From the Department of Life Science, Kwangju
Institute of Science and Technology; Buk-Gu, Gwangju 500-712, Korea,
and § Department of Biology, Kyungpook National
University, Daegu 702-701, Korea
Received for publication, December 9, 2002, and in revised form, January 28, 2003
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ABSTRACT |
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Nitric oxide (NO) causes apoptosis and
dedifferentiation of articular chondrocytes by the modulation of
extracellular signal-regulated kinase (ERK), p38 kinase, and protein
kinase C (PKC) Chondrocytes are a unique cell type in which the differentiated
phenotype is reversible. The phenotype of chondrocytes is regulated by
the balance between anabolic and catabolic reactions of molecules,
which are involved in the maintenance of cartilage homeostasis (1).
Differentiated chondrocytes both in vivo and in
vitro dedifferentiate into fibroblastic cells upon exposure to
interleukin-1 NO is generally believed to be an important mediator of the
dedifferentiation and apoptosis of articular chondrocytes in arthritic cartilage (5, 8, 9). NO is produced in chondrocytes by the action of
proinflammatory cytokines, such as interleukin-1 NSAIDs such as aspirin and indomethacin have been used to relieve pain
and inflammation in arthritic cartilage. NSAIDs exert their effects
primarily by the inhibition of cyclooxygenase (COX), a key enzyme that
converts arachidonic acid to prostaglandin (PG) (19). In addition
to the alleviation of inflammation, some NSAIDs also modulate
homeostasis of chondrocyte and cartilage such as matrix molecule
synthesis. For example, several NSAIDs, such as sodium salicylate,
inhibit proteoglycan synthesis but others, such as nimesulide, induce
cartilage matrix synthesis, whereas others including piroxicam have no
effect on matrix synthesis (20, 21). In addition, some NSAIDs, such as
nimesulide and ibuprofen, have a protective effect in
staurosporine-induced apoptosis of chondrocytes (22). Several lines of
evidence suggest that some of the effects of NSAIDs are independent of
the inhibition of COX (23). Indeed, it has been shown that NSAIDs
modulate COX-independent signaling pathways, such as Ras (24), NF Isolation of Rabbit Articular Chondrocytes and Culture
Conditions--
Articular chondrocytes were isolated from knee joint
cartilage slices of 2-week-old New Zealand White rabbits by enzymatic digestion as described previously (28). Briefly, cartilage slices were
dissociated enzymatically for 6 h in 0.2% collagenase type II
(381 units/mg solid; Sigma) in Dulbecco's modified Eagle's medium.
After collecting individual cells by brief centrifugation, the cells
were resuspended in culture medium supplemented with 10% (v/v) fetal
bovine calf serum, 50 µg/ml streptomycin, and 50 units/ml penicillin,
and then plated on culture dishes at a density of 5 × 104 cells/cm2. The medium was replaced every 2 days after plating, and cells reached confluence at ~5 days of
culture. Cells from day 4 cultures were treated with various
concentrations of the indicated pharmacological reagents for 1 h
before SNP treatment. These reagents included various NSAIDs, PD98059
(Calbiochem) to inhibit MAP kinase kinase-1/-2 (29), SB203580
(Calbiochem) to inhibit p38 kinase (30),
N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone
(Bachem, Heidelberg, Germany) to inhibit caspase-3 (31), or SN50
(Biomol, Plymouth Meeting, PA) to inhibit nuclear translocation of
NF Cartilage Explants Culture--
Rabbit joint cartilage explants
(~125 mm3) were cultured in Dulbecco's modified Eagle's
medium in the absence or presence of various pharmacological reagents
as indicated in each experiment. The cartilage explants were fixed in
4% paraformaldehyde for 24 h at 4 °C, dehydrated with graded
ethanol, embedded in paraffin, and sectioned into 4-µm slices as
described previously (33). The sections were stained by standard
procedures using Alcian blue to determine differentiation status of
chondrocytes. Apoptotic cells were determined by the procedure
described below.
Determination of Caspase-3 Activity--
Activation of caspase-3
was determined by measuring the absorbance of the cleaved synthetic
substrate of caspase-3, Ac-Asp-Glu-Val-Asp-chromophore p-nitroaniline, as described previously (16). Briefly,
chondrocytes were lysed on ice for 10 min in the cell lysis buffer
provided in the Clontech A ApoAlert CPP32
colorimetric assay kit. The lysates were reacted with 50 µM Ac-Asp-Glu-Val-Asp-chromophore
p-nitroaniline in a reaction buffer (0.1 M
HEPES, 20% glycerol, 10 mM DTT, and protease inhibitors,
pH 7.4). The mixtures were maintained at 37 °C for 1 h in a
water bath and subsequently analyzed in an enzyme-linked immunosorbent
assay reader. The enzyme activity was calculated from a standard curve
prepared using p-nitroanaline. The relative levels of pNA
were normalized against the protein concentration of each extract.
