From the Unité Mixte de Recherche CNRS
7079 Physiology and Physiopathology Laboratory, University Paris 6,
7 quai St. Bernard, Bât A, Paris 75252 cedex 5 and
§ Department of Rheumatology, Unité Formation de
Recherche Saint-Antoine, Paris 75012, France
Received for publication, November 13, 2002, and in revised form, February 7, 2003
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ABSTRACT |
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Extracellular ATP is a pro-inflammatory mediator
involved in the release of prostaglandin from articular chondrocytes,
but little is known about its effects on intracellular signaling. ATP
triggered the rapid release of prostaglandin E2
(PGE2) by acting on P2Y2 receptors in rabbit
articular chondrocytes. We have explored the signaling events involved
in this synthesis. ATP significantly increased arachidonic acid
production, which involved the activation of the 85-kDa
cytosolic phospholipase A2 (cPLA2)
but not a secreted form of PLA2, as demonstrated by various
PLA2 inhibitors and translocation experiments. We also showed that ATP induced the phosphorylation of p38 and ERK1/2 mitogen-activated-protein kinases (MAPKs). Both PD98059, an
inhibitor of the ERK pathway, and SB203580, an inhibitor of p38 MAPK,
completely inhibited the ATP-induced release of PGE2.
Finally, dominant-negative plasmids encoding p38 and ERK transfected
alone into the cells impaired the ATP-induced release of
PGE2 to about the same extent as both plasmids transfected
together. These results suggest that PGE2 production
induced by ATP requires the activation of both ERK1/2 and p38 MAPKs.
Thus, ATP acts via P2Y2-purine receptors to recruit
cPLA2 by activating both ERK1/2 and p38 MAPKs and
stimulates the release of PGE2 from articular chondrocytes.
Articular cartilage is an avascular, aneural tissue consisting of
chondrocytes embedded in an extracellular matrix. Healthy cartilage is
maintained in a state of dynamic equilibrium by matrix synthesis and
matrix degradation by the chondrocytes. Any dysregulation with
increased degradation and/or inadequate synthesis leads to loss of
tissue structure and function, as in rheumatoid arthritis (RA)1 and osteoarthritis
(OA). The proinflammatory cytokines interleukin-1 The production of PGE2 involves a cascade of three enzyme
reactions. First, AA is liberated from its phospholipid storage sites
by a phospholipase A2 (PLA2), it is then acted
on by cyclooxygenases and finally by PGE synthase to produce
PGE2. PGE2 stimulates the catabolism of
chondrocytes, having antiproliferative and proapoptotic effects (12).
An increased release of PGE2 might therefore modify the
local balance between anabolism and catabolism, resulting in matrix
degradation and cartilage destruction. Hence, by stimulating the
release of PGE2 from articular chondrocytes, ATP could
amplify the joint destruction in articular diseases. However, the
intracellular pathways activated by ATP in articular chondrocytes
remains largely unknown. The present study identifies the signaling
cascade and intracellular events leading to the release of
PGE2 stimulated by ATP in articular chondrocytes.
Materials--
All reagents were purchased from Sigma-Aldrich
(St. Quentin Fallavier, France), unless stated otherwise. Fetal bovine
serum was from Invitrogen (Cergy Pontoise, France). Type II
collagenase, trypsin, and hyaluronidase were from Roche Diagnostics
(Meylan, France). BAPTA-AM and
1-palmitoyl-2-(10-pyrenyldecanoyl)-sn-glycero-3-monomethylphosphatidic acid were from Interchim (Montluçon, France). Anti-mouse
cPLA2 monoclonal antibody was from Tébu for Santa
Cruz Biotechnology (Le Perray en Yvelines, France), anti-rabbit
phospho-ERK, and anti-rabbit phospho-p38 antibodies were obtained from
New England BioLabs (Beverly, MA), and anti-rabbit total ERK antibody
was from Upstate Biotechnology Inc. (Lake Placid, NY). AACOCF3,
SB203580, and PD98059 were obtained from Calbiochem (Meudon, France).
[5,6,8,9,11,12,14,15-3H]Arachidonic acid (specific
activity, 216 Ci/mmol) and the ECL Western blot analysis system were
purchased from Amersham Biosciences (Orsay, France). The nitrocellulose
membranes for Western blotting were obtained from Schleicher and
Schuell (Dassel, Germany). Agarose was from Eurobio (Les Ulis, France),
kaleidoscope prestained standards were from Bio-Rad (Ivry sur Seine,
France), and DNA markers were from Eurogentec (Seraing, Belgium).
Chondrocyte Cultures--
All the experiments were performed
according to protocols approved by the French/European ethical
committee. Three-week-old female Fauve de Bourgogne rabbits were
killed, and the shoulders, knees, and femoral heads were dissected out
under sterile conditions (13). The articular cartilage was removed, cut
into small pieces, and digested at 37 °C with 0.05% hyaluronidase
in PBS for 15 min, then with 0.2% trypsin for 30 min, and finally with
0.2% collagenase for 90 min. The chondrocytes were then washed with
Hams' F-12 medium for 60 min. The suspended chondrocytes were seeded
in 60-mm dishes (1.5 × 105 cells per dish) or 100-mm
dishes (5 × 105 cells per dish) in Hams' F-12 medium
supplemented with 10% fetal calf serum. The cells were maintained at
37 °C in 5% CO2, and the culture medium was changed
every 2-3 days. The cells were almost confluent in 6-7 days.
