• Top
• Abstract
• Introduction
• Materials and methods
• Statistical a...
• Results
• Discussion
• Conclusion
• Abbreviations
• Competing interests
• Authors' contributions
• Acknowledgements
• References

Research article

Increased expression of lipocalin-type prostaglandin D₂ synthase in osteoarthritic cartilage

Nadia Zayed¹ , Xinfang Li¹ , Nadir Chabane¹ , Mohamed Benderdour² , Johanne Martel-Pelletier¹ , Jean-Pierre Pelletier¹ , Nicolas Duval³ and Hassan Fahmi¹

¹Osteoarthritis Research Unit, Research Centre of the University of Montreal Hospital Center (CR-CHUM), Notre-Dame Hospital, 1560 Sherbrooke Street East, J.A. DeSève Pavilion, Y-2628, and Department of Medicine, University of Montreal, Montreal, QC, H2L 4M1, Canada

²Research Centre, Sacré-Coeur Hospital, 5400, Gouin Boulevard West, Montreal, QC, H4J 1C5, Canada

³Centre de Convalescence, de Charmilles Pavillon, 1487 des Laurentides Boulevard, Montreal, QC, H7M 2Y3, Canada

author email corresponding author email

Arthritis Research & Therapy 2008, 10:R146doi:10.1186/ar2581

The electronic version of this article is the complete one and can be found online at: http://arthritis-research.com/content/10/6/R146

Received:	12 September 2008
Revisions requested:	17 October 2008
Revisions received:	2 December 2008
Accepted:	18 December 2008
Published:	18 December 2008

© 2008 Zayed et al.; licensee BioMed Central Ltd.
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Introduction

Prostaglandin D synthase (PGDS) is responsible for the biosynthesis of PGD and J series, which have been shown to exhibit anti-inflammatory and anticatabolic effects. Two isoforms have been identified: hematopoietic- and lipocalin-type PGDS (H-PGDS and L-PGDS, respectively). The aims of this study were to investigate the expressions of H-PGDS and L-PGDS in cartilage from healthy donors and from patients with osteoarthritis (OA) and to characterize their regulation by interleukin-1-beta (IL-1β) in cultured OA chondrocytes.

Methods

The expressions of H-PGDS and L-PGDS mRNA and protein in cartilage were analyzed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry, respectively. Chondrocytes were stimulated with IL-1β, and the expression of L-PGDS was evaluated by real-time RT-PCR and Western blotting. The roles of de novo protein synthesis and of the signalling pathways mitogen-activated protein kinases (MAPKs), nuclear factor-kappa-B (NF-κB), and Notch were evaluated using specific pharmacological inhibitors.

Results

L-PGDS and H-PGDS mRNAs were present in both healthy and OA cartilage, with higher levels of L-PGDS than H-PGDS (> 20-fold). The levels of L-PGDS mRNA and protein were increased in OA compared with healthy cartilage. Treatment of chondrocytes with IL-1β upregulated L-PGDS mRNA and protein expressions as well as PGD₂ production in a dose- and time-dependent manner. The upregulation of L-PGDS by IL-1β was blocked by the translational inhibitor cycloheximide, indicating that this effect is indirect, requiring de novo protein synthesis. Specific inhibitors of the MAPK p38 (SB 203580) and c-jun N-terminal kinase (JNK) (SP600125) and of the NF-κB (SN-50) and Notch (DAPT) signalling pathways suppressed IL-1β-induced upregulation of L-PGDS expression. In contrast, an inhibitor of the extracellular signal-regulated kinase (ERK/MAPK) (PD98059) demonstrated no significant influence. We also found that PGD₂ prevented IL-1β-induced upregulation of L-PGDS expression.

Conclusions

This is the first report demonstrating increased levels of L-PGDS in OA cartilage. IL-1β may be responsible for this upregulation through activation of the JNK and p38 MAPK and NF-κB signalling pathways. These data suggest that L-PGDS might have an important role in the pathophysiology of OA.

Introduction

Osteoarthritis (OA) is the most common joint disorder and is a leading cause of disability throughout the world [1]. It can cause pain, stiffness, swelling, and loss of function in the joints. Pathologically, OA is characterized by progressive degeneration of articular cartilage, synovial inflammation, and subchondral bone remodeling. These processes are thought to be largely mediated through excess production of proinflammatory and catabolic mediators. Among these mediators, interleukin-1-beta (IL-1β) has been demonstrated to be predominantly involved in the initiation and progression of the disease [2-4]. One mechanism through which IL-1β exerts its effects is by inducing connective tissue cells, including chondrocytes, to produce matrix metalloproteinases (MMPs), aggrecanases, reactive oxygen species, and prostaglandins (PGs) [2].

The biosynthesis of PGs involves multiple enzymatically regulated reactions. The process is initiated through the release of arachidonic acid (AA) from the cell membrane by phospholipases. Subsequently, AA is converted to an intermediate substrate PGH₂ by the actions of cyclooxygenase (COX). Two distinct isoforms have been identified: COX-1 is constitutively expressed, whereas COX-2 is induced by various stimuli such as proinflammatory cytokines and growth factors [5]. Once formed by COX-1 or COX-2, the unstable PGH₂ intermediate is metabolized by specific PG synthase enzymes to generate the classical bioactive PGs, including PGE₂, PGD₂, PGF₂α, PGI₂, and thromboxane [6].

