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Departments of
*
Bone and Cartilage Biology and
Cardiovascular Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406
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Abstract |
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 Top Abstract IntroductionMethods and MaterialsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMethods and MaterialsResultsDiscussionReferences |
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play a predominant role during inflammatory
responses and autoimmune disease (1). Much attention has been paid to
the role of cytokines in rheumatoid arthritis
(RA),2 and there may be
a role for these cytokines in the development of osteoarthritis (OA)
(2, 3, 4). It is now generally accepted that cytokines can induce
proteoglycan degradation, inhibit proteoglycan synthesis, and induce
matrix metalloproteinases (5, 6, 7, 8, 9, 10), resulting in damage to both
cartilage and bone. One of the mechanisms by which cytokines elicit
their proinflammatory effects is by the stimulation of the production
of nitric oxide. Nitric oxide (NO) is a multifunctional messenger
molecule generated by a family of enzymes termed the NO synthases
(NOS). The inducible form of nitric oxide synthase (iNOS) releases high
levels of NO in response to IL-1 and TNF. IL-1 has been shown to induce
the release of NO from chondrocytes and cartilage of a number of
species (11, 12, 13, 14), and NO may activate matrix metalloproteinases
responsible for proteoglycan degradation in articular cartilage (15).
Stimulation of NO production in bone by proinflammatory cytokines
indicates that NO may be involved as a mediator of bone disease in RA
and postmenopausal osteoporosis (16).
SB 203580
(4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)imidazole;
Fig. 1
) is a member of a new series of
pyridinyl imidazole compounds that inhibit IL-1 and TNF- production
from LPS-stimulated human monocytes and the human monocyte cell line
THP-1 with IC50 values of 50 to 100 nM (17, 18). The term
CSAID has been coined for these compounds, and they have shown activity
in a number of animal models of acute and chronic inflammation,
including collagen- and adjuvant-induced arthritis (19, 20). The
molecular target of SB 203580 has been identified as a pair of closely
related mitogen-activated protein kinase homologues, termed CSAID
binding proteins (CSBPs) (17), p38 (21), or RK (22). Binding of the
pyridinyl imidazole compounds to CSBP in THP-1 cytosol correlates with
their ability to inhibit cytokine synthesis in response to various
stimuli (17). It has been shown that several cell types express p38
kinase, and of particular relevance is the finding that human articular
chondrocytes express p38 proteins that are time-dependently activated
by IL-1 (23).
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Methods and Materials |
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TopAbstractIntroductionMethods and MaterialsResultsDiscussionReferences |
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SB 203580 was synthesized at SmithKline Beecham Pharmaceuticals
(Fig. 1
). Recombinant human IL-1 (which was used in all the
experiments), the metalloproteinase inhibitor BB94, and the GST-ATF2
were also prepared at SmithKline Beecham. DMEM and Ham’s F-12 medium
were obtained from Life Technologies (Grand Island, NY). FBS was
obtained from HyClone (Logan, UT). The media were supplemented with 100
U/ml of penicillin, 100 µg/ml of streptomycin, 2.5 µg/ml
amphotericin B, and 2 mM L-glutamine (Life
Technologies). Dulbecco’s PBS was obtained from Life Technologies and
contained 2x antibiotics. L-ascorbic acid,
NG-monomethyl-L-arginine
(L-NMMA), pronase E from Streptomyces griseus,
and hyaluronidase type V were obtained from Sigma (St. Louis, MO);
collagenase D from Clostridium histolyticum was purchased
from Boehringer Mannheim (Indianapolis, IN). [3H]arginine
was obtained from Amersham (Arlington Heights, IL). SeaPlaque agarose
was obtained from FMC Bioproducts (Rockland, ME). Other standard buffer
components and chemicals, unless specifically indicated otherwise, were
obtained from Sigma.
Cartilage and chondrocyte cultures
Carpal metacarpal joints of calves (0–3 mo old) were obtained from Covance (Denver, PA). Full thickness articular cartilage slices were aseptically collected and placed in Dulbecco’s PBS with 2x antibiotics for 30 min. Cartilage disks (5–7 mg) were dissected from the cartilage using a sterile leather punch (Libertyville Saddle Shop, Libertyville IL). Disks were transferred to 96-well flat-bottom plates (Nunc, Copenhagen, Denmark) containing DMEM supplemented with antibiotics and 10% FCS. This medium was changed 48 to 72 h later to DMEM with 0.5% FCS containing 25 µg/ml ascorbic acid, and samples to be tested were added 24 h later. NO levels in the cartilage explant supernatants were determined 72 h following the addition of the samples being evaluated.
