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. Author manuscript; available in PMC: 2009 Nov 1.
Published in final edited form as: Arthritis Rheum. 2008 Nov;58(11):3530–3540. doi: 10.1002/art.23958

Prostaglandin D2 inhibits interleukin-1β-induced matrix metalloproteinase-1 and -13 production by human osteoarthritic chondrocytes

Nadia Zayed 1, Hassan Afif 1, Nadir Chabane 1, Leandra Mfuna-Endam 1, Mohamed Benderdour 2, Johanne Martel-Pelletier 1, Jean-Pierre Pelletier 1, Rajender K Motiani 3, Mohamed Trebak 3, Nicolas Duval 4, Hassan Fahmi 1
PMCID: PMC2657141  NIHMSID: NIHMS64511  PMID: 18975308

Abstract

Objective

To investigate the effects of prostaglandin (PG) D2 on interleukin-1β (IL-1)-induced matrix metalloproteinase (MMP)-1 and MMP-13 expression in human chondrocytes and the signalling pathways involved in the effects of PGD2.

Methods

Chondrocytes were stimulated with IL-1 ± PGD2 and MMP-1 and MMP-13 protein expression was evaluated by ELISA. mRNA expression and promoter activity were analyzed by real-time RT-PCR and transient transfections, respectively. The role of the PGD2 receptors, D prostanoid receptor 1 (DP1) and chemoattractant-receptor-like molecule expressed on Th2 cells (CRTH2), was evaluated using specific agonists and antibody blocking experiments. The contribution of the cAMP/PKA pathway was determined using cAMP elevating agents and PKA inhibitors.

Results

PGD2 dose-dependently decreased IL-1-induced MMP-1 and MMP-13 protein and mRNA expression as well as their promoter activation. DP1 and CRTH2 are expressed and functional in chondrocytes. The effect of PGD2 was mimicked by the selective DP1 agonist BW245C, but not by the CRTH2 selective agonist DK-PGD2. Furthermore, treatment with an anti-DP1 antibody reversed the effect of PGD2, indicating that the inhibitory effect of PGD2 is mediated by DP1. The cAMP elevating agents, 8-Br-cAMP and forskolin, suppressed IL-1-induced MMP-1 and MMP-13 expression, and the PKA inhibitors, KT5720 and H-89, reversed the inhibitory effect of PGD2, suggesting that the effect of PGD2 is mediated by the cAMP/PKA pathway.

Conclusion

PGD2 inhibits IL-1-induced MMP-1 and MMP-13 production by chondrocytes through the DP1/cAMP/PKA signalling pathway. These data also suggest that modulation of PGD2 levels in the joint may have therapeutic potential in the prevention of cartilage degradation.

INTRODUCTION

The destruction of articular cartilage is a typical pathological characteristic of arthritic diseases such as osteoarthritis (OA) and rheumatoid arthritis (RA). The degradative process is believed to be largely mediated by proteases belonging to the metalloproteinase (MMP) class of enzymes (1). Among these, MMP-1 and MMP-13 are considered to be of particular interest since they directly degrade the components of the cartilage matrix including aggrecan and collagen (2-4). Proinflammatory cytokines such as interleukin-1β (IL-1), are known to strongly induce the production of MMP-1 and MMP-13 by articular joint cells, including chondrocytes (4; 5).

Many inhibitors of MMP activity have been developed over the past 25 years and have been shown to inhibit cartilage and bone destruction in several animal models of OA (6; 7) and RA (8; 9). However, results from clinical trials failed to demonstrate a beneficial effect of MMP activity inhibition on the progression of joint damage in arthritis (10-12). Therefore, an understanding of the factors and pathways that regulate MMP-1 and -13 expression is of major importance to understand and possibly to prevent cartilage damage in arthritis.

In addition to MMPs, prostaglandins (PGs) also play an important role in cartilage metabolism and inflammation associated with arthritis (13) and inhibitors of PG production are widely used in the treatment of OA and RA. PGs are formed from arachidonic acid through the action of cyclooxygenase (COX). COX converts arachidonic acid to an intermediate substrate PGH2, which is further metabolized by specific terminal synthases to generate PGE2, PGD2, PGF2α, PGI2 and thromboxane (13). PGD2 is involved in the regulation of multiple physiological and pathological processes, including sleep, nociception, vasodilatation, bronchoconstriction, and bone metabolism. In addition, PGD2 has been shown to display anti-inflammatory effects in several models of inflammation (14-17). PGD2 elicits its downstream effects by activating two plasma membrane receptors, the D prostanoid receptor (DP) 1 (18) and chemoattractant-receptor-like molecule expressed on Th2 cells (CRTH2), also known as DP2 (19). DP1 stimulation results in an increase in intracellular levels of cAMP and subsequent activation of protein kinase A (PKA) (20), while CRTH2 activation leads to calcium mobilization (19).

