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* Stress Signaling Unit, Laboratory of Cellular and Molecular Biology, National Institute on Aging, Intramural Research Program, National Institutes of Health, Baltimore, MD 21224; and
GlaxoSmithKline Pharmaceuticals, King of Prussia, PA 19406
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, through complex signaling
mechanisms. Mitogen-activated protein (MAP) kinases play a critical
role in this process. In the present study, we have addressed the role
of MAP kinase phosphatase-1 (MKP-1) in regulating proinflammatory
cytokine production using RAW264.7 macrophages. Analysis of MAP kinase
activity revealed a transient activation of c-Jun N-terminal kinase
(JNK) and p38 after LPS stimulation. Interestingly, MKP-1 was induced
concurrently with the inactivation of JNK and p38, whereas blocking
MKP-1 induction by triptolide prevented this inactivation. Ectopic
expression of MKP-1 accelerated JNK and p38 inactivation and
substantially inhibited the production of TNF- and IL-6. Induction
of MKP-1 by LPS was found to be extracellular signal-regulated kinase
dependent and involved enhanced gene expression and increased protein
stability. Finally, MKP-1 expression was also induced by
glucocorticoids as well as cholera toxin B subunit, an agent capable of
preventing autoimmune diseases in animal models. These findings
highlight MKP-1 as a critical negative regulator of the macrophage
inflammatory response, underscoring its premise as a potential target
for developing novel anti-inflammatory
drugs. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
IL-1, IL-6, and IL-8, which induce inflammation and recruit other
immune cells, e.g., neutrophils and T lymphocytes (3).
Although these proinflammatory cytokines are beneficial to the host
defense, they can also trigger pathological conditions when expressed
in excess (4). For example, massive stimulation of
macrophages after a severe Gram-negative bacterial infection leads to
excessive production of proinflammatory cytokines, including TNF-
and IL-1, and the development of fatal septic shock syndrome,
characterized by fever, disseminated intravascular coagulation, and
multiple organ failure (4, 5). In addition, higher levels
of proinflammatory cytokines are also implicated in a variety of
chronic inflammatory diseases including rheumatoid arthritis,
psoriasis, and Crohn’s disease (4). Moreover, recent
studies suggest that macrophage hyperactivity and the overproduction of
TNF- contribute to autologous cell destruction and T cell-mediated
autoimmune diseases (6).
In macrophages, the biosynthesis of cytokines, especially TNF-, is
regulated at multiple levels and involves a multitude of signal
transduction pathways (2). It has been shown that LPS, a
component of Gram-negative bacterial cell walls, binds to LPS binding
protein (LBP).3 CD14,
a macrophage/monocyte-specific receptor protein, presents the LPS-LBP
complexes to Toll-like receptor 4 (2). Interaction
of Toll-like receptor 4 with the LPS-LBP-CD14 complexes triggers a
multitude of signaling events, including those that culminate in the
activation of both the mitogen-activated protein (MAP) kinases and the
transcription factor NF-
B, which ultimately leads to increased
TNF- expression via both transcriptional and posttranscriptional
mechanisms (2, 7).
Particularly important in the regulation of TNF- expression is an
AU-rich element (ARE) residing in the 3' untranslated region of
the TNF- mRNA that represses TNF- expression
posttranscriptionally (7, 8). MAP kinases, including
extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase
(JNK), and p38, have been shown to target this ARE to increase TNF-
biosynthesis in response to LPS stimulation (9). The
critical role of p38 in TNF- biosynthesis has been well established
(10). Selective inhibition of p38 using specific imidazole
compounds such as SB2305080 substantially decreases the translation of
TNF- in LPS-stimulated macrophages. Furthermore, inactivation of the
gene for MAP kinase-activated protein kinase-2 (MAPKAPK-2)
(11), a downstream target of p38, abolishes LPS-triggered
TNF- production and renders mice resistant to endotoxin-induced
septic shock (12). Moreover, deletion of the TNF- ARE
bypasses the requirement of p38/MAPKAPK-2 for LPS-induced TNF-
production (13, 14). JNK also appears to play a role in
relieving the ARE-mediated translational silencing of TNF- mRNA,
because glucocorticoids have been shown to inhibit LPS-induced JNK
activation and to reduce TNF- production by macrophages (7, 13, 15, 16). More recently, using transgenic mice, Dumitru et
al. (8) demonstrated that LPS-triggered ERK activation is
required for the nucleocytoplasmic transport of TNF- mRNA via a
mechanism that involves the TNF- ARE.
