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B-Responsive Genes1
Department of Adult Oncology and Joint Center for Radiation Therapy, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B, activate heat shock transcription factor 1 and suppress
cytokine gene expression in activated monocytes and macrophages.
Because our preliminary studies indicated that these effects could be
mimicked by inhibitors of signal transduction, we have studied the
effects of NSAIDs on signaling molecules potentially downstream of LPS
receptors in activated macrophages. Our findings indicate that
ribosomal S6 kinase 2 (RSK2), a 90-kDa ribosomal S6 kinase with a
critical role as an effector of the RAS-mitogen-activated protein
kinase pathway and a regulator of immediate early gene transcription is
a target for inhibition by the NSAIDs. NSAIDs inhibited the activity of
purified RSK2 kinase in vitro and of RSK2 in mammalian cells and
suppressed the phosphorylation of RSK2 substrates cAMP response element
binding protein (CREB) and I-B
in vivo. Additionally, NaSal
inhibited the phosphorylation by RSK2 of CREB and I-B on residues
crucial for their transcriptional activity in vivo and thus repressed
CREB and NF-B-dependent transcription. These experiments suggest
that RSK2 is a target for NSAIDs in the inhibition of monocyte-specific
gene expression and indicate the importance of RSK2 and related kinases
in cell regulation, indicating a new area for anti-inflammatory
drug discovery. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1ß, IL-6,
IL-8, IL-10, and ICAM-1 in monocytes activated by the proinflammatory
LPS (1, 2). A potential mechanism that may underlie
inhibition of monocyte gene expression by NSAIDs is transcriptional
repression due to the perturbation of the activity of the transcription
factors NF-B and heat shock transcription factor 1 (HSF1)
(3, 4, 5). NaSal and other NSAIDs inhibit the activation of
NF-B and simultaneously activate HSF1 to a DNA binding form at
identical drug doses (2, 5). The effect of NaSal on HSF is
highly conserved and is observed in human, murine,
Drosophila, and yeast and is mimicked by at least nine other
members of the NSAIDs, indicating an evolutionarily conserved mechanism
(4, 6, 7, 8). Regulatory mechanisms underlying the
transcriptional activation of both HSF1 and NF-B are similar and
involve protein phosphorylation (for review, see Ref. 9).
In addition, activation of both factors undergo conversion from latent
cytoplasmic forms to a nuclear trans-activating factors
(reviewed by Refs. 9 and 10). Negative
regulation of NF-B involves the influence of inhibitory subunits,
including the protein I-B, which binds NF-B and inhibits
nuclear localization (10). Activation of NF-B by
proinflammatory agonists involves the phosphorylation of I-B,
which targets it for degradation by the proteosome and releases NF-B
to migrate to the nucleus and activate the promoters of target genes
(10).
We have examined whether a common signaling pathway involved in the
activity of both factors may underlie the effects of NSAIDs on gene
expression in human monocytes by studying intermediates involved in the
signal transduction cascade that leads to NF-B activation after
exposure of monocytes to LPS. Our previous studies showed that tyrosine
kinase inhibitors herbimycin A and AG126, which inhibit LPS induction
of tyrosine phosphorylation (11, 12), mimic the effects of
NaSal in inducing the binding of HSF1 to its response element and
inhibiting the binding of NF-B to consensus B elements in primary
human monocytes (X. Wang, A. Asea, B. Chu, X. Zhang, and S. K.
Calderwood, manuscript in preparation). This suggested the existence of
one or more signaling pathways that activate NF-B and repress HSF1
that are inhibited by exposure to NSAIDs. We therefore examined protein
kinases that could potentially function in a signaling cascade
downstream of LPS and proximal to HSF1 and NF-B and found that
mitogen-activated protein kinase-activating kinase (MAPKAP K2) and the
90-kDa ribosomal S6 kinase (RSK2) are both inhibited by NaSal at
concentrations that activate HSF1 and inhibit the induction of NF-B
to nuclear factors able to bind DNA in in vitro EMSA experiments (X.