Most studies to date addressing the role of PGs in chondrocyte metabolism have focused on PGE2, while much less is known about the role of PGD2 in this process. In the present study we investigated the effect of PGD2 on MMP-1 and MMP-13 production by human chondrocytes. We demonstrated that PGD2 inhibits IL-1-induced MMP-1 and MMP-13 production. We also showed that this effect is mediated through the DP1/cAMP/PKA signalling pathway.

MATERIALS AND METHODS

Reagents

Recombinant human (rh) IL-1β was obtained from Genzyme (Cambridge, MA). PGD2, BW245C, 13,14-dihydro-15-keto-PGD2 (DK-PGD2), and anti-DP1 antibody were from Cayman Chemical Co. (Ann Arbor, MI). Anti-CRTH2 antibody was from BD Pharmingen (Mississauga, ON, Canada). 8-Bromo-cAMP (8-Br-cAMP), 3-isobutyl-1-methylxanthine (IBMX), and the PKA inhibitors H-89 and KT5720 were from Calbiochem, EMD Biosciences (San Diego, CA). Cycloheximide, ionomycin and forskolin were from Sigma-Aldrich Canada (Oakville, ON, Canada). Dulbecco’s modified Eagle’s medium (DMEM), penicillin and streptomycin, fetal calf serum (FCS), and TRIzol® reagent were from Invitrogen (Burlington, ON, Canada). All other chemicals were purchased from either Sigma-Aldrich Canada or Bio-Rad (Mississauga, ON, Canada).

Chondrocyte isolation and treatment

Articular cartilage samples from femoral condyles and tibial plateaus were obtained from OA patients undergoing total knee replacement (n = 46, mean ± SD age: 67 ± 17 years). All OA patients were diagnosed according to the criteria developed by the American College of Rheumatology Diagnostic Subcommittee for OA (21). At the time of surgery, the patients had symptomatic disease requiring medical treatment in the form of non-steroidal anti-inflammatory drugs (NSAIDs) or selective COX-2 inhibitors. Patients who had received intraarticular injection of steroids were excluded. The Clinical Research Ethics Committee of Notre-Dame Hospital approved the study protocol and the use of human articular tissues.

Chondrocytes were released from cartilage by sequential enzymatic digestion as previously described (22). Briefly, small pieces of cartilage were incubated with 2 mg/ml pronase for 1 hour followed by 1 mg/ml collagenase (type IV; Sigma-Aldrich) for 6 hours at 37°C in DMEM and antibiotics (100 U/ml penicillin, 100 μg/ml streptomycin). The digested tissue was briefly centrifuged and the pellet was washed. The isolated chondrocytes were seeded at high density in tissue culture flasks and cultured in DMEM supplemented with 10% heat-inactivated FCS.

Confluent chondrocytes were detached by trypsinization, seeded at 3.5×105cells per well in 12-well culture plates (Costar, Corning, NY) or at 7×105 cells per well in 6-well culture plates in DMEM supplemented with 10% FCS, and cultivated at 37°C for 48 hours. Cells were washed and incubated for an additional 24 hours in DMEM containing 0.5% FCS, before stimulation with either IL-1 alone or in combination with PGD2, BW-245C, DK-PGD2, 8Br-cAMP or forskolin, (The drugs were added at the same time as IL-1). In another set of experiments chondrocytes were pretreated for 30 minutes with cycloheximide, IBMX, PKA inhibitors (H89, KT-5720), anti-DP1 or anti-CRTH2 antibody before stimulation with IL-1 or PGD2. The level of MMP proteins released in supernatants was determined 24 hours after stimulation, whereas the level of MMP mRNA was determined at 8 hours. Only first passaged chondrocytes were used.

Cartilage explants were placed in the wells of a 24-well plate (80-100 mg/well) and maintained in DMEM with 0.5% FCS for 72 hours. The explants were washed and incubated for an additional 24 hours in the same medium before stimulation with IL-1 alone or co-stimulation with IL-1 and PGD2 (IL-1 and PGD2 were added simultaneously) for 72 hours. Conditioned media were harvested and the levels of MMP proteins were determined by ELISA.

MMP-1, MMP-13 and cAMP determinations

The levels of MMP-1 and MMP-13 in conditioned media were determined by specific enzyme-linked immunosorbent assays (ELISA, R&D Systems Inc, Minneapolis, MN). The intracellular levels of cAMP were measured using a cAMP enzyme immunoassay (EIA) from R&D systems Inc. All measurements were performed in duplicate.