The activities of all MAP kinases are regulated via reversible phosphorylation of the conserved threonine and tyrosine residues in their tripeptide TXY signature motifs (17). In mammalian cells, inactivation of MAP kinases is primarily conducted by a family of dual-specificity MAP kinase phosphatases (MKPs), with MKP-1 being the archetype (18). Because the expression of MKPs can be regulated by MAP kinases, it has been suggested that MKPs play an important role in the feedback control of MAP kinase signaling (18, 19). The results from the present study strongly suggest that MKP-1 is a critical negative regulator in macrophage signaling in response to inflammatory stimuli and is responsible for switching off the production of proinflammatory cytokines. Finally, we demonstrate that MKP-1 is induced by certain anti-inflammatory drugs/agents and propose that MKP-1 could be a target for developing novel anti-inflammatory drugs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RAW264.7 cells were cultured in DMEM (Invitrogen, San Diego, CA)
supplemented with 10% FCS (HyClone Laboratories, Logan, UT) at 37°C
in a humidified atmosphere containing 5% CO2.
Cells were transfected with MKP-1 expression constructs together with
pcDNA3 (Invitrogen) using FuGENE6 transfection reagent (Roche,
Indianapolis, IN) or Lipofectin (Invitrogen) according to the
manufacturer’s specifications. Cells were selected in medium
containing G418, and resistant clones were isolated. LPS
(Escherichia coli 055:B5; Calbiochem, La Jolla, CA) and
cholera toxin B subunit (CTB; Sigma-Aldrich, St. Louis, MO) were
dissolved in serum-free medium and added to the medium at the indicated
concentrations. In experiments determining MKP-1 stability, 10 µg/ml
cycloheximide (CHX; Calbiochem) was added to the culture medium, either
alone or in combination with LPS. Dexamethasone (Sigma-Aldrich) and
triptolide (Calbiochem) were dissolved in DMSO and added to the culture
at the concentrations indicated. The MAP/ERK kinase (MEK)
inhibitor U0126 (Promega, Madison, WI), and the p38 inhibitor SB203580
(Calbiochem) were dissolved in DMSO and added to the medium at a final
concentration of 10 µM 30 min before the addition of LPS. To isolate
proteins, cells were harvested in a lysis buffer containing 10 mM HEPES
(pH 7.4), 50 mM 
-glycerophosphate, 1% Triton X-100, 10% glycerol,
2 mM EDTA, 2 mM EGTA, 1 mM DTT, 10 mM NaF, 1 mM
Na3VO4, 20 nM
microcystin-LR, 2 µM leupeptin, 2 µM aprotinin, and 1
mM PMSF.
Expression vectors
pSR-Flag-MKP-1, which expresses full-length MKP-1
tagged with three Flag epitopes at its amino terminus, has been
previously described (20). pSR-Flag-MKP-1
C was
created by introducing a premature stop codon in the open reading frame
of MKP-1 through site-directed mutagenesis (QuickChange; Stratagene, La
Jolla, CA) to delete the last 54 aa from the C terminus. The bacterial
expression vector for GST-tagged p38 has been described previously
(21).
Western blotting and ELISA
Western blot analysis was conducted essentially as previously
described using ECL reagent (Amersham Pharmacia Biotech, Piscataway,
NJ) (22). MKP-1 was detected using a rabbit polyclonal Ab
(Santa Cruz Biotechnology, Santa Cruz, CA). Phosphorylated ERK and p38
were detected using rabbit polyclonal Abs from Cell Signaling
Technology (Beverly, MA). Phosphorylated JNK was detected using
a rabbit polyclonal Ab purchased from Promega. ERK1/2 was detected
using either a monoclonal anti-pan ERK (BD Transduction
Laboratories, Lexington, KY) or a polyclonal Ab recognizing both ERK1
and ERK2 (Cell Signaling Technology). Total JNK was detected using a
mAb recognizing both JNK1 and JNK2 (BD PharMingen, San Diego, CA).