RNA Isolation and Complementary DNA Synthesis--
Total RNA was
isolated from cultured rabbit articular chondrocytes using the Qiagen
RNeasy Mini kit (Qiagen, Courtaboeuf, France) following the supplier's
instructions. Cells were released from dishes by trypsinization, and
the resulting cells were suspended in the lysis buffer plus
2-mercaptoethanol at 600 µl/5 × 106 cells. The RNA
concentration was determined spectrophotometrically at 260 nm. The
quality of the RNA preparation was assessed by estimating the 18 S/28 S
rRNA ratio on a denaturing gel. Two micrograms of RNA was
reverse-transcribed to cDNA in a 20-µl reaction volume containing
5 µM random hexamers, 0.5 mM dNTPs, and 10 mM dithiothreitol, and 200 units of Moloney murine leukemia
virus reverse transcriptase was dissolved in the supplied buffer. The
reactions were allowed to proceed for 1 h at 37 °C and stopped
by heating at 65 °C for 10 min.
PCR--
A set of forward and reverse primers specific to the
P2Y1 and P2Y2 receptor subtypes was
custom-synthesized by Oligoexpress (Paris, France).
Oligonucleotides for P2Y1 were 5'-CGGTCCGGGTTCGTCC-3' (forward, sense) and 5'-CGGACCCCGGTACCT-3' (reverse, antisense) as
originally designed by Ayyanathan et al. (14). The
oligonucleotides for P2Y2 were
5'-CGTCATCCTTGTCTGTTACGTGCT-3' (forward, sense) and
5'-CTACAGCCGAATGTCCTTAGTG-3' (reverse, antisense) as originally designed by Bowler et al. (15). The sequences used to
amplify the glyceraldehyde-3-phosphate dehydrogenase fragment were
glyceraldehyde-3-phosphate dehydrogenase forward (sense),
5'-CCATGGAGAAGGCTGGGG-3', and glyceraldehyde-3-phosphate dehydrogenase
reverse (antisense), 5'-CAAAGTTGTCATGGATGACC-3'. The PCR
reactions were performed in 50-µl final volumes containing 5.0 µl
of cDNA, 400 ng of each primer, 0.5 mM dNTPs, 2.5 units of red Taq DNA polymerase dissolved in the supplied buffer.
Samples were initially denatured for 2 min at 94 °C and then
amplified for 40 cycles as follows: denaturation at 94 °C for 1 min,
annealing at 54 °C for 1 min, and extension at 72 °C for 90 s. After the final PCR cycle, extension was allowed to proceed
for 10 min at 72 °C. Products were run on a preparative 2%
agarose gel, stained with ethidium bromide, and photographed.
PGE2 Assay--
Aliquots of supernatants from
stimulated chondrocytes cultures were collected and the
PGE2 concentrations were measured using a specific enzyme
immunoassay kit (Cayman Chemical, Ann Arbor, MI). The assay was
performed according to the manufacturer's instructions. PGE2 production was evaluated in duplicate, and amounts
were taken from a standard curve of PGE2. The sensitivity
of the assay allowed the detection of up to 15 pg/ml. When necessary
the samples were diluted in the assay buffer.
Assessment of Arachidonic Acid Mobilization--
Release of
3H from cultures prelabeled with [3H]AA was
used to assess the response to stimulants. Confluent cultures were
placed in quiescent medium (Hams' F-12 medium containing 0.1% bovine fatty acid-free serum albumin) for 24 h before they were labeled for 4 h with 1 µCi/ml [3H]arachidonic acid.
Cultures were washed once with phosphate-buffered saline (PBS)
containing 0.1% free fatty acid albumin and twice with PBS alone.
Cells were then incubated at 37 °C in fresh Hams' F-12 medium
supplemented with 0.1% free fatty acid albumin plus agonists,
antagonists, or vehicle. The supernatants were removed at the indicated
times and centrifuged at 10 000 × g for 10 min to
remove floating cells and/or cell debris. The cells were washed with
Triton 5% and scraped off. The radioactivity of cells and media was
quantified by scintillation counting. The results were normalized and
expressed as a percentage of the mean of the basal release.
sPLA2 Assay--
sPLA2 activity was
measured by the selective fluorometric assay of Radvanyi et
al. (16) as modified by Pernas et al. (17). The
PLA2 activity secreted into the medium was measured on
100-µl samples using 2 nmol of fluorescent substrate
1-palmitoyl-2-(10-pyrenyldecanoyl)-sn-glycero-3-monomethylphosphatidic acid. 100% hydrolysis of the substrate was measured using 0.1 unit of
PLA2 from Naja naja.
Sample Preparation for Protein Studies--
Cells were grown to
confluence in 75-cm2 flasks or 100-mm dishes and rendered
quiescent by starving them of serum for 24 h. The starving medium
contained 0.1% bovine serum albumin. Cells were stimulated by adding
medium containing or lacking agonists at 37 °C for the indicated
time, unless otherwise indicated. The exposure to agonists or vehicle
was terminated by rapidly washing twice with 5 ml of ice-cold PBS. For
MAPK studies, cells were scraped into 500-µl lysis buffer (20 mM Tris, pH 7.6, 0.15 M NaCl, 2 mM
EDTA, 1% Triton, 10% glycerol, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 2 mM sodium
pyrophosphate, 1 mM p-aminoethylbenzenesulfonyl fluoride, 10 µg/ml leupeptin, 1 µg/ml antipain, 1 µg/ml
pepstatin A), kept on ice for 20 min, and then vortexed for 1 min. The
cell debris was removed by centrifugation at 13,000 × g for 10 min at 4 °C, and the total protein content was
determined photometrically using bicinchoninic acid (Micro-BCA kit,
Pierce). For cPLA2 studies, the homogenization buffer was
40 mM Tris, pH 7.4, 0.25 M sucrose, 2 mM EDTA, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 2 mM sodium
pyrophosphate, 1 mM p-aminoethylbenzenesulfonyl
fluoride, 10 µg/ml leupeptin, 1 µg/ml antipain, 1 µg/ml
pepstatin A. The chondrocytes were disrupted by sonication, and the
resulting suspension was then ultracentrifuged at 105,000 × g for 1 h at 4 °C (in a Beckman SW 50.1 rotor), and
the supernatant was recovered (cytosolic fraction). The pellet was
suspended in 0.1 ml of homogenization buffer (membrane fraction). The
protein content of the broken cells, cytosolic and membrane fractions,
was determined using the Micro-BCA kit with bovine serum albumin as the standard.