Western blot analysis

Chondrocytes were lysed in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1 mM PMSF, 10 μg/ml each of aprotinin, leupeptin, and pepstatin, 1% NP-40, 1 mM Na3VO4, and 1 mM NaF). Lysates were sonicated on ice and centrifuged at 12 000 rpm for 15 minutes. The protein concentration of the supernatant was determined using the bicinchoninic acid method (Pierce, Rockford, IL). Twenty μg of total cell lysate was subjected to SDS-polyacrylamide gel electrophoresis and electrotransferred to a nitrocellulose membrane (Bio-Rad). After blocking in 20 mM Tris-HCl pH 7.5 containing 150 mM NaCl, 0.1% Tween 20, and 5% (w/v) non-fat dry milk, blots were incubated overnight at 4°C with the primary antibody and washed with a Tris buffer (Tris-buffered saline (TBS) pH 7.5, with 0.1% Tween 20). The blots were then incubated with horseradish peroxidase-conjugated secondary antibody (Pierce), washed again, incubated with SuperSignal Ultra Chemiluminescent reagent (Pierce), and exposed to Kodak X-Omat film (Eastman Kodak Ltd, Rochester, NY).

RNA extraction and real-time quantitative PCR

Total RNA was isolated using the TRIzol® reagent (Invitrogen) according to the manufacturer’s instructions. To remove contaminating DNA, isolated RNA was treated with RNase-free DNase I (Ambion, Austin, TX). The RNA was quantitated using the RiboGreen RNA quantitation kit (Molecular Probes, Eugene, OR), dissolved in diethylpyrocarbonate (DEPC)-treated-H2O and stored at -80°C until use. One μg of total RNA was reverse-transcribed using Moloney Murine Leukemia Virus reverse transcriptase (Fermentas, Burlington, ON, Canada) as detailed in the manufacturer’s guidelines. One fiftieth of the reverse transcriptase reaction was analyzed by traditional PCR or real-time quantitative PCR. The following primers were used: MMP-1, sense 5′-CTGAAAGTGACTGGGAAACC-3′ and antisense 5′-AGAGTTGTCCCGATGATCTC-3′; MMP-13, sense 5′-CTT AGA GGT GAC TGG CAA AC-3′ and antisense 5′-GCC CAT CAA ATG GGT AGA AG-3′; glyceraldehyde-3- phosphate dehydrogenase (GAPDH), sense 5′-CAGAACATCATCCCTGCCTCT-3′ and antisense 5′-GCTTGACAAAGTGGTCGTTGAG -3′; DP1, sense 5′-GCAACCTCTATGCGATGCAC and antisense 5′-CAAGGCTCGGAGGTC TTCT-3′, and CRTH2, sense 5′-CCTCTGTGCCCAGAGCCCCACGATGTCGGC-3′ and antisense 5′-CACGGCCAAGAAGTAGGTGAAGAAG-3′

Quantitative PCR analysis was performed in a total volume of 50 μl containing template DNA, 200 nM of sense and antisense primers, 25 μl of SYBR® Green master mix (QIAGEN, Mississauga, ON, Canada) and uracil-N-glycosylase (UNG, 0.5 Unit, Epicentre Technologies, Madison, WI). After incubation at 50°C for 2 minutes (UNG reaction), and at 95°C for 10 minutes (UNG inactivation and activation of the AmpliTaq Gold enzyme), the mixtures were subjected to 40 amplification cycles (15 sec at 95°C for denaturation and 1 minutes for annealing and extension at 60°C). Incorporation of SYBR® Green dye into PCR products was monitored in real time using a GeneAmp 5700 Sequence detection system (Applied Biosystems, Foster City, CA) allowing determination of the threshold cycle (CT) at which exponential amplification of PCR products begins. After PCR, dissociation curves were generated with one peak, indicating the specificity of the amplification. A threshold cycle (CT value) was obtained from each amplification curve using the software provided by the manufacturer (Applied Biosystems).

Relative mRNA expression in chondrocytes was determined using the ΔΔCT method, as detailed in the manufacturer’s guidelines (Applied Biosystems). A ΔCT value was first calculated by subtracting the CT value for the housekeeping gene GAPDH from the CT value for each sample. A ΔΔCT value was then calculated by subtracting the ΔCT value of the control (unstimulated cells) from the ΔCT value of each treatment. Fold changes compared with the control were then determined by raising 2 to the -ΔΔCT power. Each PCR reaction generated only the expected specific amplicon as shown by the melting-temperature profiles of the final product and by gel electrophoresis of test PCR reactions. Each PCR was performed in triplicate on two separate occasions for each independent experiment.