Total p38 was detected using a mAb (BD Transduction Laboratories).
Flag-tagged MKP-1 was detected using a mAb against Flag (M2)
(Berkeley Antibody, Richmond, CA). In the experiments for determining
the stability of MKP-1, Western blot films were scanned using a
Scanmaker 5 (MicroTek, Carson, CA) and the images were analyzed
using ImageMaster 1D Elite software (Pharmacia Biotech, Uppsala,
Sweden). TNF- and IL-6 in the culture medium were determined using
ELISA kits (R&D Systems, Minneapolis, MN) according to the
manufacturer’s recommendations.
GST pull down
GST-p38 was produced in Escherichia coli as previously described (21). RAW264.7 cell lysates containing 200 µg of total protein were incubated with 4 µg of GST-p38 prebound to glutathione-Sepharose beads (Amersham Pharmacia Biotech) for 1 h at 4°C. The beads were washed twice with 1 ml of lysis buffer and twice with 1 ml of lysis buffer supplemented with 200 mM NaCl. The proteins were separated by electrophoresis and analyzed by Western blotting using the anti-MKP-1 Ab (Santa Cruz Biotechnology).
Northern blot analysis
Total RNA was isolated with STAT-60 (Tel-Test, Friendswood, TX). Northern blot analysis was performed using mouse MKP-1 cDNA as a probe as described previously (23, 24). The membrane was stripped and reprobed with an oligonucleotide corresponding to 18S rRNA (22).
Immune complex kinase assays
MAPKAPK-2 activity was measured by immune complex kinase assay
as previously described (20). Briefly, endogenous
MAPKAPK-2 was immunoprecipitated from 500 µg of RAW264.7 cell lysate
using 3 µg of the rabbit polyclonal antiserum (kindly provided by J.
Huot, l’Université Laval, Quebec, Canada) and protein
A-Sepharose (Amersham Pharmacia Biotech). The kinase activity in the
MAPKAPK-2 immune complexes was assayed using
[
-32P]ATP and recombinant heat shock protein
25 (StressGen Biotechnologies, Victoria, BC, Canada) as a
substrate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The kinetics of MAP kinase activation in subconfluent RAW264.7
cells stimulated with 100 ng/ml LPS were examined by Western blotting
using Abs specifically recognizing phosphorylated ERK, JNK, and p38
(Fig. 1
A). JNK and p38 were
rapidly activated by LPS, reaching their maximal activities within 15
min. Their activities then decreased and plunged to nearly basal levels
by 60 min. ERK was also potently activated. In contrast to JNK and p38,
ERK activity decreased only modestly after achieving maximal activation
but was sustained at relatively high levels throughout the time period
examined. MAPKAPK-2, the downstream target of p38, was also activated
in response to LPS with kinetics similar to those seen for p38 MAP
kinase (Fig. 1B). These observations raised the possibility
that a MKP(s) that preferentially inactivates JNK and p38 could be
induced in response to LPS and prompted us to directly examine the role
of MKPs in this process.
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C). In
unstimulated cells, the MKP-1 protein level was very low, essentially
below the limit of detection. In response to LPS stimulation, MKP-1
protein increased dramatically, reaching its maximal level between 1
and 2 h, and then decreased slightly. The reciprocal relationship
between MKP-1 and the two stress-activated MAP kinases, p38 and JNK,
supports the notion that MKP-1 may play a critical role in the
inactivation of these MAP kinases. Blocking LPS-triggered MKP-1 induction with diterpenoid triepoxide triptolide prevents the inactivation of MAP kinases
To further understand the relationship between MKP-1 induction and
MAP kinase inactivation, we screened various chemical compounds for
agents that block the induction of MKP-1 by LPS. We found that
triptolide, a diterpenoid triepoxide (27), potently
blocked MKP-1 induction by LPS in a dose-dependent manner (Fig. 2A). This dose-dependent
blockade of MKP-1 protein accumulation was associated with a reciprocal
increase in the levels of phosphorylated/active JNK and p38. Triptolide
pretreatment also prevented the modest decrease in ERK phosphorylation
that normally occurred after 
60 min after LPS stimulation (Fig. 2A). To ascertain that triptolide did not act by
neutralizing LPS or by merely slowing down the signal transduction
process, we examined the activation kinetics of these kinases over a
2-h time period (Fig. 2B). LPS stimulated MAP kinase
activation in both control and the triptolide-pretreated cells within
15 min. Triptolide completely blocked MKP-1 induction and prevented the
inactivation of MAP kinases (Fig. 2B). These observations
strengthen the link between MKP-1 induction and the inactivation of MAP
kinases, especially JNK and p38.