Determination of Apoptosis--
We have shown previously that
NO-induced death of primary culture of articular chondrocytes is caused
by apoptosis, as demonstrated by DNA fragmentation and terminal
deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) (16). In
this study, apoptotic cells were quantified by counting surviving cells
using an MTT assay kit (Roche Molecular Biochemicals) according to the
manufacturer's protocol. Apoptosis of articular chondrocytes in
cartilage explant culture was determined by TUNEL assay according to
the manufacturer's protocol (Roche Molecular Biochemicals).
Immunoprecipitation and Kinase Assays--
The activity of
PKC NF PGE2 Assay--
PGE2
production in articular chondrocytes was determined by measuring the
levels of cellular and secreted PGE2 by using a PGE2 assay kit (Amersham Biosciences). Briefly,
chondrocytes were plated in standard 96-well microtiter plates at a
density of 2 × 104 cells/well. After treatment with
various pharmacological reagents, as indicated in each experiment, the
total cell lysate was used to quantify the amount of PGE2
according to the manufacturer's protocol. PGE2 levels were
calculated against a standard curve of PGE2.
Western Blot Analysis--
Whole-cell lysates were prepared by
extracting proteins using a buffer containing 50 mM
Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, and 0.1%
SDS supplemented with protease inhibitors and phosphatase inhibitors as
described above. The proteins were size-fractionated by
SDS-polyacrylamide gel electrophoresis and transferred to a
nitrocellulose membrane. Proteins were detected using the following
antibodies: type II collagen from Chemicon (Temecula, CA); rabbit
anti-p53 polyclonal antibody and phosphorylation-specific antibody for
ERK from New England Biolabs (Beverly, MA); PKC NSAIDs Block NO-induced Apoptosis of Primary Culture Articular
Chondrocytes--
Direct production of NO by SNP treatment causes
dedifferentiation and apoptosis of articular chondrocytes (16-18). To
examine the effects of NSAIDs on articular chondrocyte function, we
first examined the effects of NSAIDs on NO-induced apoptotic cell
death. Consistent with our previous reports, SNP treatment in rabbit articular chondrocytes caused apoptosis in a dose-dependent
manner, which was demonstrated by the reduction of cell viability
determined by MTT assay (Fig.
1A). The reduction of cell
viability was detectable as early as 3 h after SNP treatment (data
not shown). The effects of NSAIDs on the survival of articular
chondrocytes were examined by pretreating chondrocytes with various
concentrations of NSAIDs for 1 h before exposure to 1.5 mM SNP for 18 h. As shown in Fig. 1B, all
of the examined NSAIDs, including indomethacin, ketoprofen, ibuprofen,
sulindac sulfide, and flurbiprofen, blocked chondrocyte apoptosis in a
dose-dependent manner. These results indicate that NSAIDs
block NO-induced apoptosis of articular chondrocytes.
NSAIDs Block NO-induced Dedifferentiation of Primary Culture
Articular Chondrocytes--
We also examined the effects of NSAIDs on
NO-induced dedifferentiation of articular chondrocytes. NO production
caused loss of the differentiated phenotype of articular chondrocytes,
as demonstrated by the reduction of sulfated proteoglycan accumulation and type II collagen expression (16). Pretreatment of chondrocytes with
indomethacin blocked the NO-induced decrease in sulfated proteoglycan
accumulation as determined by Alcian blue staining (Fig.
2A, top) and type
II collagen expression as determined by Western blot analysis (Fig.
2A, bottom). Treatment of chondrocytes with
indomethacin alone did not affect the accumulation of sulfated proteoglycan or type II collagen expression (Fig. 2A). Other
NSAIDs examined in this study (i.e. ketoprofen, ibuprofen,
sulindac sulfide, and flurbiprofen), with effects similar to those of
indomethacin, also blocked the NO-induced decrease in proteoglycan
accumulation and type II collagen expression in a
dose-dependent manner (Fig. 2B). These results
indicate that NSAIDs inhibit not only NO-induced apoptosis but also
dedifferentiation of primary culture articular chondrocytes.
NSAIDs Block NO-induced Apoptosis and Dedifferentiation of
Articular Chondrocytes during Cartilage Explants Culture--
Because
it is possible that the responses of chondrocytes in monolayer culture
may differ from their responses in a 3-dimensional natural matrix, we
examined whether NO production causes apoptosis and dedifferentiation
of chondrocytes during cartilage explants culture and whether NSAIDs
also block NO-induced apoptosis and dedifferentiation. Similar to the
effects on chondrocytes cultured on plastic, NO production caused
apoptotic cell death and inhibition of sulfated proteoglycan synthesis
in chondrocyte in cartilage explants as demonstrated by TUNEL assay and
Alcian blue staining, respectively (Fig.