Plasmids and transient transfection

The human MMP-1 promoter/luciferase reporter plasmid (pMMP1-0.5kb-Luc) was kindly provided by Dr. C.E. Brinckerhoff (Dartmouth Medical School, Hanover, NH). The human MMP-13 promoter (pMMP-13-1.6kb-Luc) has been previously described (22). β-galactosidase reporter vector under the control of SV40 promoter (pSV40-β-galactosidase) was from Promega (Madison, WI). Transient transfection experiments were performed using FuGene-6 (1 μg DNA: 4 μl FuGene 6) (Roche Applied Science, Laval, QC, Canada) according to the manufacturer’s recommended protocol. Briefly, chondrocytes were seeded 24 hours prior to transfection at a density of 6.105 cells/well in 6-well plates and transiently transfected with 1 μg of the reporter construct and 0.5 μg of the internal control pSV40-β-galactosidase. Six hours later, the cells were rinsed in phosphate-buffered saline and changed to medium containing 0.5% FCS for an additional 18 hours. The cells were then treated with IL-1 in the absence or presence of PGD2 for 18 hours. In these conditions, transfection efficiency typically ranges between 40% and 50%. After harvesting, luciferase activity was determined and normalized to β-galactosidase activity (22). All of the transfection experiments were repeated at least three times in duplicate.

Calcium mobilization assay

Ca2+ mobilization assays were performed as previously described(23). Briefly, coveslips with attached chondrocytes were mounted in a Teflon chamber and incubated at 37°C for 50 minutes in culture media containing 5 μM Fura-2 AM (Molecular Probes, Eugene, OR). Cells were then washed 3 times and bathed in HEPES-buffered saline solution (140 mM NaCl, 4.7 mM KCl, 2 mM CaCl2, 1.13 mM MgCl2, 10 mM glucose, and 10 mM HEPES, pH 7.4) for at least 10 minutes before Ca2+ measurements were made. Baseline was established a few minutes in the presence of extracellular Ca2+, before addition of PGD2 where indicated. Fura-2 fluorescence at an emission wavelength of 510 nm was induced by exciting Fura-2 alternately at 340 and 380 nm. Fluorescence images of several cells were recorded and analyzed with a digital fluorescence imaging system (InCyt Im2; Intracellular Imaging Inc., Cincinnati, OH). The data are expressed as the ratio of Fura-2 fluorescence due to excitation at 340 nm to that due to excitation at 380 nm.

Statistical analysis

Data are expressed as the mean ± SEM. Statistical significance was assessed by the 2-tailed Student’s t-test. P values less than 0.05 were considered statistically significant.

Results

PGD2 inhibits IL-1-induced MMP-1 and MMP-13 protein production in human chondrocytes and cartilage explants

To examine the effects of PGD2 on MMP-1 and MMP-13 protein release, cultured human chondrocytes were stimulated with IL-1 (100 pg/ml) in the absence or presence of increasing concentrations of PGD2 (1-10 μM), and the amount of MMP-1 and MMP-13 proteins in conditioned media were measured by ELISA. As expected, IL-1 strongly enhanced the production of both MMP-1 and MMP13. Treatment with PGD2 inhibited IL-1-induced MMP-1 and MMP-13 production in a dose-dependent manner (Fig. 1A). The utilized concentrations of PGD2 did not affect chondrocyte viability as judged by the trypan blue exclusion assay and the MTT salt assay (data not shown). Next, we evaluated the effect of PGD2 on IL-1-induced MMP-1 and MMP-13 protein production in cartilage explants. As shown in Fig. 1B, PGD2 dose-dependently suppressed IL-1-induced MMP-1 and MMP-13 release from cartilage explants.

Figure 1.

Figure 1

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Inhibition by PGD2 of IL-1-induced MMP-1 and MMP-13 protein release in cultured chondrocytes and cartilage explants A, Chondrocytes were stimulated with either IL-1 alone (100 pg/ml) or co-stimulated with IL-1 and increasing concentrations of PGD2 for 24 h. B, Cartilage explants were stimulated with IL-1 alone (1 ng/ml) or co-stimulated with IL-1 and increasing concentrations of PGD2 for 72 h. The levels of MMP-1 and MMP-13 proteins in conditioned media were measured by ELISA. Results are expressed as the percentage of control, considering 100% as the value of cells or explants treated with IL-1 alone and represent the mean ± SEM of four independent experiments. * p< 05 compared with cells or explants treated with IL-1 alone.

PGD2 inhibits IL-1-induced MMP-1 and MMP-13 expression at the transcriptional level

To further characterize the effect of PGD2 on IL-1-induced MMP-1 and MMP-13 expression, we measure"dthe steady-state level of MMP-1 and MMP-13 mRNAs by real-time PCR. As shown in Fig. 2A, IL-1 dramatically increased the expression of MMP-1 and MMP-13 mRNAs (32-fold and 26-fold respectively). Consistent with its effects on protein expression, PGD2 dose-dependently suppressed the induction of MMP-1 and MMP-13 mRNAs by IL-1. Next, we evaluated the effect of PGD2 on IL-1-mediated activation of MMP-1 and MMP-13 promoters. Chondrocytes were transiently transfected with the human MMP-1 or MMP-13 promoter-luciferase reporter gene and stimulated with IL-1 in the absence or presence of PGD2. As shown in Fig. 2B, IL-1 activated MMP-1 and MMP-13 promoters and this activation was decreased in a dose-dependent manner by PGD2. Together, these data suggest that PGD2 suppress IL-1-induced MMP-1 and MMP-13 expression at the transcriptional level.