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To understand the mechanism(s) mediating MKP-1 induction by LPS,
Northern blot analysis was performed. MKP-1 mRNA levels were virtually
undetectable in unstimulated control cells. In response to LPS
treatment, MKP-1 mRNA was potently induced within 30 min, reaching its
maximal levels after 60 min (Fig. 3A). Similar to what was
observed at the protein level (Fig. 1A), a significant
decrease in MKP-1 mRNA level was observed at later time points. To
examine the role of ERK and p38 in MKP-1 induction by LPS, we exploited
the pharmacological inhibitors for these pathways. Pretreatment of
cells with either U0126, an inhibitor specific for MEK1/2, or SB203580,
a well-established inhibitor for p38, did not alter basal MKP-1 mRNA
levels (Fig. 3B). U0126 (10 µM) substantially attenuated
MKP-1 induction, decreasing MKP-1 mRNA by 65%, whereas SB203580 (10
µM) had little effect on MKP-1 induction by LPS. Surprisingly,
compared with MKP-1 mRNA in cells pretreated with U0126 alone, MKP-1
mRNA levels in cells pretreated with both U0126 and SB203580 were
significantly lower (Fig. 3B). Similar to what was observed
at the MKP-1 mRNA levels, U0126 also potently inhibited the MKP-1
increase at the protein level, resulting in an 72% reduction in
MKP-1 protein (Fig. 3C). Pretreatment of cells with SB203580
also moderately inhibited the MKP-1 protein increase (33% reduction at
the MKP-1 protein levels). Pretreatment of cells with a
combination of U0126 and SB203580 almost abolished the LPS-triggered
MKP-1 protein induction. Consistent with the notion that MKP-1 is
responsible for the dephosphorylation of p38 in these cells, blockage
of MKP-1 induction by either U0126 alone or a combination of U0126 and
SB203580 resulted in a significant increase in phosphorylated p38 (Fig. 3C). These results indicate that MKP-1 induction in response
to LPS is primarily regulated by the ERK pathway with p38 playing only
a minor role.
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and IL-6
To examine the effect of MKP-1 on the production of
proinflammatory cytokines, a mammalian vector expressing Flag-tagged
MKP-1 was transfected into RAW264.7 cells by Fugene6 or Lipofectin to
establish cells stably expressing Flag-MKP-1. After selection in medium
containing G418, the colonies were pooled (hereafter referred to as
pools F and N for pools established using Fugene6 or Lipofectin,
respectively). Western blotting using an Ab against the Flag tag
indicated that MKP-1 expression levels were higher in pool F than in
pool N (Fig. 4A). The
ectopically expressed MKP-1 was compared with the endogenous protein
levels through Western blotting using an Ab against MKP-1. Even in pool
F, the absolute level of ectopically expressed MKP-1 was moderate at
best, which was estimated to be less than 30% of the endogenous
protein found in LPS-stimulated cells (Fig. 4A). The effect
of elevated MKP-1 expression on MAP kinase activities was examined by
Western blotting. As indicated in Fig. 4B, LPS stimulation
at a dose of 50 ng/ml potently activated all three MAP kinases within
15 min. At the 15-min time point, there was little difference in either
the phospho-JNK or the phospho-p38 levels between the cell pool stably
transfected with an empty vector and the two MKP-1-expressing cell
pools. However, by 60 min, the levels of both phospho-JNK and
phospho-p38 were only slightly reduced in the cell pool carrying the
empty vector. In contrast, the amounts of both phospho-JNK and
phospho-p38 were reduced to almost basal levels in pool F. Although
less prominent than in pool F, the level of phospho-JNK was also
considerably lower in pool N than in the cells carrying the empty
vector (Fig. 4B). However, there was no significant
difference in the phospho-ERK levels between the cells carrying the
vector and any of the stable MKP-1-expressing pools (data not shown).