There is a growing body of evidence suggesting that PGD₂ may have protective effects in OA and possibly other chronic articular diseases. For instance, treatment with PGD₂ enhances the expression of the cartilage-specific matrix components collagen type II and aggrecan [7] and prevents chondrocyte apoptosis [8]. In addition, we have recently shown that PGD₂ inhibits the induction of MMP-1 and MMP-13, which play an important role in cartilage damage [9]. Thus, PGD₂ can mediate its chondroprotective effects not only through chondrogenesis enhancement, but also through inhibition of catabolic events. PGD₂ was also shown to exhibit anti-inflammatory properties. Indeed, increased levels of PGD₂ are observed during the resolution phase of inflammation and the inflammation is exacerbated by COX inhibitors [10,11]. The anti-inflammatory role of PGD₂ is supported by studies using PGD₂ synthase-deficient and transgenic mice. The knockout animals show impaired resolution of inflammation, and transgenic animals have little detectable inflammation [12]. In addition, retroviral delivery of PGD₂ synthase suppresses inflammatory responses in a murine air-pouch model of monosodium urate monohydrate crystal-induced inflammation [13]. Some effects of PGD₂ can be mediated by its dehydration end product, 15d-PGJ₂ (15-deoxy-delta12,14-PGJ₂), which has been shown to exhibit potent anti-inflammatory and anticatabolic properties [14]. PGD₂ exerts its effects principally by binding and activating two plasma membrane receptors, the D prostanoid receptor (DP) 1 [15] and chemoattractant-receptor-like molecule expressed on Th2 cells (CRTH2), also known as DP2 [16]. The effects of the PGD₂ metabolite 15d-PGJ₂ are mediated through mechanisms independent of and dependent on nuclear peroxisome proliferator-activated receptor-gamma (PPARγ) [14,17,18].

The biosynthesis of PGD₂ from its precursor PGH₂ is catalyzed by two PGD synthases (PGDSs): one is gluthatione-independent, the lipocaline-type PGDS (L-PGDS), and the other is glutathione-requiring, the hematopoietic PGDS (H-PGDS) [19]. L-PGDS (also called β-trace) is expressed abundantly in the central nervous system [20,21], the heart [22], the retina [23], and the genital organs [24]. H-PGDS is expressed mainly in mast cells [25], megakaryocytes [26], and T-helper 2 lymphocytes [27]. So far, little is known about the expression and regulation of L-PGDS and H-PGDS in cartilage. To better understand the role of PGD₂ in the joint, we investigated the expressions of H-PGDS and L-PGDS in healthy and OA cartilage. Moreover, we explored the effect of IL-1β, a key cytokine in the pathogenesis of OA, on L-PGDS expression in cultured chondrocytes.

Materials and methods

Reagents

Recombinant human IL-1β was obtained from Genzyme (Cambridge, MA, USA). Cycloheximide (CHX) was purchased from Sigma-Aldrich Canada (Oakville, ON, Canada). SB203580, SP600125, PD98059, SN-50, and N-[N-(3,5-diflurophenylacetate)-L-alanyl]-(S)-phenylglycine t-butyl ester (DAPT) were from Calbiochem (now part of EMD Biosciences, Inc., San Diego, CA, USA). PGD₂ was from Cayman Chemical Company (Ann Arbor, MI, USA). Dulbecco's modified Eagle's medium (DMEM), penicillin and streptomycin, foetal calf serum (FCS), and TRIzol^® reagent were from Invitrogen (Burlington, ON, Canada). All other chemicals were purchased from either Bio-Rad Laboratories (Mississauga, ON, Canada) or Sigma-Aldrich Canada.

Specimen selection and chondrocyte culture

Healthy cartilage and synovial fluids were obtained at necropsy, within 12 hours of death, from donors with no history of arthritic diseases (n = 13, mean ± standard deviation [SD] age of 64 ± 17 years). To ensure that only healthy tissue was used, cartilage specimens were thoroughly examined both macroscopically and microscopically. OA cartilage and synovial fluids were obtained from patients undergoing total knee replacement (n = 32, mean ± SD age of 67 ± 16 years). All OA patients were diagnosed on criteria developed by the American College of Rheumatology Diagnostic Subcommittee for OA [28]. At the time of surgery, the patients had symptomatic disease requiring medical treatment in the form of nonsteroidal anti-inflammatory drugs or selective COX-2 inhibitors. Patients who had received intra-articular injections of steroids were excluded. The Clinical Research Ethics Committee of Notre-Dame Hospital (Montreal, QC, Canada) approved the study protocol and the informed consent form.

Chondrocytes were released from cartilage by sequential enzymatic digestion as previously described [29]. Briefly, this consisted of 2 mg/mL pronase for 1 hour followed by 1 mg/mL collagenase for 6 hours (type IV; Sigma-Aldrich Canada) at 37°C in DMEM and antibiotics (100 U/mL penicillin and 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. At confluence, the chondrocytes were detached, seeded at high density, and allowed to grow in DMEM, supplemented as above. The culture medium was changed every second day, and 24 hours before the experiment, the cells were incubated in fresh medium containing 0.5% FCS. Only first-passaged chondrocytes were used.

