From the Nuclear Signalling Laboratory, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom
Received for publication, June 22, 2000, and in revised form, November 6, 2000
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ABSTRACT |
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Tumor necrosis factor- Monocytes and macrophages play a pivotal role in inflammation and
immune regulation. Upon activation, monocytes produce and release many
inflammatory mediators such as
IL-1,1 IL-6, IL-8, TNF- Lipopolysaccharide (LPS), a component of the cell wall of Gram-negative
bacteria, is a very potent inducer of TNF- Recent work indicates that Toll-like receptors, a family of human
receptors related to Drosophila Toll, mediate LPS-induced signal transduction and that this response is dependent on LBP and
enhanced by CD14 (6-9).
The involvement of all three mitogen-activated protein (MAP) kinase
subtypes, c-Jun N-terminal kinase (10, 11), p38 (12-14), and the
extracellular signal-regulated kinase (ERK) p42/44 (13, 15), in
LPS-induced cytokine production and release has been documented
extensively. However, the precise role of these MAP kinase subtypes in
the processes of transcriptional induction and/or translation of
cytokine transcripts remains in contention. In particular, although the
production of human TNF- The differences outlined above may arise from the use of different cell
types or different experimental methods to measure gene induction. The
low numbers of primary monocytes obtainable by purification from human
blood is a restrictive factor in studying the role of MAP kinases in
TNF- Materials--
Lymphoprep was from Nycomed Pharma (Oslo,
Norway). RPMI 1640 medium and Hanks' balanced salt solution
were purchased from Life Technologies, Inc. FCS was obtained from
Globepharm (Guildford, UK). LPS (Escherichia coli
serotype 055:B5) and TPA were from Sigma. SB203580 was a gift from
J. Souness (Aventis Pharma Ltd., Dagenham, UK). PD98059 was
obtained from Alexis (Nottingham, UK). U0126 was purchased from Promega
(Southampton, UK).
The following monoclonal antibodies were used: fluorescein
isothiocyanate-conjugated rabbit anti-mouse immunoglobulins and CD54
were from Dako (Glostrup, Denmark); CD14 (Hb246) and CD3 (UCHT-1) were
from ATCC; CD19 (BU12) was a gift of D. Hardie (University of
Birmingham, UK), CD58 was from Roche Molecular Biochemicals. Rabbit
anti-phospho-p38 antibody was purchased from New England Biolabs
(Hitchin, UK). Dynabeads M-450 sheep anti-mouse IgG were from Dynal
(Oslo, Norway). [ Cell Culture--
THP-1 cells were grown in RPMI 1640 medium
with 10% fetal calf serum and 20 µM
Human peripheral blood monocytes were freshly prepared from the buffy
coat fraction of 1 unit of donor blood. Mononuclear cells were
separated on Ficoll Lymphoprep (400 g, 30 min) and the remaining red
blood cells were lysed for 15 min in 0.01 M Tris-HCl
containing 8.3 g/liter ammonium chloride. Mononuclear cells were then
allowed to adhere (5 × 106 cells/ml) in 10%
FCS-RPMI. After 90 min at 37 °C, 6% CO2, nonadherent cells were removed, and adherent cells were recovered by a 30-min treatment in 0.02% EDTA. Monocytes were then depleted of contaminating T and B cells using CD3 and CD19 monoclonal antibodies followed by
sheep anti-mouse IgG-coated Dynabeads.
Monocyte purity was assessed by fluorescence-activated cell sorting in
a Becton Dickinson FACSscan. Cells were <3% CD3 or CD19 and >90%
CD14. Cells were rendered quiescent by an overnight incubation
in 0.5% FCS-RPMI before stimulation for MAPK analysis or Northern blots.
p38 Activation by Western Blot--
THP-1 or TPA-differentiated
THP-1 cells were lysed in ice-cold lysis buffer consisting of 20 mM Hepes, pH 8, 5 mM EDTA, 10 mM
EGTA, 5 mM NaF, 10% glycerol, 1 mM
dithiothreitol, 400 mM KCl, 0.4% Triton X-100, 20 mM sodium
Analysis of p38 activation was performed using specific phospho-p38
antibodies. Cell extracts were loaded on a 10% SDS-polyacrylamide gel
and transferred to polyvinylidene difluoride membranes (Immobilon P,
Millipore). Membranes were blocked overnight at 4 °C in 5% skimmed
milk in TBST (20 mM Tris-HCl, pH 8, 137 mM NaCl containing 0.1% Tween 20). After several washes,
membranes were probed with phospho-p38 antibodies diluted in 5%
skimmed milk in TBST for 1 h at room temperature. After washes,
membranes were incubated with the appropriate horseradish
peroxidase-conjugated secondary antibody for 1 h, and blots were
then developed using a chemiluminescent detection.
Northern Blot Analysis--
Total cellular RNA in THP-1 cells
was isolated according to the method of Chomczynski and Sacchi (28).