Taken together, these results indicate that a moderate amount of
ectopically expressed MKP-1 accelerates the inactivation of p38 and JNK
in a dose-dependent manner.
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B, bottom panel). Interestingly, the ectopically
expressed MKP-1 increased substantially after LPS stimulation in all
stable MKP-1-expressing pools. It should be pointed out that
Flag-tagged MKP-1 was expressed under a heterologous promoter in an
expression cassette that contains only the MKP-1 open reading frame and
lacks both the 3' and 5' untranslated regions (20). The
marked increase in Flag-MKP-1 protein after LPS stimulation in pool F
and pool N strongly suggests that MKP-1 protein is also regulated
posttranslationally.
The effect of increased MKP-1 expression on the production of TNF-
and IL-6 was examined by ELISA. Compared with cells transfected with an
empty plasmid, the pool population expressing the higher level of
Flag-MKP-1 (i.e., pool F) exhibited a profoundly attenuated response to
LPS stimulation, as shown by the 70% reduction in TNF- secretion.
A less prominent but nevertheless significant decrease in TNF-
secretion was also observed in pool N (Fig. 5A). In addition to TNF-,
LPS-stimulated IL-6 biosynthesis was also inhibited in the cell pools
expressing Flag-MKP-1 (Fig. 5B). IL-6 biosynthesis appeared
to be more sensitive to MKP-1. IL-6 biosynthesis was almost completely
abolished in pool F. The ectopically expressed MKP-1 in pool N also
substantially inhibited IL-6 production (Fig. 5B), although
it only slightly inhibited TNF- production (Fig. 5A).
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To examine whether the stability of MKP-1 protein was influenced
by LPS, cells from pool F were treated with either CHX alone or CHX
together with LPS and were harvested at different time points (Fig. 6A). The decay of Flag-MKP-1
was studied by Western blotting using Flag Ab. In the absence of LPS,
Flag-MKP-1 degraded rapidly with a half-life of 50 min. LPS
treatment resulted in a significant increase in MKP-1 stability,
extending its half-life to 200 min. Pretreatment with U0126
substantially accelerated the degradation of Flag-MKP-1 in
LPS-stimulated cells, reducing the half-life to close to 60 min (Fig. 6B). In contrast, SB203580 had little effect on MKP-1
stability. These results indicate that posttranslational stabilization
of MKP-1 plays a significant role in MKP-1 induction by LPS and that
ERK plays an important role in mediating this stabilization
process.
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C). MKP-1C lacks the last 54 aa in
its C terminus, which includes the two serine residues shown to be
phosphorylated by ERK and a putative docking motif for ERK binding. The
stability of MKP-1C in control and LPS-stimulated cells was examined
by Western blotting (Fig. 7). In
unstimulated cells, the half-life of MKP-1C was comparable to that
of the full-length protein. In response to LPS stimulation, its
half-life was increased by less than twofold, from 47 to 78 min,
indicating that the C-terminal domain of MKP-1 plays an important role
in mediating its LPS-induced stabilization. This result also suggests
that in addition to the phosphorylation sites targeted by ERK in the
MKP-1 C terminus, other domains of MKP-1 may also play a role in
LPS-stimulated stabilization.
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Glucocorticoids are well-known anti-inflammatory and
immune-suppressant agents that can block the production of TNF-, at
least in part, by inhibiting JNK activity (13, 15). In
mast cells, glucocorticoids have been shown to inhibit ERK activity by
increasing MKP-1 transcription and decreasing MKP-1 protein degradation
(29). To investigate whether in macrophages enhanced MKP-1
expression is involved in the anti-inflammatory activity of
glucocorticoids, RAW264.7 cells were treated with dexamethasone over a
2-day period. MKP-1 protein was first pulled down using GST-p38 and
then was analyzed by Western blotting (Fig. 8A). MKP-1 protein was
elevated after a 4-h dexamethasone treatment and lasted for the entire
time period examined; solvent (DMSO) alone had little effect.