3). In addition, pretreatment of
chondrocytes with indomethacin blocked the NO-induced apoptosis and
decrease in sulfated proteoglycan accumulation (Fig. 3), indicating
that indomethacin blocks NO-induced apoptosis and dedifferentiation of
chondrocytes cultured either on plastic or in a three-dimensional natural matrix.
Modulation of NO-induced Activation of ERK and p38 Kinase by
Indomethacin--
We next investigated the molecular mechanism of
NSAID modulation of apoptosis and dedifferentiation. We used
indomethacin as an NSAID and first examined the possible modulation of
NO-induced activation of the MAP kinase subtypes ERK-1/2 and p38
kinase, because these MAP kinase subtypes have opposite effects
on NO-induced apoptosis and dedifferentiation (16). SNP treatment
caused transient activation of ERK-1/2 and p38 kinase as determined by
Western blot analysis and in vitro kinase assay,
respectively (Fig. 4A, top). Treatment of chondrocytes with indomethacin before SNP
treatment slightly but consistently enhanced NO-induced ERK-1/2
activation (Fig. 4A, middle). In contrast,
NO-induced activation of p38 kinase was blocked by indomethacin
treatment in a dose-dependent manner (Fig. 4A,
bottom). Inhibition of NO-induced ERK-1/2 activation with 20 µM PD98059 resulted in the potentiation of apoptosis and a blockade of dedifferentiation, whereas inhibition of p38 kinase with
20 µM SB203580 blocked apoptosis with the potentiation of dedifferentiation (Fig. 4, B and C). The present
results are consistent with our previous observation (16) and indicate
that NO-induced ERK activation causes dedifferentiation and inhibits
apoptosis, whereas activation of p38 kinase induces apoptosis and
inhibits dedifferentiation. Therefore, the inhibitory effects of
indomethacin on NO-induced apoptosis and dedifferentiation with the
potentiation and inhibition of ERK and p38 kinase, respectively,
suggest that inhibition of apoptosis by indomethacin is caused by
blockade of p38 kinase and potentiation of ERK. In contrast, the above results also suggest that modulation of MAP kinase subtypes by indomethacin is not responsible for the inhibition of NO-induced dedifferentiation, because the potentiation of ERK and inhibition of
p38 kinase is a condition that enhances dedifferentiation.
Indomethacin Blocks NO-induced Inhibition of PKC NSAIDs Inhibit Apoptosis and Dedifferentiation Independent of the
Expression of COX-2 and Production of PGE2--
Because
NSAIDs block COX-2 activity and PGE2 production and because
PGE2 is known to regulate differentiation of chondrocytes (34, 35), we examined whether the inhibition of apoptosis and
dedifferentiation by NSAIDs is caused by their ability to block COX-2
activity and PGE2 production. NO-induced COX-2 expression (Fig. 7A, upper)
and PGE2 production (Fig. 7B) were blocked by all of the examined NSAIDs, and the effects of indomethacin on COX-2
expression were dose-dependent (Fig. 7A,
bottom). Although a low concentration of indomethacin
(i.e. 70 µM) did not significantly block COX-2
expression (Fig. 7A), it completely blocked PGE2
production (Fig. 7C), indicating that COX-2 was blocked at
this concentration. Interestingly, although PGE2 production
was completely blocked at 70 µM indomethacin, the effects
on apoptosis and proteoglycan synthesis were observed at higher
concentrations of indomethacin (Fig. 7C), suggesting that
the ability of indomethacin to modulate apoptosis and dedifferentiation
is independent of the inhibition of COX-2 and PGE2
production.
This possibility was further examined by the specific inhibition of
COX-2 with NS398. As shown in Fig.
8A, NS398 (5 µM)
completely blocked NO-induced PGE2 production but did not
affect apoptosis and accumulation of sulfated proteoglycans. In
addition, treatment of chondrocytes with various concentrations of
PGE2 in the absence or presence of SNP did not affect cell
viability, accumulation of sulfated proteoglycan (Fig. 8B),
or type II collagen expression (Fig. 8D, top).
The COX-2- and PGE2-independent regulation of apoptosis and
dedifferentiation were further demonstrated by inhibiting ERK-1/2 and
p38 kinase, which block NO-induced COX-2 expression (Fig.
8D, bottom) and PGE2 production (Fig.
8C). Although inhibition of both MAP kinase subtypes blocked
PGE2 production, NO-induced apoptosis and dedifferentiation
were conversely regulated by ERK-1/2 and p38 kinase (Fig.
8C). Taken together, these results indicate that the ability
of indomethacin to block NO-induced apoptosis and dedifferentiation is
independent of the inhibition of COX-2 activity and PGE2
production.