Bovine chondrocytes were isolated from bovine cartilage as described previously for human chondrocytes (24, 25). Cartilage was cut into small pieces, and chondrocytes were liberated by sequential treatment with hyaluronidase (0.2% in DMEM without FCS) for 30 min, pronase E (0.25% in DMEM without FCS) for 30 min, and collagenase D (0.2% in DMEM with 10% FCS) for 20 h at 37°C. No effort was made to separate different layers of cartilage. Cells were washed twice in DMEM with 10% FCS. Viability was determined by trypan blue exclusion.
For NO production and isolation of RNA, monolayer cultures of chondrocytes were used. Chondrocytes were seeded into six-well tissue culture grade plates (Corning, Cambridge, MA) at a concentration of 2 x 106 cells/ml (4 ml/well) in Ham’s F-12 containing 10% FCS, 50 µg/ml ascorbic acid, and antibiotic/antimycotic. Cells were allowed to adhere and were cultured for 72 h at 37°C in 5% CO2. The medium was then replenished, and the chondrocytes were treated with log doses of SB 203580 for 30 min followed by stimulation with IL-1. The cells were incubated at 37°C in a 5% CO2 atmosphere for either 4 or 24 h. At the end of the incubation, the extracellular medium was removed and saved for evaluation of nitrite levels, and the cells were homogenized in Triazol reagent (Molecular Research Center, Cincinnati, OH; 0.5 ml/well) for the isolation of RNA.
For culture of chondrocytes in agarose, a previously described method was used (26). Briefly, a 2% (w/v) solution of SeaPlaque agarose in Ham’s F-12 medium containing antibiotics but without FCS was prepared by heating to 80°C. The agarose was cooled and mixed with an equal volume of Ham’s F-12 with 20% FCS. A chondrocyte suspension was added to the agarose to a final density of 8 x 105 cells/ml, and 0.5-ml aliquots were distributed into 24-well plates. Plates were incubated at 37°C for 30 min to allow the cells to settle and then at room temperature until the agar gelled. Liquid nutrient (1 ml of Ham’s F-12 containing 20% FCS and 25 µg/ml ascorbic acid) was added and was replaced every other day.
Immune complex kinase assays
Bovine chondrocytes were established in 10-cm tissue culture
petri dishes in DMEM with 10% FCS (10 ml, 1 x
106/ml) and stimulated with 20 ng/ml of IL-1 for
varying periods of time. The cells were washed twice in PBS,
solubilized on ice in lysis buffer (20 mM Tris-HCl (pH 7.4), 150 mM
NaCl, 1% Triton X-100, 10% glycerol, 2 mM EDTA, 25 mM
ß-glycerophosphate, 20 mM NaF, 1 mM sodium orthovanadate, 2 mM sodium
pyrophosphate, 1 mM PMSF, 10 µg/ml leupeptin, and 5 U/ml aprotinin),
and centrifuged at 15,000 x g for 20 min at 4°C.
Endogenous kinases were precipitated from cell lysates using
anti-p38 (17) or anti-MAPKAP kinase-2 Abs (supplied by Dr.
Jacques Landry, Quebec, Canada) bound to protein A-agarose for 4 h
at 4°C. The beads were washed twice with lysis buffer and twice with
kinase buffer (25 mM HEPES (pH 7.4), 25 mM MgCl2, 25 mM
ß-glycerophosphate, 100 mM sodium orthovanadate, and 2 mM DTT). The
immune complex kinase assays were initiated by the addition of 25 µl
of kinase buffer containing 2 µg of GST-ATF2 for p38 or 2 µg of the
small heat shock protein 27 (hsp27; StressGen Biotechnology, Ontario,
Canada) for MAPKAP kinase-2 as substrate and 50 µM
[
-32P]ATP (20 Ci/mmol; Amersham). After 30 min at
30°C, the reaction was stopped by the addition of SDS sample buffer,
and the phosphorylated products were resolved by SDS-PAGE and
visualized by PhosphorImager (Molecular Dynamics, Sunnyvale, CA). For
the in vitro inhibition experiment, SB 203580 was added directly to the
immune complexes before the kinase assay was performed. For inhibition
in intact cells, the cells were pretreated with SB 203580 before
stimulation with IL-1. Escherichia coli expressing GST-ATF2
was provided by Dr. Roger Davis, Worcester, MA). Recombinant GST-ATF2
was expressed in E. coli and purified over
glutathione Sepharose 4B (Pharmacia, Uppsala, Sweden) chromatography
according to the manufacturer’s instructions.
