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INTRODUCTION |
p38 mitogen-activated protein kinase
(MAPK)1 is a member of the
MAPK family and is activated by the inflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor (TNF), by bacterial lipopolysaccharide (LPS), and by a range of cellular stresses (1-5).
Although originally characterized as a stress or inflammatory kinase,
it is likely to have diverse functions because it is also activated in
platelets by thrombin and collagen (6) and in T cells upon activation
by various stimuli (7) and is constitutively active in liver (8, 9).
Little is known about the physiological functions it controls. One
substrate is MAPK-activated protein kinase-2 (MAPKAPK-2) (10, 11),
which in turn phosphorylates the small heat shock protein hsp27 (12)
and the cAMP-response element binding protein (13). Other putative
targets are the MAPK integrating kinase (14, 15) and the transcription
factors CHOP (16), myocyte enhancer factor 2C (17), and activating transcription factor 2 (4).
Besides the original p38 MAPK (called
), a closely similar
form
has been described (18) as well as two more distantly related enzymes
that also contain the TGY motif: stress-activated protein kinase 3 (or
p38
) (19-21) and stress-activated protein kinase 4 (or p38
)
(22-24). The p38
and p38
MAPKs are inhibited by a class of
pyridinyl imidazole compounds of which the best characterized is SB
203580 (11). These were first identified as inhibitors of TNF (and
IL-1) production by LPS-activated monocytes (25) and were later shown
to inhibit p38 MAPK (5, 11). The pyridinyl imidazoles inhibited TNF
(and IL-1) protein production with relatively little effect on the
levels of mRNA induced, and it was suggested that they worked
mainly by inhibiting translation (25, 26). Our laboratory used SB
203580 to investigate the role of p38 MAPK in cellular responses to
IL-1 (27). We found that the drug strongly inhibited the induction of
cyclooxygenase-2 (COX-2), collagenase (matrix metalloproteinase 1), and
stromelysin (matrix metalloproteinase 3) by IL-1 at the protein and
mRNA levels within the range of its IC50 for p38 MAPK
(0.1-0.5 µM) (27). There was comparatively little
inhibition of secondary cytokine production; IL-6 was only inhibited
30-50% (at 1 µM), and IL-8 was unaffected. The
inhibition of induction by IL-1 of COX-2 and matrix metalloproteinase
in fibroblasts at the mRNA level was in contrast to the inhibition
of TNF and IL-1 protein at the translational level in LPS-activated
monocytes. However, a recent report showed that LPS induction of COX-2
mRNA was inhibited by SB 203580 in monocytes (28). It was possible
that p38 MAPK regulated the expression of different genes at different
levels, depending upon the cell type and the nature of the stimulus.
We have therefore investigated the effects of SB 203580 on the
induction of both COX-2 and TNF in monocytes stimulated by LPS and
found each to be similarly inhibited at both the protein and mRNA
levels. We then went on to show that in the case of COX-2, the p38 MAPK
inhibitor impaired transcription and destabilized the mRNA.
Doubts have recently been raised concerning the specificity of SB
203580 (29, 30) because it can inhibit certain isoforms of
c-jun N-terminal kinase (JNK), another MAPK family member
activated by the same stimuli as p38 MAPK. We have therefore checked
the sensitivity of the LPS-activated JNKs in human monocytes to the inhibitor.
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EXPERIMENTAL PROCEDURES |
Materials--
Lymphoprep was from Nycomed Pharma A.S. (Oslo,
Norway). Fluorochrome-conjugated anti-CD-14 monoclonal antibodies were
from Becton Dickinson (Oxford, United Kingdom).
4-(4-Fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridinyl) imidazole
(SB 203580) was from Calbiochem-Novabiochem, Ltd. (Nottingham, United
Kingdom). LPS from Salmonella typhimurium was from
Sigma-Aldrich Company, Ltd. (Poole, United Kingdom) and was used at a
concentration of 10 ng ml
1. Sheep anti-rabbit MAPKAPK-2
antibody was from Upstate Biotechnology, Inc. (Lake Placid, NY). The
rabbit antiserum to the C-terminal peptide of p38
MAPK
(ISFVPPLDQEEMES) has been described previously (6). Rabbit anti-human
COX-2 antibody was from Oxford Biomedical Research, Inc. (Milwaukee,
WI). Recombinant human hsp27 was from Bioquote, Ltd. (York, United Kingdom).