Electrophoresis and Western Blotting--
Aliquots (20-50 µg)
of clarified total proteins, for the MAPK studies, and of membrane and
cytosol fraction, for cPLA2 studies, were separated by
SDS-PAGE, on 10% or 7.5% running gels, in 25 mM Tris-HCl,
190 mM glycine, 0.1% SDS, pH 8.3. The separated proteins were electroblotted on to Protran BA83 nitrocellulose membranes (Schleicher & Schuell). The membranes were soaked in 10 mM
Tris-HCl, pH 7.5, 100 mM NaCl, 0.1% Tween 20, 5% serum
albumin at room temperature for at least 2 h. MAPKs activation was
assayed by incubating the nitrocellulose blots overnight at 4 °C
with an antiserum that recognizes only the phosphorylated forms of p42
and p44 MAPK or p38 MAPK. Stripped blots were also probed with an
antiserum recognizing both the unphosphorylated (inactive) and
phosphorylated (active) forms to check the application of equal amounts
of lysate.
The membranes were washed and incubated with goat anti-rabbit IgG
conjugated to horseradish peroxidase. Immunoreactive bands were
detected using the ECL Western blotting detection system, and Biomax
MR1 film (Sigma) exposed for 10-90 s. When necessary, the blots were
stripped for 40 min at 52 °C in a Tris buffer containing 62.5 mM Tris, 2% SDS, 100 mM mercaptoethanol, 0.1%
sodium azide adjusted to pH 7.4. cPLA2 was immunodetected
in an analogous manner, using the appropriate antibodies.
Dominant Negative Plasmids of MEK and p38
Overexpression--
The chondrocytes were transfected using the
calcium phosphate DNA coprecipitation method. Cells were changed by
Dulbecco's modified Eagle's medium before transfection. Cells were
incubated for 4 h with the transfection mixture containing 4 µg
of an expression vector for dominant negative MEK (DN-MEK, a gift from
J. Pouysségur (18)) and/or 4 µg of an expression vector for
dominant negative p38 MAPK (DN-p38, a gift from B. Dérijard).
Constant amounts of DNA were maintained by using control empty vector.
The cells were then shocked with HBS buffer (21 mM HEPES,
pH 7.1, 16 mM dextrose, 0.8 mM
NA2HPO4, 5 mM KCl, and 137 mM NaCl) containing 15% glycerol for 90 s. The
cultures were then maintained 3 days in Hams' F-12 supplemented with
10% fetal calf serum before already described starvation and ATP
stimulation. Supernatants were collected, and the PGE2
concentrations were measured as described above. Transfection
experiments were performed in triplicate and repeated two times with
two different preparations of plasmids.
Statistical Analysis--
All data are presented as means ± S.E. unless otherwise indicated. Data populations were tested for
Gaussian distribution and equal standard deviations. Student's
t test was used when the data satisfied these criteria.
Otherwise, non-parametric Mann-Whitney tests were used. We used
GraphPad InStat version 3.05 for Windows 95/NT, GraphPad software (San
Diego, CA, available at www.graphpad.com). p values
of < 0.05 were considered to be significant.
Induction by ATP of an Immediate PGE2 Release from
Articular Chondrocytes--
Extracellular ATP (10 µM to
1 mM) caused a significant rise in the PGE2
released from rabbit articular chondrocytes; 10 µM ATP
stimulated PGE2 release 1.9 ± 0.3-fold
(n = 7), and the maximal effect was reached with 100 µM ATP (4.3 ± 0.3-fold, n = 29)
(Fig. 1A). PGE2
production increased after only 2 min of stimulation and continued to
rise to a maximum after 1 h (Fig. 1B). Longer stimulation of ATP (24 h) caused no significant additional increase in
PGE2 release over that obtained at 1 h. We therefore
used 100 µM ATP for all the following experiments.
Involvement of a P2Y2 Purinergic Receptor in
ATP-dependent PGE2 Release--
Other purine
and pyrimidine derivatives were tested to further characterize the
release of PGE2 triggered by ATP. The effect of ATP was
mimicked by ATP Stimulation of AA Release from Articular Chondrocytes by
ATP--
Because the first step in the PGE synthesis is the liberation
of AA from its phospholipid storage sites by PLA2, we
assessed the effect of ATP on AA release. Cultured articular
chondrocytes were incubated with 1 µCi/ml [3H]AA and
then with ATP and other nucleotides. ATP (100 µM)
stimulated the transient release of [3H]AA, the initial
increase peaked 2-5 min post stimulation and then declined (data not
shown). AA was measured 5 min after stimulation in subsequent
experiments, unless noted. Maximal stimulation with ATP (100 µM) increased the release by 158.1 ± 27.2%
(n = 19) times that of the control. The concentration
dependence of the effect of ATP on [3H]AA release is
shown in Fig. 3A. Chondrocytes
were incubated with other nucleotides and antagonists to confirm the
involvement of the P2Y purinergic receptor in ATP-evoked
[3H]AA mobilization. The agonists UTP and ATP Activation of cPLA2 by Extracellular ATP--
The
release of AA is largely a PLA2-mediated process. Studies
using different PLA2 inhibitors were performed, and
sPLA2 activity was assayed (Fig.