NO production in articular chondrocytes plays a central role in
the pathophysiology of arthritis (5, 8, 9, 36). High levels of
nitrite/nitrate are found in the synovial fluid and serum of arthritis
patients (37), and it has been shown that NO causes loss of a
differentiated phenotype and apoptosis of articular chondrocytes (6, 7,
12-14). We have previously shown that direct production of NO causes
apoptosis and dedifferentiation of articular chondrocytes by the
activation of ERK-1/2 and p38 kinase and inhibition of PKC and -
. In this study, we investigated the effects
and mechanisms of non-steroidal anti-inflammatory drugs (NSAIDs), such
as indomethacin, ketoprofen, ibuprofen, sulindac sulfide, and
flurbiprofen, in NO-induced apoptosis and dedifferentiation of
articular chondrocytes. We found that all of the examined NSAIDs
inhibited apoptosis and dedifferentiation. NO production in
chondrocytes caused activation of ERK-1/2 and p38 kinase, which
oppositely regulate apoptosis and dedifferentiation. NO
production also caused inhibition of PKC
and -
independent of and
dependent on, respectively, p38 kinase, which is required for apoptosis
and dedifferentiation. Among the signaling molecules modulated by NO,
NSAIDs blocked NO-induced activation of p38 kinase, potentiated ERK
activation, and blocked inhibition of PKC
and -
. NSAIDs also
inhibited some of the apoptotic signaling that is downstream of p38
kinase and PKC, such as NF
B activation, p53 accumulation, and
caspase-3 activation. The inhibitory effects of NSAIDs on apoptosis and
dedifferentiation were independent of the inhibition of cyclooxygenase
(COX)-2 and prostaglandin E2 (PGE2)
production, as evidenced by the observation that specific inhibition of
COX-2 activity and PGE2 production or exogenous PGE2 did not affect NO-induced apoptosis and
dedifferentiation. Taken together, our results indicate that NSAIDs
block NO-induced apoptosis and dedifferentiation of articular
chondrocytes by the modulation of ERK, p38 kinase, and PKC
and -
in a manner independent of their ability to inhibit COX-2 and
PGE2 production.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(2, 3), retinoic acid (4), or nitric oxide
(NO)1 (5). Although the
molecular mechanism is not yet clear, dedifferentiation of articular
chondrocytes is believed to play a role in the pathophysiology of
arthritis. In addition to dedifferentiation, increased apoptotic death
of chondrocytes was observed in arthritic cartilage, and apoptosis is
closely related to cartilage destruction (6, 7), indicating that
chondrocyte apoptosis plays an important role in the pathogenesis
of arthritis.
. NO production in
chondrocytes causes activation of matrix metalloproteinases (10),
decreased production of interleukin-1 receptor antagonists (11),
inhibition of proteoglycan synthesis and type II collagen expression
(12, 13), and apoptosis of chondrocytes (6, 7, 14). Indeed, inhibition
of NO production protects against damage of cartilage and chondrocytes
in a number of experimental models. For instance, in experimentally
induced osteoarthritis in a range of animal species, a significant
correlation was observed between the level of NO and the prevalence of
apoptotic cells in cartilage tissue (7). Moreover, inhibition of NO
resulted in reduced articular cartilage damage and apoptotic cell
death (11, 15). In our previous studies, we have shown that direct production of NO by treating chondrocytes with an NO donor, sodium nitroprusside (SNP), induces apoptosis and dedifferentiation of primary culture articular chondrocytes (16-18). NO-induced apoptosis and dedifferentiation of articular chondrocytes were regulated by
opposite functions of mitogen-activated protein (MAP) kinase subtypes,
extracellular signal-regulated protein kinase (ERK), and p38 kinase
(16). NO-induced activation of ERK-1/2 induces dedifferentiation with
the inhibitory effects on apoptosis, whereas activation of p38 kinase
induces apoptosis and is responsible for the maintenance of
differentiated phenotype. In addition to MAP kinase signaling, NO
production caused the inhibition of protein kinase C (PKC)
and -
activities (17). The inhibition of PKC
activity is caused by
inhibition of its expression, which is independent of MAP kinase
signaling. In contrast, PKC
activity is blocked as a result of p38
kinase activation, and inhibition of PKC
activity is followed by
proteolytic cleavage by caspase-3. We also found that p38 kinase
induces NO-induced apoptosis by accumulating p53 via
NF
B-dependent transcription and stabilization by serine
15 phosphorylation (18).
B
(25), activator protein-1 (26), ERK (26), p38 kinase (27), and others.
Because the role of NSAIDs in the maintenance of homeostasis and
apoptosis of articular chondrocytes is not clearly understood, although
the action of NSAIDs in inflammation is clear, we investigated the
function of various NSAIDs in NO-induced dedifferentiation and
apoptosis and characterized the molecular mechanism of NSAIDs action in
articular chondrocytes.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B (32). In some experiments, where indicated, chondrocytes from
day 3 cultures were infected with either control adenovirus or
adenovirus containing wild-type rabbit PKC
or mouse PKC
that was
inserted into a cosmid cassette, pAxCAwt. Infected cells were cultured
in complete medium for 24 h and were then treated with 1.5 mM SNP for an additional 24 h.