Nitrite determination
NO production was measured by estimating the stable NO metabolite, nitrite, in conditioned medium using a spectrophotometric method based on the Griess reaction (27). Following culture of the cartilage explants/chondrocytes for the times indicated, 100 µl of the culture supernatants or sodium nitrite standard dilutions were mixed with 50 µl of Griess reagent (1% sulfanilamide, 0.1% naphthyl ethylenediamine dihydrochloride, and 1.25% H3PO4) and incubated for 10 min at room temperature. Nitrite concentrations were determined by measuring absorbance at 550 nm in an ELISA reader (Molecular Devices, Sunnyvale, CA). The detection limit of the test was 2 µM NO2-. Values are expressed as micromolar concentrations of nitrite released per 106 chondrocytes or per milligram of cartilage.
Determination of iNOS activity
Inducible NOS activity was evaluated in monolayer cultures of bovine chondrocytes. The cells were plated (4 x 106 in 2 ml) in 12-well plastic dishes in Ham’s F-12 with 10% FCS containing 50 µg/ml ascorbic acid. The cells were incubated for 72 h, at which time the medium was replenished. IL-1 (20 ng/ml) and varying concentrations of SB 203580 were then added for an additional 24 h. Following the incubation period, the cells were washed twice in medium and then incubated in fresh medium for a further 20 min. During this period a second set of wells that had been treated with IL-1 alone was treated with similar doses of SB 203580 to determine the compound’s effect on the already induced enzyme. [3H]arginine (3 µCi/ml) was added to the cultures for 20 min, and incorporation of labeled arginine was terminated by rapid aspiration of extracellular medium and two washes (1 ml) using ice-cold PBS. Cells were lysed by addition of 1 ml 0.4 N HClO4, and the intracellular extract was neutralized with K2CO3 and 50 mM Tris-HCl buffer (pH 7.4). Aliquots (100 µl) of the neutralized intracellular extract were used to measure [3H]arginine uptake into the cells. To separate [3H]citrulline from [3H]arginine, 400-µl aliquots of the extracts were applied to 1-ml columns of Dowex AG50WX-8 (Na+ form) as described previously (28, 29). The columns were washed three times with 1 ml of water, and the radioactive material in the flow-through fractions (i.e., that containing almost exclusively [3H]citrulline) was quantified by scintillation spectroscopy. All measurements were made in duplicate, and the data are presented as the recovery of radiolabeled [3H]citrulline. The disintegrations per minute are corrected for the number of counts per 4 x 106 cells.
Isolation of chondrocyte RNA and Northern blot analysis
Total RNA was isolated from chondrocytes treated with IL-1 and SB 203580 by a modified guanidine isothiocyanate extraction using Tri-Reagent (MRC, Cincinnati, OH) according to the manufacturer’s instructions. RNA concentration and purity were determined spectrophotometrically. All RNA samples had an A260/A280 ratio >1.8. Approximately 10 µg of the RNA samples were electrophoresed in 1% agarose gels containing 2.5% formaldehyde, 20 mM 3-(N-morpholino)propanesulfonic acid, 5 mM sodium acetate, and 1 mM EDTA, pH 7.0. After electrophoresis, the RNA was transferred to positively charged nylon membranes (Bio-Rad, Hercules, CA). Membranes were prehybridized for 1 to 2 h at 68°C with ExpressHyb (Clontech, Palo Alto, CA). Hybridization was performed under identical conditions as prehybridization with addition of 32P-labeled (Amersham) specific probe for bovine iNOS. The 372-bp probe was provided by Dr. T. Jungi (35) (University of Berne, Berne, Switzerland) and corresponds to nucleotides 682 to 1053 of the human iNOS cDNA (30). Radiolabel on the blots was analyzed and integrated using phosphorimaging.