4, A-C), to identify the
specific PLA2 isoforms involved in ATP-induced
[3H]AA mobilization. Because the release of
PGE2 is a good index of AA synthesis that is more sensitive
than [3H]AA mobilization, we tested the effect of
PLA2 inhibitors on PGE2 release. BEL, a
compound that selectively inhibits iPLA2 at low
concentration (0.5-5 µM), rather than cPLA2,
had no effect on ATP-induced PGE2 release, eliminating
iPLA2 (Fig. 4A). The acute effect of the ATP on
PGE2 release did not favor the implication of
sPLA2. We confirmed this by assaying sPLA2 with
a specific fluorescent substrate (Fig. 4B). As expected, the
sPLA2 activity in the supernatants from ATP-stimulated
cells was unchanged. Because IL-1 ATP-induced Phosphorylation of ERK1/2 and p38 MAPKs in
Articular Chondrocytes--
We next attempted to identify the
signaling pathways activated downstream of the P2Y receptor and
underlying the cPLA2 activation and subsequent ATP-induced
release of PGE2. Western blotting using an antibody that
specifically recognized the phosphorylated (active) forms of ERK1/2 and
p38 MAPKs was used. Exposure to ATP caused the rapid phosphorylation of
both the ERKs and p38 MAPKs. The maximal effect was obtained with 100 µM ATP, as for ATP-induced PGE2 release (Fig.
5A). The phosphorylations of
ERKs and p38 MAPKs were detectable within 2 min, and the peak
was reached after 5-10 min. Both responses to ATP were transient and
returned to control level within 30 min (Fig. 5C). Other
compounds (ATP Requirement of MAPKs for cPLA2 Activation by
ATP--
The concomitant stimulation of PGE2 release and
activation of ERK1/2 and p38 MAPKs by ATP indicates that MAPKs must be
activated for ATP to trigger the release of PGE2. We
therefore estimated by Western immunoblotting the concentrations of
specific inhibitors of p38 MAPK (SB203580) and MEK1/2 (PD98059) needed
to prevent the activation of p38 or ERKs MAPKs (Fig.
6A). We then tested the useful
concentrations of SB203580 and PD98059 on the ATP-evoked PGE2 release. PD98059 (20 µM) totally
inhibited the release of PGE2 from cells stimulated with
ATP. Similarly, SB203580 (5 µM) inhibited p38 MAPK and
prevented the ATP response (Fig. 6B). Lastly, PD98059
greatly reduced AA mobilization in response to ATP, whereas SB203580
inhibited the release of AA to the same extent. Treatment with both
PD98059 and SB203580 resulted in no further decrease in AA mobilization
(Fig. 6C). To further confirm that ERK and p38 activation
are needed for the ATP-induced PGE2 release, chondrocytes were transitorily transfected with a DN-MEK and/or DN-p38 mutants. First, we checked using Western blot analysis, whether the DN-MEK and
the DN-p38 mutants reduced significantly the ATP-induced
phosphorylation of ERK1/2 and p38, respectively (data not shown). In
agreement with our results using the inhibitors, overexpression of a
DN-MEK, a DN-p38, or the DN-MEK and DN-p38 added together reduced
ATP-induced PGE2 release by 28 ± 7, 27 ± 7, and
33 ± 5%, respectively (Fig. 6D). The rates of
inhibition observed in these experiments were consistent with the
DNA transfer efficiency previously observed in these cells (21).
Taken together these results imply that p38 and ERK1/2 MAPKs act in
concert on cPLA2 activity.
The results of this study demonstrate that ATP stimulates the
rapid, transient release of PGE2 from rabbit articular
chondrocytes in primary culture by acting on a P2Y2
purinoceptor. Stimulation of the P2Y2 receptor led to the
activation of ERK1/2 and p38 MAPK and increases intracellular calcium.
These three intracellular pathways act in concert to stimulate the
cPLA2 enzyme, resulting in the liberation of AA, which is
the rate-limiting step for the ATP-stimulated production of
PGE2 (Fig. 7).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(IL-1
) and
tumor necrosis factor
(TNF-
) are implicated in this phenomenon.
They act on chondrocytes to increase the production of the major
cartilage-degrading proteinases, the matrix metalloproteinases, by
inhibiting the synthesis of cartilage proteoglycans and type II
collagen and by stimulating the release of reactive oxygen species and
prostaglandins at the sites of inflammation (1). ATP could be another
key mediator, because it acts as an extracellular messenger in several
systems. It affects many biological processes, including smooth muscle
contraction, neurotransmission, the immune response, platelet
aggregation, and inflammation (2). ATP can be released by healthy cells
via anionic channels and vesicular secretion (3) or after cell lysis
due to trauma or inflammation (4). Recently, Graff et al.