RNA from human blood monocytes was purified using the Purescript RNA
isolation kit (Gentra system, Minneapolis, MN). Aliquots containing 5 µg of RNA were resolved on 1% agarose gels containing 0.41 M formaldehyde. RNA was transferred onto nylon membranes
(Hybond N+, Amersham Pharmacia Biotech) by capillary
transfer in 50 mM NaOH. cDNA probes for TNF-
TNF- TNF- Flow Cytometry Analysis--
Cells (106 cells/ml) in
RPMI containing 10% rabbit serum and 0.1% NaN3 were
incubated for 30 min at 4 °C with murine monoclonal antibodies
followed by a 30-min incubation with fluorescein isothiocyanate rabbit
anti-mouse IgG. After washing, cells were fixed in 3.7% formaldehyde
in Hanks' balanced salt solution and analyzed using a FACScan flow
cytometer (Becton Dickinson).
Differentiated THP-1 Cells Show Stronger Responsiveness to LPS
Stimulation: Analysis of TNF-
We next analyzed levels of TNF-
Time-course analysis showed that normal THP-1 cells produce no
detectable TNF- TPA-induced Differentiation of THP-1 Cells Increases Expression of
CD14 and CD54 Cell Surface Markers--
To confirm that differentiated
THP-1 cells mimic human monocytes more closely than undifferentiated
cells, we next determined the levels of expression of two monocyte
surface markers, CD14 and CD54, in undifferentiated and
TPA-differentiated cells by FACScan and compared it with
expression levels in purified human blood monocytes. This showed that
TPA treatment enhanced the expression of monocyte markers in THP-1
cells (Fig. 2). Undifferentiated THP-1
cells very weakly express the LPS receptor CD14; only 13.1% of the
cells are positive. By contrast, 42.2% of differentiated THP-1 cells
express the CD14 marker compared with 94.7% in purified blood
monocytes. The same observation was made in analyses of the adhesion
molecule CD54 (ICAM-1). CD54 is not detectably expressed in normal
THP-1 cells but is expressed in 53.9% of the TPA-differentiated cells
compared with 61.3% in purified blood monocytes. This confirms indications from the TNF- LPS Induces p38 Activation in TPA-differentiated THP-1 Cells and
Blood Monocytes--
Because TPA differentiation increases the
expression of the LPS receptor CD14, we then compared the ability of
LPS to activate p38 MAP kinase in THP-1 and TPA-differentiated THP-1
cells to that in purified blood monocytes. For positive controls,
stimuli such as anisomycin, UV radiation, and hyperosmotic stress,
which are known to strongly activate p38, were used. As expected,
anisomycin, UV radiation, or osmotic stress using sodium chloride or
sorbitol strongly induced p38 activation in normal THP-1 cells (Fig.
3) showing that the p38 cascade is
present and activated normally in these cells. However, LPS stimulation
only produced very weak and barely detectable activation of p38 in
these cells. By contrast, in TPA-differentiated THP-1 cells, as in
purified blood monocytes, all of the five stimuli tested, including
LPS, proved to be very efficient in activating the p38 pathway. This
finding reiterates at the signal transduction level all previous
indications that the differentiated cells more closely resemble mature
monocytes than the undifferentiated cells.
Inhibition of ERK or p38 MAP Kinase Cascades Differentially Affects
TNF-
Human monocytes were purified from buffy coat preparations as described
(see "Experimental Procedures") and pretreated for 30 min with the
indicated concentrations of the p38 inhibitor SB203580 (Fig.
4A) or the MEK1/2 inhibitor
PD98059 (Fig. 4B), followed by a 16-h incubation with 10 ng/ml LPS. Cell supernatants were then harvested and assayed for the
presence of TNF- Inhibition of ERK or p38 MAP Kinase Pathways Reduces LPS-stimulated
TNF- Combinations of SB203580 and PD98059 Produce Virtual Ablation
of TNF-
This observation was then confirmed using purified human blood
monocytes (Fig. 6B). Again, more than 90% inhibition of
TNF-
Recently, a new MEK inhibitor, U0126, has become commercially
available. We studied the effect of U0126 on LPS-induced TNF- Inhibition of p38 MAPK Destabilizes TNF-
In the absence of inhibitors, TNF- In their seminal paper, Lee et al. (12) identify p38
MAP kinase as the primary target of anti-inflammatory pyridinyl
imidazole drugs, previously shown to inhibit LPS-stimulated production
of pro-inflammatory cytokines such as IL-1 and TNF- In this report we have re-examined, using pyridinyl imidazole
compounds, the role of p38 MAPK in LPS-induced TNF- The p38 inhibitor SB203580 and the ERK pathway inhibitor PD98059 have
been reported to act individually and in combination to reduce
LPS-stimulated secretion of TNF- The effect of p38 inhibition on TNF- Wang et al. (31), studying the effect of another p38
inhibitor SB202190 on cytokine production by reverse
transcriptase-polymerase chain reaction, also suggested that specific
mRNA destabilization represents an important site of action for the
cytokine suppressive effect of p38 inhibitors. However, there are
crucial differences between our work and that of Wang et al.