[
-32P]ATP, [
-32P]UTP,
[
-32P]dCTP, Ready To Go DNA labeling kit, Hybond N
membrane, Vistra Enhanced Chemifluorescence kit, and a Mono Q column
were all from Amersham Pharmacia Biotech. RNasin was from Promega
(Southampton, United Kingdom), DNase I was from Life Technologies, Inc.
(Paisley, United Kingdom), and RNeasy kits were from Qiagen, Ltd. (West
Sussex, United Kingdom). Polyvinylidene difluoride membrane was from
DuPont, Ltd. (Stevenage, United Kingdom).
A construct expressing human glutathione S-transferase-c-Jun
(1-135) was a kind gift of J. R. Woodgett (Ontario Cancer Institute, Toronto, Canada). A plasmid containing full-length human COX-2 cDNA
(from D. Fitzgerald, College of Surgeons, Dublin, Ireland) was used for
Northern blots (EcoRI/EcoNI 800-base pair
fragment) and nuclear run-ons. Human GAPDH probe for Northern blots was a gift from C. Clarke (the Walter and Eliza Hall Institute, Melbourne, Australia), and human GAPDH (1.2 kb) for nuclear run-ons was from Diane
Watling (Imperial Cancer Research Fund, London, United Kingdom). A
plasmid containing the mouse
-actin cDNA (1.5 kb) and
pBluescript plasmid were from Stratagene, Ltd. (Cambridge, United Kingdom).
Isolation and Culture of Human Monocytes--
For each
experiment, human peripheral blood monocytes were freshly prepared from
the buffy coat fraction of a unit of blood from a single donor.
Mononuclear cells were prepared by Ficoll-Hypaque centrifugation on a
Lymphoprep gradient, and monocytes were isolated by centrifugal
elutriation on a Beckman JE6 elutriator (High Wycombe, United Kingdom)
using RPMI 1640 medium with L-glutamine and 1% heat-inactivated fetal calf serum. Monocyte purity was routinely assessed by fluorescence-activated cell-sorting forward/side scatter measurements in a Becton Dickinson FACScan, and cells of >80% purity
with
10% T cells were collected. Monocytes collected in this way
were typically found to be 75% pure as judged by flow cytometry using
fluorochrome-conjugated anti-CD-14 monoclonal antibodies. Monocytes
were cultured in the same medium used for elutriation at 37 °C in a
humidified atmosphere containing 5% CO2.
p38 MAPK and MAPKAPK-2 Kinase Assays--
Cells (5 × 106) were stimulated for the time period indicated with 10 ng ml
1 LPS and lysed for 10 min on ice in 1 ml of lysis
buffer (20 mM HEPES, pH 7.4, 50 mM sodium
-glycerophosphate, 2 mM EGTA, 1% Triton X-100, 10%
glycerol, 150 mM NaCl, 10 mM NaF, 1 mM sodium orthovanadate, 2 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 3 µg ml
1
aprotinin, 10 µM E64, and 2 µg ml
1
pepstatin A). Samples were clarified by centrifugation at 4 °C for
10 min at 16,000 × g. p38 MAPK was immunoprecipitated
by the addition of 5 µl of rabbit antiserum to the C terminus of p38 MAPK. Samples were incubated at 4 °C for 1 h with mixing before the addition of 30 µl of a 50% slurry of protein A beads in lysis buffer, and samples were incubated for an additional 2 h. The beads were washed four times in 1 ml of wash buffer (50 mM
Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 25 mM sodium
-glycerophosphate, 10 mM sodium
tetrapyrophosphate, 1 mM sodium orthovanadate, 2 mM DTT, 1% Triton X-100, 0.5% sodium deoxycholate, and
0.1% SDS) and twice in 1 ml of kinase assay buffer (20 mM
HEPES, pH 7.5, 20 mM sodium
-glycerophosphate, 200 mM NaCl, 2 mM DTT, 10 mM MgCl2, 10 mM NaF, 0.1 mM sodium
orthovanadate, 0.5 mM EDTA, 0.5 mM EGTA, and
0.05% BRIJ 35). p38 MAPK was assayed using 1 µg of recombinant
MAPKAPK-2 as substrate in kinase assay buffer with 20 µM
ATP and 4 µCi of [
-32P]ATP in an assay volume of 30 µl. Samples were incubated at room temperature for 20 min with
agitation, and the reaction was stopped by the addition of 4×
SDS-polyacrylamide gel electrophoresis sample buffer. Samples were
separated by 10% SDS-polyacrylamide gel electrophoresis (31). Gels
were stained with Coomassie Brilliant Blue R-250, and phosphorylation
of the substrate was measured on a phosphorimager (Fuji FLA-2000; Fuji,
Tokyo, Japan).