Statistical analysis
Comparisons between treated and control groups were determined by Student’s t test, with p < 0.05 considered significant. All experiments were performed at least three times, and the results shown are those from representative experiments.
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Results |
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TopAbstractIntroductionMethods and MaterialsResultsDiscussionReferences |
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IL-1 induced a rapid activation of p38 MAP kinase in the
chondrocyte cultures (Fig. 2A). A fourfold
activation of p38 was achieved by a 5-min treatment with IL-1. At 60
min postactivation, p38 activity was still increased threefold over the
basal level. We also analyzed the activation of MAPKAP kinase-2, a
physiologic substrate of p38, by IL-1 in the same cell lysate. MAPKAP
kinase-2 was activated fivefold within 5 min after treatment with IL-1,
but unlike P38, its activity level fell rapidly. At 60 min, MAPKAP
kinase-2 activity was only twofold over the basal level (Fig. 2A). Immunoblots verified that the same amount of
kinase was immunoprecipitated from different samples (data not
shown).
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B, SB 203580 inhibited the activity of p38 in a
dose-dependent manner, with an IC50 value of about 0.1
µM. Complete inhibition occurred at 1 and 10 µM. While SB 203580
inhibits the activity of p38 MAP kinase, it has no effect on the
activation of p38 by upstream kinase in response to IL-1 or any other
stimuli (31). It has been shown that MAPKAP kinase-2 is a physiologic
substrate of p38 in vivo (32). The activity of MAPKAP kinase-2 itself
is not inhibited by SB 203580, but activation of the enzyme by p38 MAP
kinase in cells is inhibited by SB 203580 (31). Therefore, to show that
SB 203580 inhibits p38 MAP kinase in bovine chondrocytes, we pretreated
the chondrocytes with SB 203580 at different concentrations before
activation with IL-1 and then assayed for MAPKAP kinase-2 activity
using hsp27 as substrate. As shown in Figure 2B, SB 203580
inhibited the activation of MAPKAP kinase-2 in a dose-dependent manner
with an IC50 value of about 0.4 µM, suggesting that SB
203580 treatment results in the inhibition of p38 MAP kinase activity
within the cell. Almost complete inhibition was achieved at 1 and 10
µM. Similar IC50 values have been reported in several
earlier reports for other cell types (31, 32, 33). NO production by bovine articular cartilage and chondrocytes
Bovine articular cartilage produces large amounts of NO following
stimulation with IL-1 (34). In these studies cartilage explants were
cultured for 48 h in DMEM with 10% FCS and then transferred to
medium containing 0.5% FCS. Twenty-four hours later the cultures were
stimulated with 20 ng/ml of IL-1, and SB 203580 was added at doses
ranging from 0.15 to 10 µM. The explants were incubated for an
additional 72 h, and secreted NO was determined in the
supernatants. In the presence of IL-1 there was a two- to threefold
increase in NO production compared with that in the untreated control
cultures. This increase was inhibited by SB 203580 with an
IC50 value that ranged from 0.6 to 1 µM, and maximal
inhibition was observed at 5 to 10 µM. The pooled results of two
experiments are shown in Figure 3. For
comparison, in this culture system, IL-1-induced NO production was
inhibited by L-NMMA at 500 µM (L-NMMA is a
specific competitive inhibitor of all isoforms of NOS) and by SB 203580
(5 µM), but not by the metalloproteinase inhibitor BB94 (Fig. 4). For SB 203580 to significantly
inhibit IL-1 induced NO production in cartilage explants, the compound
had to be present early during the incubation period. Maximum
inhibition was obtained when the compound was added during the first
4 h of culture with IL-1. The effect became weaker over the next
4 h and was lost by 24 h (Fig. 5).
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To show that the activity of SB 203580 was due to its ability to
inhibit the synthesis of iNOS and not to a direct effect on NOS
activity, we examined the ability of the compound to inhibit
IL-1-induced conversion of radiolabeled arginine to citrulline during
or after the iNOS synthetic phase. As shown in Figure 7, SB 203580 inhibited nitrite production
when the compound was added to the cell cultures in combination with
IL-1 (Fig. 7A), but not when it was added at the time
of iNOS assay (Fig. 7B). No significant effects of SB
203580 on arginine uptake into cells were observed (data not shown).
This would indicate that the compound has no direct effect on NOS
enzyme activity directly, but has a dose-related effect on its
induction.