(31). First, they used the p38 inhibitor either 2 h before or
instead of the transcriptional inhibitor actinomycin D, and thus, their
studies do not totally exclude an indirect effect of SB202190 on
cytokine mRNA levels that involves transcription. In our
experiments, transcription was halted with 5,6-dichlorobenzimidazole
riboside (DRB) prior to the addition of the p38 inhibitor, allowing the
conclusion that pyridinyl imidazole compounds affect TNF- Recent data suggest a role for the p38 pathway in the
post-transcriptional regulation of several cytokines (26, 32, 33). A
key factor of TNF- (TNF-
) is a potent
proinflammatory cytokine whose synthesis and secretion are implicated
in diverse pathologies. Hence, inhibition of TNF-
transcription or
translation and neutralization of its protein product represent major
pharmaceutical strategies to control inflammation. We have studied the
role of ERK and p38 mitogen-activated protein (MAP) kinase in
controlling TNF-
mRNA levels in differentiated THP-1 cells and
in freshly purified human monocytes. We show here that it is possible
to produce virtually complete inhibition of
lipopolysaccharide-stimulated TNF-
mRNA accumulation by using a
combination of ERK and p38 MAP kinase inhibitors. Furthermore,
substantial inhibition is achievable using combinations of 1 µM of each inhibitor, whereas inhibitors used
individually are incapable of producing complete inhibition even at
high concentrations. Finally, addressing mechanisms involved, we show
that inhibition of p38 MAP kinase selectively destabilizes TNF-
transcripts but does not affect degradation of c-jun
transcripts. These results impinge on the controversy in the literature
surrounding the mode of action of MAP kinase inhibitors on TNF-
mRNA and suggest the use of combinations of MAP kinase inhibitors
as an effective anti-inflammatory strategy.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
or arachidonic acid metabolites, as well as the anti-inflammatory
mediators IL-10, soluble TNF receptor, and IL-1 receptor antagonist.
Among the different products released, TNF-
is thought to be one of
the most important mediators of inflammatory disease (1), and therefore
the understanding of molecular mechanisms of TNF-
gene induction is
of considerable medical interest.
production and release in
human monocytes. Two proteins, CD14 and LPS-binding protein (LBP), are
implicated in mediating the cellular response to LPS (2-5). CD14 is a
55-kDa glycoprotein found as a glycosyl phosphatidylinositol-anchored
membrane protein (mCD14) and as a soluble form (sCD14) in plasma. LBP
is a 60-kDa plasma protein that binds to LPS and functions primarily to
accelerate the binding of LPS to CD14 (5). However, because CD14 lacks
transmembrane and intracellular domains, the mechanism by which the LPS
signal is transduced across the plasma membrane is not fully understood.
in response to LPS is clearly regulated at
both transcriptional and post-transcriptional levels (16, 17), the role
of the three MAP kinase subtypes, especially of p38 MAP kinase, within
these processes has been quite controversial. The major advance in this
area was the discovery of pyridinyl imidazole compounds, such as
SB203580, which was identified by a high-throughput screen for
compounds that would inhibit the release of proinflammatory cytokines
including TNF-
. These compounds function by binding specifically to
and inhibiting p38 MAP kinase (12). However, in the study of Lee
et al., it was claimed that inhibition of p38
primarily affected the translation of the TNF-
transcripts but not
the level of TNF-
mRNA in these cells. By contrast, we have
shown that induction of several immediate-early genes is clearly
inhibited at the transcriptional level by these compounds (18, 19). In
addition, pyridinyl imidazoles have now been reported to affect the
LPS-stimulated transcription of several cytokine genes including
IL-1
(20, 21). Manthey et al. (20) reproduced earlier
findings (12, 22, 23) that p38 inhibitors appeared to suppress TNF-
protein levels more than TNF-
mRNA. By contrast, Dean et
al. (14) using these inhibitors to study LPS-stimulated
cyclooxygenase-2 and TNF-
production, came to the very opposite
conclusion and found no discrepancy between the suppression of TNF-
protein and mRNA levels, suggesting that inhibition of mRNA
accumulation might account completely for the effect of this compound
on TNF-
secretion in human blood monocytes. A further complication
is the very recent indication of a novel mode of control of TNF-
transcript levels, namely through modulation of their sensitivity to
degradation (24-26).
gene regulation and necessitates the use of model systems.
However, cell lines such as U937 or THP-1, commonly used as models of
human monocytes to overcome this problem, represent quite immature
stages of monocyte differentiation and may not accurately reflect the
situation in mature monocytes. Here, we show first that although THP-1
cells are a poor model for studying LPS-stimulated TNF-
mRNA
induction, they become more sensitive to LPS stimulation after
differentiation with TPA and compare well with mature human monocytes
purified from human blood. Moreover, we show in TPA-differentiated
THP-1 cells and in purified blood monocytes that individual blockade of
p38 or ERK pathways does certainly affect TNF-
transcript levels,
although neither inhibitor is capable of completely blocking TNF-
mRNA induction. Most importantly however, using a combination of
these inhibitors it is possible to ablate TNF-
induction totally at the mRNA level. Finally, we show that the inhibition of p38 MAP kinase selectively destabilizes TNF-
transcripts without affecting other inducible transcripts such as c-jun. These results are
discussed in relation to the controversy in the literature outlined
above and to the possible use of combinations of MAP kinase inhibitors at reduced concentrations as a therapeutic strategy.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]CTP was purchased from ICN
Pharmaceuticals (Irvine, CA). The random primed DNA labeling kit was
obtained from Roche Molecular Biochemicals.