Furthermore, it should be noted that this increase in MKP-1 protein was
detected in cells treated with dexamethasone at a dose as low as 25 nM.
Northern blotting analysis also indicated that dexamethasone increased
MKP-1 mRNA in RAW264.7 cells (Fig. 8B).
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and IL-6
(30, 31). However, the signaling mechanism(s) involved are
not fully understood. We examined the effect of CTB on MKP-1 protein
expression. Treatment of RAW264.7 cells with CTB potently increased
MKP-1 protein levels within 2 h, and the effect lasted up to
8 h (Fig. 9A).
Pretreatment of cells with CTB significantly increased the MKP-1
protein levels and attenuated the LPS-stimulated activation of all
three major MAP kinases (Fig. 9B). Interestingly, compared
with the cells pretreated with CTB, cells that did not receive CTB
pretreatment accumulated a higher level of MKP-1 at the 45-min time
point (Fig. 9B, bottom panel), likely due to the
more robust ERK activation in these cells. This inhibitory effect of
CTB on MAP kinases was also reflected in the production of both TNF-
and IL-6. CTB pretreatment substantially inhibited TNF- production
(>75%) and completely abolished IL-6 biosynthesis in response to LPS
stimulation, although CTB alone slightly increased the biosynthesis of
TNF- and had no effect on IL-6 (Fig. 9C). These findings
suggest that MKP-1 induction could be a potential mechanism accounting
for the immune-modulating properties of CTB. The findings may also be
relevant to the pathogenic property of V. cholerae.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). MKP-1 was
found to interact both in vitro and in vivo with representative members
of all three major MAP kinase subfamilies (data not shown). More
importantly, a block in MKP-1 induction by the pharmacological
inhibitor triptolide prevented the inactivation of both JNK and p38
that normally occurs at 60 min after LPS stimulation (Fig. 2). An
increase of MKP-1 expression, even to a modest level, significantly
accelerated the inactivation of JNK and p38 (Fig. 4), resulting in a
substantial decrease in TNF- production and a complete inhibition of
IL-6 biosynthesis in LPS-stimulated cells (Fig. 5). Taken together,
these results strongly support a critical role for MKP-1 in the
termination of signals that govern the biosynthesis of the
proinflammatory cytokines.
Despite the fact that MKP-1 can play a pivotal role in the
dephosphorylation of ERK MAP kinases in some cell systems (18, 19), the primary targets of MKP-1 in terminating proinflammatory
cytokine biosynthesis do not appear to be the ERK MAP kinases in the
present model system. This notion is supported by two observations.
First, maximal MKP-1 induction did not correlate with a substantial
dephosphorylation of ERK in LPS-stimulated cells. MKP-1 expression was
maximally induced by LPS after 60 min, a time when JNK and p38 were
almost completely inactivated, but a substantial amount of ERK activity
still remained (Fig. 1, A and C). Second, ectopic
expression of MKP-1 did not significantly alter the time course of
LPS-triggered ERK activation (data not shown), but significantly
attenuated proinflammatory cytokine biosynthesis (Fig. 5). Our data are
consistent with the conclusion by Franklin and Kraft (25)
that among all three major MAP kinase subfamilies, MKP-1 prefers
members of the p38 and JNK subfamilies as substrates. However, we
neither exclude a role for MKP-1 in the inactivation of ERK MAP kinases
nor rule out the possibility that other MKPs, such as phosphatase of
activated cells-1, may also participate in the inactivation of
p38 and JNK MAP kinases.