MAPKAPK-2 immunoprecipitation was performed in an identical fashion,
but with 5 µg of sheep anti-rabbit MAPKAPK-2 antibody and protein G
beads. Samples were assayed for kinase activity as described above
using 1 µg of recombinant human hsp27 as substrate.
Mono-Q Anion Exchange Chromatography and JNK
Assays--
108 monocytes were lysed in 1.0 ml of Buffer A
(20 mM Tris-HCl, pH 8.5, 20 mM sodium
-glycerophosphate, 10 mM NaF, 2 mM DTT, 0.1 mM sodium orthovanadate, 0.02% sodium azide, 1 mM EDTA, and 1 mM EGTA) containing 1% Triton
X-100, 1 mM phenylmethylsulfonyl fluoride, 3 µg
ml
1 aprotinin, and 10 µM E64 for 10 min on
ice. The extract was clarified and filtered (0.2 µm), and 100 µl
(approximately 500 µg of protein) were applied to a 100 µl Mono Q
column equilibrated with Buffer A containing 0.05% BRIJ 35. The column
was washed with 5 column volumes of Buffer A, and protein was eluted
with a 3-ml gradient of Buffer A containing 0-0.5 M NaCl.
100-µl fractions were collected, and 10 µl of each were assayed for
JNK activity by mixing with 10 µl of glutathione
S-transferase-c-Jun (1-135) (300 µg ml
1)
and 10 µl of assay buffer (150 mM Tris-HCl, pH 7.4, 30 mM MgCl2, 60 µM ATP, and 0.4 µCi µl
1 [
-32P]ATP) and incubated for
20 min at room temperature. The reaction was stopped with 10 µl of
4× SDS-polyacrylamide gel electrophoresis sample buffer, and the
samples were electrophoresed. Phosphorylation of substrate was assessed
as for p38 MAPK and MAPKAPK-2.
Nuclear Run-On Assay--
Monocytes (3 × 107
cells/assay point) were washed once with phosphate-buffered saline and
resuspended in ice-cold lysis buffer (0.3 M sucrose, 0.5%
Nonidet P-40, 1 mM DTT, 1.5 mM
MgCl2, 3 mM CaCl2, and 10 mM Tris-HCl, pH 7.5) and disrupted in a loosely fitting
Dounce homogenizer. The lysate was layered onto a 30% sucrose cushion
and centrifuged at 3,000 revolutions/min for 10 min at 4 °C. The
nuclear pellet was resuspended in 200 µl of 50 mM
Tris-HCl, pH 8.3, 5 mM MgCl2, 0.1 mM EDTA, and 40% glycerol and stored in liquid nitrogen.