-mercaptoethanol.
Differentiated THP-1 cells were obtained by treatment with 5 nM TPA for 2 days and then starved overnight in 0.5%
FCS-RPMI in the presence of 5 nM TPA before stimulation.
-glycerophosphate, 1 µM
microcystin, 0.2 µM okadaic acid, 0.1 mM
sodium orthovanadate, and proteases inhibitors. Lysates were incubated
on ice for 10 min and cleared by centrifugation at 13,000 × g for 10 min. Protein concentrations were measured in the
supernatants by the Bradford method (27), and equal amounts of proteins
were loaded onto electrophoresis gels.
,
c-jun, and GAPDH were labeled with 50 µCi of
[
-32P]CTP by random priming. GAPDH mRNA was
detected using a 1-kilobase. PstI fragment of murine
cDNA in pBluescript KS (Stratagene) kindly provided by D. R. Edwards (University of East Anglia, Norwich, UK). TNF-
mRNA was
detected using a 738-base pair EcoRI fragment of human
TNF-
cDNA clone in pBluescript SK+, kindly provided
by D. R. Katz (University College London). c-jun mRNA was detected using a 749-base pair
EcoRI/SacII fragment derived from the mouse
c-jun pAH119 plasmid (generously provided by R. Bravo,
European Molecular Biology Laboratory, Heidelberg, Germany).
and c-jun mRNA were visualized by
autoradiography and quantified using a PhosphorImager and ImageQuant
software (Molecular Dynamics, Sunnyvale, CA) and corrected by reference
to the corresponding GAPDH reading to compensate for slight variation
in loading.
Release by ELISA--
THP-1, TPA-differentiated THP-1,
or freshly purified blood monocytes (105 cells/well) were
incubated for 16-18 h with 10 ng/ml of LPS, unless differently
noted, in RPMI containing 1% FCS. TNF-
levels were detected
using the Duoset kit (Genzyme Diagnostic, Cambridge, MA) according to
the manufacturer's instructions.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mRNA and Protein Levels--
The
human monocytic cell line THP-1 corresponds to an immature stage of
monocyte differentiation and thus does not represent an ideal model of
human monocytes for studying cytokine regulation. Several studies have
shown that TPA treatment of THP-1 cells resulted in a more
differentiated phenotype in terms of adherence, loss of proliferation,
or expression of surface markers (29, 30). To study the levels at which
TNF-
production is regulated, we first compared LPS-induced TNF-
mRNA and protein in TPA-differentiated and nondifferentiated THP-1
cells. Normal THP-1 cells or THP-1 cells differentiated for 2 days with
5 nM TPA were rendered quiescent and stimulated with 100 ng/ml of LPS, and TNF-
gene induction was evaluated by Northern blot
analysis (Fig. 1A). In THP-1
cells, LPS only weakly induces TNF-
transcripts, whereas after
differentiation with TPA, much higher levels of TNF-
mRNA are
seen in response to LPS (Fig. 1A). In the differentiated
cells, TNF-
mRNA was detectable 30 min after LPS treatment.
Maximal induction was observed between 1 and 2 h, and transcript
levels declined thereafter, with barely detectable levels remaining
after 6 h. We have shown further that this response is detectable
in the presence of translational inhibitors (data not shown), and this
finding, together with the transient response, characterizes the
TNF-
gene as an immediate-early gene in response to LPS
stimulation.
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Fig. 1.
TNF- mRNA and
protein analysis in undifferentiated and TPA-differentiated THP-1
cells. A, normal (
) or TPA-differentiated (
)
THP-1 cells were treated with 100 ng/ml LPS for up to 8 h. TNF-
gene induction in response to LPS was evaluated at different time
points by Northern blot (5 µg of total RNA). Quantification of the
radioactivity shown on these blots was performed by phosphorimaging,
corrected for loading against the corresponding GAPDH signals, and
expressed as fold-stimulation over the signal in unstimulated cells
(time = 0). B, THP-1 (
) or TPA-differentiated THP-1
cells (
) were treated with various concentrations of LPS. TNF-
released in the medium was measured by ELISA after a 16-h incubation
period. C, THP-1 (
) or TPA-differentiated THP-1 cells
(
) were treated with 100 ng/ml LPS for the times indicated, and the
levels of TNF-
protein secreted in the culture medium were measured
by ELISA. Data are the means of quadruplicate culture supernatants ± S.E. and are representative of two independent experiments.
protein released in the culture
medium of these two cell systems in response to LPS. Dose-response analysis showed that normal THP-1 cells secreted only low levels of
TNF-
protein when stimulated with up to 1 µg/ml of LPS, and we
could only detect significant amounts of TNF-
in these supernatants using 5 µg/ml of LPS (Fig. 1B). By contrast, the
TPA-differentiated cells secreted large quantities of TNF-
protein
following treatment with concentrations of LPS as low as 1 ng/ml, the
lowest concentration tested (Fig. 1B); this was approaching
the maximal level and showed no significant further increase at
higher concentrations.