Wang, A. Asea, B. Chu, X. Zhang, and S. K. Calderwood,
manuscript in preparation). However, only RSK2 catalyzed the
phosphorylation of I-B in vitro, suggesting RSK2 as a signal
transduction intermediate in the response to LPS, which is targeted by
NaSal (X. Wang, A. Asea, B. Chu, X. Zhang, and S. K. Calderwood,
manuscript in preparation). We have therefore studied RSK2 as a
potential target for NSAIDs in vivo. Here we show that the activity of
purified RSK2 is inhibited by a wide spectrum of NSAIDs in
vitro and that these drugs inhibit the activity of cellular RSK2
in human monocytes and murine fibroblasts. Subsequent studies in vivo
indicated that RSK2 overexpression leads to the phosphorylation of at
least two target proteins, CREB and I-B, on sites crucial for
trans-activation by CREB and NF-B and that NaSal inhibits
such phosphorylation. As NaSal has been shown previously to repress the
promoters of monocyte genes, we examined the potential role of RSK2 in
gene transcription and NaSal as a transcriptional inhibitor (2, 13). Inhibition of CREB and phosphorylation in RSK2-transfected
cells was correlated with repression of CREB- and NF-B-responsive
promoters.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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pMT2.HA-RSK2 was a gift from Dr. M. E. Greenberg (Harvard
Medical School, Boston, MA); pFA-CREB and pFR-Luc were purchased from
Stratagene (La Jolla, CA); anti-RSK and p65 Abs, purified PP2A1,
and MAPKAP K2 were purchased from Upstate Biotechnology (Lake Placid,
NY); anti-CREB and anti-IB were purchased from New
England Biolabs (Beverly, MA). Chromatographic materials were purchased
from Pharmacia (Piscataway, NJ).
Cells and cell culture
NIH 3T3 mouse embryonic fibroblasts and HeLa S3 human carcinoma cells were obtained from the American Type Culture Collection (Manassas, VA), grown in DMEM containing 10% bovine calf serum (NIH 3T3) or Ham’s F-10 plus 10% FCS (HeLa S3), and passaged at a 1:10 ratio using trypsin/EDTA.
Isolation and enrichment of human monocyte
PBMC were isolated from freshly drawn peripheral venous blood (Kraft Family Blood Center, Dana-Farber Cancer Institute, Boston, MA) using the Ficoll-Paque separation technique as previously described (14). Monocytes were enriched from the PBMC fraction by negative depletion using magnetic beads coated with appropriate mAbs (15). Briefly, using a monocyte isolation kit, (Miltenyi Biotec, Auburn, CA), PBMC were treated with FcR blocking agent followed by hapten-Ab mixture containing monoclonal hapten-conjugated CD3 (mouse Ig2A), CD7 (mouse Ig2A), CD19 (mouse IgG1), CD45RA (mouse IgG1), CD56 (mouse IgG2b), and anti-IgE (mouse IgG2A) Abs and incubated for 5 min at 4°C. Cells were washed twice in PBS and incubated in FcR blocking buffer and MACs anti-hapten microbeads. After a 5-min incubation at 4°C, cells were washed and passed through a column attached to a magnet. Unlabeled monocytes (CD45+CD14+) eluted from the column were >98% pure, as judged by flow cytometry.
Protein extraction immunoprecipitation and Western blot analyses
Proteins were extracted from cells following treatment with ice-cold RIPA buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 2.5% deoxycholate, 2 mM EGTA, 1 mM leupeptin, 1 mM aprotinin, 10 mM NaF, and 1 mM PMSF), and samples were cleared by centrifugation at 15,000 x g for 20 min at 4°C. For immunoprecipitation experiments, supernatant was carefully removed and incubated with primary Ab for 1–2 h on ice, and immunoprecipitates were collected with protein A-Sepharose beads (Pharmacia) for 30 min at 4°C. The precipitate was washed three times with PBS, boiled in SDS-PAGE sample buffer before electrophoresis on 12% SDS-polyacrylamide gels, and transferred to Immobilon polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membrane was blocked by incubation in TBS buffer supplemented with 5% nonfat dry milk (Bio-Rad, Hercules, CA) and 0.1% Tween 20 for 1 h at room temperature, washed three times in TBS buffer, and incubated overnight with the appropriate primary Ab at 4°C. Membranes were washed three times with TBS buffer and incubated with the appropriate alkaline phosphate-conjugated secondary Ab for 1 h at room temperature. Detection of proteins was achieved by the enhanced chemiluminescence system.