(5) demonstrated that chondrocytes continuously release ATP under
resting conditions. In addition, the enhanced ATP release due to
mechanical loading of chondrocytes suggests that ATP is involved in the
pathogenesis of OA (5). This is supported by the finding that the
synovial fluids of patients affected by OA or RA contain high
concentrations of ATP (6). Leong et al. (7, 8) reported that
ATP triggered the degradation of cultured explants of bovine nasal
cartilage and stimulated PGE2 production by articular
chondrocytes. IL-1
and TNF-
both interact synergistically with
extracellular ATP (9, 10). Finally, there are metabotropic (P2Y)
receptors on the membranes of articular tissues. The P2Y2
subtype was recently found on human articular chondrocytes (11).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of ATP on PGE2
release. A, release of PGE2 in response to
increasing concentrations of ATP for 1 h. Chondrocytes were
maintained in medium alone (open bars) or in medium
supplemented with indicated concentrations of ATP (filled
bars) prior to measuring PGE2 in cell supernatants.
Error bars denote S.E. n = 7 (1-10
µM ATP), n = 4 (1 mM ATP),
n = 23 and 29, respectively, for control and 100 µM ATP. The asterisks denote a statistically
significant difference from control values. ***, p < 0.001; **, p < 0.01. B, time course of
PGE2 increase in response to 100 µM ATP.
Chondrocytes were challenged with ATP for the indicated time prior to
measuring PGE2 release. The basal release of
PGE2 recorded in the absence of ATP was subtracted, and the
data have been averaged from four separate experiments. Error
bars denote S.E.
S, the non-hydrolysable analog of ATP, suggesting
that ATP itself is active and does not require hydrolysis. ADP was less
effective than ATP in inducing PGE2 release (Fig. 2A), whereas AMP and adenosine
had no significant effect. Suramin, an ATP-receptor antagonist, used at
500 µM blocked the release of PGE2 in
response to 100 µM ATP (percent inhibition: 89.1 ± 1.4%, n = 4). Reactive Blue 2 (RB-2), a P2Y
purinoceptor subtype inhibitor, also strongly inhibited the effect of
ATP (78.5 ± 3.6%, n = 9), suggesting that the
P2Y purinergic receptor is involved in PGE2 release (Fig.
2B). Finally, UTP was as effective as ATP in inducing
PGE2 release, suggesting that a P2Y2
purinoceptor is implicated in the response of articular chondrocytes to
ATP. The finding that the RT-PCR detected P2Y2 receptor
mRNA but not P2Y1 receptor mRNA in rabbit articular
chondrocytes supports this idea (Fig. 2C).
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Fig. 2.
Implication of a P2Y2-like
receptor in the ATP-induced PGE2 synthesis.
A and B, effects of purinergic receptor agonists
and antagonists on PGE2 production. A,
chondrocytes were stimulated with 100 µM of the indicated
compounds for 1 h. n = 6 or 7 but for control and
ATP for which n = 23 and 29, respectively. ***,
p < 0.001 compared with control (Student t
test). B, effect of P2 purine receptor antagonists on
ATP-induced PGE2 release. Chondrocytes were incubated with
500 µM suramin (dotted bar) or 30 µM RB-2 (hatched bar) for 15 min and then
stimulated with 100 µM ATP for 1 h.
n = 4 and 9, respectively, for suramin and RB-2. ***,
p < 0.001; *, p < 0.05 compared with
control (Student t test). C, total RNA from
cultured chondrocytes was reverse-transcribed, and the resulting
cDNA was PCR-amplified using the indicated primers (see
"Experimental Procedures"). Lane Mw contains molecular
weight markers. Primers specific for glyceraldehyde-3-phosphate
dehydrogenase were used to amplify a positive control, similar in all
reactions, and confirming the integrity of cDNA in each sample.
Shown is a representative image of four independent experiments.
S caused
the release of [3H]AA with the same potency as ATP, and
RB-2 inhibited ATP-induced [3H]AA mobilization (Fig.
3B). These results are consistent with the activation of the
P2Y2 receptor.
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Fig. 3.
Release of [3H]arachidonic acid
induced by ATP. AA released (A) in response to
increasing concentrations of ATP and (B) after purinergic
receptors agonists and inhibitors. Confluent cells were labeled with 1 µCi/ml [3H]AA for 4 h and then stimulated by the
indicated concentrations of ATP in A and by 100 µM of the indicated compounds in B. At the end
of the incubation, the medium was removed and counted as described
under "Experimental Procedures." Data are expressed as percent
increase in [3H]AA release compared with control cells.
Each point is the mean of at least two independent
experiments performed in duplicate. Values represent the means ± S.E. ***, p < 0.001; **, p < 0.01 compared with control (Student t test).
strongly stimulates
sPLA2 production in this model (13, 19), we used
supernatants from chondrocytes stimulated with IL-1
as positive
controls (Fig. 4B). We used AACOCF3, a specific inhibitor of
cPLA2, to test the involvement of cPLA2 in
ATP-stimulated PGE2 release. AACOCF3 inhibited the
ATP-induced PGE2 release dose dependently (Fig.
4C). Because cPLA2 activity is
calcium-dependent, we preincubated the cultured
chondrocytes with the permeable calcium chelator, BAPTA-AM, to prevent
the changes in intracellular calcium that occur when P2Y are activated
in articular chondrocytes (7) and confirmed by us (data not shown). ATP
did not significantly stimulate the release of AA or PGE2
by BAPTA-loading chondrocytes (Fig. 4D). Finally,
cPLA2 must be translocated for it to become active. We
therefore investigated its translocation to the cell membrane in
response to ATP, to confirm that it accounted for the action of ATP.