RNA extraction and reverse transcriptase-polymerase chain reaction

Total RNA from homogenized cartilage or stimulated chondrocytes was isolated using the TRIzol^® reagent (Invitrogen) in accordance with the manufacturer's instructions. To remove contaminating DNA, isolated RNA was treated with RNase-free DNase I (Ambion, Inc., Austin, TX, USA). The RNA was quantitated using the RiboGreen RNA quantitation kit (Molecular Probes, Inc., now part of Invitrogen Corporation, Carlsbad, CA, USA), dissolved in diethylpyrocarbonate (DEPC)-treated H₂O, and stored at -80°C until use. One microgram of total RNA was reverse-transcribed using Moloney murine leukemia virus reverse transcriptase (RT) (Fermentas, Burlington, ON, Canada), as detailed in the manufacturer's guidelines. One fiftieth of the RT reaction was analyzed by real-time quantitative polymerase chain reaction (PCR) as described below. The following primers were used: L-PGDS [GeneBank: NM000954], sense 5'-AACCAGTGTGAGACCCGAAC-3', antisense 5'-AGGCGGTGAATTTCTCCTTT-3'; H-PGDS [GeneBank: NM014485], sense 5'-CCCCATTTTGGAAGTTGATG-3', antisense 5'-TGAGGCGCATTATACGTGAG-3; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [GeneBank: NM002046], sense 5'-CAGAACATCATCCCTGCCTCT-3', antisense 5'-GCTTGACAAAGTGGTCGTTGAG-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 units; Epicentre Biotechnologies, Madison, WI, USA). 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; Qiagen), the mixtures were subjected to 40 amplification cycles (15 seconds at 95°C for denaturation and 1 minute 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, USA), allowing the determination of the threshold cycle (C_T) at which exponential amplification of PCR products begins. After PCR, dissociation curves were generated with one peak, indicating the specificity of the amplification. A C_T value was obtained from each amplification curve using the software provided by the manufacturer (Applied Biosystems).

Relative amounts of mRNA in healthy and OA cartilage were determined using the standard curve method. Serial dilutions of internal standards (plasmids containing cDNA of target genes) were included in each PCR run, and standard curves for the target gene and for GAPDH were generated by linear regression using log (C_T) versus log (cDNA relative dilution). The C_T values were then converted to number of molecules. Relative mRNA expression in cultured chondrocytes was determined using the ΔΔC_T method, as detailed in the guidelines of the manufacturer (Applied Biosystems). A ΔC_T value was first calculated by subtracting the C_T value for the housekeeping gene GAPDH from the C_T value for each sample. A ΔΔC_T value was then calculated by subtracting the ΔC_T value of the control (unstimulated cells) from the ΔC_T value of each treatment. Fold changes compared with the control were then determined by raising 2 to the -ΔΔC_T power. Each PCR generated only the expected specific amplicon as shown by the melting-temperature profiles of the final product and by gel electrophoresis of test PCRs. Each PCR was performed in triplicate on two separate occasions for each independent experiment.

Immunohistochemistry

Cartilage specimens were processed for immunohistochemistry as previously described [29]. The specimens were fixed in 4% paraformaldehyde and embedded in paraffin. Sections (5 μm) of paraffin-embedded specimens were deparaffinized in toluene and were dehydrated in a graded series of ethanol. The specimens were then preincubated with chondroitinase ABC (0.25 U/mL in phosphate-buffered saline [PBS] pH 8.0) for 60 minutes at 37°C, followed by a 30-minute incubation with Triton X-100 (0.3%) at room temperature. Slides were then washed in PBS followed by 2% hydrogen peroxide/methanol for 15 minutes. They were further incubated for 60 minutes with 2% healthy serum (Vector Laboratories, Burlingame, CA, USA) and overlaid with primary antibody for 18 hours at 4°C in a humidified chamber. The antibody was a rabbit polyclonal anti-human L-PGDS (United States Biological Inc., Swampscott, MA, USA), used at 10 μg/mL. Each slide was washed three times in PBS pH 7.4 and stained using the avidin-biotin complex method (Vectastain ABC kit; Vector Laboratories). The colour was developed with 3,3'-diaminobenzidine (DAB) (Vector Laboratories) containing hydrogen peroxide. The slides were counterstained with eosin. The specificity of staining was evaluated by using antibody that had been preadsorbed (1 hour at 37°C) with a 20-fold molar excess of recombinant human L-PGDS (Cayman Chemical Company) and by substituting the primary antibody with nonimmune rabbit IgG (Chemicon International, Temecula, CA, USA), used at the same concentration as the primary antibody. The evaluation of positive-staining chondrocytes was performed using our previously published method [29]. For each specimen, six microscopic fields were examined under × 40 magnification. The total number of chondrocytes and the number of chondrocytes staining positive were evaluated, and the results were expressed as the percentage of chondrocytes staining positive (cell score).

Western blot analysis

Chondrocytes were lysed in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA [ethylenediaminetetraacetic acid], 1 mM PMSF [phenylmethylsulphonyl fluoride], 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 revolutions per minute for 15 minutes. The protein concentration of the supernatant was determined using the bicinchoninic acid method (Pierce, Rockford, IL, USA). Twenty micrograms of total cell lysate was subjected to SDS-PAGE and electrotransferred to a nitrocellulose membrane (Bio-Rad Laboratories). After blocking in 20 mM Tris-HCl pH 7.5 containing 150 mM NaCl, 0.1% Tween 20, and 5% (wt/vol) nonfat dry milk, blots were incubated overnight at 4°C with the primary antibody and washed with a Tris buffer (Tris-buffered saline 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, finally, exposed to Kodak X-Omat film (Eastman Kodak Company, Rochester, NY, USA). Bands on the films were scanned using the imaging system Chemilmager 4000 (Alpha Innotech Corporation, San Leandro, CA, USA), and the intensity of the L-PGDS bands was normalized by dividing them by the intensity of the β-actin band of the corresponding sample.