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To determine the level at which SB 203580 regulated iNOS, the
effect on iNOS mRNA was examined using Northern analysis. Chondrocytes
were established in monolayers in six-well tissue culture plates (for
72 h), treated with SB 203580, and stimulated with IL-1 (100
ng/ml) for 4 or 24 h, and then RNA was isolated as described in
Materials and Methods. Following electrophoresis of the RNA
and blotting onto nylon membranes, the blots were hybridized with the
labeled iNOS probe and analyzed using the PhosphorImager. Figure 8A shows that there was a
large increase in expression of iNOS mRNA following treatment with IL-1
and that this was inhibited in a dose-related manner by SB 203580.
Similar results were obtained at both 4 and 24 h, with slightly
more inhibition being obtained with SB 203580 at the 4 h point as
measured by the PhosphorImager counts (Fig. 8B).
Normalization of the PhosphorImager counts based on the 28S ribosomal
RNA were within 10% of the data shown in Figure 8B. The
inhibition of induced message was in agreement with the NO levels in
the 24-h cell supernatants of the IL-1-stimulated bovine chondrocytes
used to obtain the RNA (Fig. 8C).
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Discussion |
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TopAbstractIntroductionMethods and MaterialsResultsDiscussionReferences |
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(IC50 = 0.1–1 µM) (17, 18). SB 203580 as well as
other members in this series of compounds have shown efficacy in
several animal models of inflammation where cytokines play a definitive
role. Examples are collagen-induced arthritis in the DBA/LacJ mouse
(20, 36); adjuvant-induced arthritis in the Lewis rat, in which
protective effects on both bone and cartilage have been observed (20);
and mouse models of endotoxin shock (37, 38). In addition, these
compounds are active in the fetal rat long bone resorption assay, thus
demonstrating a direct therapeutic effect on bone integrity (39). The
role of cytokines in these models is well documented, and inhibition of
TNF- and IL-1 by the CSAID compounds is well established. SB 203580
inhibits TNF- synthesis at the translational rather than the
transcriptional level (40, 41), and the target of this and other CSAID
compounds is known as CSBP/p38 kinase (17). SB 203580 is a highly
selective and potent inhibitor of p38 MAP kinase (32). One of the
physiologic substrates of CSBP/p38 is MAPKAP kinase-2, which, in turn,
phosphorylates hsp27. SB 203580 inhibits the activation of MAPKAP
kinase-2 with an IC50 of about 0.4 µM, and subsequently
the phosphorylation of hsp27 in stress-stimulated cells is inhibited by
this compound (32). In this study we report for the first time that
there is clearly an elevation in specific p38 kinase activity in bovine
chondrocytes treated with IL-1 and that the CSAID inhibitor SB 203580
inhibits this activity and blocks the production of the proinflammatory
enzyme iNOS.
One of the documented proinflammatory actions of IL-1 and TNF- is
the induction of NO from a variety of tissues and cell types, including
bone, cartilage, and cartilage-derived chondrocytes (12, 34, 42). In
fact, the induction of iNOS by IL-1 results in copious amounts of NO
that can contribute to tissue regulation and damage, and the
spontaneous production of NO has been observed in OA-affected
chondrocytes (43). NO has also been shown to mediate the IL-1-induced
inhibition of proteoglycan synthesis in rat articular cartilage in
vitro (44). In recent studies, the knockout of iNOS has been shown to
block both the IL-1-induced inhibition of proteoglycan biosynthesis and
the release of cartilage proteoglycan in the zymosan-induced arthritis
model (W. van den Berg, unpublished observations). The role of NO in
vivo in inflammatory disease has also been well documented, and
inhibitors of iNOS have been shown to protect against both inflammation
and cartilage matrix loss in a number of experimental models, including
inflammatory arthritides (45, 46, 47, 48). Together, these data suggest that
direct inhibition of iNOS or inhibition of iNOS production could be
therapeutically beneficial in diseases such as OA and RA, in which
there is both significant cartilage proteoglycan loss as well as
inflammatory components.
