF). When inhibition of the activity of tyrosine phosphatases was required, 1 mM sodium orthovanadate was included. For PKC detection experiments, 1 mM EGTA and 2 mM EDTA were added to 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. The proteins from cell lysates (50–100 µg) were boiled at 95°C in Laemli SDS-loading buffer and separated by 10% SDS-PAGE (unless otherwise stated) and electrophoretically transferred to nitrocellulose membranes (Hybond-ECL; Amersham, Arlington Heights, IL). The membranes were blocked in 2% BSA in TBS-0.1% Tween 20 (TBS-T) for 3 h at room temperature and then incubated with the primary Ab. For MKP-1 immunoblotting, incubation was performed for 1 h at room temperature with rabbit IgG anti-mouse MKP-1 (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA). For the recognition of PKC isozymes, incubation was done overnight at 4°C with rabbit IgG selective for each mouse PKC isoform (1:1000) in the presence or absence of specific competitor peptides (1:1000) (Abs and peptides were kindly provided by Dr. P. J. Parker, Imperial Cancer Research Fund, London, U.K.). For some experiments, we used equivalent Abs purchased from Life Technologies (Grand Island, NY). After three washes of 15 min each in TBS-T, the membranes were incubated with peroxidase-conjugated anti-rabbit IgG Ab (Cappel, Durham, NC) (1:5000) for 1 h. After three washes of 15 min with TBS-T, enhanced chemiluminescence detection was performed (Amersham Life Science, London, U.K.) and the membranes were exposed to x-ray films (Amersham).
Determination of ERK activity by in-gel-kinase assay
This assay was performed as previously described
(30). Briefly, 50 µg of total protein were separated by
12.5% SDS-PAGE in the presence of 0.1 mg/ml of myelin basic protein
(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 described previously (31) with some modifications. The cells were lysed by scraping in cold hypotonic buffer T10 (10 mM Tris-HCl, pH 7.5, 1 mM EGTA, 10 mM NaCl), containing inhibitors of proteases (1 µg/ml aprotinin, 1 µg/ml leupeptin, 86 µg/ml iodoacetamide, 1 mM PMSF) and 1 mM sodium orthovanadate. The cell lysates were centrifuged at 100,000 x g for 30 min at 4°C and the supernatants were collected (cytosol fraction). The pellets were resuspended in cold buffer T10 containing 1% Triton X-100 and homogenized on ice (15–20 strokes with a Dounce homogenizer). To allow PKC extraction from cell membranes, the samples were left for 1 h at 4°C and then centrifuged at 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, passed through a 19-gauge needle five times, and boiled at 100°C for 5 min. Insoluble material was removed by centrifugation at 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 anti-PKC isoforms Abs in the presence or absence of competitor peptide.
Measurement of PKC activity
This assay was performed as previously described
(32) with modifications. Specific Abs against PKC (Life
Technologies) were used to immunoprecipitate this isoform from
subcellular fractions (2 µg of Ab per 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, 1 mM EGTA)
supplemented with protease inhibitors and 1 mM sodium orthovanadate,
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). A
25Ser-substituted peptide obtained from the
pseudosubstrate region of PKC (Calbiochem) was used as the substrate
for the phosphorylation assay because it represents an appropiate
substrate for measuring PKC activity (33). The reaction
was conducted for 10 min at 30°C. Each sample was spotted on a
phosphocellulose filter (Whatman 3 MM) and subjected to five washes of
30 min each in 5% TCA, 10 mM sodium pyrophosphate. Radioactivity was
counted by liquid scintillation using a Packard Tri-Carb 1400
scintillation counter (Meriden, CT).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A). Maximal expression was
observed at 45–60 min and then it gradually decayed to basal levels
within 2–3 h. Synthesis of the MKP-1 protein tightly correlated with
the time course of MKP-1 mRNA expression (Fig. 1B). The
expression of MKP-1 was also analyzed after the stimulation of
macrophages with different concentrations of LPS for 45 min (Fig. 1, C and D). The induction of MKP-1 by LPS was dose
dependent and reached a plateau at 100 ng/ml of LPS. Therefore, in
additional experiments we used 100 ng/ml and 1 ng/ml of LPS for
saturant and subsaturant conditions, respectively.