Thawed nuclei were added to 200 µl of reaction mixture (10 mM Tris-HCl, pH 8.0, 10 mM MgCl2,
300 mM KCl, 5 mM DTT, 40 units of RNasin, 1 mM each of ATP, CTP, and GTP, and 125 µCi of
[
-32P] UTP (800 Ci mmol
1)) and incubated
at 30 °C for 30 min. Reactions were stopped by the addition of 20 µl of 7.2 mg ml
1 DNase I and an additional 10-min
incubation at 30 °C. 420 µl of 100 mM Tris, pH 7.4, 50 mM EDTA, 2% SDS, 100 µg ml
1 yeast tRNA,
and 400 µg ml
1 proteinase K were added, followed by
incubation at 37 °C for 45 min. Nuclear RNA was isolated by
phenol/chloroform extraction and precipitated from 840 µl of the
aqueous phase with 600 µl of isopropanol and 200 µl of 7.5 M ammonium acetate. RNA was then resuspended in 100 µl of
10 mM Tris-HCl, pH 8.0, and 1 mM EDTA, made 0.2 M with respect to NaOH, and incubated on ice for 10 min. The solution was neutralized with HEPES and precipitated with ethanol.
RNA was dissolved in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA and hybridized to slot blots (plasmids linearized
with EcoRI and UV-cross-linked to Hybond N membrane) in 50%
formamide, 5× saline sodium phosphate EDTA, 2.5× Denhardt's
solution, 0.1% SDS, and 0.2 mg ml
1 salmon sperm DNA.
Blots were washed twice for 10 min in 2× SSC and 0.1% SDS at
37 °C, washed for 30 min in 2× SSC and 0.1% SDS at 50 °C, and
then washed twice for 10 min in 2× SSC at 37 °C. Blots were then
washed for 30 min in 2× SSC and 10 µg ml
1 RNase A at
37 °C, followed by two washes in 2× SSC and 0.1% SDS at 37 °C.
Signals were quantified in a phosphorimager.
Northern Blot--
Monocyte total RNA (4 µg) was
electrophoresed on denaturing formaldehyde/1% agarose gels with 0.41 M formaldehyde (32). RNA was capillary transferred to a
Hybond N membrane according to Sambrook et al. (33) and
fixed by UV cross-linking. cDNA probes (50 ng) for COX-2, TNF-
,
and GAPDH were labeled with 50 µCi of [
-32P]dCTP by
random priming. Prehybridization (2 h) and hybridization (overnight)
were performed at 42 °C in 10 mM Tris-HCl containing 50% (v/v) formamide, 4× saline sodium phosphate EDTA, 5× Denhardt's solution, 0.1% SDS, 1 mM EDTA, and 0.1 mg
ml
1 heat-denatured salmon sperm DNA. Blots were washed
three times for 30 min at 42 °C with 5× SSC and 0.1% SDS, 2× SSC
and 0.1% SDS, and 0.1× SSC and 0.1% SDS. Signals were quantified in
a phosphorimager.
Western Blot--
Cell lysates (total protein, 50 µg) were
electrophoresed on 10% SDS-polyacrylamide gels (31) and blotted on
polyvinylidene difluoride membrane. Membranes were treated according to
the instructions of the Enhanced Chemifluorescence kit.
TNF-
Enzyme-linked Immunosorbent Assay--
TNF-
enzyme-linked immunosorbent assay was performed as described by Foey
et al. (34).
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RESULTS |
Induction of COX-2 Protein and mRNA by LPS Is Inhibited by the
p38 MAPK Inhibitor SB 203580--
Monocytes were untreated or
pretreated for 1 h with increasing concentrations of SB 203580. They were then stimulated for 4 h with LPS (with the inhibitor
remaining present on the pretreated cells). Cells were then
immunoblotted for COX-2 protein (Fig. 1A), or RNA was extracted and
Northern blotted for COX-2 mRNA (Fig. 1B). SB 203580 inhibited both protein and mRNA in a similar dose-dependent fashion. Significant inhibition was seen at
0.1 µM (33% for protein and 50% for mRNA); this was
stronger at 1 µM (55% and 72%, respectively), with some
additional increase at 10 µM. The results are
representative of three separate experiments with different batches of
cells.

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Fig. 1.
Inhibition of induction of COX-2 and TNF by
SB 203580 in LPS-treated human monocytes. Monocytes were treated
with SB 203580 at the doses indicated for 1 h and then left
untreated or treated with LPS for an additional 4 h. A,
Western blot of monocyte lysates for COX-2 protein. B,
Northern blot of monocyte RNA for COX-2 mRNA. C,
monocytes were treated as described in A, but were treated
with LPS for 16 h and Northern blotted for TNF mRNA. See
"Experimental Procedures" for details.