11β-PGF₂α and PGD₂ assays

The levels of 11β-PGF2α in hyaluronidase-treated synovial fluids and of PGD₂ in chondrocyte supernatants were determined using competitive enzyme immunoassays from Cayman Chemical Company. Assays were performed according to the manufacturer's recommendation.

Statistical analysis

Data are expressed as the mean ± standard error of the mean (SEM). Statistical significance was assessed by the two-tailed Student t test. P values of less than 0.05 were considered significant.

Results

Expressions of L-PGDS and H-PGDS in healthy and osteoarthritis cartilage

We first analyzed the levels of L-PGDS and H-PGDS mRNAs in healthy and OA cartilage using real-time quantitative RT-PCR. As shown in Figure 1, cartilage predominantly expresses L-PGDS mRNA, and its levels of expression were approximately threefold higher in OA cartilage compared with healthy cartilage. In contrast to L-PGDS, there was no statistically significant difference in the levels of H-PGDS mRNA between OA and healthy cartilage (Figure 1). In preliminary experiments, we showed that the amplification efficiencies of tested genes and GAPDH were similar. The efficiencies for the amplification of each gene and the reference were approximately equal, ranging between 1.95 and 2.

Next, we used immunohistohemistry to analyze the localization and the expression level of L-PGDS and H-PGDS proteins in healthy and OA cartilage. As shown in Figures 2a and 2b, the immunostaining for L-PGDS was located in the superficial and upper intermediate layers of cartilage. Statistical evaluation for the cell score revealed a clear and significant increase in the number of chondrocytes staining positive for L-PGDS in OA cartilage (43% ± 6%, mean ± SEM) compared with healthy cartilage (20% ± 4%, mean ± SEM). The specificity of the staining was confirmed using antibody that had been preadsorbed (1 hour at 37°C) with a 20-fold molar excess of the recombinant protein (Figure 2c) or nonimmune control IgG (data not shown). Using several commercially available antibodies directed against human H-PGDS, we were unable to detect H-PGDS protein expression in OA or healthy cartilage. Together, these data indicate that the expression level of L-PGDS is increased in OA cartilage.

To assess the level of PGD₂ in synovial fluids from OA and healthy donors, we quantified its major stable metabolite, 11β-PGF₂α. We measured this metabolite because PGD₂ is unstable in vivo [30] and quantification of PGD₂ in synovial fluid can be unreliable. We found a higher level of 11β-PGF₂α in OA synovial fluid when compared with healthy synovial fluid (Figure 3), indicating that the production of PGD₂ is higher in OA synovial fluids. Together, these data indicate increased expression and activity of L-PGDS in OA tissues.

Interleukin-1-beta induces L-PGDS expression in chondrocytes

IL-1β plays a major role in the cartilage physiology and in the pathogenesis of OA [2]; therefore, we examined its effects on the expression of L-PGDS in cultured OA chondrocytes. Cells were treated with IL-1β (100 pg/mL) for different time periods, and the levels of L-PGDS mRNA were quantified using real-time RT-PCR. IL-1β-induced changes in gene expression were evaluated as fold over control (untreated cells) after normalization to the internal control gene, GAPDH. As shown in Figure 4a, treatment with IL-1β (100 pg/mL) enhanced L-PGDS mRNA expression in a time-dependent manner. L-PGDS mRNA expression started to gradually increase 24 hours post-stimulation with IL-1β and remained elevated until 72 hours. The induction of L-PGDS mRNA by IL-1β was also dose-dependent. A significant increase at concentrations as low as 10 pg/mL was observed and the maximal effect was reached at 100 pg/mL (Figure 4b). To determine whether changes in mRNA levels were paralleled by changes in L-PGDS protein levels, we performed Western blot analysis. Consistent with its effects on L-PGDS mRNA, treatment with IL-1β led to a dose- and time-dependent increase in the L-PGDS protein expression (Figure 4c, d). To establish whether the IL-1β-induced increase in L-PGDS expression corresponded with an increase in PGDS activity, we measured PGD₂ levels in conditioned media. As shown in Figures 4e and 4f, the increased expression of L-PGDS protein was accompanied by a time- and dose-dependent increase in PGD₂ production.

The upregulation of L-PGDS mRNA expression in chondrocytes requires de novo protein synthesis

The lag period required for IL-1β to induce L-PGDS mRNA in chondrocytes contrasts with those required for other IL-1β-inducible genes, the expression of which starts as early as 2 to 6 hours and reaches a maximum at 8 to 18 hours. This suggests that de novo protein synthesis is required for IL-1β-induced L-PGDS expression. To evaluate this possibility, we examined the impact of the protein synthesis inhibitor CHX. Chondrocytes were stimulated with IL-1β in the absence or presence of CHX, and the levels of L-PGDS mRNA were analyzed by real-time PCR. As shown in Figure 5, treatment with CHX prevented IL-1β-mediated upregulation of L-PGDS mRNA expression. This suggests that, to upregulate L-PGDS expression in chondrocytes, IL-1β must induce the synthesis of one or more proteins.