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shows that the blockage of ERK-1/2
activation by PD98059, a specific inhibitor of MAPK kinase (MEK)-1, did
not alter the LPS-induced expression of MKP-1 at either the mRNA or
protein levels. This indicates that LPS induces the expression of MKP-1
through a mechanism that is independent of the activation of the late
elements of the ERK cascade (MEK1 and ERK-1/2).
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, C and D), a dose that saturates
the binding of LPS to its high-affinity receptor, the molecule CD14
(34). Although other molecules may also transduce signals
at very high doses of LPS (1–10 µg/ml), our results suggest that the
signaling pathway leading to MKP-1 induction is triggered after the
recognition of LPS by CD14. The fact that the induction of MKP-1 by LPS
was dependent on the presence of serum (data not shown) supports this
hypothesis. In fact, the recognition of LPS by CD14 requires the
previous association of LPS with the serum protein LBP (LPS-binding
protein) (35). Binding of LPS to CD14 results in the
tyrosine phosphorylation of several cellular proteins (8, 36). To determine the involvement of tyrosine kinases in the
induction of MKP-1 by LPS, the cells were preincubated with genistein,
a general inhibitor of this type of kinases (Fig. 3). A significant decrease of the
expression of MKP-1 mRNA was observed. As a control of the effect of
genistein, we also analyzed the LPS-induced expression of TNF- and
IL-1ß. As previously reported (37), the expression of
these cytokines was inhibited by this treatment (Fig. 3). Thus, our
results suggest that the LPS-induced expression of MKP-1 is dependent
on the activation of tyrosine kinases.
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A). Besides, 8-Br-cAMP
was unable to increase the induction of MKP-1 when macrophages were
stimulated with a subsaturant concentration of LPS. Because sustained
activation of PKA has been associated with repression of the
LPS-induced expression of TNF- in macrophages (40), the
expression of TNF- mRNA was determined as a control of the
activation of PKA by 8-Br-cAMP. Our results show that the addition of
8-Br-cAMP blocked the LPS-induced expression of TNF- (Fig. 4A). Furthermore, the activation of PKA was inhibited by
pretreating the cells with KT5720. This treatment did not affect the
induction of MKP-1 mRNA and protein by either saturant or subsaturant
concentrations of LPS (Fig. 4, B and C). The
implication of a cGMP-dependent pathway was also tested. However,
8-Br-cGMP neither induced the expression of MKP-1 nor increased its
induction by a subsaturant dose of LPS (Fig. 4A). Besides,
the inhibition of c-GMP-dependent protein kinase G (PKG) with KT5823
did not alter the induction of MKP-1 by LPS (Fig. 4, B and
C). Our results allow us to conclude that the LPS signaling
events that lead to MKP-1 induction are not dependent on the activation
of PKA or PKG.
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A).
The cells were also treated with two natural activators of PKC, PLC and
sphyngomyelinase. Fig. 5B shows that the expression of MKP-1
was induced in macrophages by both treatments. All these results
indicate that the activation of PKC is sufficient to induce the
expression of MKP-1 in macrophages. The cells were also treated with
ionomycin, an ionophore that promotes the mobilization of intracellular
Ca2+, thus inducing the activation of
Ca2+-dependent PKC isoforms. However, this
treatment neither induced the expression of MKP-1 nor increased the
induction of this phosphatase by PMA, thus suggesting that
Ca2+ mobilization alone does not induce the
expression of MKP-1 in macrophages (Fig. 5A).
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, C and D).
GF109203X also appears to block the activation of Rsk-2 and p70 S6
kinase (44). However, it is unlikely that the blockage of
MKP-1 induction is due to the inhibition of any of these molecules. In
fact, Rsk-2 is activated by ERK-1 and -2 (45), and, as we
have shown above, specific blockage of this cascade with PD98059 did
not inhibit the MKP-1 induction by LPS. In contrast, rapamycin, a
selective inhibitor for p70 S6 kinase (46), did not alter
MKP-1 expression in response to LPS (data not shown). Besides,
calphostin C, another PKC inhibitor not related to GF109203X, also
inhibited the LPS-induced expression of MKP-1 (Fig. 5E),
further supporting the involvement of PKC in this process.
We further explored the effect of the inhibition of PKC on the pattern
of ERK activation. In bone marrow macrophages, LPS induced the
activation of ERK-1/2 within the first 15 min. ERK activity remained
high until 30 min of LPS stimulation and decayed drastically thereafter
(Fig. 5F). The major part of ERK-1/2 inactivation correlated
with the time course of expression of the MKP-1 protein (Fig. 1).