MKP-1 is potently induced after LPS stimulation through mechanisms
mediated, in a large part, by the ERK pathway. This is an intriguing
result, considering that MKP-1 preferentially acts on p38 and JNK. The
induction of MKP-1 involves both increased MKP-1 mRNA levels,
presumably due to elevated gene transcription (Fig. 3), and enhanced
protein stability (Fig. 6). LPS-induced increases in MKP-1 mRNA are
substantially inhibited by the MEK inhibitor U0126 (Fig. 3),
illustrating the importance of the ERK pathway in MKP-1 induction. The
role of p38 in MKP-1 induction is less clear. Although the p38
inhibitor SB203580 alone did not show a significant inhibitory effect
on MKP-1 mRNA induction, in combination with the MEK inhibitor U0126 it
did attenuate MKP-1 mRNA induction (Fig. 3B), and by itself
it significantly reduced the LPS-triggered increase in MKP-1 protein
(Fig. 3C). These observations suggest that p38 plays a minor
role in mediating MKP-1 mRNA induction by LPS. The fact that the
induction of MKP-1 expression is mediated primarily by the ERK pathway
raises a very interesting possibility that MKP-1, and potentially also
other MKPs, may serve as a critical mediator for interplay and
cross-talk between the various MAP kinase pathways (Fig. 4C). A finding that also illustrates this possibility has
been reported for MKP-M, another MKP family member isolated from
macrophages (35). MKP-M has been shown to primarily act on
JNK, although its induction is primarily mediated by p38. Our finding
that MKP-1 induction by LPS is primarily mediated by the ERK pathway is
different from a previous observation made by Valledor et al.
(36) using mouse bone marrow macrophages. Although LPS
also potently induced MKP-1 in those cells, MKP-1 induction was
resistant to PD98051, another MEK inhibitor (36). It is
unclear whether this discrepancy may be due to the differences between
primary bone marrow macrophages and the established macrophage cell
line used here or due to differences in experimental methodologies.
Future studies using primary macrophages will address this issue.
In addition to up-regulating MKP-1 mRNA levels, LPS also substantially
stabilizes the MKP-1 protein. Presumably, enhanced MKP-1 protein
stability in addition to increased MKP-1 transcription offers more
leverage for the ERK pathway to dominate the biological program and
prevent overproduction of proinflammatory cytokines. Previously, in
fibroblasts as well as in Xenopus oocytes (28),
MKP-1 has been shown to be stabilized through phosphorylation mediated
by ERK MAP kinases. Our experiments using stable MKP-1-expressing cells
provide unequivocal support for this conclusion. In the stable cell
pools, MKP-1 expression was driven by a heterologous expression
cassette. Because this cassette only contains an open reading frame
from MKP-1, the increase in ectopically expressed MKP-1 after LPS
stimulation is very likely due to an increase in protein stability.
This is supported by an approximately fourfold increase in MKP-1
half-life after LPS stimulation (Fig. 6A). Such an increase
in half-life after LPS stimulation explains the accelerated
inactivation of JNK and p38 in the two cell pools that express moderate
amounts of MKP-1 (Fig. 4B). The fact that U0126
significantly shortens the half-life of MKP-1 confirms the conclusion
that ERK plays an important role in the MKP-1 protein increase seen
after LPS treatment. However, ERK MAP kinases are unlikely to be the
only mediators involved in MKP-1 stabilization, because U0126 did not
completely prevent the LPS-triggered increase in Flag-MKP-1 stability
(Fig. 6B). Another interesting finding from this study is
that LPS-induced stabilization of MKP-1 is not solely mediated by the C
terminus domain of MKP-1 (Fig. 7). The involvement of the MKP-1
C-terminal domain in enhancing its stability is not surprising because
two serine residues, serine 359 and serine 364, have been shown to be
phosphorylated by ERK (28). The fact that a C-terminal
truncated MKP-1 mutant can undergo a compromised (compared with the
full-length protein) but nevertheless significant increase in protein
half-life suggests that other domains of MKP-1 contribute to its
stabilization (Fig. 7), possibly via phosphorylation by ERK or other
protein kinases. Recently, threonine 168 has been identified as a third
phosphorylation site targeted by ERK (37). Whether
threonine 168 plays a role in LPS-stimulated MKP-1 stabilization
remains to be examined.
Our findings that MKP-1 acts as a negative inhibitor of inflammatory
cytokine production may have important implications in understanding
the action of some anti-inflammatory drugs and immune-modulatory
agents. Glucocorticoids are potent anti-inflammatory drugs that can
inhibit the production of proinflammatory cytokines (7).