JNK and p38 MAPKs and NF-κB pathways contribute to interleukin-1-beta-induced upregulation of L-PGDS

IL-1β exerts its effects acting through activation of the mitogen-activated protein kinase (MAPK) (extracellular signal-regulated kinase [ERK], c-jun N-terminal kinase [JNK], and p38) and nuclear factor-kappa-B (NF-κB) signalling cascades [31-35]. To evaluate the potential contribution of these pathways in IL-1β-induced L-PGDS expression, we used specific pharmacological inhibitors. Chondrocytes were pretreated for 30 minutes with selective inhibitors for the above pathways and then stimulated or not with IL-1β for 48 hours. As shown in Figure 6a, pretreatment with the p38 MAPK inhibitor SB203580 (1 μM), the JNK MAPK inhibitor SP600125 (10 μM), or the NF-κB inhibitor SN-50 (1 μM) suppressed IL-1β-induced upregulation of L-PGDS expression. In contrast, pretreatment with the p42/44 MAPK inhibitor PD98059 (10 μM) had no effect on IL-1β-induced upregulation of L-PGDS. The concentration of the MAPK and NF-κB inhibitors used for these experiments had no significant effect on cell viability as indicated by the results of the MTT (3- [4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay (data not shown). These results suggest that the activation of JNK and p38 MAPK as well as NF-κB is essential to the induction of L-PGDS by IL-1β in chondrocytes.

The Notch signalling pathway regulates diverse cellular processes, including proliferation, differentiation, and apoptosis [36], and was reported to contribute to the regulation of L-PGDS expression [37]. To determine whether this pathway participates in IL-1β-induced L-PGDS expression in human chondrocytes, we assessed the effect of DAPT. DAPT is a γ-secretase inhibitor, which blocks cleavage of the intracellular domain of all Notch proteins, and is widely used to evaluate the effect of Notch inhibition [36]. As shown in Figure 6b, pretreatment with DAPT dose-dependently prevented IL-1β-induced L-PGDS protein expression, indicating the involvement of Notch signalling in this process. Notch inhibition was confirmed by transcriptional inhibition of its direct target gene, Hes1 (data not shown).

PGD₂ downregulated L-PGDS expression

To further characterize the regulation of L-PGDS expression in cartilage, we examined the effect of PGD₂, the end product of L-PGDS. Chondrocytes were stimulated with IL-1β in the absence or presence of increasing concentrations of PGD₂ for 48 hours, and the expression of L-PGDS was evaluated by Western blotting. As shown in Figure 7, treatment with PGD₂ dose-dependently reduced IL-1β-induced L-PGDS expression.

Discussion

This is the first report to demonstrate the presence of L-PGDS in human cartilage and to show that its levels are elevated in OA cartilage compared with healthy cartilage. The proinflammatory cytokine IL-1β upregulated, whereas PGD₂ downregulated, the expression of L-PGDS in cultured chondrocytes. These findings suggest that L-PGDS may be implicated in the pathogenesis of OA.

In healthy cartilage, L-PGDS immunostaining was located in only a few cells in the superficial and middle zones. By contrast, in OA cartilage, the cell score was significantly higher, particularly in cartilage areas showing significant damage (fibrillation). Given the anti-inflammatory and anticatabolic roles of PGD₂, it is reasonable to speculate that the upregulation of L-PGDS may act as a sort of chondroprotective mechanism. Increased expression of L-PGDS was described in other diseases such as atherosclerosis [22], multiple sclerosis [38], diabetes [39] essential hypertension [40], and Tay-Sachs and Sandhoff diseases [41]. Thus, L-PGDS expression is upregulated in many pathologies.

The enhanced expression of L-PGDS in the superficial and middle zones of cartilage could potentially be due to the increased level of the proinflammatory cytokine IL-1β in these zones. Indeed, IL-1β, which plays pivotal roles in the initiation and progression of OA, has been shown to accumulate in these zones [42-46]. To prove this hypothesis, we performed cell culture experiments. Our results revealed that exposure to IL-1β led to a time- and concentration-dependent upregulation of L-PGDS expression and PGD₂ production. The upregulation of L-PGDS expression by IL-1β was blocked by CHX, suggesting that this effect of IL-1β requires de novo protein synthesis and would be consistent with an indirect stimulatory mechanism.

The delayed induction of L-PGDS by IL-1β in chondrocytes is consistent with the recently reported anti-inflammatory and anticatabolic properties of PGD₂. Indeed, the production of PGD₂ is markedly elevated during the resolution of inflammation in carrageenan-induced pleurisy in rats, and exogenous PGD₂ significantly reduces neutrophil levels in the inflammatory exudates [10,11]. Enhanced production of PGD₂ was also described during the resolution phase of the wound-healing process [47]. Cipollone and colleagues [48] examined the expression of L-PGDS in atherosclerotic arteries and found lower expression of L-PGDS and higher expression of microsomal prostaglandin E synthase-1 (mPGES-1) in symptomatic plaques and found higher expression of L-PGDS and lower expression of mPGES-1 in asymptomatic ones. This suggests that the balance between PGD₂ and PGE₂ contributes to the pathology of atherosclerosis and that a shift toward PGD₂ synthesis may have an anti-inflammatory role. This is supported by the observation that increased biosynthesis of PGD₂ is associated with reduced production of PGE₂ in several in vitro studies [49,50]. Recently, two separate studies demonstrated anti-inflammatory properties of PGD₂ in an air-pouch model of inflammation induced by monosodium urate monohydrate crystals [13,51]. Moreover, H-PGDS knockout mice fail to resolve a delayed-type hypersensitivity reaction [12]. In addition to its anti-inflammatory effects, PGD₂ was shown to induce the expression of collagen type II and aggrecan [7], to prevent apoptosis [8], and to inhibit the induction of MMP-1 and MMP-13 [52] in chondrocytes. Together, these data and those from the present study favour the hypothesis that the upregulation of L-PGDS expression in chondrocytes may be part of a negative feedback control of inflammatory and catabolic responses activated by IL-1β in the joint.