In addition to inducing iNOS and the production of NO in
cartilage/chondrocytes, IL-1 has been shown to induce the activation of
p38 kinase in both human (23) and rabbit (49) articular cartilage. In
the studies described here we have investigated the effect of IL-1 on
the p38 kinase in bovine articular chondrocytes and also its effects on
NO production and iNOS expression. IL-1 activated CSBP/p38 kinase
within 5 min of treatment, and the level of activation was maintained
over a 60-min period. In contrast, activation of MAPKAP kinase-2 peaked
5 min following the addition of IL-1 and then rapidly decreased. SB
203580 inhibited the activity of p38 MAP kinase in vitro when added
directly to the p38 kinase assay (Fig. 2B, upper
panel). It also inhibited the activity of p38 in cells;
this was shown by the inhibition of activation of MAPKAP kinase-2 by
p38 when SB 203580 was added to the cell cultures (Fig. 2B, lower panel). The IC50
value of inhibition of p38 MAP kinase when the compound was either
added directly to the kinase assay or to the cells was about 0.1 to 0.4
µM, similar to that described in previous reports (31, 32, 33).
To profile the effect of SB 203580 further in the bovine
cartilage/chondrocyte assays, we examined its effects on NO production.
The compound effectively inhibited IL-1-stimulated NO release into the
culture medium with an IC50 value of approximately 0.6 µM
(Fig. 3). SB 203580 appeared to inhibit only the inducible levels of NO
(
Figs. 3–5) (A. Badger, unpublished observations). To effectively
inhibit NO production, SB 203580 needed to be added to the cultures
within 4 h of IL-1. After this time the compound began to lose its
inhibitory activity, indicating that its site of action is probably
during the enzyme induction period (Fig. 5). The fact that IL-1 effects
are still inhibited 2 h following induction may well be due to
transport and availability in explant cultures. However, in separate
experiments, a significant amount of p38 remained active for up to 4 to
6 h after IL-1 treatment (data not shown). A similar profile of
drug action was obtained in the case of HIV-1 LTR transcriptional
activation, where the key role of p38 activity occurred some 2 to
4 h after cytokine and UV stimulation (33). Measurement of p38
kinase activity and its inhibition by SB 203580 could not be performed
in cartilage explants due to the technical difficulty in quickly
isolating the active p38 enzyme from this tissue. However, as
chondrocytes are the only cell type residing in cartilage, we used
cartilage-derived chondrocytes for these experiments. Modulation of p38
MAP kinase (Fig. 2) and the inhibitory effects of SB 203580 on
IL-1-induced NO production were clearly demonstrated in
cartilage-derived chondrocytes grown in agarose (Fig. 6) and monolayer
culture (Fig. 8C).
Experiments to determine whether SB 203580 affected iNOS activity in
chondrocytes, measured by the conversion of arginine to citrulline,
resulted in the finding that inhibition occurred with an
IC50 between 0.1 and 1 µM, and that the compound was
inactive when added to the assay mixture, indicating inhibition of
enzyme induction but not its activity (Fig. 7). We extended this
observation to show that IL-1-induced expression of iNOS mRNA was
inhibited in a dose-dependent manner by SB 203580 at both 4 and 24
h following activation with IL-1 (Fig. 8). Inhibition of iNOS mRNA at
the 24 h point paralleled the inhibition of NO production in the
supernatant of the cells used to prepare the RNA (Fig. 8). Since both
p38 activity and iNOS expression are inhibited with comparable
IC50 values (
0.5 µM), we hypothesize that p38 MAP
kinase resides in the signal transduction pathway of IL-1-mediated NO
induction. Activation of p38 MAP kinase appears to be an early event,
whereas NO induction is a downstream late event mediated by
IL-1.
Our results are consistent with those of a similar report in which the spontaneous activation of iNOS in chondrocytes was inhibited by SB 203580 (A. Amin, unpublished observations). In contrast to our findings described here, Guan et al. (50) found that an inhibitor of p38 MAP kinase, SC68376, enhanced NO biosynthesis in rat primary mesangial cells by increasing iNOS mRNA expression, protein expression, and nitrite production. A difference in the cell system (rat mesangial cells vs bovine articular chondrocytes) may explain the discrepancy between the two results. In addition, SC68376 is a relatively less well-characterized p38 inhibitor, with an IC50 value of 2 to 5 µM, compared with SB 203580, which has been extensively characterized and exhibits an IC50 value of at least 10-fold lower. It is possible that SC 68376 has other inhibitory activity and thus complicates the interpretation of the results.