Immunoblot assay
Cells were washed three times in ice-cold Dulbecco’s PBS and dissolved in Laemmli sample buffer, and samples ere fractionated by 10% SDS-PAGE before immunoblot assay with anti-HSF1 Abs using procedures similar to those described previously (16).
In vitro kinase assays
RSK2 was purified from rabbit muscle, using a preparation scheme
described previously, as a 90-kDa polypeptide by SDS-PAGE analysis and
was identified by an Ab that recognizes RSK2, but not RSK1
(17). Although free of RSK1 and RSK3, some of the
enzymatic activity in the extract could possibly be due to a trace
contaminant. However, purified RSK2 contained negligible activity for
MAPK, MAPKAP K2, or PKC activity assessed using enzyme-specific
peptides. RSK2 (0.2 U) was incubated for 20 min at 30°C with 0.25 mM
peptide substrate (RRRLSSLRA; 95% pure) and 0.5 µCi
[
-32P]ATP (New England Nuclear, Boston, MA)
in 25 µl of GNM buffer (60 mM ß-glycerophosphate, 30 mM
p-nitrophenylphosphate, 25 mM MOPS, 15 mM
MgCl2, 150 mM ATP, 0.1 mM sodium orthovanadate, 5
mM EGTA, and 1 mM DTT, pH 7.0). Phosphopeptides were isolated on p81
filters (Pierce, Rockford, IL), washed in ice-cold 75 mM phosphoric
acid, and assayed by Cherenkov counting. Purified MAPKAP K2 (0.1 U) was
assayed with synthetic peptide KKPLNRTLSVASLPGLamide (3)
and 0.5 µCi of [-32P]ATP (New England
Nuclear) as described above for RSK2. To test for cross-reactivity of
the purified MAPKAP K2 with RSK2, controls were incubated with 0.1 mM
H7, which inhibits RSK2 but not MAPKAP K2 (3). Recombinant
HSF1 (13) and GST-I-B were incubated (5
µg/reaction) with RSK2 and assayed as described above.
In vivo immunokinase assay
Cells were washed in ice-cold PBS and lysed in MGGE buffer (30
mM 4-morpholinepropanesulfonic acid (pH 7.0), 10% glycerol, 80 mM
ß-glycerophosphate, 20 mM, EGTA, 0.1 mM
Na3VO4, 10 mM
MgCl2, 0.5% Triton X-100, 5 mM DTT, 1.0 mM PMSF,
and 1 mM leupeptin). RSK2 was then immunoprecipitated from supernatants
after a 15-min spin at 15,000 x g using anti-RSK2
antiserum (60 min at 4°C). Immune complexes were collected on protein
A-Sepharose beads and washed in GNM buffer (described above),
supplemented with 1 mM PMSF, 1 mM leupeptin, and 30 ng/ml of the
protein kinase A inhibitor peptide (Sigma). The immune complexes were
then assayed by incubation with RSK2 substrate peptide and
[-32P]ATP (New England Nuclear) as described
above (see In vitro kinase assays).
Transient transfection and transcriptional reporter gene assay
For transfection experiments, cells were dispensed into six-well
plates (Costar, Cambridge, MA) at 2.2 x 105
cells/well and left for 20–24 h before liposome-mediated transfection
with reporter plasmids (B-Luc and pFR-Luc), expression plasmids
(pMT2.HA-RSK2 and pFA-CREB), and control (pCMV-lacZ
expression plasmids) as previously described (18).