Interestingly, the inhibition of PKC with GF109203X lead to a
significant prolongation of ERK activity during the macrophage response
to LPS (Fig. 5F), correlating with the ability of the drug
to inhibit MKP-1 expression (Fig. 5, C and D).
These results reinforce the importance of MKP-1 as a negative regulator
of ERK activity in LPS-stimulated macrophages. We were also interested
in determining which PKC isoform was responsible for MKP-1 induction in
LPS-stimulated macrophages. At least 11 PKC isoforms have been
described, including , ßI, ßII, , , , , ,
µ, /
, and (9, 10). However, only a subset of
PKC isozymes are expressed in a given tissue and at a certain stage of
differentiation (10). In previous studies, we used
specific Abs to determine the expression of PKC isozymes in bone
marrow-derived macrophages. We found that only PKCßI, , and
were present in these cells, whereas the rest of the isozymes were not
detected (47). Among the three isoforms expressed in bone
marrow macrophages, PKC is not effectively inhibited by treating the
cells with GF109203X at the doses used in our experiments
(48), thus suggesting that the LPS-induced expression of
MKP-1 is not mediated by this isoform. In support of this hypothesis,
MKP-1 induction was not affected by wortmannin (data not shown), an
inhibitor of phosphatidylinositol 3-kinase. This enzyme catalyzes the
production of phosphatidylinositol (3, 4, 5)-trisphosphate, an activator
of PKC (13).
To determine which of the PKC isoforms, ßI or , regulated the
expression of MKP-1, macrophages were treated with PMA for 12 h
before the addition of LPS. Prolonged exposure to PMA completely
down-regulated PKCßI but had only a partial down-modulating effect on
PKC (Fig. 6A). Accordingly,
this treatment had only a low inhibitory effect on the induction of
MKP-1 mRNA by LPS (Fig. 6B). Furthermore, Gö6976, a
selective inhibitor of conventional PKC isoforms, including PKCßI
(48), did not alter the LPS-induced expression of this
phosphatase (Fig. 6C). Taken together, these results
indicate that the activation of PKCßI is not necessary for the
induction of MKP-1 by LPS and thus suggest that PKC is the main PKC
isoform involved in this process.
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, and in response to LPS. Fig. 7 shows that PKCßI and were mainly
present in the cytosol in unstimulated cells. Whereas PMA induced the
translocation of PKCßI to the membrane and cytoskeleton fractions
(Fig. 7B), LPS, at the concentrations used in our
experiments, did not induce the translocation of either PKCßI or
to any of these compartments (Fig. 7A). In contrast, PKC
was present in the membrane fraction of both unstimulated and
LPS-treated cells, thus being able to interact with its cofactors once
a peak of DAG is generated in response to LPS.
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, we immunoprecipitated PKC from the membrane fraction of
macrophages stimulated or not with LPS and analyzed its capability to
phosphorylate the specific susbtrate
25Ser-substituted PKC peptide in
vitro. The PKC activity detected in the membrane fraction of
nonstimulated macrophages was equivalent to the background signal
detected in the cytosolic fraction of these cells (data not shown).
Fig. 7C shows that LPS induced significant activation of
PKC within the first 15 min of stimulation. These results further
correlate PKC activation with the induction of MKP-1 in bone marrow
macrophages.
Moreover, the cells were treated with antisense oligonucleotides
specific for each of the three PKC isoforms ßI, , or .
Antisense oligonucleotides directed against PKCßI or , although
blocking the expression of these isoforms (Fig. 8A), did not inhibit the
induction of MKP-1 by LPS (Fig. 8B). In contrast,
oligonucleotides specific for PKC efficiently blocked the expression
of this isoform and significantly inhibited the induction of MKP-1 in
response to LPS. Besides, this treatment lead to an increase in the
levels of ERK-1/2 activity after 70 min of exposure to LPS in
comparison to those observed in control cells and in cells treated with
specific oligonucleotides against PKCßI or (Fig. 8D).