The production of PGD₂ by chondrocytes is of particular interest since PGD₂ is readily converted to 15d-PGJ₂, a potent antiarthritic agent [14]. 15dPGJ₂ downregulates the expression of a number of inflammatory and catabolic mediators involved in the pathogenesis of OA, including IL-1β, tumour necrosis factor-alpha, inducible nitric-oxide synthase, and MMPs [14]. Moreover, many in vivo studies support a protective effect of 15d-PGJ₂ and other PPARγ ligands in experimental animal models of OA [53,54]. Thus, the increased expression of L-PGDS can lead to the production of a PPARγ ligand in the joint. In contrast to classical PGs, which induce their effects through binding to cell surface G protein-coupled receptors, 15d-PGJ₂ induces most of its effects through the nuclear receptor PPARγ. We have previously shown that PPARγ expression is reduced in OA cartilage and that IL-1β downregulates its expression in chondrocytes [29], which may interfere with the protective effect of the PGD₂ metabolite 15d-PGJ₂. Therefore, the increased expression of L-PGDS observed in our study may represent a compensatory mechanism to counter the reduced expression of PPARγ in OA and to limit local inflammatory and catabolic responses. Also, it should be noted that 15d-PGJ₂ can induce many of its effects independently of PPARγ [14,17,18]. In addition, PGD₂ can directly exert protective effects in OA before being metabolized into 15d-PGJ₂. Indeed, we have recently demonstrated that human chondrocytes express functional DP1 and CRTH-2 and that PGD₂ downregulates MMP-1 and MMP-13 expressions through activation of the DP1 pathway [9].

To elucidate the mechanisms by which IL-1β upregulates L-PGDS expression, we evaluated the roles played by downstream signalling cascades using specific pharmacological inhibitors. We found that JNK and p38 MAPK inhibitors blocked IL-1β-induced L-PGDS upregulation, whereas an inhibitor of the ERK MAPK was without effect. We also found that NF-κB blockade caused a significant decrease in IL-1β-induced upregulation of L-PGDS protein expression. These findings support the hypothesis that the JNK and p38 MAPKs as well as the NF-κB pathways are involved in the upregulation of L-PGDS expression by IL-1β. Our results are concordant with previous reports that implicate activation of MAPKs (JNK and p38) and NF-κB in the upregulation of L-PGDS in leptomeningel cells [55], endothelial cells [56], and macrophages [57]. The activation of JNK and p38 MAPK and of NF-κB pathways in chondrocytes has been shown to cause activation of their downstream transcription factors, including activation protein-1 (AP-1) and NF-κB [31-35]. Interestingly, the promoter region of the human L-PGDS contains binding sites for NF-κB and AP-1 [55,56]. Therefore, one could speculate that upregulation of L-PGDS expression by IL-1β could be mediated by AP-1 and NF-κB. Our results also demonstrate that the Notch signalling pathway positively contributes to IL-1β-induced L-PGDS expression in chondrocytes because DAPT, a Notch signalling inhibitor, blocked this process. These findings contrast with previous data showing that the Notch pathway downregulates L-PGDS expression in the brain-derived TE671 cells [37]. The reasons for these discrepancies are presently unclear but are most likely due to cell-type differences or to differences in experimental conditions.

We also found that PGD₂ inhibits IL-1β-induced L-PGDS expression. These results suggest that PGD₂ may exert a negative feedback mechanism to downregulate L-PGDS expression and activity. Given that the levels of L-PGDS are elevated in OA cartilage and that IL-1β upregulated its expression in chondrocytes, it is possible that the IL-1β effect prevails over that of PGD₂ in vivo during advanced stages of the disease. Indeed, the OA cartilage specimens used in this study were from donors with long-established OA. Further studies are clearly warranted to determine the expression profile of L-PGDS over the course of OA in animal models of the disease.

The concentrations of PGD₂ used to suppress IL-1β-induced L-PGDS expression are likely to be much higher than those produced in synovial fluids. However, it should be noted that, like other eicosanoids, PGD₂ functions as an autocrine and paracrine molecule and can readily reach pharmacological levels in the microenvironment of cells that produce it.

Conclusion

Our study has demonstrated for the first time that L-PGDS is upregulated in OA cartilage. The proinflammatory cytokine IL-1β may be responsible for this upregulation via a mechanism that seems to involve the activation of the JNK and p38 MAPK and NF-κB signalling pathways. These results suggest that the increased expression of L-PGDS may play a protective role against articular inflammation and cartilage damage.