Membrane and cytosol fractions were prepared from control and
ATP-stimulated articular chondrocytes and immunoblotted using an
anti-cPLA2 antibody. The control membrane fractions
contained no detectable cPLA2, but there was
cPLA2 immunoactivity in the cytosol fraction. The membranes
isolated from chondrocytes exposed to ATP were enriched in
cPLA2 (Fig. 4E). These data demonstrate the
pivotal role of cPLA2 in the ATP-evoked release of AA.
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Fig. 4.
Lack of involvement of iPLA2 and
sPLA2 and activation of cPLA2 in the ATP
response. A, the cells were incubated with the
indicated concentrations of BEL for 15 min and then in 100 µM ATP for 1 h. The supernatants were assayed for
their PGE2 content. BEL had no significant influence on the
ATP stimulation. B, enzymatic activity of sPLA2
measured in the supernatants from control (open bar);
ATP-stimulated (filled bar) and IL-1 -stimulated
(hatched bar) chondrocytes. Cells were treated for 1 h
with 100 µM ATP or for 24 h with 10 ng/ml IL-1
.
No significant difference was observed between control and ATP.
C, effect of AACOCF3, a cPLA2 inhibitor, on
ATP-stimulated PGE2 production. Chondrocytes were treated
with 100 µM ATP for 1 h alone or with ATP plus the
indicated concentrations of AACOCF3. The bar graph
represents the means ± S.E. of two independent experiments
performed in duplicate. ***, p < 0.001; **,
p < 0.01 compared with ATP alone (Mann Whitney test).
D, effect of calcium chelation on ATP-induced AA and
PGE2 release. Chondrocytes were incubated with vehicle
(open bar), with 100 µM ATP (filled
bar), or with 50 µM BAPTA/AM for 30 min prior to
stimulation with 100 µM ATP (hatched bar).
Results are expressed as percent of the control for AA release and in
nanograms/mg of protein for PGE2 release. Each
point is the mean of at least two independent experiments
performed in duplicate. Values are the means ± S.E. ***,
p < 0.001; **, p < 0.01 compared with
control (Student t test). E,
anti-cPLA2 immunoblot of chondrocyte membrane
(bottom) and cytosol (top) fractions. Articular
chondrocytes were incubated with 100 µM ATP for the
indicated time. Cytosolic and membrane fractions were prepared, and
equal amounts (50 µg) of fractions were separated by SDS-PAGE,
transferred to nitrocellulose, blotted with anti-cPLA2
antibody, and visualized by ECL.
S, UTP, and ADP), which also triggered
PGE2 release, also stimulated ERK1/2 and p38 MAPK
activation (Fig. 5B). We investigated the effects of the
purinergic receptor inhibitors suramin and RB-2 to see if the
P2Y2 purinoceptor was involved in ERKs and p38 MAPKs
activation induced by ATP. Preincubation with 500 µM
suramin totally prevented the activation of either MAPK by ATP, whereas
30 µM RB-2 antagonized the activation of both MAPKs by
ATP (Fig. 5D). The [Ca2+]i
increases in response to ATP in mammalian articular chondrocytes
(20). We tested the effect of BAPTA on the MAPK activation induced by
ATP to determine the interplay between the various components of the
signaling pathway activated by ATP in articular chondrocytes. The
phosphorylation of ERK1/2 and p38 MAPK triggered by ATP did not require
an increase in calcium, because ERK1/2 and p38 were significantly
activated in chondrocytes treated with BAPTA-AM (Fig.
5E).
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Fig. 5.
Effect of ATP on ERK and p38 MAPKs
phosphorylation. A-E, cell lysates were analyzed by a
10%-gradient SDS-PAGE. The proteins were transferred to nitrocellulose
and blotted with anti-phospho p38 MAPK antibody or anti-phospho ERK
antibody. In the lower panel, the blots were stripped and
reprobed with anti-native ERK antibody. The p42 and p44 forms of ERK
are abbreviated as p42 and p44, whereas the
corresponding phosphorylated ERK forms are abbreviated as
P-p42 and P-p44. Each blot is
representative of two to five independent experiments. A,
chondrocytes were challenged with increasing concentrations of ATP for
10 min. B, purinergic receptor agonists were applied on
chondrocytes for 10 min at 100 µM. C, temporal
activation by 100 µM ATP. D, effects of
purinergic receptor antagonists on ATP-induced MAPK phosphorylation.
E, chondrocytes were stimulated with ATP with or without
pretreatment with 50 µM of the calcium chelator
BAPTA-AM.
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Fig. 6.
Effect of MAPK inhibitors on ATP-induced MAPK
phosphorylation and on AA and PGE2 releases.
A, cells were incubated for 15 min with vehicle ( ),
PD98059, or SB203580 at the indicated concentrations and then
stimulated with 100 µM ATP for 10 min. The lysates were
separated by 10% SDS-PAGE and assessed by Western blotting.
Top, the membrane was immunolabeled with an anti-phospho-p38
antibody. Bottom, Western blot showing the
anti-phospho-p42/p44 signal. Each membrane was stripped and probed with
antibodies that cross-reacted with total p42/p44 MAPK to ensure equal
protein loading. B and C, chondrocytes were
preincubated with 20 µM PD98059 (dotted bars),
5 µM SB203580 (hatched bars), or without any
inhibitors (filled bar) for 15 min prior to exposure to 100 µM ATP for 1 h, PGE2 (B) or
AA (C) release were assayed. The bar graphs are
the means ± S.E. of three independent experiments performed in
duplicate.***, p < 0.001; *, p < 0.05 compared with control (Mann Whitney test). D, effect of the
overexpression of a DN-MEK vector (4 µg) and a DN-p38 vector (4 µg)
alone or in combination was evaluated on the ATP-induced
PGE2 release. The results are expressed as the percentage
of basal PGE2 release recorded when cells were transfected
with the control empty vector. **, p < 0.01; *,
p < 0.05 compared with ATP stimulated and control
empty vector transfected cells (Student t test). There was
no significant difference (ns) between plasmids encoding p38
or ERK transfected alone and both plasmids transfected together.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 7.