B-Luc was made by inserting four contiguous consensus B elements
into the polycloning site of the pGL3-Promoter vector (Promega). All
treatments contained the same amount of DNA. Transfectants were
incubated overnight in a 2.5% CO2/air
atmosphere. Cell extracts were prepared as described in detail, and
luciferase (Luc) and ß-galactosidase assays were performed as
previously described (19). Reporter gene activity was
normalized to ß-galactosidase activity, which was used as an internal
transfection efficiency control. Results are expressed as the fold
increase in activity over the control value.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We first examined the effects of NSAIDs shown previously to induce
HSF1 and repress NF-B on the activity of RSK2 purified from rabbit
muscle. We investigated the ability of the NSAIDs to inhibit the
stimulation of phosphorylation of a peptide substrate for RSK2 (Fig. 1
A). RSK2 activity was
inhibited by drugs from a number of classes of NSAID, including the
salicylates NaSal and aspirin (Asp); the arylproprionic acid ibuprofen
(Ibu); the arylacetic acids indomethacin, sulindac, and diclofenac; the
oxicam piroxicam; and the pyrazolidinedione phenylbutazone (Fig. 1A). The drugs inhibited RSK2 at concentrations (Fig. 1A) shown previously to activate HSF1 and to inhibit NF-B
(2, 4). Most of the drugs inhibited RSK2 at concentrations
between 0.1 and 1 mM, although Asp (3 mM) and NaSal (20 mM) required
higher concentrations (Fig. 1A). Even though it required
higher concentrations for inhibition of RSK2 activity, we used NaSal in
most of the subsequent studies shown here due to its low toxicity in
tissue culture (NaSal could be used at concentrations up to 100 mM
without significant killing of NIH 3T3 cells). We next went on to
examine the dose dependency of RSK2 inhibition by NaSal and Asp (Fig. 1B). RSK2 activity was inhibited by 50% at a concentration
of 20 mM NaSal, while Asp, which inhibits HSF1 and NF-B activity at
a lower concentration than NaSal (4, 5), was also a more
effective RSK2 inhibitor, reducing RSK2 activity by 50% at 
2.5 mM
(Fig. 1B). Kinetic analysis of RSK2 inhibition was then
performed using a double-reciprocal plot of RSK2 activity against the
peptide substrate concentration, either with no inhibitor or with 20 or
50 mM NaSal (Fig. 1C). The plots do not share a common
intercept on the 1/Vo axis characteristic of a noncompetitive
inhibitor, indicating that NaSal interacts with RSK2 to cause
allosteric inhibition (Fig. 1C).
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We next examined the effectiveness of NaSal as an inhibitor of the
ability of RSK2 to catalyze phosphorylation of protein substrates in
vitro (Fig. 2). As the DNA binding
activities of NF-B and HSF1 are modulated by NaSal in cells, we
first determined whether HSF1 and I-B were phosphorylated by RSK2
in vitro in a NaSal-sensitive manner (5, 6). RSK2
stimulated the phosphorylation of both HSF1 (Fig. 2A,
lane 2) and I-B (lane 4), and this
activity was inhibited by NaSal (Fig. 2B, lanes 3
and 5) at concentrations that lead to the activation of HSF1
and the inhibition of NF-B binding to DNA in vivo (20
mM). Because previous studies showed that RSK kinases
phosphorylate I-B at Ser32 and that
site-directed mutagenesis at this site blocks the response to
activators (20, 21), we determined whether
Ser32 phosphorylation was inhibited by NaSal and
Ibu (Fig. 2B). Using Abs specific for I-B
phosphorylated at Ser32, we found that RSK2
catalyzed the phosphorylation of I-B on
Ser32, an effect prevented by incubation with
NaSal and Ibu (Fig. 2B). Recent studies indicate that HSF1
is phosphorylated in vitro by RSK2 on at least three residues (S.
K. Calderwood, unpublished observations), which may account for the
greater incorporation of 32P into HSF1 compared
with I-B, which is apparently phosphorylated at only one site
(Fig. 2B). However the nature of the amino acids
phosphorylated in HSF1 after incubation with RSK2 is not yet known. We
also investigated the effect of NaSal on phosphorylation of a
well-characterized RSK2 substrate protein, the transcription factor
cAMP response element binding protein (CREB) (22).
Incubation with RSK2 resulted in the phosphorylation of murine CREB
protein immunoprecipitated from NIH 3T3 cells, and this effect was
inhibited by NaSal (Fig. 2C). As CREB is a
well-characterized substrate for RSK2, we used this protein as a
primary model for examining the effects of NaSal on RSK2 activity in
subsequent studies in tissue culture.