It has been reported that suppression of TNF- production in primary
macrophages by glucocorticoids is mediated through the inhibition of
JNK (8, 13, 15). Recently, it has been demonstrated that
MKP-1 is potently induced by dexamethasone in mast cells
(29). Our results indicate that MKP-1 expression can also
be induced by dexamethasone in macrophages at both the protein and mRNA
levels (Fig. 8). This is consistent with previous findings indicating
that the MKP-1 promoter region habors several putative
glucocorticoid-response elements (29). Therefore, the
results presented here strongly suggest that the induction of MKP-1 by
glucocorticoids may be responsible for the inhibition of JNK and, at
least in part, for the suppression of TNF- production in
macrophages. Very recently, TGF-, a cytokine with
anti-inflammatory activity, has also been shown to induce MKP-1 in
RAW264.7 cells (38). Consistent with our findings,
induction of MKP-1 by TGF- is also mediated by ERK. It should be
pointed out that MKP-1 induction is probably not the only mechanism via
which the anti-inflammatory cytokines/drugs exert their effects.
For example, the anti-inflammatory cytokine IL-10 has been shown to
inhibit the p38 pathway and decrease TNF- biosynthesis
(39). It has been proposed that IL-10 may interfere with
the MAP kinase kinase-6 signals to inhibit the p38 cascade
(39). Consistent with such a hypothesis, preliminary
results from our laboratory indicate that MKP-1 is not induced by IL-10
in our system (data not shown).
MKP-1 can also be induced by CTB (Fig. 9), an immune-modulatory agent
(30, 31). Coupled to relevant auto antigens, CTB has been
shown to effectively prevent the development of T cell-mediated
autoimmune diseases, including encephalomyelitis, arthritis, and type I
diabetes, in animal models (32, 33, 34). Recently, it has been
reported that pretreatment of macrophages and monocytes with CTB
diminishes the LPS-induced proinflammatory response, leading to a
significant decrease in the production of TNF- and IL-6 (30, 31). Because cytokines such as TNF- and IL-6 are critical in
the implementation of the T cell response, attenuating their production
in macrophages by CTB could have a major impact on the adaptive immune
response. In this report, we have demonstrated that CTB potently
induced MKP-1, abolished the LPS-elicited MAP kinase activation, and
abrogated the secretion of both TNF- and IL-6 (Fig. 9), thereby
providing a mechanistic explanation for its inhibitory effect on the
innate immune system. The higher MKP-1 level in cells treated with LPS
alone in comparison with that in cells treated with CTB and LPS at the
45-min time point was likely due to the higher ERK activity in these
cells (Fig. 9B). Thus, it is possible that, in addition to
MKP-1, some ERK-selective phosphatases may also be induced by CTB. The
relatively higher p38 activity in cells treated with LPS alone in
comparison to cells treated with both CTB and LPS at the 45-min time
point may be explained by the kinetic differences in MKP-1 protein
accumulation between the two populations (Fig. 9B). In cells
pretreated with CTB, a moderate amount of MKP-1 existed before LPS
stimulation and the amount did not substantially increase after LPS
stimulation (Fig. 9, A and B). In contrast, in
cells that received no CTB pretreatment, the MKP-1 protein level was
very low and only started to increase after 30 min (Fig. 1C). It is possible that in cells pretreated with CTB, the
moderate amount of MKP-1 over a long period of time dephosphorylated
more p38 than the MKP-1 acutely accumulated over a short period of time
in cells that received no CTB pretreatment. Our results also indicate
that CTB has immune-suppressive properties independent of the action of
the cholera toxin A chain. Such an inhibitory effect of CTB on
macrophages may offer V. cholerae protection against the
host innate immunity and may be relevant to the pathophysiology of this
infectious pathogen. In conclusion, the data presented here highlight
MKP-1 as a negative regulator of the macrophage inflammatory response
and as a potential target for the development of anti-inflammatory
and anti-rheumatic drugs.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Yusen Liu, Laboratory of Cellular and Molecular Biology, National Institute on Aging, 5600 Nathan Shock Drive, Baltimore, MD 21224. E-mail address: yusen-liu{at}nih.gov
3 Abbreviations used in this paper: LBP, LPS binding protein; MAP, mitogen-activated protein; ARE, AU-rich element; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MAPKAPK-2, MAP kinase-activated protein kinase-2; MKP, MAP kinase phosphatase; CTB, cholera toxin B subunit; CHX, cycloheximide; MEK, MAP/ERK kinase.
Received for publication July 23, 2002. Accepted for publication September 25, 2002.
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