Abbreviations

15d-PGJ₂: 15-deoxy-delta12,14-PGJ₂; AA: arachidonic acid; AP-1: activation protein-1; CHX: cycloheximide; COX: cyclooxygenase; CRTH2: chemoattractant-receptor-like molecule expressed on Th2 cells; C_T: threshold cycle; DAPT: N-[N-(3,5-diflurophenylacetate)-L-alanyl]-(S)-phenylglycine t-butyl ester; DMEM: Dulbecco's modified Eagle's medium; DP: D prostanoid receptor; ERK: extracellular signal-regulated kinase; FCS: foetal calf serum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; H-PGDS: hematopoietic-type prostaglandin D synthase; IL-1β: interleukin-1-beta; JNK: c-jun N-terminal kinase; L-PGDS: lipocalin-type prostaglandin D synthase; MAPK: mitogen-activated protein kinase; MMP: matrix metalloproteinase; mPGES-1: microsomal prostaglandin E synthase-1; NF-κB: nuclear factor-kappa-B; OA: osteoarthritis; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PG: prostaglandin; PGDS: prostaglandin D synthase; PPARγ: peroxisome proliferator-activated receptor-gamma; RT: reverse transcriptase; RT-PCR: reverse transcriptase-polymerase chain reaction; SD: standard deviation; SEM: standard error of the mean; UNG: uracil-N-glycosylase.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

NZ conceived the study and designed and carried out cell and real-time RT-PCR experiments and some immunohistochemistry experiments. NC contributed to the study design and carried out immunoassays and some cell experiments. XL carried out some cell experiments and data analysis. MB participated in the study design and data analysis. JM-P, J-PP, and ND helped to obtain tissues and participated in the study design and some immunohistochemistry experiments. HF conceived, designed, and coordinated the study, carried out some cell experiments, and drafted the manuscript. All authors read and approved the final manuscript.

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).

References

1. Lawrence RC, Helmick CG, Arnett FC, Deyo RA, Felson DT, Giannini EH, Heyse SP, Hirsch R, Hochberg MC, Hunder GG, Liang MH, Pillemer SR, Steen VD, Wolfe F: Estimates of the prevalence of arthritis and selected musculoskeletal disorders in the United States.

   Arthritis Rheum 1998, 41:778-799. PubMed Abstract | Publisher Full Text

2. Goldring MB: The role of cytokines as inflammatory mediators in osteoarthritis: lessons from animal models.

   Connect Tissue Res 1999, 40:1-11. PubMed Abstract | Publisher Full Text

3. Pelletier JP, Martel-Pelletier J, Abramson SB: Osteoarthritis, an inflammatory disease: potential implication for the selection of new therapeutic targets.

   Arthritis Rheum 2001, 44:1237-1247. PubMed Abstract | Publisher Full Text

4. Goldring MB, Berenbaum F: The regulation of chondrocyte function by proinflammatory mediators: prostaglandins and nitric oxide.

   Clin Orthop Relat Res 2004, S37-46. PubMed Abstract | Publisher Full Text

5. Smith WL, Langenbach R: Why there are two cyclooxygenase isozymes.

   J Clin Invest 2001, 107:1491-1495. PubMed Abstract | Publisher Full Text | PubMed Central Full Text

6. Martel-Pelletier J, Pelletier JP, Fahmi H: Cyclooxygenase-2 and prostaglandins in articular tissues.

   Semin Arthritis Rheum 2003, 33:155-167. PubMed Abstract | Publisher Full Text

7. Jakob M, Demarteau O, Suetterlin R, Heberer M, Martin I: Chondrogenesis of expanded adult human articular chondrocytes is enhanced by specific prostaglandins.

   Rheumatology (Oxford) 2004, 43:852-857. PubMed Abstract | Publisher Full Text

8. Relic B, Benoit V, Franchimont N, Ribbens C, Kaiser MJ, Gillet P, Merville MP, Bours V, Malaise MG: 15-deoxy-delta12,14-prostaglandin J2 inhibits Bay 11–7085-induced sustained extracellular signal-regulated kinase phosphorylation and apoptosis in human articular chondrocytes and synovial fibroblasts.

   J Biol Chem 2004, 279:22399-22403. PubMed Abstract | Publisher Full Text

9. Zayed N, Afif H, Chabane N, Mfuna-Endam L, Benderdour M, Martel-Pelletier J, Pelletier JP, Motiani RK, Trebak M, Duval N, Fahmi H: Inhibition of interleukin-1beta-induced matrix metalloproteinases 1 and 13 production in human osteoarthritic chondrocytes by prostaglandin D(2).

   Arthritis Rheum 2008, 58:3530-3540. PubMed Abstract | Publisher Full Text

10. Gilroy DW, Colville-Nash PR, Willis D, Chivers J, Paul-Clark MJ, Willoughby DA: Inducible cyclooxygenase may have anti-inflammatory properties.

    Nat Med 1999, 5:698-701. PubMed Abstract | Publisher Full Text

11. Ianaro A, Ialenti A, Maffia P, Pisano B, Di Rosa M: Role of cyclopentenone prostaglandins in rat carrageenin pleurisy.

    FEBS Lett 2001, 508:61-66. PubMed Abstract | Publisher Full Text

12. Trivedi SG, Newson J, Rajakariar R, Jacques TS, Hannon R, Kanaoka Y, Eguchi N, Colville-Nash P, Gilroy DW: Essential role for hematopoietic prostaglandin D₂ synthase in the control of delayed type hypersensitivity.