Figure 2.

Figure 2

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PGD2 down-regulates IL-1-induced MMP-1 and MMP-13 expression at the transcriptional level and does not involve de novo protein synthesis. A, Chondrocytes were treated with 100 pg/ml IL-1 in the absence or presence of increasing concentrations of PGD2 for 8 h. Total RNA was isolated, reverse transcribed into cDNA, and MMP-1 and MMP-13 mRNAs were quantified using real-time PCR. The housekeeping gene GAPDH was used for normalization. All experiments were performed in triplicate, and negative controls without template RNA were included in each experiment. B, Chondrocytes were co-transfected with 1 μ/well of either the MMP-1 (pMMP1-0.5kb-Luc) or the MMP-13 (pMMP-13-1.6kb-Luc) promoter and 0.5 μ/well of the internal control pSV40-β-galactosidase, using FuGene 6 transfection reagent. Six hours later, the cells were washed and changed to medium containing 0.5% FCS for an additional 18 hours. Transfected cells were then treated with 100 pg/ml IL-1 in the absence or presence of increasing concentrations of PGD2 for 18 h. Luciferase activity values were determined and normalized to β-galactosidase activity. All experiments were repeated at least three times in duplicate. C. Chondrocytes were incubated with cycloheximide (10 μ/ml) for 30 minutes prior to stimulation with 100 pg/ml IL-1 in the absence or presence of 10 μM PGD2 for 8 hours. Total RNA was isolated, reverse transcribed into cDNA, and MMP-1 and MMP-13 mRNAs were quantified using real-time PCR. Results are expressed as -fold changes, considering 1 as the value of untreated cells, and represent the mean ± SEM of 4 independent experiments. *p<0.05 compared with cells treated with IL-1 alone.

Role of protein synthesis in PGD2-mediated inhibition of IL-1-induced MMP-1 and MMP-13 expression

To determine whether the effect of PGD2 on IL-1-induced MMP-1 and MMP-13 expression is direct or indirect, we tested the impact of the protein synthesis inhibitor cycloheximide (CHX). Chondrocytes were pretreated with CHX for 30 minutes and stimulated with IL-1 alone or in combination with PGD2 for 8 hours. The levels of MMP-1 and MMP-13 mRNAs were analyzed by real-time PCR. Pretreatment with CHX did not affect PGD2-mediated inhibition of IL-1-induced MMP-1 and MMP-13 expression (Fig. 2C), suggesting that the suppressive effect of PGD2 was a direct primary effect through pre-existing factors and was not dependent on de novo protein synthesis.

DP1 and CRTH-2 are expressed and functional in human chondrocytes

To determine the mechanisms involved in PGD2-mediated down-regulation of IL-1-induced MMP-1 and -13 expression, we first examined the expression of mRNA corresponding to the two plasma membrane PGD2 receptors DP1 and CRTH2. RT-PCR analysis demonstrated the presence of both DP1 and CRTH-2 mRNAs in three chondrocyte populations derived from three different donors (Fig. 3A. These results were confirmed by Western blot analysis using specific anti-DP1 and anti-CRTH2 antibodies (Fig. 3B). Human osteoblasts express both DP1 and CRTH2 and were used as positive controls.

Figure 3.

Figure 3

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DP1 and CRTH2 are expressed and functional in chondrocytes.A, Total RNA of chondrocytes from three different donors was reverse transcribe"dand amplified by PCR using specific primers for DP1, CRTH2, and GAPDH. PCR in the absence of RT and with RNA from human osteoblasts were used as negative and positive controls, respectively. B, Cell lysates from chondrocytes obtained from three different donors were analyzed for DP1 and CRTH2 protein expression by Western blotting using specific anti-DP1 and anti-CRTH2 antibodies. Protein extracts from human osteoblasts were used as positive control. C. Effect of PGD2 on intracellular levels of cAMP. Chondrocytes were pre-treated with 10 μM IBMX for 30 minutes followed by incubation with PGD2 (10 μM) for the indicated time periods. cAMP levels were measured by EIA. Results are expressed as pmol/106 cells and represent the mean ± SEM of four independent experiments. *p<0.05 compared with untreated cells. D. Effect of PGD2 on Ca2+ mobilization in chondrocytes. Chondrocytes were loaded with Fura-2 and stimulated with 10 μM PGD2 in a nominally Ca2+ free solution, followed by replenishment of calcium (2 mM) to the extracellular milieu where indicated. Ionomycin was added as a control at the end of the experiment to assess the maximum Fura-2 signal in the cells. The changes in intracellular levels of Ca2+ were monitored as described in Materials and Methods. Traces obtained with individuals cells (upper panel) and the average (lower panel) are presented. The data shown are representative of four independent analyses from separate donors, each showing similar results.