, PKC
, and p38 kinase was determined by an immune complex
kinase assay. Briefly, cell lysates were prepared in lysis buffer
containing 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM
-glycerol
phosphate, protease inhibitors [10 µg/ml leupeptin, 10 µg/ml
pepstatin A, 10 µg/ml aprotinin, and 1 mM
4-(2-aminoethyl) benzenesulfonyl fluoride], and phosphatase inhibitors
(1 mM NaF and 1 mM
Na3VO4). The cell lysates were precipitated
with polyclonal antibodies against p38 kinase, PKC
(Santa Cruz
Biotechnology), or PKC
(BD Transduction Laboratories, Lexington,
KY). Immune complexes were collected by protein A Sepharose beads and
resuspended in 20 µl of kinase reaction buffer containing 25 mM Tris-HCl, pH 7.5, 5 mM
-glycerol
phosphate, 2 mM dithiothreitol, 0.1 mM sodium
orthovanadate, 10 mM MgCl2,
[
-32P]ATP, and 1 µg of substrate (activating
transcription factor-2) for p38 kinase or myelin basic protein for
PKC
and -
. The phosphorylation of substrates was detected by autoradiography.
B Luciferase Assay--
NF
B activity was determined by a
reporter gene assay. Briefly, chondrocytes were transfected with
plasmid containing luciferase and three tandem repeats of serum
response element or a control vector. Transfection of the expression
vector was performed by using LipofectAMINE Plus as described
previously (18). The transfected cells were cultured in complete medium
for 24 h and used to determine luciferase activity using an assay
kit from Promega. Luciferase activity was normalized against
-galactosidase activity.
, PKC
, and ERK-1/2
from BD Transduction Laboratories; and p38 kinase from Santa Cruz
Biotechnology Inc. Blots were developed using a peroxidase-conjugated
secondary antibody and an enhanced chemiluminescence system.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
NSAIDs inhibit NO-induced apoptosis of
primary culture articular chondrocytes. Articular chondrocytes
were treated with 1.5 mM SNP for the indicated time periods
(A) or were treated with the indicated concentrations
(micromolar) of various NSAIDs, including indomethacin
(Indo), ketoprofen (Keto), ibuprofen
(Ibup), sulindac sulfide (Suli), and flurbiprofen
(Flur), for 1 h and then exposed to 1.5 mM
SNP for 18 h (B). Cell viability was determined by an
MTT assay. Data are represented as mean values with S.D.
(n = 4).
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Fig. 2.
NSAIDs inhibit NO-induced dedifferentiation
of primary culture articular chondrocytes. Articular chondrocytes
were treated with the indicated concentrations (micromolar) of
indomethacin (Indo) (A) or ketoprofen
(Keto), ibuprofen (Ibup), sulindac sulfide
(Suli), and flurbiprofen (Flur) (B)
for 1 h and then exposed to 1.5 mM SNP for 18 h.
Accumulation of sulfated glycosaminoglycans was determined by Alcian
blue staining (top) and expression of type II collagen was
determined by Western blot analysis (bottom). The data
represent mean values with S.D. and results of a typical experiment
selected from at least four independent experiments.
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Fig. 3.
NSAIDs block NO-induced apoptosis and
dedifferentiation of articular chondrocytes during cartilage
explants culture. Cartilage explants were untreated or treated
with 1.5 mM SNP for 72 h in the absence or presence of
280 µM indomethacin. Apoptotic cells were determined by
TUNEL assay, and synthesis of sulfated proteoglycan was determined by
Alcian blue staining. The data represent results of a typical
experiment selected from at least four independent experiments.
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Fig. 4.
Indomethacin modulates NO-induced activation
of p38 kinase and ERK-1/2. A, chondrocytes were treated
with 1.5 mM SNP for the indicated time periods
(top) or cells were treated with 1.5 mM SNP for
12 h (middle and bottom) in the presence of
indicated concentrations (micromolar) of indomethacin
(Indo). NO-induced activation of ERK-1/2 and p38 kinase in
SNP-treated chondrocytes was determined by Western blot analysis or a
kinase assay, respectively. pATF-2, activating
transcription factor 2. B-C, chondrocytes were
treated with 1.5 mM SNP for 18 h in the absence or
presence of 20 µM PD98059 or SB202190; then, cell
viability, accumulation of sulfated glycosaminoglycan, and expression
of type II collagen were determined by MTT assay, Alcian blue staining,
and Western blot analysis, respectively. The data represent mean values
with S.D. (B and C) (n = 4) or a
typical result (A).
