Concomitant Recruitment of ERK1/2 and p38 MAPK Signalling Pathway Is Required for Activation of Cytoplasmic Phospholipase A2 via ATP in Articular Chondrocytes*

Francis BerenbaumDagger §, Lydie HumbertDagger , Gilbert BereziatDagger , and Sylvie ThirionDagger

From the Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-1beta (IL-1beta ) and tumor necrosis factor alpha  (TNF-alpha ) 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-1beta and TNF-alpha 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).

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


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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.

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 ATPgamma 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.

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 ATPgamma 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).

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-1beta strongly stimulates sPLA2 production in this model (13, 19), we used supernatants from chondrocytes stimulated with IL-1beta 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-1beta -stimulated (hatched bar) chondrocytes. Cells were treated for 1 h with 100 µM ATP or for 24 h with 10 ng/ml IL-1beta . 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.

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 (ATPgamma 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.

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.


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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

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).


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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-1beta -mediated signal transduction and fine-tunes the transcription of genes involved in inflammatory responses in the human central nervous system (30). Conversely, IL-1beta 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-beta acts after a delay of several hours. The release of PGE2 from rabbit articular chondrocytes is maximally stimulated after incubation with IL1- beta  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-1beta 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-1beta -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-1beta , TNF-alpha , 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-1beta , interleukin-1beta ; TNF-alpha , tumor necrosis factor alpha ; 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; ATPgamma S, adenosine 5'-O-(thiotriphosphate); RT, reverse transcription.

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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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