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TNF Protein and mRNA Induced by LPS Are Both Inhibited by SB
203580--
Monocytes were treated for 1 h with SB 203580 or left
untreated and further treated for 16 h with LPS or left untreated.
Secreted TNF protein, which was measured by an enzyme-linked
immunosorbent assay, was inhibited in a dose-dependent
fashion by SB 203580 (42% at 0.1 µM and 67% at 1 µM). TNF mRNA showed a sensitivity to the inhibitor
that was similar to that seen for the protein (Fig. 1C). In
this experiment, LPS stimulation increased the levels of GAPDH
mRNA. LPS is a very strong monocyte activator, and some batches of
cells show up-regulation of housekeeping genes. TNF protein and
mRNA showed similar sensitivities to the inhibitor at a
concentration of 2 µM (62% and 65%, respectively) in
two separate experiments with different batches of cells using a 4-h LPS stimulation (data not shown).
Inhibition of p38 MAPK by SB 203580 in LPS-treated
Monocytes--
Fig. 2A shows
the activity of p38 MAPK immunoprecipitated from lysates made from
cells at various times after the addition of LPS. Activity increased
6-fold by 10 min and then returned to resting levels after 1 h. It
was important to compare the degree of inhibition of p38 MAPK activity
with COX-2 mRNA induction. Inhibition of p38 MAPK in monocytes by
treatment with SB 203580 (which is a reversible inhibitor) was measured
by immunoprecipitating its substrate, MAPKAPK-2, from cell lysates and
assaying the latter's activity on hsp27. Monocytes were pretreated
with the inhibitor for 20 min or left untreated, and cells were then
treated with LPS for 10 min (Fig. 2B, left
panel). Monocytes were also treated with LPS for 4 h and
either treated with the inhibitor for the final 30 min of stimulation
or left untreated (Fig. 2B, right panel). MAPKAPK-2 was
immunoprecipitated from the lysates. At both time points,
i.e. at the peak of p38 activation at 10 min and after it
had returned to resting levels at 4 h, the MAPKAPK-2 activity was
reduced by about 60% in cells treated with 0.1 µM and by
80% in cells treated with 1 µM. The degree of inhibition of the MAPKAPK-2 correlated well with the effect on COX-2 gene expression (Fig. 1, A and B).

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Fig. 2.
Activity of p38 MAPK in LPS-treated monocytes
and its inhibition by SB 203580. A, p38 activity in
monocytes at different times after LPS stimulation. Left
panel, lysates from untreated monocytes were immunoprecipitated
with antiserum raised against p38 MAPK C-terminal peptide
(I) or preimmune serum (PI) and assayed for p38
MAPK activity using MAPKAPK-2 as substrate. Right panel, the
time course of p38 MAPK activity assayed in an identical fashion.
B, MAPKAPK-2 activity in LPS-stimulated cells treated with
SB 203580. Left panel, monocytes were pretreated with the
indicated dose of SB 203580 for 20 min and then treated with LPS for 10 min. Right panel, monocytes were treated with LPS for 4 h and were also treated with the indicated dose of SB 203580 for the
final 30 min of LPS stimulation. MAPKAPK-2 activity was
immunoprecipitated from lysates and assayed on recombinant human hsp27
substrate. MAPKAPK-2 activity was not inhibited by 1 µM
SB 203580 when the agent was added directly to the kinase assay (data
not shown).
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Effect of SB 203580 on JNK Activity in LPS-treated
Monocytes--
Certain forms of JNK, particularly the
splice
variants of JNK-2, are sensitive to micromolar concentrations of SB
203580 (29, 30). We chromatographed lysates of monocytes that had been
treated with LPS for 20 min on a Mono-Q anion exchanger and measured
JNK activity in the fractions eluted with a 0-0.5 M NaCl gradient (Fig. 3A). As is
characteristic of the enzyme in lysates of activated cells (29, 35),
two major peaks were eluted. These were tested for sensitivity to SB
203580 (note that p38 MAPK itself elutes after the JNK peaks at about
0.4 M NaCl (2, 3)). The activity of the first peak (which
contains the short ~46-kDa JNKs) was unaffected by the inhibitor at
concentrations up to 10 µM (Fig. 3B, left
panel). The activity of the second peak (which contains the
full-length ~54-kDa JNKs) was unaffected by 0.1 µM but
was significantly inhibited (24%) at 2 µM (Fig. 3B, right panel). The data shown in Fig. 3B are
means of three experiments on three different batches of monocytes.