These results indicate that PKC is specifically involved in the
induction of the phosphatase MKP-1 and thus in the negative regulation
of ERK activity in LPS-stimulated macrophages.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In fibroblasts, the activation of ERK-1/2 is sufficient to induce the expression of MKP-1, thus promoting the attenuation of their own cascade in a direct negative feedback loop (50). However, in bone marrow macrophages, the induction of MKP-1 by LPS takes place even when ERK activation is blocked. Thus, LPS seems to activate two independent pathways, one leading to ERK-1/2 activation and the other one determining the expression of their specific phosphatase. Also in contrast to what has been reported in fibroblasts (21, 50), a cAMP-dependent pathway is not involved in the induction of MKP-1 in macrophages.
Our results suggest that LPS leads to MKP-1 induction through a pathway that involves the activation of intracellular tyrosine kinases and PKC. Although we have not identified the tyrosine kinases involved in this response, the members of the src family of tyrosine kinases are likely candidates for two reasons. First, p53/56lyn is associated with CD14 in macrophages (51). Second, p53/56lyn, p58/64hck, and p59c-fgr are all transiently activated upon stimulation of macrophages with LPS (15, 51). However, some LPS responses occur in the absence of Hck, Fgr, and Lyn (52), which suggests that their activity can be replaced either by other members of the same family or by totally distinct tyrosine kinases.
In bone marrow macrophages, PKC activation is sufficient to induce the
expression of MKP-1. This has been also described in some other cell
types (21, 53, 54). By using two unrelated PKC inhibitors,
we have shown that PKC is also involved in the induction of MKP-1 by
LPS. Although PKC has been extensively implicated in the control of
several LPS-induced events (6, 7, 8, 55, 56, 57, 58, 59), it is still
unclear which isoform(s) are involved in each of these effects. In
previous studies, we found that bone marrow macrophages express PKC
isoforms ßI, , and (47). Although LPS shows high
structural similarities with ceramide (60), a second
messenger that activates PKC (12), and despite the fact
that MKP-1 expression is induced in macrophages stimulated with
sphyngomyelinase, an upstream activator of PKC, it is unlikely that
this isoform mediates the induction of MKP-1 by LPS for several
reasons. First, the PKC inhibitor that blocks MKP-1 expression,
GF109203X, was used at doses that inhibit conventional and novel PKCs,
but not the atypical isoforms, including PKC (48).
Second, the use of wortmannin to inhibit PI3K, an upstream regulator of
PKC, did not affect MKP-1 induction by LPS. Third, we could not
detect translocation of PKC in response to LPS. And finally,
oligonucleotides against PKC did not block MKP-1 induction by LPS.
All these observations suggest that PKC is not involved in the LPS
signaling pathway that leads to MKP-1 expression.
Several observations support the involvement of PKC rather than
PKCßI in the induction of MKP-1 by LPS. First, GF109203X inhibits
conventional PKC isoforms better than novel ones (48).
Concentrations of up to 1 µM of GF109203X completely inhibit the
activation of conventional PKCs, including ßI, whereas concentrations
of up to 5 µM are needed to completely block novel PKC isoforms,
including . In our hands, maximal inhibition of MKP-1 was reached at
concentrations of 3–5 µM of GF109203X. Second, prolonged treatment
of macrophages with PMA causes only a low inhibition of MKP-1 induction
by LPS. In bone marrow macrophages, this treatment leads to a complete
depletion of PKCßI, but not of PKC. Similarly, in three
macrophagic cell lines and in several other cell systems, PKC has
been also shown to be resistant to prolonged PMA treatment (6, 61, 62, 63, 64). Third, Gö6976, a selective inhibitor of
conventional PKCs, does not block MKP-1 induction by LPS. And finally,
treatment of these cells with antisense oligonucleotides specific for
PKC, and not with those specific for PKCßI, significantly inhibits
the induction of MKP-1 by LPS. All these results suggest that, in bone
marrow macrophages, PKC is specifically involved in the induction of
the phosphatase MKP-1 by LPS. In previous studies, we had observed that
MKP-1 expression was also induced in macrophages stimulated with M-CSF
(47). A strong parallelism has been observed between the
mechanism used by M-CSF and LPS to induce MKP-1 expression, although
both agents trigger totally opposed effects in macrophage biology
(proliferation vs activation). During the macrophage response to M-CSF,
MKP-1 induction was similarly mediated through a PKC-dependent pathway,
and, of all the PKC isoforms detected in bone marrow macrophages,
PKC was the main candidate to mediate MKP-1 expression. These,
together with the data shown in the present report, suggest that, in
bone marrow macrophages, PKC plays a major role in the control of
MKP-1 expression.