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We next examined the ability of NSAIDs to inhibit cellular RSK2 in
vivo. As RSK2 is largely inactive under resting conditions, we examined
two cell types in which RSK2 can be induced by upstream signaling
cascades (22). We first investigated the effects of LPS on
RSK2 activity in freshly recovered human monocytes isolated from
peripheral venous blood (Fig. 3A). Exposure to LPS caused an
2-fold increase in cellular RSK2 activity measured by immunokinase
assay, and this increase was inhibited by exposure to NaSal (Fig. 3A). Thus, doses of NaSal that inhibit the activity of
purified rabbit RSK2 in vitro inhibit cellular RSK2 in human monocytes
(Fig. 1
, A and B, and 2A). However,
these experiments do not indicate whether the effects of NaSal are due
to direct interactions of the drug with the enzyme or to indirect
effects on upstream signaling, cellular phosphatases, or other
regulatory interactions. RSK2 is activated downstream of the
extracellularly regulated kinase (ERK) MAPK pathway and is a direct
substrate for ERK (23, 24). As NaSal has been shown to
inhibit LPS-induced cellular ERK activity, we examined these
interactions further using mitogen-activated fibroblasts in which RSK2
is activated downstream of ERK. As NaSal has been shown not to inhibit
epidermal growth factor (EGF) stimulation of ERK in fibroblasts and
12-O-tetradecanoylphorbol-13-acetate (TPA) stimulation of
ERK in PMN, we examined the effects of NaSal and other NSAIDs on RSK2
activated by EGF and TPA in murine fibroblasts. RSK2 activity was
strongly stimulated by both TPA (Fig. 3B) and EGF (Fig. 3C) in NIH 3T3 murine fibroblasts. TPA- and EGF-induced RSK2
activity was inhibited by NaSal, Asp, and Ibu (Fig. 3, B and
C). The possibility that inhibition of RSK2 by NSAIDs may be
independent of the effects of ERK activity was confirmed by experiments
in which TPA-induced cellular ERK activity was not significantly
inhibited by 10 or 5 mM NaSal, 1 mM Asp, or 1 mM Ibu (Fig. 3D), concentrations previously demonstrated to drastically
inhibit TPA-induced cellular RSK2 activity (Fig. 3C),
indicating that RSK2 is indeed the primary target for NSAIDs.
Interestingly, TPA-induced ERK activity was inhibited by 20 mM NaSal, 3
mM Aps, and 2 mM Ibu (Fig. 3D), suggesting the possibility
that at high concentrations NSAIDs may target multiple kinases.
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We next examined the effects of exposure to NaSal on
phosphorylation of intracellular CREB, a well-characterized RSK2
substrate in murine NIH 3T3 fibroblasts. We investigated the
phosphorylation of CREB at a critical residue, serine 133, within the
transcriptional activation domain that is phosphorylated in vivo by
RSK2 (22). Exposure to TPA markedly increased CREB
phosphorylation on Ser133, and this effect was
partially reversed by exposure to 1 mM NaSal and completely inhibited
by 10- and 20-mM concentrations of the drug (Fig. 4A). Total CREB protein
levels, as determined by immunoblot, remained constant. The
Ser133-specific Ab used here also cross-reacts
with ATF-1 phosphorylated at a serine residue conserved within the
trans-activation domain of CRE binding proteins
(25). As with CREB, TPA-induced ATF-1 phosphorylation is
inhibited by 20 mM NaSal (Fig. 4A).
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B, lane
6), an effect that was increased by exposure to serum
(lane 7) and was inhibited by 20 mM NaSal
(lane 8). Relative levels of phospho-GAL4 CREB are
indicated in the densitometer scan in Fig. 4C. Also evident
in the immunoblot are native phospho-CREB and phospho-ATF-1, the level
of each of which was activated by serum exposure (Fig. 4B,
lane 7) and inhibited by NaSal (Fig. 4D,
lane 8). Fig. 4D shows the relative levels of
RSK2 in controls and in cells overexpressing HA-RSK2 with and without
serum exposure. RSK2 and HA levels increased markedly in the
transfectants, and these levels were not altered by exposure to serum
or NaSal (Fig. 4, B and C). Total cellular RSK2
kinase activity determined by immunokinase assay was elevated 2-fold in
serum-treated cells and RSK2 transfectants and was markedly increased
when RSK2 expression was combined with serum stimulation (Fig. 4F, lane 7). NaSal inhibited the increases in
RSK2 activity observed in RSK2 transfectants with or without serum
stimulation (Fig. 4F, lanes 5 and 8).