Inhibition of the transcriptional regulation of iNOS and the subsequent production of NO by cartilage-derived chondrocytes by SB 203580 indicate that the CSAID compounds may be useful for therapy of disease states in which NO has been shown to play a proinflammatory role. In addition to the experimental models already discussed, additional evidence for the role of NO is provided by studies showing spontaneous production of this mediator by primary synovial cultures from both RA and OA patients, which may contribute to the pathology of these diseases (51). In addition, an increased expression of blood mononuclear cell iNOS has been described in RA patients (52). Thus, the CSAID compounds could be efficacious therapeutic agents by way of their ability to inhibit cytokines such as TNF and IL-1 and by inhibiting the downstream signal transduction pathways initiated by IL-1 and or TNF, including their ability to modulate iNOS expression. Given the central role that IL-1 plays in the pathology of RA and OA, an inhibitor of both IL-1 synthesis as well as IL-1 signal transduction and action would be of immense utility in the management of these debilitating diseases by controlling the inflammatory contribution as well as the effects on cartilage matrix biosynthesis and degradation.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: RA, rheumatoid arthritis; OA, osteoarthritis; NO, nitric oxide; iNOS, inducible nitric oxide synthase; SB 203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)imidazole; CSBP, CSAID binding protein; RK, reactivating kinase; GST, glutathione-S-transferase; ATF2, activating transcription factor 2; L-NMMA, NG-monomethyl-L-arginine; MAPK, mitogen-activated protein kinase; MAPKAPK, mitogen-activated protein kinase-activated protein kinase; hsp, heat shock protein.
Received for publication October 6, 1997. Accepted for publication March 6, 1998.
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References |
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TopAbstractIntroductionMethods and MaterialsResultsDiscussionReferences |
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on proteoglycan metabolism of human articular chondrocytes cultured in agarose. J. Rheumatol. 21:314.[Medline]
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G.-J. Feng, H. S. Goodridge, M. M. Harnett, X.-Q. Wei, A. V. Nikolaev, A. P. Higson, and F.-Y. Liew Extracellular Signal-Related Kinase (ERK) and p38 Mitogen-Activated Protein (MAP) Kinases Differentially Regulate the Lipopolysaccharide-Mediated Induction of Inducible Nitric Oxide Synthase and IL-12 in Macrophages: Leishmania Phosphoglycans Subvert Macrophage IL-12 Production by Targeting ERK MAP Kinase J. Immunol., December 15, 1999; 163(12): 6403 - 6412. [Abstract] [Full Text] [PDF]
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J. P. Lian, R. Huang, D. Robinson, and J. A. Badwey Activation of p90RSK and cAMP Response Element Binding Protein in Stimulated Neutrophils: Novel Effects of the Pyridinyl Imidazole SB 203580 on Activation of the Extracellular Signal-Regulated Kinase Cascade J. Immunol., October 15, 1999; 163(8): 4527 - 4536. [Abstract] [Full Text] [PDF]
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M. Delgado, E. J. Munoz-Elias, R. P. Gomariz, and D. Ganea Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase-Activating Polypeptide Prevent Inducible Nitric Oxide Synthase Transcription in Macrophages by Inhibiting NF-{kappa}B and IFN Regulatory Factor 1 Activation J. Immunol., April 15, 1999; 162(8): 4685 - 4696. [Abstract] [Full Text] [PDF]
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L. A. Marshall, M. J. Hansbury, B. J. Bolognese, R. J. Gum, P. R. Young, and R. J. Mayer Inhibitors of the p38 Mitogen-Activated Kinase Modulate IL-4 Induction of Low Affinity IgE Receptor (CD23) in Human Monocytes J. Immunol., December 1, 1998; 161(11): 6005 - 6013. [Abstract] [Full Text] [PDF]
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Y.-Z. Wang, P. Zhang, A. B. Rice, and J. C. Bonner Regulation of Interleukin-1beta -induced Platelet-derived Growth Factor Receptor-alpha Expression in Rat Pulmonary Myofibroblasts by p38 Mitogen-activated Protein Kinase J. Biol. Chem., July 14, 2000; 275(29): 22550 - 22557. [Abstract] [Full Text] [PDF]
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is the IL-1-treated control; 
indicates the
SB 203580- plus IL-1-treated groups. Data are the mean ± SD
of four replicates per group. * indicates p < 0.05; **
indicates p < 0.01; *** indicates p <
0.001.