Activation of DP1 by PGD2 leads to increased intracellular cAMP levels (20), whereas activation of CRTH2 by PGD2 results in intracellular Ca2+ mobilization (19). To determine whether DP1 and CRTH2 are functional in chondrocytes, we examined the capacity of PGD2 to modulate the intracellular levels of cAMP and to induce Ca2+ mobilization. As shown in Fig. 3C, treatment with PGD2 enhanced intracellular levels of cAMP (Fig. 3C) in a time dependent-manner, suggesting that DP1 is functional in chondrocytes. To test whether PGD2 can induce Ca2+ mobilization in chondrocytes, cells were first stimulated with PGD2 in a nominally Ca2+ free solution to assess Ca2+ release from internal stores, followed by replenishment of Ca2+ (2 mM) to the extracellular milieu, to evaluate calcium entry. Ionomycin was added as a control at the end of Ca2+ imaging experiment to assess the maximum Fura-2 signal in the cells. The results shown in Fig. 3D indicate that CRTH2 is functional in chondrocytes and are consistent with the activation of a receptor coupled to a phosphoinositide-specific phospholipase, inducing Ca2+ release from the internal stores and Ca2+ entry through plasma membrane channels.

As shown in Fig. 3C and D, treatment with PGD2 enhanced intracellular levels of cAMP (Fig. 3C) and induced Ca2+ mobilization (Fig. 3D) in chondrocytes. These results indicate that chondrocytes express functional DP1 and CRTH2 and suggest that PGD2 might modulate IL-1-induced MMP-1 and MMP-13 production through activation of either PGD2 receptor.

Inhibition of IL-1-induced MMP1 and MMP-13 expression by PGD2 is mediated by the DP1, but not the CRTH2, receptor

To determine which of these receptors is responsible for the observed effect of PGD2, we examined the effects of BW245C, a specific DP1 agonist (24; 25), and DK-PGD2, a specific CRTH2 agonist (26), on IL-1-induced MMP-1 and MMP-13 production. The results show that BW245C dose-dependently prevented IL-1-induced MMP-1 and MMP-13 production (Fig. 4). In contrast, DK-PGD2 was without effect on IL-1-induced MMP-1 and MMP-13 expression (Fig. 4). These data suggest that PGD2 modulates IL-1-induced MMP-1 and MMP-13 gene expression through activating the DP1 receptor.

Figure 4.

Figure 4

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Effects of DP1 and CRTH2 agonists on IL-1-induced MMP-1 and MMP-13 production. Chondrocytes were treated with 100 pg/ml IL-1 in the absence or presence of increasing concentrations of BW245C or DK-PGD2 for 24 h. The levels of MMP-1 and MMP-13 in conditioned media were evaluated by ELISA. Results are expressed as the percentage of control, considering 100% as the value of cells treated with IL-1 alone, and represent the mean ± SEM of four independent experiments. *p<0.05 compared with cells treated with IL-1 alone.

To confirm the involvement of DP1 in the inhibitory effect of PGD2, chondrocytes were treated with different dilutions of a DP1 polyclonal antibody and then stimulated with IL-1 in the absence or presence of PGD2 and the levels of MMP-1 and MMP-13 were evaluated in conditioned media. As shown in Fig 5A, treatment with the DP1 polyclonal antibody reversed the inhibitory effect of PGD2 in a dose-dependent manner. In contrast, treatment with an anti-CRTH2 antibody had no effect on PGD2-mediated down-regulation of IL-1-induced MMP-1 and MMP-13 expression. The ineffectiveness of the anti-CRTH2 antibody to prevent the effect of PGD2 on MMP-1 and MMP-13 release was not due to its inability to block CRTH2 since it completely blocked PGD2-induced Ca2+ mobilization (Fig. 5B). Taken together these data strongly suggest that the inhibitory effect of PGD2 on MMP-1 and MMP-13 production is mediated by DP1, but not CRTH2, activation

Figure 5.

Figure 5

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PGD2 inhibits IL-1-induced MMP-1 and MMP-13 production via DP1. A. Chondrocytes were pre-treated with different dilutions (1:1000, 1:500 and 1:100) of anti-DP1 or anti-CRTH2 antibodies for 30 minutes, then stimulated with IL-1 (100 pg/ml) in the absence or presence of PGD2 (10 μM) for 24 h. The levels of MMP-1 and MMP-13 in conditioned media were evaluated by ELISA. Results are expressed as the percentage of control, considering 100% as the value of cells treated with IL-1 alone, and represent the mean ± SEM of four independent experiments. *p<0.05 compared with cells treated with IL-1 + PGD2. B. Inhibition of PGD2- induced Ca2+ mobilization by the anti-CRTH2 antibody. Fura-2-loaded chondrocytes were either directly subjected to Ca2+ mobilization assay (upper panel) or pretreated with the anti-CRTH2 antibody (final 1/100) at room temperature for 20 minutes, then subjected to the assay (lower panel). These measurements were performed in continuous presence of extracellular Ca2+ (2 mM). Arrows indicate the time of addition of PGD2 (10 μM) or inomycin (10 μM). Changes in intracellular levels of Ca2+ were monitored as described in Materials and Methods. Traces represent average from (6-14) cells per condition and are representative of at least four independent experiments.