We also examined the effects of exposure to NaSal on the
phosphorylation of I-B in cells exposed to TPA and overexpressing
RSK2. TPA activated I-B phosphorylation of
Ser32, and this effect was inhibited by NaSal
(data not shown). We also examined the effects of NaSal on I-B
phosphorylation after treatment with another stimulus, TNF-, in a
TNF--responsive cell line (HeLa cells). TNF- activated
phosphorylation of I-B on Ser32, and this
effect was inhibited by exposure to NaSal (data not shown).
RSK2-dependent activation of CREB- and B-responsive promoters is
inhibited by sodium salicylate
Having examined the effects of NaSal on CREB phosphorylation by
RSK2 in vivo, we next attempted to determine whether these effects on
CREB phosphorylation translated into changes in transcriptional
activation by CREB. We therefore examined the effects of NaSal on the
activation of a GAL4-Luc reporter construct by the GAL4-CREB hybrid
protein with or without RSK2 expression using the conditions described
previously in Fig. 4, B–F. Overexpression of RSK2 increased
GAL4-Luc activation by the hybrid transcription of GAL4-CREB (3- to
4-fold), an effect that was increased by exposure to serum and
inhibited by incubation with NaSal (Fig. 5A). Transcriptional
activation of GAL4-CREB (Fig. 5A) thus paralleled its
phosphorylation state (Fig. 4B), indicating that activation
of CREB in transfectants is repressed by NaSal through a mechanism that
may include direct noncompetitive inhibition of the phosphotransferase
activity of RSK2.
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B in cells cotransfected with a
B-Luc promoter-reporter construct and an RSK2 expression vector
(Fig. 5B). RSK2 expression activated the B-driven
promoter, and this effect was blocked by exposure to NaSal (Fig. 5B). The inhibitory effects of NaSal on the CREB- and
B-responsive promoters were specific and were not due to general
effects on transcription, as indicated in control experiments showing
that the constitutively active CMV immediate-early gene control
promoter was neither activated by RSK2 expression nor inhibited by
NaSal (Fig. 5C). Thus, the effects of NaSal were specific
for the activation of transcription by RSK2 (Fig. 5C).
Transcriptional repression by NaSal is not limited to the experimental
CREB- and NF-B-responsive promoters examined above, and both the
IL1B and c-fms promoters are repressed by NaSal
(Y. Xie and S. K. Calderwood, manuscript in preparation). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and 2) (2).
Although we have not addressed the structural mechanisms involved in
RSK2 inhibition by NaSal in any depth, the analysis in Fig. 1 suggests
that the drugs can interact directly with the enzyme in such a way as
to inhibit catalytic activity. The RSK kinases are unusual in
containing two highly conserved kinase domains (26, 27).
The N-terminal domain, which is apparently active in phosphorylating
exogenous substrates of RSK2 is structurally related to protein kinase
C and the catalytic domains of protein kinases A and G
(27). The C-terminal domain, which contains sequence
similarities with calcium- and calmodulin-activated kinase II and
MAPKAP K2, does not directly phosphorylate exogenous substrates but,
instead, plays a role in autophosphorylation and apparently is an
essential regulatory subunit of RSK2 (27). As NaSal
inhibits MAPKAP K2 activity at similar doses as RSK2 inhibition (data
not shown), it may be reasonable to infer that NaSal interacts with the
C-terminal domain of RSK2 to block a regulatory activity required for
RSK2 function. The functional domain in RSK2 inhibited by NaSal may be
involved in autophosphorylation, as indicated by the finding that NaSal
inhibits RSK2 autophosphorylation in vitro and blocks the mobility
shift accompanying activation (which may involve autophosphorylation)
in vivo (Fig. 4, B–E). However, further studies will be
required to determine more precisely the mechanism of RSK2 inhibition
by NaSal.