Proposed cascade of the signal transduction
pathway leading to PGE2 production by extracellular ATP in
articular chondrocytes. Micromolar concentrations of
extracellular ATP binds to the P2Y2 purine receptor, which
increases intracellular calcium and causes the phosphorylation of p38
and ERK1/2 MAPKs. The resulting phosphorylation and translocation of
the cPLA2 increase its catalytic activity, release AA, and,
consequently, increase the release of PGE2. Selective
inhibitors of the various steps are indicated in
boxes.
Release of Endogenous ATP in Articular Chondrons in Vivo-- ATP appears to be an extracellular signal in a wide variety of systems, where it is involved in both physiological and physiopathological conditions (reviewed in Ref. 22). ATP is continuously released by chondrocytes at rest in articular chondrons (5), and this release is increased by mechanical loads and constraints (5). The concentrations of ATP increase into the synovial fluid during inflammation (6). The concentration of ATP reached at the site of release and in the vicinity of the surrounding cells is hardly to determine experimentally. In the nervous system where the action of ATP was extensively studied, a local extracellular ATP concentration reaching 40 µM in a diameter of 20 µm around the site of liberation was reported (23). In synovial fluid the ATP concentrations occur up to 217 nM (6). However, given the architecture of the chondrons where chondrocytes are organized in cluster surrounded by a complex pericellular matrix, it is likely that upper extracellular concentrations of ATP could be reached in the vicinity of the same or neighboring chondrocytes, allowing its paracrine and/or autocrine effect. The half-life of extracellular ATP in articular cartilage is a crucial parameter when considering its action on cartilage in vivo. ATP is rapidly hydrolyzed by the ecto-nucleotidases in the synovial fluid (24); it has a half-life of 2-3 h in bovine articular chondrocytes (25) and 15 min in chondrocytes from chick embryo sternum (26). Although ATP is rapidly hydrolyzed in cartilage, the time course of the effects reported in this study appears to be compatible with ATP having a proinflammatory action in vivo. The release of PGE2 in response to ATP is rapid, starting just 2 min after exposure and reaching a maximum after 1 h. Also, ATP is hydrolyzed more slowly in synovial fluids from RA patients, suggesting that ATP has a stronger proinflammatory action in RA (24).
Stimulation of PGE2 Release by ATP via P2Y2 Purinoceptor in Chondrocytes-- The effects of the purinergic receptor antagonists demonstrated in this study indicate that a G-protein-coupled P2 receptor is involved in the PGE2 production induced by ATP. A P2Y2 receptor may well be involved, because ATP and UTP are equally effective in causing PGE2 release in our model, and this pharmacological profile fits well with a P2Y2 receptor subtype (27). This is supported by our detection by RT-PCR of P2Y2 transcripts. ADP stimulates partially the PGE2 release under our conditions. ADP is generally believed to be the most potent endogenous ligand for P2Y1 receptors and mostly ineffective on P2Y2 receptors (27). Nevertheless, we did not detect P2Y1 transcripts in rabbit chondrocytes by RT-PCR, confirming the findings of Koolpe et al. (11) in human articular chondrocytes. Thus, extracellular ATP releases PGE2 by activating a P2Y2-like purinergic receptor in rabbit articular chondrocytes.
ATP as a Mediator of Articular Inflammation in Vivo--
ATP
caused the fast, transient release of PGE2. Studies on the
PGE2 concentrations in animal models of arthritis have
demonstrated the importance of this prostanoid in arthritis progression
(28). This has been confirmed by studies on PGE receptor EP4/
null mice, which showed a profound decrease in the intensity of the inflammation and in markers of joint destruction (29). Thus, ATP and
proinflammatory cytokines in articular tissue may well act
synergistically and complementarily, because there is a true interplay
between the classic inflammatory mediators and ATP in other systems. In
astrocytes, P2Y modulates IL-1
-mediated signal transduction and
fine-tunes the transcription of genes involved in inflammatory
responses in the human central nervous system (30). Conversely, IL-1
increases the transcription and function of P2Y2 receptors
in vascular smooth muscle cells (31) and in mammalian astrocytes (32).
In our system, ATP rapidly stimulates PGE2 release, within
2-5 min, with a decline after 1 h. By contrast, IL1-
acts
after a delay of several hours. The release of PGE2 from
rabbit articular chondrocytes is maximally stimulated after incubation
with IL1-
for 24 h (13). We believe that ATP plays a role in
initiating the inflammatory process in the joint diseases.
Releases of AA and PGE2 Induced by ATP Involve
cPLA2 Activation--
ATP mobilizes AA in articular
chondrocytes in parallel with the release of PGE2. The
range of concentrations and the pharmacological profiles are consistent
with the activation of a P2Y2-like receptor. Three subtypes
of PLA2: sPLA2 (secretory PLA2),
cPLA2 (cytoplasmic PLA2), and iPLA2
(Ca2+-independent PLA2) could be implicated in
the release of AA from its phospholipid storage sites (33). We find
that cPLA2 is involved in the AA production evoked by ATP,
whereas sPLA2 and iPLA2 are not. IL-1
increases the release of AA from rabbit articular chondrocytes via the
IIA sPLA2 but not via cPLA2 (for instance see
Ref. 13). However, this involves the transcription of the type IIA
sPLA2 gene and so needs a few hours. We proposed that
ATP-induced cPLA2 activity, but not type IIA
sPLA2 activity, triggered the early release of AA, relayed
then by IL-1
-induced type IIA sPLA2 activity but not
cPLA2 activity.