Because the peaks contain similar amounts of JNK activity, we estimate
that total JNK activity might be inhibited by 10-15% in cells treated with 2 µM SB 203580. The inhibition of COX-2 expression
by SB 203580 is therefore consistent with its effect on p38 MAPK and not on JNK.

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Fig. 3.
Inhibition of monocyte JNKs by SB 203580. A, anion exchange chromatography of JNK activity in
LPS-treated monocytes. Monocytes were stimulated with LPS (20 min),
lysed, and chromatographed on a Mono Q column with a 0-0.5
M NaCl gradient. JNK activity in the fractions was assayed
as described under "Experimental Procedures." B,
inhibition of JNK activity peaks by SB 203580. Fractions from the two
peaks of JNK activity in A were pooled and assayed as
described above in the presence of the indicated concentrations of SB
203580. The results shown are the mean values from three batches of
cells chromatographed independently. Error bars are not indicated where
they are too small to be shown.
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Transcription of COX-2 Is Inhibited by SB 203580--
The effect
of p38 MAPK blockade on both the protein and mRNA levels of COX-2
could be due to decreased transcription and/or reduced mRNA
stability. We tested for an effect on transcription by performing
nuclear run-on assays. Monocytes were left untreated or pretreated for
1 h with 2 µM SB 203580 and then further incubated for 4 h in the presence or absence of LPS. Nuclei were isolated as
described in "Experimental Procedures," and COX-2 transcription was
measured by nuclear run-on assay. Fig.
4A shows a representative experiment. COX-2 transcription was stimulated 12-fold, and this was
inhibited by 74% in nuclei from cells treated with 2 µM
SB 203580. Data from three independent experiments are combined in Fig.
4B, which shows an average 5-fold stimulation of COX-2
transcription by LPS with a mean inhibition of 60% by 2 µM SB 203580. The inhibitor had a similar effect when the
run-on assay was performed after only a 30-min (rather than a 4-h)
stimulation with LPS (data not shown). These results strongly suggested
that inhibiting p38 MAPK interfered with the transcription of
COX-2.

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Fig. 4.
Inhibition of LPS-induced COX-2 transcription
by SB 203580 as determined by nuclear run-on assays. A,
monocytes were left untreated or pretreated for 1 h with 2 µM SB 203580 and either untreated (control) or stimulated
with LPS for 4 h. Nuclei were isolated, and a nuclear run-on assay
was performed as described under "Experimental Procedures."
B, the results from three separate experiments are
summarized.
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Inhibition of p38 MAPK Destabilizes COX-2 mRNA--
To examine
the stability of COX-2 mRNA in human monocytes, actinomycin D was
added to cells after 4 h of LPS treatment, and COX-2 mRNA
levels were measured from the time of addition up to 90 min (Fig.
5A). The predominant 4.6-kb
and the minor 2.8-kb transcripts decayed at a similar slow rate
(t1/2 = 110 min). Simultaneous addition of SB
203580 (2 µM) and actinomycin D to the cells after a 4-h
stimulation with LPS resulted in a rapid decrease
(t1/2 = 30 min) in COX-2 mRNA levels (Fig.
5A). In cells that were not treated with actinomycin D or
the p38 MAPK inhibitor, COX-2 mRNA levels were roughly constant for
1 h after a 4-h LPS stimulation (Fig. 5B), showing that
a steady state had been reached. The decay observed in cells treated
only with actinomycin D indicated that in LPS-treated monocytes, COX-2
mRNA is continually being degraded and transcribed. When SB 203580 (2 µM) alone was added to cells treated with LPS for
4 h, there was a rapid decay of COX-2 mRNA that occurred at a
similar rate to that for cells treated with the inhibitor and
actinomycin D (Fig. 5B). This indicated that transcription
was not needed for SB 203580 to destabilize COX-2 mRNA.