protein over a 48-h incubation period using 100 ng/ml LPS (Fig. 1C), the same stimulus as used for analysis of transcript levels shown above. By contrast, after TPA
differentiation, these cells release detectable quantities of TNF-
cytokine as early as 2 h after LPS-stimulation. The level of
secreted protein peaks at around 6 h and diminishes to a quite
stable but submaximal level over 48 h. Dose-response and time
course analyses were also performed in freshly isolated monocytes from
human blood (discussed further below). TNF-
mRNA and protein
levels compared well with those seen in TPA-differentiated THP-1
cells (data not shown). These studies revealed a very marked
enhancement of TNF-
response in the TPA-differentiated cells,
especially when stimulated with lower concentrations of LPS. Thus,
TPA-differentiated THP-1 cells are a better model for events in
purified human monocytes than the nondifferentiated THP-1 cells, in
particular for the study of TNF-
mRNA and protein.
expression studies above that
TPA-differentiated THP-1 cells are a better model for human monocytes
than the undifferentiated cells. Because CD14 is part of the LPS
receptor, the increased levels of CD14 seen here may explain why the
differentiated cells mount a stronger response to LPS than
undifferentiated cells.
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Fig. 2.
Expression of CD14 and CD54 on the surface of
THP-1, TPA-differentiated THP-1 cells, and purified blood
monocytes. The results are expressed as mean fluorescence
intensity (MFI) and as the percentage of positive cells
compared with second antibody only (open curves). Comparable
results were obtained in two separate experiments.
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Fig. 3.
Activation of p38 in THP-1,
TPA-differentiated THP-1 cells, and purified blood monocytes.
Cells were serum-starved overnight in RPMI-0.5% FCS and then left
untreated (Un) or treated for 30 min with
subinhibitory anisomycin (sAn, 50 ng/ml), UV (400 J/m2), LPS (100 ng/ml), NaCl (0.5 M), or
sorbitol (0.5 M). p38 phosphorylation was then assessed by
Western blotting.
Protein Released by Human Monocytes--
Lee et al.
(12) showed that pyridinyl imidazole compounds that inhibit p38 MAPK
are very potent inhibitors of LPS-induced TNF-
secretion in human
monocytes. Further, PD98059, a MEK inhibitor that blocks ERK
activation, also causes a marked inhibition of LPS-stimulated TNF-
release in human blood monocytes (13). To investigate the mechanism of
action of these two inhibitors, we first carried out dose-response
analyses to assess the level of inhibition of TNF-
protein released
by purified blood monocytes.
protein. These data confirmed that both
inhibitors inhibit LPS-induced TNF-
release from human monocytes in
a dose-dependent manner. PD98059 only partially blocked
TNF-
production (47% inhibition using 50 µM PD98059).
In agreement with previous data, the p38 inhibitor SB203580 was shown
to be a very powerful inhibitor of TNF-
release, since it blocked
more than 77% of the TNF-
production at a concentration of 120 nM (Fig. 4A). No obvious loss of viability of
human monocytes cells was detected at the concentrations of inhibitors
used.
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Fig. 4.
Effect of SB203580 and PD98059 on
TNF- release in purified human blood
monocytes. A, freshly purified blood monocytes were
pretreated for 30 min with Me2SO (
) or various
concentrations of SB203580 (
) before a 16-h incubation with 10 ng/ml
LPS. TNF-
levels were then measured by ELISA. Data are the means of
quadruplicate culture supernatants ± S.E. and are representative
of at least two separate experiments. B, the same conditions
as in A, using different concentrations of PD98059
(
).
mRNA in Purified Human Monocytes--
The inhibition seen
with these compounds, particularly the p38 inhibitor, has previously
been ascribed principally to translational inhibition of TNF-
transcripts, with minimal effect on the levels of transcripts (12, 22,
23). We next asked whether the presence of SB203580 or PD98059 would
have any effect on the level of TNF-
mRNA in purified human
monocytes. These experiments were done in triplicate, but because of
the difficulty of obtaining purified monocytes in sufficient numbers
for mRNA analysis, only a subset of data points corresponding to
the protein data from Fig. 4, A and B, was chosen
for mRNA analysis (Fig. 5,
A and B). This showed that over the concentration
range selected, SB203580 produced increasing and pronounced inhibition
of TNF-
transcript levels, contradicting the proposal that
SB203580 acts primarily at the level of translation. It is
important to note that a reduction in secreted TNF-
protein (Fig.
4A) was always more substantial than its effects on
transcript levels (Fig. 5A). This finding is in agreement
with published data indicating TNF-
translation as another point of
action of this inhibitor. However, contrary to the prevailing view, the
data showed that SB203580 also has a clear
concentration-dependent effect on TNF-
transcript
levels, and this must also contribute to the overall loss of TNF-
production. By contrast, the milder reduction of TNF-
protein seen
with varying concentrations of PD98059 (Fig. 4B) corresponds
well with a reduction in transcripts (Fig. 5B) and
conceivably could account for most of the reduced protein levels seen
under these conditions. Although strict comparisons of protein and
transcripts levels are not safe, the data suggest that as opposed to
SB203580, which affects TNF-
production at both mRNA and
translational levels, the less marked effects of PD98059 may occur
principally at the mRNA level.
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Fig. 5.