Influence of ERK1/2 and p38 MAPKs on the
ATP-dependent Activation of cPLA2 and
PGE2 Release--
The activation of p38 MAPK by ATP in two
different cell types has been reported recently (34, 35). We have now
demonstrated the ATP-dependent activation of p38 MAPK in
chondrocytes. ATP rapidly and transiently activated p38 MAPK, by acting
on the P2Y2-like receptor borne by articular chondrocytes.
Inhibition of p38 MAPK by SB203580 totally abolishes the action of ATP
on PGE2 release, strongly suggesting that p38 MAPK must be
activated for ATP to stimulate PGE2 synthesis. The p38
MAPKs family can relay multiple inflammatory responses, because they
are essential for the production of proinflammatory cytokines
such as IL-1, TNF-
, and IL-6 (for review see Ref. 36) or
the induction of enzymes like inducible nitric-oxide synthase (37),
cyclooxygenase 2 (38), and PGE synthase,2 particularly in RA
tissues (39). Pharmacological evidence indicates that p38 MAPK is one
of the most validated targets for the future treatment of RA (39) and
OA (40). The induction of p38 MAPK by ATP demonstrated in this study
strongly suggests that ATP regulates other key functions driven by p38
MAPK in articular chondrocytes, in addition to its action on
PGE2 release.
We show that the activation of P2Y2 receptors by ATP leads to the activation of the ERK1/2 MAPK in articular chondrocytes, as it does in other cell types (for review see Ref. 41). PD98059, a specific inhibitor of MEK (MAPK/ERK kinase), the immediate upstream kinase of ERK1/2, totally inhibits the release of PGE2 by chondrocytes. Each specific MAPK inhibitor did not affect the activation of the other MAPK but did prevent significant activation of the whole cascade. Therefore, ATP causes the release of PGE2 in articular chondrocytes by activating both the ERK1/2 and p38 MAPK. Because giving PD98059 and SB203580 separately or in combination caused no difference in the released amount of PGE2 or AA, and because overexpression of DN-MEK and DN-p38 separately or in combination inhibited in the same range the PGE2 release, we can infer that ERK1/2 and p38 act concomitantly rather than cooperatively to mediate the ATP-dependent activation of cPLA2 and release of PGE2, as in macrophages (42) and platelets (43). These effects might be due to each MAPK phosphorylating a different site among the several consensus sites on cPLA2 or to the integration of the phosphorylation of both MAPK by an upstream enzyme, which then activates cPLA2, as in cardiomyocytes (34). Finally, calcium chelation did not prevent the activation of ERK1/2 and p38 MAPKs by ATP; therefore, the activation of the MAPKs is not due to increased intracellular calcium, as in HEK-293 cells (44) or in response to ATP in PC12 cells (45). Thus, ATP activates three distinct intracellular signaling pathways that act independently and simultaneously (Fig. 7).
In conclusion, published data indicate that extracellular purines are a
rapid intercellular communication signal in many systems, including
articular tissue. A significant amount of ATP is released during
inflammation and mechanical loading of cartilage under pathophysiological conditions. ATP contributes to the destructive process by activating signaling pathways involved in articular pathology, PGE2, AA, p38, and ERK1/2 MAPKs, especially in
the early stage of the diseases. Although further in vitro
and in vivo studies are necessary to reveal the complex role
of ATP in cartilage metabolism, the modulation of signaling pathways
triggered by extracellular ATP could clearly help to reduce the joint
destruction that occurs in articular diseases.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Dr. Jean-Denis Troadec for critically reading the manuscript and for helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by CNRS, University Pierre et Marie Curie, the Association de Recherche sur la Polyarthrite, and the Société Française de Rhumatologie.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.
¶ To whom correspondence should be addressed. Tel.: 33-1-44-27-26-92; Fax: 33-1-44-27-51-40; E-mail: sylvie.thirion@snv.jussieu.fr.
Published, JBC Papers in Press, February 18, 2003, DOI 10.1074/jbc.M211570200
2 K. Masuko-Hongo, F. Berenbaum, L. Humbert, C. Salvat, M. G. Attur, M. N. Dave, A. Sautet, M. B. Goldring, A. R. Amin, and S. Thirion, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
RA, rheumatoid
arthritis;
OA, osteoarthritis;
MAPK, mitogen-activated protein
kinase;
ERK, extracellular-regulated protein kinase;
p38 MAPK, p38
mitogen-activated kinase;
JNK, c-Jun NH2-terminal kinase;
MEK1/2, MAPK/ERK kinase;
PGE2, prostaglandin
E2;
AA, arachidonic acid;
IL-1, interleukin-1
;
TNF-
, tumor necrosis factor
;
PLA2, phospholipase A2;
sPLA2, secretory
PLA2;
cPLA2, cytoplasmic PLA2;
iPLA2, Ca2+-independent PLA2;
BAPTA-AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid/acetoxymethyl ester;
RB-2, Reactive Blue 2;
BEL, bromoenol
lactone;
AACOCF3, arachidonyl trifluoromethyl ketone;
P2Y, metabotropic receptors;
PBS, phosphate-buffered saline;
DN, dominant
negative;
ATP
S, adenosine 5'-O-(thiotriphosphate);
RT, reverse transcription.
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