and -
Activity--
A previous study from our lab (17) indicated that
inhibition of PKC
and -
activities is required for NO-induced
apoptosis and dedifferentiation of articular chondrocytes. We also
demonstrated that inhibition of PKC
is caused by activation of p38
kinase, whereas inhibition of PKC
is independent of MAP kinase
signaling (17). We therefore examined whether indomethacin blocks
NO-induced dedifferentiation and apoptosis by modulating PKC
and
-
signaling. Consistent with our previous results (17), expression
and activity of PKC
and -
decreased over time in SNP-treated
chondrocytes (Fig. 5A). The
NO-induced decrease in expression and activity of PKC
and -
was
completely blocked by the pretreatment of indomethacin in a
dose-dependent manner (Fig. 5B). Moreover,
ectopic expression of PKC
or -
by adenovirus infection blocked
NO-induced dedifferentiation (Fig. 5, C and D)
and apoptosis (Fig. 5E). Therefore, the above results
suggest that the inhibitory effects of indomethacin on NO-induced
dedifferentiation are caused by the blockade of NO-induced inhibition
of PKC
and -
activity, whereas the inhibition of apoptosis by
indomethacin is caused by its ability to modulate both MAP kinase
subtypes and PKC
and -
. Consistent with the inhibition of
apoptosis, indomethacin also blocked signaling molecules downstream of
p38 kinase during NO-induced apoptosis, such as activation of NF
B,
which was demonstrated by the inhibition of I
B degradation
(Fig. 6A) and NF
B reporter
gene assay (Fig. 6B), the accumulation of p53 (Fig.
6A), and the activation of caspases-3 (Fig.
6C).
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Fig. 5.
Indomethacin blocks the NO-induced inhibition
of PKC and -
activities. A and B, indomethacin blocks
the NO-induced inhibition of PKC
and -
activity. Articular
chondrocytes were treated with 1.5 mM SNP for the indicated
time periods (A) or were treated with 1.5 mM SNP
for 18 h in the presence of the indicated concentration
(micromolar) of indomethacin (Indo) (B). Levels
of PKC
and -
proteins were determined by Western blot
(WB) analysis and the activities of PKC
and -
proteins
were determined by a kinase assay. C-E,
chondrocytes were infected with control adenovirus (control
and SNP) or adenovirus containing PKC
or -
cDNA.
Infected cells were cultured in complete medium for 24 h and
treated with 1.5 mM SNP for an additional 24 h.
Expression of type II collagen (Coll-II), and PKC
was
determined by Western blot analysis (C). Accumulation of
sulfated glycosaminoglycan was determined by Alcian blue staining
(D). Cell viability was determined by an MTT assay
(E). The data shown represent a typical result
(A-C) or the mean value with S.D. (D
and E) from at least four independent experiments.
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Fig. 6.
Inhibition of NO-induced
NF B activation, p53 accumulation, and
caspase-3 activation by indomethacin. Articular chondrocytes were
treated with 1.5 mM SNP for 18 h in the presence of
the indicated concentration (micromolar) of indomethacin
(Indo) (A), 1.5 mM SNP for 18 h
in the absence or presence of 50 µg/ml SN50 peptide (B),
or 20 µM
N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone
(DEVD) (C). Degradation of I
B and accumulation
of p53 protein were determined by Western blot analysis (A).
NF
B activity (B) and caspase-3 activity (C)
were determined as described under "Experimental Procedures." Data
represent results of a typical experiment (A) or mean values
with standard deviation (B and C)
(n = 4).
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Fig. 7.
Inhibition of NO-induced COX-2 expression and
PGE2 production by NSAIDs. Chondrocytes
were untreated (control) or were treated with 1.5 mM SNP for 18 h in the absence or presence of 1 mM ketoprofen (Keto), 1 mM ibuprofen
(Ibup), 0.1 mM sulindac sulfide
(Suli), 1 mM flurbiprofen (Flur), or
of the indicated concentration (micromolar) of indomethacin
(Indo). Expression of COX-2 protein was determined by
Western blot analysis (A) and production of PGE2
was determined by using a PGE2 assay kit (B).
Cell viability and accumulation of sulfated glycosaminoglycan were
determined by an MTT assay or Alcian blue staining, respectively
(C). The data in A represent results of a typical
experiment conducted five times, and the data in B and
C represent mean values with S.D. from at least four
independent experiments.
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Fig. 8.
Inhibition of NO-induced apoptosis and
dedifferentiation by NSAIDs is independent of the blockade of COX-2
expression and PGE2 production. Articular
chondrocytes were treated with 1.5 mM SNP for 18 h in
the absence or presence of 5 µM NS398 (A), the
indicated concentration (in nanograms per milliliter) of
PGE2 (B), 20 µM PD98059, 20 µM SB202190, or 200 ng/ml PGE2 (C
and D). The production of PGE2, cell viability,
and the accumulation of sulfated glycosaminoglycan were determined as
described under "Experimental Procedures" (A-C).