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Fig. 5.
p38 MAPK inhibitor destabilizes COX-2
mRNA in the presence (A) or absence (B) of
actinomycin D. A, monocytes were either left untreated or
treated with LPS for 4 h, and actinomycin D (final concentration,
5 µg ml 1) was then added with ( ) or without SB
203580 (final concentration 2 µM; ). RNA was isolated
at the times shown and Northern blotted for COX-2 mRNA and GAPDH
mRNA as described under "Experimental Procedures." The graph
shows COX-2 mRNA levels normalized to GAPDH mRNA levels for
three separate experiments with different batches of cells. Error bars
are not shown where they are smaller than the symbol. B,
monocytes were left untreated or treated with LPS for 4 h. They
were then left untreated ( ), or 2 µM SB 203580 was
added ( ). RNA was isolated at the times shown and Northern blotted
as described in A. The graph shows COX-2 mRNA levels
normalized to GAPDH mRNA levels for two separate experiments.
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|
 |
DISCUSSION |
We found that the p38 MAPK inhibitor prevented the LPS induction
of both COX-2 and TNF protein, and that the degree of inhibition of
protein and mRNA for both were very similar. We concluded that there was little evidence for p38 MAPK regulating translation for the
expression of both these gene products in primary monocytes. Our
additional studies were restricted to COX-2, and these indicated that
p38 MAPK is required for optimal transcription of the gene, and that it
is needed for stabilization of the mRNA. The importance of this
second finding is that it suggests a novel function for p38 MAPK
that has not previously been considered.
These conclusions are dependent on the inhibitor being specific for
this protein kinase. The original description of SB 203580 suggested
that it might be a highly specific inhibitor of p38 MAPK (11). Its
IC50 was about 0.5 µM, and it did not affect the activity of a wide range of protein kinases (including JNK and
extracellular signal-regulated kinase) and phosphatases up to a
concentration of 100 µM (11). However, two recent reports stress that caution is needed in the use of SB 203580 (29, 30). In the
first, the
splice variants of JNK-2 were shown to be completely
inhibited by 10-20 µM SB 203580 (30). The
splice variants of JNK-2 and JNK-1 were inhibited about 50%, whereas the
forms of JNK-1 were resistant to the drug (30). This raised the
possibility that SB 203580 may significantly inhibit JNKs in cells,
depending upon the isoforms expressed. An example of this is a recent
report that certain chromatographic forms of cardiac myocyte JNKs are
strongly inhibited by 10 µM SB 203580 (29).
To establish that the effects of SB 203580 on COX-2 gene expression
were due to the inhibition of p38 MAPK, we compared the suppression of
MAPKAPK-2 activity with the suppression of COX-2 protein and mRNA.
We also investigated the sensitivity of human monocyte JNKs activated
by LPS to the inhibitor. At 0.1 µM SB203580, there was
about a 50% inhibition of both COX-2 mRNA and MAPKAPK-2 activity
in monocytes, which is consistent with the effects on COX-2 gene
expression being due to p38 MAPK blockade.
The LPS-activated JNKs in human monocytes were relatively insensitive
to the inhibitor. The second of the two chromatographically distinct
forms was inhibited about 10-20% at 1-2 µM SB 203580, whereas the first peak was unaffected. We therefore concluded that the
effects of SB 203580 at concentrations up to 1-2 µM were unlikely to be due to the inhibition of JNK isoforms, and the effects
on COX-2 gene expression were probably due to the inhibition of p38 MAPK.
Our investigation of LPS-induced COX-2 in monocytes was prompted partly
by the discrepancy between the finding that SB 203580 inhibited COX-2
mRNA induced by IL-1 in fibroblasts (27) and the suggestion that it
might be acting as a translational inhibitor of TNF production in
monocytes (25). Young et al. (25) reported a 50%
suppression of TNF mRNA in monocytes treated with LPS in the
presence of 5 µM SKF 86002 (a pyridinyl imidazole very
similar to SB 203580) but a 90% inhibition of secreted TNF protein.