We have also shown a significant prolongation of the time course of ERK
activity in macrophages treated with the PKC inhibitor GF109203X or
with antisense oligonucleotides against PKC, correlating with the
capability of these compounds to inhibit MKP-1 expression. Although we
cannot discard the involvement of other phosphatases different to
MKP-1, specially because partial inactivation of ERK-1/2 still took
place in those experimental conditions, our results allow us to
conclude that PKC has an important function in the control of the
pattern of ERK activity by inducing the expression of MKP-1.
PKC activation has been frequently associated to its translocation to
the membrane or the cytoskeleton (9, 10). Surprisingly, we
have detected PKC at the membranal fraction of unstimulated
macrophages. This confirms previous reports in which the constitutive
association of PKC to the membrane was also described in other cell
types and in a macrophagic cell line (10, 65). However,
our results reveal that the presence of PKC at this compartment does
not mean that PKC is constitutively active in unstimulated
macrophages. In fact, treatment with LPS is required to detect
significant activation of membrane-bound PKC, thus correlating the
activation of this isoform with the induction of MKP-1 in
LPS-stimulated macrophages.
Although we cannot exclude the activation of PKC isoforms other than
PKC, studies with blocking Abs showed that activation of PKC
accounts for the majority of PKC activity in murine peritoneal
macrophages treated with LPS (66). Moreover, stimulation
of human monocytes with LPS results in the specific activation of a
Ca2+-independent isoform of PKC (7).
These and our observations predict a crutial role for PKC in the
biology of macrophages. Several reports suggest that generation of DAG
by LPS takes place through the action of a phosphatydilcholine-specific
form of PLC (67). Phosphatydilcholine-PLC activity does
not generate IP3, a second messenger that induces
the release of Ca2+ from intracellular storage
sites (68). In fact, in accordance with previously
published work (69, 70, 71), we have not detected mobilization
of intracellular Ca2+ in bone marrow macrophages
treated with 1–100 ng/ml of LPS (A.F.V. et al., manuscript in
preparation). Because conventional PKCs require the presence of
Ca2+ for full activation (9, 10),
the absence of Ca2+ mobilization would explain
the lack of translocation of PKCßI from the cytosolic to particulate
fractions in response to the doses of LPS used in our experiments.
Although we can assume that PKC is required for the induction of
MKP-1 by LPS, we cannot discard the involvement of other signaling
molecules that have not been analyzed in this report. In fact, we did
not get a total inhibition of MKP-1 expression in macrophages treated
with either specific inhibitors or with oligonucleotides against
PKC. Besides, the induction of this phosphatase by PMA was
substantially lower than that mediated by LPS. These observations
suggest that PKC is required for this process but some other
mechanism may participate in the induction of MKP-1 by LPS. We are
currently analyzing the involvement of other pathways that regulate
MKP-1 expression.
In this report, we have studied the mechanisms that control the
expression of MKP-1 in bone marrow macrophages stimulated with LPS. It
should be emphasized that LPS induces the expression of MKP-1 in an
ERK-independent manner. Therefore, in macrophages stimulated with LPS,
ERK-1/2 do not attenuate their own activation by inducing MKP-1. LPS
ensures this negative control by triggering an alternative pathway that
is activated via the CD14 molecule and specifically requires the action
of tyrosine kinases and PKC. The results of this report demonstrate
the key role of PKC in the control of the duration of MAPK activity
during the macrophage response to LPS.
|
Acknowledgments |
|---|
cDNA, Dr.
Rose Wilson (Glaxo Research and Development Limited, Greenford, U.K.)
for the IL-1ß-containing plasmid, and Dr. José A.
García-Sanz (CNB, Madrid, Spain) for the plasmid containing
ß-actin and for helpful advice about some protocols. We also thank
Martin Cullell-Young for reviewing the manuscript. |
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Antonio Celada, Departament de Fisiologia, Facultat de Biologia, Av. Diagonal 645, 08028 Barcelona, Spain. E-mail:
3 Abbreviations used in this paper: PLC, phospholipase C; CRE, cAMP response element; DAG, diacylglycerol; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; MKP-1, MAPK phosphatase-1; PKA, protein kinase A; PKC, protein kinase C; PKG, protein kinase G; 8-Br-cAMP. 8-bromo-cAMP; 8-Br-cGMP, 8-bromo-cyclic GMP.
Received for publication June 16, 1999. Accepted for publication October 8, 1999.
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