Expression of type II collagen and COX-2 was determined by Western blot
analysis (D). The data in A-C represent mean
values with S.D.; data in D represent results of a typical
experiment (n = 4).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and -
(16-18). In this study, we investigated the effects of various NSAIDs
on NO-induced apoptosis and dedifferentiation of articular
chondrocytes cultured on plastic or in a 3-dimensional natural matrix
(i.e. explants culture) and found that all of the examined
NSAIDs inhibit apoptosis and dedifferentiation in both conditions
independent of the inhibition of COX-2 expression and PGE2
production. As summarized in Fig. 9, we
also demonstrated that the inhibitory effects of NSAIDs on apoptosis
are caused by their ability to potentiate NO-induced ERK activation, to
inhibit activation of p38 kinase, and to block inhibition of PKC
and -
. In contrast, the inhibition of NO-induced dedifferentiation by
NSAIDs is caused by the blockade of PKC
and -
signaling but not
the modulation of ERK-1/2 and p38 kinase signaling.
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Fig. 9.
Schematic summary of NSAIDs modulation of
signaling pathway during NO-induced dedifferentiation and apoptosis of
articular chondrocytes. NSAIDs inhibit NO-induced apoptosis by
blocking NO-induced activation of p38 kinase, potentiating ERK
activation, and blocking inhibition of PKC and -
, whereas the
inhibition of NO-induced dedifferentiation by NSAIDs is caused by the
blockade of PKC
and -
signaling but not the modulation of ERK-1/2
and p38 kinase. The inhibitory effects of NSAIDs on apoptosis and
dedifferentiation are independent of their ability to block COX-2
activity and PGE2 production.
The cellular effects of NSAIDs are exerted primarily by the inhibition of COX and PGE2 production. PGE2 is known to regulate differentiation (34, 35) and apoptosis (14, 38) of chondrocytes depending on the experimental system. Because NO production in chondrocytes causes COX-2 expression and PGE2 production, it is possible that NSAIDs modulate NO-induced apoptosis and dedifferentiation by inhibiting COX-2. However, our current results clearly indicate that the inhibitory effects of NSAIDs on NO-induced apoptosis and dedifferentiation are independent of the inhibition of COX-2 and PGE2 production. This conclusion is clearly demonstrated by the specific inhibition of COX-2 activity and by the observation that PGE2 production or exogenous PGE2 did not affect NO-induced apoptosis and dedifferentiation. In addition, the concentration of indomethacin that is required for the inhibition of NO-induced apoptosis and dedifferentiation is higher than that needed for the inhibition of PGE2 synthesis. This is consistent with observations that the COX-independent actions of NSAIDs such as inhibition of cell cycle progression (39, 40), induction of apoptosis (41-43), and inhibition of angiogenesis (44, 45), require high concentrations of NSAIDs that are 100- to 1000-fold higher than those needed to inhibit prostaglandin synthesis.
Our results indicated that the inhibition of NO-induced apoptosis
of chondrocytes by NSAIDs is related to the potentiation of ERK
activation, blockade of p38 kinase activation, blockade of PKC and
-
inhibition, and inhibition of downstream apoptotic signaling,
including NF
B, p563, and caspase-3, as summarized in Fig. 9. The
modulation of MAP kinase subtypes by indomethacin (i.e.
inhibition of p38 kinase and potentiation of ERK) seems to be involved
in the inhibition of NO-induced apoptosis based on the observation that
NO-induced activation of p38 kinase induces apoptosis, whereas ERK
activation inhibits apoptosis. The inhibition of p38 kinase and
apoptosis by indomethacin is consistent with the inhibition of
apoptotic signaling molecules located downstream of p38 kinase, such as
PKC
, NF
B, p53, and caspase-3 (Fig. 9). In contrast to the
inhibition of apoptosis, the inhibitory effects of indomethacin on
NO-induced dedifferentiation are attributable to its ability to block
NO-induced inhibition of PKC
and -
, but not its ability to
modulate MAP kinase subtypes. This conclusion is based on the
observations that NO-induced inhibition of PKC
and -
is required
for the induction of dedifferentiation as well as apoptosis and that
the potentiation and inhibition of ERK and p38 kinase, respectively,
are the signaling events leading to the potentiation of
dedifferentiation (Fig. 9). Modulation of ERK and p38 kinase by
indomethacin is independent of its ability to block COX activity,
consistent with previously reported results. Indeed, many studies have
indicated that COX-independent effects of NSAIDs are exerted by the
modulation of ERK and p38 kinase. For example, NSAIDs such as aspirin
and sodium salicylate exert their effects by the inhibition ERK-1/2
(26, 45-47), whereas p38 kinase is activated by sodium salicylate in
human fibroblasts and is associated with induction of apoptosis
(27).
Our current results clearly indicate that indomethacin blocked the
NO-induced inhibition of PKC and -
activity that is required for
the induction of apoptosis and dedifferentiation of SNP-treated chondrocytes (17). Because the activity of PKC
is blocked as a
result of p38 kinase activation (17), it is likely that the effects of
indomethacin on PKC
are caused by the inhibition of p38 kinase
signaling. However, the possibility that indomethacin directly
regulates PKC
activity cannot be ruled out, although no evidence
supports the direct action of NSAIDs in the regulation of PKC isoforms.