Effect of SB203580 and PD98059 on
TNF- gene induction, analyzed by Northern
blot, in purified human blood monocytes. A, freshly
purified blood monocytes were pretreated for 30 min with
Me2SO (
) or various concentrations of SB203580 (
)
followed by stimulation with 100 ng/ml LPS for 1 h. Total cellular
RNA was then isolated using the Purescript RNA isolation kit and
analyzed by Northern blot using cDNA probes for TNF-
and GAPDH.
Quantification of radioactivity was performed by phosphorimaging,
corrected for loading against the corresponding GAPDH signals, and
expressed as a percent ± S.E. of the signal measured with LPS
only. Quantification of the data represents the average of three
independent experiments. B, the same conditions as in
A, using different concentrations of PD98059 (
).
Induction at the mRNA Level--
We next
investigated whether combinations of the two inhibitors described above
might produce more pronounced inhibition of TNF-
transcript levels.
Because of the difficulty of obtaining sufficient quantities of
purified monocytes for these studies, we first analyzed these phenomena
in TPA-differentiated THP-1 cells, shown above to be a good model of
human monocytes. Cells were pretreated with different concentrations of
SB203580, PD98059, or a combination of both compounds as indicated
(Fig. 6A) for 60 min before
LPS stimulation (100 ng/ml, 60 min). Used on its own, each inhibitor at
the highest concentration tested blocked slightly more than 50% of
LPS-stimulated TNF-
mRNA accumulation. Note that SB203580 was
less efficient in inhibiting TNF-
mRNA expression in
differentiated THP-1 cells than in purified human monocytes, (50%
inhibition compared with >75% inhibition using 10 µM
SB203580). More interestingly, we observed that a combination of
SB203580 (10 µM) and PD98059 (50 µM) almost
completely ablated TNF-
mRNA accumulation in response to LPS,
suggesting that p38 and ERK pathways are both necessary for maximal
TNF-
gene induction.
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Fig. 6.
Effect of combinations of SB203580 and
PD98059 on TNF- gene induction, analyzed by
Northern blot. A, TPA-differentiated THP-1 cells were
pretreated for 30 min with Me2SO (
,
) or with
SB203580 (SB,
), PD98059 (PD,
), or a
combination of both compounds (SB + PD,
) at the
concentrations indicated followed by stimulation with 100 ng/ml LPS for
1 h. Total cellular RNA (5 µg) was then isolated and analyzed by
Northern blot using cDNA probes for TNF-
and GAPDH. The
quantification of radioactivity shown on these blots was performed by
phosphorimaging, corrected for loading against the corresponding GAPDH
signals, and expressed as fold-stimulation over the signal in control
unstimulated cells. Data are representative of three independent
experiments. B, blood monocytes were freshly purified and
pretreated for 30 min with Me2SO (
), 10 µM
SB203580 (SB), 50 µM PD98059 (PD),
or a combination of both compounds at these concentrations (SB
+ PD) followed by stimulation with 100 ng/ml LPS for
1 h. Cellular RNA was isolated and analyzed as detailed in
A. Data are representative of three independent
experiments.
gene induction was obtained using a combination of the two
inhibitors. We then carried out repeated analyses of the effect
of these inhibitors on LPS-stimulated TNF-
mRNA levels in
several different preparations of human monocytes and
TPA-differentiated THP-1 cells, subjecting the data to quantitative and
statistical analyses (Table I). Over the
course of several experiments that were in close agreement, LPS-induced
TNF-
mRNA levels were inhibited on average by 53.7% in
TPA-differentiated THP-1 cells and by more than 79% in blood monocytes
(75-86%) using the p38 inhibitor. Further, these experiments confirmed our findings that in purified human monocytes, a combination of ERK and p38 MAP kinase inhibitors almost completely blocks LPS-induced TNF-
mRNA accumulation (Table I).
Inhibition of TNF- mRNA levels
gene induction compared with cells
treated with LPS only and correspond to the average of at least three
independent experiments.
gene
induction in differentiated THP-1 cells. As shown in Fig. 7, the new MEK inhibitor used alone
slightly inhibits LPS-stimulated TNF-
mRNA accumulation, but
when it was used in combination with SB203580, complete inhibition was
again observed using a 10 µM concentration of each
compound. In combination, at concentrations of 1 µM for
each compound, an inhibition of 84.1 ± 10.8% (n = 3) of LPS-induced TNF-
mRNA was obtained.
View larger version (38K):
[in a new window]
Fig. 7.
Effect of SB203580 and U0126 on
TNF- gene induction analyzed by Northern
blot. TPA-differentiated THP-1 cells were pretreated for 30 min
with Me2SO (
,
) or with SB203580 (
), U0126
(
) or with a combination of both compounds (SB + U,
) at the
concentrations indicated, followed by stimulation with 100 ng/ml LPS
for 1 h. Total cellular RNA was then isolated and analyzed as
detailed in Fig. 6. Data are representative of three separate
experiments.
mRNA but Not c-jun
mRNA--
Contrary to previous work, these data show that TNF-
mRNA levels are considerably reduced by MAP kinase inhibitors.