Further work was carried out in the human monocytic cell line THP 1 (26). In this cell line, TNF protein induced by LPS was inhibited by SKF 86002, whereas mRNA induction was unaffected. Furthermore, TNF
mRNA accumulated in a pre-ribosomal compartment (26). It may be
that different mechanisms predominate in the immortalized cell line and
primary monocytes. Working with SB 203580 at low doses, we have found
no discrepancy between the suppression of TNF protein and mRNA. It
is possible that p38 MAPK may regulate TNF and COX-2 by similar mechanisms.
We found that p38 MAPK activity is required for the full induction of
COX-2 transcription. Analysis of the COX-2 promoter by means of
transfection of reporter constructs has implicated several
transcription factors in its regulation. These experiments have
generally been carried out on transformed cell lines using a variety of
stimuli (e.g. phorbol ester, IL-1, TNF, LPS, and growth
factors). The transcription factors implicated include NF
B (36),
NF-IL-6 (36-38), c-jun (39), signal transducer and activator of transcription 5 (40), and NF-1 (41). A number of reports
have emphasized the importance of NF
B in the induction of COX-2 in
response to LPS in cells of monocyte-macrophage lineage (42-45). The
relative importance of different transcription factors for COX-2
expression in primary monocytes is not known.
It has recently been suggested that the inhibition of p38 MAPK by SB
203580 blocks the activation of NF
B reporter constructs in cells by
a mechanism unrelated to either the phosphorylation and degradation of
I
B or nuclear translocation and DNA binding of NF
B (46-48).
However, in these studies, the inhibitor was used at concentrations
substantially above its reported IC50, and the observed
effects on NF
B-dependent transcription were only
partial. For example, the response of NF
B-dependent
reporter constructs to TNF was diminished less than 50% by 10 µM SB203580 (47). Because we observe stronger effects on
COX-2 expression at much lower concentrations of the inhibitor, it
seems unlikely that the inhibition of NF
B activity accounts for our
findings. However, it is difficult to examine this proposition
directly, because the study of events downstream of DNA binding
requires the transfection of primary monocytes.
The human COX-2 promoter also contains potential binding sites for
members of the cAMP response element binding protein activating transcription factor, Ets/ternary complex factor, and myocyte enhancer
factor 2 families; activating transcription factor 2 (4), Ets family
members (49, 50), and myocyte enhancer factor 2C (17) can all be
phosphorylated and activated by p38 MAPK. cAMP-responsive element
binding protein (13) is phosphorylated and activated by MAPKAPK-2,
which is directly downstream of p38 MAPK. These factors all represent
possible targets for the transcriptional regulation of the human COX-2
gene by p38 MAPK.
We have found strong evidence that p38 MAPK stabilizes COX-2 mRNA.
Although COX-2 mRNA stability was studied in stimulated cells, it
is not clear whether LPS actually regulates this, because p38 MAPK
activity had returned to resting levels after 4 h of stimulation.
It appears that a basal level of p38 MAPK activity plays a housekeeping
role in maintaining COX-2 mRNA stability. Destabilization of COX-2
mRNA is also caused by dexamethasone (51). This effect was
described in IL-1-stimulated endothelial cells and inhibited by
actinomycin D. Thus, the effects of dexamethasone and SB 203580 differ.
Sequences that determine the stability of an mRNA have been found
in the 5' untranslated region, the coding region, and the 3'
untranslated region (52). The 3' untranslated region of COX-2 contains
22 AUUUA motifs that are recognized to be important determinants of
mRNA instability (51). Both proximal and distal regions have been
identified as important in regulating the stability of transfected reporter constructs bearing the 3' untranslated region in resting cells
(53). It is possible that the instability caused by the p38 MAPK
inhibitor is mediated through the AU-rich region proximal to the coding
region that is contained in the 4.6- and 2.8-kb transcripts, both of
which we have found to be stabilized through this protein kinase.
The results of the present study provide evidence for a new role of p38
MAPK in mRNA stabilization. It will be of interest to determine
whether this process occurs in other cell types for other genes and to
identify the downstream pathway and molecular mechanisms involved.