Nevertheless, it is apparent that the blockade of the NO-induced
inhibition of PKC
by indomethacin inhibits both apoptosis and
dedifferentiation of SNP-treated chondrocytes. In contrast to PKC
,
the inhibition of PKC
expression and activity is caused by the
inhibition of its expression independent of ERK and p38 kinase
signaling (17). Based on the observation that inhibition of PKC
activity is a prerequisite for the induction of apoptosis and
dedifferentiation, the MAP kinase-independent inhibition of PKC
activity by indomethacin is also essential for the inhibitory effects
of indomethacin on apoptosis and dedifferentiation. The mechanisms of
indomethacin regulation of PKC
expression and activity remain to be
determined, although it is possible that indomethacin regulates PKC
either directly or indirectly by modulating upstream signaling events.
Nevertheless, because our present results indicated that
indomethacin-induced inhibition of p38 kinase and potentiation of ERK
is not directly involved in the inhibition of dedifferentiation as
discussed above, we conclude that the blockade of the inhibition of
PKC
and -
activities by indomethacin plays a critical role in the
inhibition of NO-induced dedifferentiation.
Consistent with the inhibition of apoptotic signaling mediators such as
p38 kinase and PKC, indomethacin also blocked their downstream
signaling molecules such as the activation of NFB, accumulation of
p53, and activation of caspase-3. Indeed, it has been shown that some
types of NSAIDs, including ibuprofen, sulindac, sulindac sulfide, and
flurbiprofen, are able to inhibit NF
B activation, whereas
indomethacin, ketoprofen, and ketorolac are ineffective (23). Using a
reporter gene assay and I
B degradation, we found that ectopic
expression of wild type PKC
or -
by adenovirus infection in
chondrocytes blocked the NO-induced activation of NF
B (48),
indicating that p38 kinase-dependent and -independent PKC
and -
regulates NF
B activation. We also found that NF
B activation is required for COX-2 expression (48). This suggests that
inhibition of PKC
and -
is an upstream signaling event leading to
NF
B activation that causes COX-2 expression as summarized in Fig. 9.
Given the inability of indomethacin to inhibit NF
B (23), it is
highly likely that the inhibitory effects of indomethacin on NF
B
activation observed in this study are caused by the inhibition of its
upstream signaling molecules (i.e. p38 kinase and PKC) rather than by a direct action on NF
B.
In summary, we found that various NSAIDs block apoptosis and
dedifferentiation of articular chondrocytes caused by NO production in
a manner independent of their ability to inhibit COX-2 and PGE2 production. The inhibitory effects of NSAIDs on
apoptosis are derived from their ability to block NO-induced activation of p38 kinase, to potentiate ERK activation, and to block inhibition of
PKC and -
, whereas the inhibition of NO-induced dedifferentiation by NSAIDs is caused by the blockade of PKC
and -
signaling but not the modulation of ERK-1/2 and p38 kinase signaling. Additionally, the effects of NO production and NSAIDs treatment on chondrocytes derived from adult rabbit joint cartilage (from 4-month-old rabbits) or
human osteoarthritic cartilage obtained from patients undergoing total
knee arthroplasty are essentially same as in growth plate chondrocytes
(from 2-week-old rabbits) (data not shown). Because NO production via
inducible NO synthase in articular chondrocytes plays a central role in
the pathophysiology of arthritis by causing inflammation, apoptosis,
dedifferentiation, and the activation of matrix metalloproteinases, our
results suggest that NSAIDs have protective effects on cartilage
damage, not only by alleviating inflammation but also by inhibiting
NO-induced apoptosis and dedifferentiation of articular chondrocytes.
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FOOTNOTES |
---|
* This work was supported by the National Research Laboratory Program (M1-0104-00-0064) from the Korea Ministry of Science and Technology and the Interdisciplinary Research Project (1999-2-207-004-5) from the Korea Science and Engineering Foundation.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.
¶ Funded by Korea Research Foundation Grant KRF-2000-015-DP0352.
To whom correspondence should be addressed. Tel.:
82-62-970-2497; Fax: 82-62-970-2484; E-mail: jschun@kjist.ac.kr.
Published, JBC Papers in Press, February 14, 2003, DOI 10.1074/jbc.M212520200
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ABBREVIATIONS |
---|
The abbreviations used are:
NO, nitric
oxide;
COX, cyclooxygenase;
ERK, extracellular signal-regulated protein
kinase;
MAP, mitogen-activated protein;
NFB, nuclear factor
B;
NSAIDs, nonsteroidal anti-inflammatory drugs;
PG, prostaglandin;
PKC, protein kinase C;
SNP, sodium nitroprusside;
TUNEL, terminal
deoxynucleotidyl transferase dUTP nick-end labeling;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium.
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