The inhibitory effect of PGD2 is mediated by the cAMP/protein kinase A (PKA) pathway

To investigate the role of the cAMP/PKA pathway in DP1-mediated inhibition of MMP-1 and MMP-13 expression by PGD2, we first examined whether elevating intracellular levels of cAMP would affect IL-1-induced MMP-1 and MMP-13 production. Chondrocytes were stimulated with IL-1 in the absence or presence of two cAMP elevating agents, 8-Br-cAMP, a membrane-permeable cAMP analog, and forskolin, an adenylate cyclase activator. Twenty four hours later, the MMP-1 and MMP-13 content in conditioned media were analyzed by ELISA. Interestingly, both 8-Br-cAMP and forskolin prevented IL-1-induced MMP-1 and MMP-13 production (Fig. 6A). 8-Br-cAMP and forskolin did not have any significant effect on cell viability as judged by the trypan blue exclusion assay (data not shown).

Figure 6.

Figure 6

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Role of the cAMP-PKA pathway in the inhibition of IL-1-induced MMP-1 and -13 by PGD2. A, Effect of cAMP elevating agents on IL-1-induced MMP-1 and MMP-13 production. Chondrocytes were treated with 100 pg/ml IL-1 in the absence or presence of 8-Br-cAMP (0.5 mM) or forskolin (10 μM) for 24 h. B, Effect of PKA inhibitors on PGD2-mediated down-regulation of IL-1-induced MMP-1 and -13 production. Chondrocytes were pre-treated with the PKA inhibitors H89 (1 μM) or KT5720 (1 μM) for 30 min before co-stimulation with IL-1 (100 pg/ml) and PGD2 (10 μM) for 24 h. The levels of MMP-1 and MMP-13 in conditioned media were evaluated by ELISA. Results are expressed as the percentage of control, considering 100% as the value of cells treated with IL-1 alone and represent the mean ± SEM of four independent experiments. * p<0.05 compared with cells treated with IL-1 alone (A) or IL-1 + PGD2 (B).

Next, we evaluated the effect of inhibitors of PKA, a major effector of cAMP actions, on PGD2-mediated suppression of IL-1-induced MMP-1 and MMP-13 production. Treatment with H89 (1 μM) or KT5720 (1 μM), two PKA inhibitors, dramatically antagonized the inhibitory effect of PGD2 on IL-1-induced MMP1 and MMP-13 protein production (Fig. 6B). Together, these data indicate that PGD2 inhibits IL-1-induced MMP-1 and MMP-13 production by activating the cAMP/PKA pathway.

DISCUSSION

In this study we demonstrate for the first time that PGD2 inhibits IL-1-induced MMP-1 and MMP-13 expression in human chondrocytes. We also provide evidence that the inhibitory effect of PGD2 is mediated by the DP1 receptor through the cAMP-PKA signalling pathway. Thus, PGD2 has the potential to prevent cartilage damage by blocking the expression of MMP-1 and MMP-13. PGD2 elicits its effects through two plasma membrane receptors, the DP1 and CRTH2 (also named DP2) (18; 19). To determine which receptor mediates the inhibitory effect of PGD2 on IL-1-induced MMP-1 and -13 expression, we first investigated the expression of DP1 and CRTH-2 in chondrocytes. We found that DP1 and CRTH2 are expressed at both the mRNA and protein levels. We also showed that treatment with PGD2 increased intracellular levels of cAMP and induced Ca2+ mobilization thereby confirming the presence of functional DP1 and CRTH2 receptors in chondrocytes. To our knowledge this is the first report showing that DP1 and CRTH2 are expressed and functinal in chondrocytes. We next investigated the effect of specific agonists of DP1 and CRTH2 on IL-1-induced MMP-1 and MMP-13 expression. Importantly, we found that the DP1 agonist BW245C, but not the CRTH2 agonist DK-PGD2, reduced IL-1-induced MMP-1 and MMP-13 expression. In line with this finding, treatment with a DP1 polyclonal antibody blocked the inhibitory effect of PGD2 on IL-1-induced MMP-1 and MMP-13 expression. Taken together these findings suggest that PGD2 down-regulates IL-1-induced MMP1 and MMP-13 expression through activation of the DP1 receptor. Although the inhibitory effect of BW245C was as potent as that of PGD2, we cannot rule out the possibility that PGD2 downregulates IL-1-induced MMP-1 and MMP-13 expression through DP1-independent mechanisms. Indeed, PGD2 can be non-enzymatically converted to 15-deoxy-Δ 12,14-PGJ2 (15d-PGJ2) (27), which has been shown to inhibit the induction of MMP-1 and MMP-13 expression in several cell types including chondrocytes (22; 28). Further studies are clearly warranted to define whether DP1-independent mechanisms are involved in the inhibitory effect of PGD2 on IL-1-induced MMP-1 and MMP-13 expression in chondrocytes.