However, Baldassare et al. (21) using nuclear run-on
analyses, provided evidence that SB203580 does not significantly affect
LPS-stimulated TNF-
gene transcription, raising the possibility that
the compound could have an effect on TNF-
transcript stability. To
address this possibility, we then analyzed the effect of SB203580 and PD98059 on TNF-
mRNA stability. Differentiated THP-1 cells were stimulated for 1 h with 100 ng/ml of LPS, following which the transcriptional inhibitor DRB (25 µg/ml) was added to the cells to
block any further transcription of the TNF-
gene. Five minutes later, MAP kinase inhibitors were added to the cells and the levels of
TNF-
, c-jun, and GAPDH mRNA over the next 90 min were
measured by Northern blotting analyses (Fig.
8A).
View larger version (30K):
[in a new window]
Fig. 8.
p38 MAPK inhibitor destabilizes
TNF- mRNA but not c-jun
mRNA. A, TPA-differentiated THP-1 cells were
either left untreated (Control) or treated for 1 h with
100 ng/ml LPS. DRB (final concentration 25 µg/ml) was then added to
the cells 5 min before the addition of Me2SO (no
inhibitor,
), 10 µM SB203580 (+ SB,
), or 50 µM PD98059 (+ PD,
). TNF-
and c-jun mRNA decay was evaluated by Northern blot from
the time of the addition of the compounds (t = 0) up to
90 min. Results were quantified by phosphorimaging and normalized to
GAPDH mRNA levels. Results are representative of three independent
experiments. B, TPA-differentiated THP-1 cells were treated
as described in A using no inhibitor (
), 10 µM SB203580 (
), or a combination of 10 µM SB203580 and 50 µM PD98050 (
).
Results are expressed as detailed in A and are
representative of five independent experiments.
transcripts decayed gradually
over 90 min (TNF-
mRNA half-life = 52 ± 7.5 min).
This decay is slightly quicker in the presence of PD98059
(t1/2 = 39.2 ± 9.4 min) but when cells are treated
with SB203580, pronounced acceleration of TNF-
transcript
degradation was obtained (t1/2 = 26.4 ± 4.8 min) and
no TNF-
mRNA could be detected after 60 min of LPS treatment. To
assess whether this effect on mRNA stability was specific to
TNF-
, we compared this with the degradation of c-jun
mRNA, also induced by LPS, by re-probing this blot for
c-jun transcripts (Fig. 8A). This showed that in
TPA-differentiated THP-1 cells c-jun transcripts decayed
more rapidly than TNF-
(c-jun mRNA
t1/2 = 33.4 ± 4.1 min without an inhibitor).
The addition of either p38 or ERK pathway inhibitors had no effect on
c-jun mRNA stability (t1/2 = 30.2 ± 4.9 min in the presence of SB203580;
t1/2 = 29.9 ± 5.7 min in the presence of
PD98059). No change was observed in GAPDH transcripts, which are
extremely stable. Finally, we asked whether a combination of SB203580
and PD98059 would have additive or synergistic effects on TNF-
mRNA stability (Fig. 8B). Over five separate
experiments, we found that the majority of the destabilizing effect
could be accounted for by the presence of SB203580 and that the
additional presence of PD98059 only very slightly accelerated this
decay (Fig. 8B). We conclude that p38 MAP kinase appears to
have the major role in stabilizing TNF-
transcripts, although a
slight effect of PD98059 was reproducibly observed. These findings are
discussed further below in the context of recent observations of novel
stability determinants, potentially regulated via MAP kinase cascades,
in the 3' untranslated regions of this gene.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. Several pieces of data have been advanced to support the idea that the inhibitory effect of these drugs on the production of cytokines, and in particular TNF-
, is mediated primarily at the translational level (12, 22, 23),
a finding that is much cited in the literature (10, 13, 15, 21).
In contrast, a recent study carried out by Dean et al. (14)
reported that pyridinyl imidazole compounds inhibit cyclooxygenase-2
and TNF-
in response to LPS at the mRNA level, suggesting that
the decrease in mRNA could fully account for the reduced protein levels.
release in
TPA-differentiated THP-1 cells as well as in purified blood monocytes.
We showed first that after differentiation for 3 days with 5 nM TPA, THP-1 cells exhibit a higher sensitivity to LPS and
display more surface markers characteristic of monocytes. TPA-differentiated THP-1 cells thus represent a model closer to human
blood monocytes for the study of LPS-induced cytokine release. We have used this model to study the role of p38 on TNF-
release and
confirmed all findings in purified blood monocytes. Our data strongly
support the idea that pyridinyl imidazole compounds inhibit TNF-
induction in response to LPS at the mRNA level. Northern blot
studies following LPS stimulation showed that in differentiated THP-1
cells, SB203580 could inhibit >50% of TNF-
mRNA; this
inhibition level reaches almost 80% in human blood monocytes. Although
this study, contradicting earlier work, shows definitively that
pyridinyl imidazole inhibitors of p38 do reduce TNF-
mRNA
levels, it is important to note that it does not contradict the earlier
model whereby the compound diminishes translation of these transcripts, an issue that has not been addressed here. In fact, the observation that SB203580 causes virtually complete loss of cytokine secreted in
the media (Fig. 4A) but incomplete (~80%) inhibition of
TNF-
mRNA (Table I) in human blood monocytes may support the
previously expressed view that the compound also affects translation of
these transcripts.
protein in human monocytes (13),
but that study did not distinguish between transcriptional, post-transcriptional, or translational modes of action. We have now
extended these findings and show here that a combination of ERK and p38
inhibitors is able to ablate LPS-stimulated TNF-
induction at the
mRNA level. Thus, both p38 and ERK pathways are necessary for
normal LPS-induced TNF-
mRNA accumulation in these cells.