Incubation of cells with NSAIDs also leads to the inhibition of
intracellular RSK2 activity (Fig. 3). These effects of the NSAIDs on
cellular RSK2 may thus reflect direct inhibition of RSK2 activity as
observed with purified RSK2 (Figs. 1 and 2). However, the drugs may
also act indirectly on regulatory pathways that impact on RSK2 activity
in the cell. RSK2 is an inducible kinase activated by the MAPK family
members ERK1 and ERK2, which are downstream of a kinase cascade induced
by multiple extracellular signals (28). In fact,
intracellular ERK activity may be inhibited by NaSal under some
circumstances (23, 24). Evidence that argues against ERK
as the primary target for NaSal rather than RSK2 in our studies is that
intermediate to low concentrations of the NSAIDs NaSal, Asp, and Ibu
did not inhibit cellular ERK activity induced by TPA (Fig. 3D). In these experiments we observed that high
concentrations of NSAIDs caused specific inhibition of TPA-induced
cellular ERK activity, indicating the possibility that at these
concentrations multiple kinases upstream of RSK2 may also be targets
for NSAIDs. Further evidence in favor of RSK2 as the primary target is
that the NSAIDs inhibit RSK2 activity induced by EGF and TPA, a
treatment that leads to the induction of ERK activity that is not
inhibited by NaSal (23, 24). Additionally, both NaSal and
Asp inhibit cellular RSK2 at concentrations (5 and 1 mM) that only
minimally affect purified RSK2 in vitro (Figs. 1, A and
B, and 2B). This discrepancy may have a number of
causes. Firstly, the distribution of the drugs across the cell membrane
may not be uniform. The NSAIDs are weak acids that distribute across
the plasma membrane according to intra- and extracellular pH (28, 29). The drugs are uncharged at lower pH and rapidly cross
membranes, while being charged and relatively immobile across membranes
at pH values above 7.0 (28, 29). An additional possibility
is that the NSAIDS affect multiple metabolic and regulatory pathways in
the cell, and these may have additive/synergistic effects on individual
cellular activities such as RSK2. Effects on upstream pathways such as
the ERK-MAPK pathway and on the activities of cellular phosphatases,
scaffold proteins, or other molecules could be involved in the
intracellular effects of the drugs. Inhibition of purified RSK2 by
NaSal occurs at concentrations of 20 mM, well above the range
achieved in patients undergoing treatment for inflammatory diseases
(for reviews, see Refs. 30 and 31). However,
significant inhibitory effects on cellular RSK2 were observed at 5 mM
NaSal and 1 mM Asp (Fig. 3B). In addition, other members of
the NSAIDs that have in common the inhibitory effect on RSK2 were
significantly more effective than NaSal and Asp in inhibiting purified
RSK2 in vitro (Fig. 1A). These, however, remain at the
extreme range of concentrations encountered in patients. These findings
may be relevant to the behavior of cells in rheumatoid arthritis
patients undergoing treatment with high concentrations of NSAIDs
(32, 33). Each of the cytokines whose expression by human
monocytes was shown previously to be inhibited by NaSal is
characteristically found at elevated levels in the synovial fluid of
rheumatoid arthritis (RA) patients (2, 34, 35). Such
cytokines, which appear to play important roles in the progression of
RA, may thus be targets for the NSAIDs used in treatment (31, 35). In support of this conjecture, recent studies have shown
that NSAIDs can reduce the concentrations of IL-6 in the synovial fluid
of RA patients and inhibit the expression of TNF- and IL-1ß in
human synovial membrane explants (16, 36). Our previous
findings indicated that NaSal and other NSAIDs inhibit the expression
of a wide spectrum of cytokines through inhibition of a number of
transcription factors involved in cytokine regulation (2).