In general, occupancy of DP1 activates adenylate cyclase and enhances intracellular cAMP levels leading to activation of PKA (20). Our results suggest that the intracellular mechanisms that mediate the inhibitory effect of PGD2 on IL-1-induced MMP-1 and MMP-13 expression involve the cAMP-PKA pathway. This is supported by the fact that stimulation of chondrocytes with PGD2 increases the intracellular levels of cAMP. Moreover, cAMP elevating agents inhibited IL-1-induced MMP-1 and MMP-13 expression. Finally, the inhibitory effect of PGD2 was blocked by inhibitors of PKA (H89 or KT5720). These data thus identified the DP1/cAMP/PKA signalling pathway as the intracellular mechanism by which PGD2 prevents MMP-1 and MMP-13 production in IL-1-stimulated chondrocytes.

PGD2 is among the most abundantly produced prostaglandin in synovial fluid (29) and can be released by several cell types present within the joint including chondrocytes (30), osteoblasts (31), synovial fibroblasts (32), and synovial mast cells (33), suggesting that PGD2 can contribute to the maintenance of cartilage homeostasis. Recently, PGD2 was shown to prevent chondrocyte apoptosis (34) and to enhance chondrogenic differentiation and hyaline cartilage matrix deposition (35). PGD2 was also reported to stimulate collagen synthesis (36), indicating that PGD2 may protect cartilage integrity not only through inhibition of MMP-1 and MMP-13 expression, but also by enhancing chondrocyte anabolic events. Furthermore, several lines of evidence suggest that PGD2 has anti-inflammatory properties. For instance, the initiation of inflammation in a number of animal models of inflammation, was shown to be associated with reduced production of PGD2, while the resolution phase was associated with increased levels of PGD2 (14; 15; 17). Recently, Trivedi et al (37) analyzed the duration and severity of delayed type hypersensitivity reaction in PGD2 synthase knockout and transgenic mice and found that knockout mice exhibit a more severe inflammatory response that failed to resolve, whereas transgenic mice had little detectable inflammation. Moreover, retrovirally mediated ectopic expression of PGD2 synthase reduces several inflammatory responses and cellular infiltration in a murine air-pouch model of monosodium urate monohydrate crystal-induced inflammation (16). Thus, in addition to its chondroprotective effects, PGD2 can also reduce inflammatory responses, suggesting that PGD2 may be of therapeutic value in arthritis.

Nonsteroidal anti-inflammatory drugs (NSAIDs) which act through COX inhibition are widely used to relieve pain and inflammation in arthritis, but NSAIDs might also have deleterious effects on cartilage. Indeed, indomethacin and diclofenac were shown to display chondrotoxic effects in animal models of OA (38; 39) and to accelerate radiographic OA progression in patients with hip and knee OA (40; 41). This is consistent with in vitro studies showing that several NSAIDs (such as indomethacin, nimesulide, ibuprofen and naproxen) inhibit the synthesis of proteoglycans and collagens by articular cartilage explants (42). These data together with the demonstration that PGD2 may have chondroprotective effects suggest that inhibition of endogenous PGD2 biosynthesis by NSAIDs may be related to their deleterious effect on cartilage and underline the limits of therapeutic approaches for the treatment of inflammatory joint diseases by inhibition of all prostaglandin biosynthesis

In conclusion, our data show that PGD2 inhibits IL-1-induced MMP-1 and MMP-13 expression by chondrocytes. This effect is mediated through DP1 and involves the cAMP/PKA pathway. These results suggest that modulation of PGD2 levels within the joint might be of therapeutic interest in arthritis.

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Acknowledgements

This work was supported by the Canadian Institutes of Health Research (CIHR) Grant MOP-84282, and the Fonds de la Recherche du Centre de Recherche du Centre Hospitalier de l’Université de Montréal (CHUM). HF is a Research Scholar of the Fonds de Recherche en Santé du Québec (FRSQ). MT is supported by an early career NIH grant (K22ES014729) and by start up funds from the Albany Medical College, NY

List of abbreviations

cAMP

cyclic adenosine monophosphate

COX

cyclooxygenase

DP1

PGD2 receptor 1

IL

interleukin

MMP

matrix metalloproteinase

OA

Osteoarthritis

PGD2

prostaglandin D2

PKA

protein kinase A

RA

rheumatoid arthritis

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