Furthermore, using a new MEK inhibitor, U0126, which is effective at
lower concentrations, together with SB203580, we observed virtually
complete ablation of TNF-
transcripts by using a combination of
inhibitors at a concentration of 1 µM each.
mRNA levels could be due to
decreased transcription and/or reduced mRNA stability. Baldassare et al. (21) performing nuclear run-on analysis showed
SB203580 did not inhibit TNF-
expression at the transcriptional
level, in contrast with the effect of this inhibitor on IL-1
transcription. Since we observed SB203580 greatly decreased TNF-
mRNA level, we assessed the effect of this compound on TNF-
mRNA stability and showed that this compound clearly destabilizes
TNF-
mRNA in response to LPS, providing evidence for a role of
p38 in TNF-
mRNA stabilization. We reproducibly observed a
slight effect of PD98059 on TNF-
mRNA stability but to a much
lesser extent than with SB203580. The combination of both inhibitors
produced only slightly accelerated destabilization compared with the
use of SB203580 alone. These data suggest that p38 MAP kinase
influences both the levels of TNF-
transcripts and their
translation, whereas the effect of ERK inhibition on TNF-
transcript
levels alone could be sufficient to account completely for its effect
on TNF-
production. Most importantly, however, this study suggests
the use of combinations of MAP kinase inhibitors at reduced
concentrations as a most efficient means of inhibiting TNF-
production.
mRNA
stability in the complete absence of any further transcription. A
second important difference is that Wang et al. (31) suggest
that this effect might be mediated through AU-rich elements and could
therefore affect all transcripts that carry these motifs within their
3' untranslated regions, including c-fos and
c-jun, in which induction is known to be inhibited by
SB203580 (18). However, this assertion did not accommodate the earlier
demonstration that SB203580 clearly did not affect the stability of
these two transcripts nor that of three other transiently induced
messages (19) in mouse fibroblasts. To eliminate cell-specific
variability, we have shown directly that under identical conditions
c-jun transcripts in the cells used here are not affected by
blockade of p38 MAP kinase, whereas TNF-
mRNA is destabilized.
Thus, our data do not agree with the assertion that SB203580
destabilizes all transcripts with AU-rich elements in their 3'
untranslated regions and argue instead for destabilization that is
selectively targeted at a subset of AU-rich element-containing labile transcripts.
regulation is the presence of AU-rich elements in
the 3' untranslated region of TNF-
mRNA (34, 35); several proteins could regulate cytokine mRNA stability by binding to the
regulatory AU-rich elements such as AUF1 (36), TIAR (35), or
tristetraprolin (24, 25). Tristetraprolin is a member of a family of
zinc finger proteins, which has been shown to interfere with cytokine
production by binding to AU-rich elements and destabilizing TNF-
mRNA (24, 25, 37) as well as granulocyte/macrophage colony-stimulating factor (38). However, little is known about the signaling pathways involved in the binding of this protein on the
AU-rich elements. In NIH-3T3 cells, tristetraprolin has been shown to
be phosphorylated rapidly on Ser residues after stimulation with
different mitogens, and p42 MAPK could in vitro phosphorylate tristetraprolin on Ser-220 (39). Since we show
here that p38 is clearly involved in controlling TNF-
mRNA
stability, it would be interesting to investigate whether
tristetraprolin can be directly or indirectly phosphorylated by p38
MAPK in human monocytes following LPS stimulation, thereby explaining
how MAP kinases may selectively regulate TNF-
mRNA stability.
Note that since the submission of this paper, Brook et al.
(40) have also reported that inhibition of p38 MAP kinase destabilizes
TNF-
transcripts.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. John Souness and
Brenda Burton (Aventis, Dagenham, UK) for many helpful discussions and
for assisting with the measurements of TNF- protein levels and to
Prof. Benjamin Chain (University College, London, UK) for providing
access to FACScan facilities.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a grant from Aventis.
§ Supported by a grant from the Cancer Research Campaign, London, UK.
¶ To whom correspondence should be addressed. Tel.: 44-1865-285-345; Fax: 44-1865-275-259; E-mail: louiscm@bioch.ox.ac.uk.
Published, JBC Papers in Press, November 13, 2000, DOI 10.1074/jbc.M005486200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
IL, interleukin;
TNF-, tumor necrosis factor
;
LPS, lipopolysaccharide;
LBP, LPS-binding protein;
MAP, mitogen-activated protein;
MAPK, MAP kinase;
ERK, extracellular signal-regulated kinase;
MEK, mitogen-activated
protein ERK kinase;
TPA, 12-O-tetradecanoylphorbol-13-acetate;
DRB, 5,6-dichlorobenzimidazole riboside;
FCS, fetal calf serum;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
ELISA, enzyme-linked
immunosorbent assay.
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