In one case, NaSal inhibits phosphorylation of CREB at a key site in
the transcriptional activation domain and prevents the induction of a
CREB-dependent promoter (Fig. 4A). As CREB plays a role in
cytokine expression, such an effect may impact on NaSal-induced
cytokine repression (37). Inhibition of an
NF-B-sensitive promoter may involve a contrasting mechanism, with
NaSal inhibiting the phosphorylation of the negative regulatory
molecule I-B by RSK2, which may prevent NF-B access to the
nucleus (Fig. 2B). RSK2 phosphorylates I-B on a
residue (Ser32) required for a response to
NF-B-activating signals (Fig. 2, E and F) and
is therefore one of the I-B kinases that include RSK1 as well as
unrelated enzymes (20, 21, 38). Although the contribution
of RSK2 to NF-B activation by proinflammatory signals has not been
widely investigated, our studies indicate that prevention of I-B
phosphorylation on Ser32 by NaSal can mediate
inhibition of the transcriptional activation of NF-B
(5). In addition, NaSal inhibits the phosphorylation of
HSF1, a repressor of cytokine genes, on as yet unknown sites and
stimulates its ability to bind DNA, although the precise mechanisms
involved in this process are unclear (9, 13, 39). RSK2 is
evidently a repressor of HSF1 activation, and NaSal may function to
reverse the repression (Figs. 1 and 2B). By inhibiting RSK2,
NaSal may thus coordinately influence the activity of at least three
transcription factors, and its ultimate effects on target promoters may
be determined by effects on individual factors or a combination of the
factors. As mentioned earlier, however, the NSAIDS appear to have
pleiotropic effects on cell regulation, and inhibition of RSK2 may
mediate only a portion of the inhibitory effects of the drugs on
cytokine gene expression. Recent studies have also shown that three of
the NSAIDs bind to the peroxisome proliferator-activated receptor-
(PPAR-), a nuclear receptor that activates genes involved in
adipogenesis but represses monocyte-specific transcription
(1). PPAR- appears to be capable of repressing both
natural promoters, such as the inducible nitric oxide synthase and
TNF- promoters and artificial promoters containing cannonical sites
for AP-1, Stat-1, and NF-B (1). The relative
contributions of RSK2 and PPAR- or other molecules to cytokine
repression by the NSAIDs is, however, not known. In addition, although
the effects of NaSal on RSK2 activation occur at concentrations well
above those needed for inhibition of prostaglandin H synthase 1 or 2,
we cannot rule out a role for prostaglandin H synthase as a necessary,
but not sufficient, component in this effect (4). In
addition to I-B, HSF1, and CREB, other cellular substrates for
RSK2 include the ribosomal S6 protein, the G subunit of glycogen
synthase, and a number of nuclear proteins, including lamin C,
c-fos, and p67SRF (22). Preventing the
phosphorylation of these proteins may also contribute to the
pharmacological actions of the NSAIDs (4, 5, 6, 30, 40, 41).
In conclusion, NaSal and other NSAIDs inhibit the 90-kDa RSK2 in vitro and in vivo at concentrations that lead to inhibition of inflammatory gene expression in monocytes. RSK2 stimulates the phosphorylation of a number of transcription factors involved in monocyte gene expression, and NaSal coordinately inhibits CREB phosphorylation and CREB-dependent transcription. Inhibition of signal transduction pathways activated by proinflammatory agonists is thus a promising strategy for the development of anti-inflammatory agents.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Stuart K. Calderwood, Dana-Farber Cancer Institute, 44 Binney Street (D810), Boston, MA 02115. E-mail address:
3 Abbreviations used in this paper: NaSal, sodium salicylate; CREB, cyclic AMP response element binding protein; HSF, heat shock factor; HSP, heat shock protein; NSAID, nonsteroidal anti-inflammatory drug; MAP, mitogen-activated protein; MAPK, MAP kinase; MAPKAP K2, mitogen-activated protein kinase-activating kinase; RSK2, ribosomal S6 kinase 2; Luc, luciferase; Asp, aspirin; Ibu, ibruprofen; ERK, extracellularly regulated kinase; EGF, epidermal growth factor; TPA, 12-O-tetradecanoylphorbol-13-acetate; ATF, activating transcription factor; HA, hemagglutinin; RA, rheumatoid arthritis; PPAR-, peroxisome proliferator-activated receptor-.
Received for publication November 5, 1998. Accepted for publication August 30, 1999.
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). RSK2
was incubated for 20 min with specific peptide substrate and 0.5 µCi
[