    Proc Natl Acad Sci USA 2006, 103:5179-5184. PubMed Abstract | Publisher Full Text | PubMed Central Full Text

13. Murakami Y, Akahoshi T, Hayashi I, Endo H, Hashimoto A, Kono S, Kondo H, Kawai S, Inoue M, Kitasato H: Inhibition of monosodium urate monohydrate crystal-induced acute inflammation by retrovirally transfected prostaglandin D synthase.

    Arthritis Rheum 2003, 48:2931-2941. PubMed Abstract | Publisher Full Text

14. Fahmi H, Pelletier JP, Martel-Pelletier J: PPARgamma ligands as modulators of inflammatory and catabolic responses on arthritis. An overview.

    J Rheumatol 2002, 29:3-14. PubMed Abstract | Publisher Full Text

15. Hirata M, Kakizuka A, Aizawa M, Ushikubi F, Narumiya S: Molecular characterization of a mouse prostaglandin D receptor and functional expression of the cloned gene.

    Proc Natl Acad Sci USA 1994, 91:11192-11196. PubMed Abstract | Publisher Full Text | PubMed Central Full Text

16. Hirai H, Tanaka K, Yoshie O, Ogawa K, Kenmotsu K, Takamori Y, Ichimasa M, Sugamura K, Nakamura M, Takano S, Nagata K: Prostaglandin D₂ selectively induces chemotaxis in T helper type 2 cells, eosinophils, and basophils via seven-transmembrane receptor CRTH2.

    J Exp Med 2001, 193:255-261. PubMed Abstract | Publisher Full Text | PubMed Central Full Text

17. Boyault S, Bianchi A, Moulin D, Morin S, Francois M, Netter P, Terlain B, Bordji K: 15-Deoxy-delta(12,14)-prostaglandin J(2) inhibits IL-1beta-induced IKK enzymatic activity and IkappaBalpha degradation in rat chondrocytes through a PPARgamma-independent pathway.

    FEBS Lett 2004, 572:33-40. PubMed Abstract | Publisher Full Text

18. Bianchi A, Moulin D, Sebillaud S, Koufany M, Galteau MM, Netter P, Terlain B, Jouzeau JY: Contrasting effects of peroxisome-proliferator-activated receptor (PPAR)gamma agonists on membrane-associated prostaglandin E₂ synthase-1 in IL-1beta-stimulated rat chondrocytes: evidence for PPARgamma-independent inhibition by 15-deoxy-Delta12,14prostaglandin J₂.

    Arthritis Res Ther 2005, 7:R1325-1337. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text

19. Urade Y, Eguchi N: Lipocalin-type and hematopoietic prostaglandin D synthases as a novel example of functional convergence.

    Prostaglandins Other Lipid Mediat 2002, 68–69:375-382. Publisher Full Text

20. Blodorn B, Mader M, Urade Y, Hayaishi O, Felgenhauer K, Bruck W: Choroid plexus: the major site of mRNA expression for the beta-trace protein (prostaglandin D synthase) in human brain.

    Neurosci Lett 1996, 209:117-120. PubMed Abstract | Publisher Full Text

21. Urade Y, Fujimoto N, Hayaishi O: Purification and characterization of rat brain prostaglandin D synthetase.

    J Biol Chem 1985, 260:12410-12415. PubMed Abstract | Publisher Full Text

22. Eguchi Y, Eguchi N, Oda H, Seiki K, Kijima Y, Matsu-ura Y, Urade Y, Hayaishi O: Expression of lipocalin-type prostaglandin D synthase (beta-trace) in human heart and its accumulation in the coronary circulation of angina patients.

    Proc Natl Acad Sci USA 1997, 94:14689-14694. PubMed Abstract | Publisher Full Text | PubMed Central Full Text

23. Beuckmann CT, Gordon WC, Kanaoka Y, Eguchi N, Marcheselli VL, Gerashchenko DY, Urade Y, Hayaishi O, Bazan NG: Lipocalin-type prostaglandin D synthase (beta-trace) is located in pigment epithelial cells of rat retina and accumulates within interphotoreceptor matrix.

    J Neurosci 1996, 16:6119-6124. PubMed Abstract | Publisher Full Text

24. Gerena RL, Irikura D, Eguchi N, Urade Y, Killian GJ: Immunocytochemical localization of lipocalin-type prostaglandin D synthase in the bull testis and epididymis and on ejaculated sperm.

    Biol Reprod 2000, 62:547-556. PubMed Abstract | Publisher Full Text

25. Urade Y, Ujihara M, Horiguchi Y, Igarashi M, Nagata A, Ikai K, Hayaishi O: Mast cells contain spleen-type prostaglandin D synthetase.

    J Biol Chem 1990, 265:371-375. PubMed Abstract | Publisher Full Text

26. Fujimori K, Kanaoka Y, Sakaguchi Y, Urade Y: Transcriptional activation of the human hematopoietic prostaglandin D synthase gene in megakaryoblastic cells. Roles of the oct-1 element in the 5'-flanking region and the AP-2 element in the untranslated exon 1.

    J Biol Chem 2000, 275:40511-40516. PubMed Abstract | Publisher Full Text

27. Tanaka K, Ogawa K, Sugamura K, Nakamura M, Takano S, Nagata K: Cutting edge: differential production of prostaglandin D₂ by human helper T cell subsets.

    J Immunol 2000, 164:2277-2280. PubMed Abstract | Publisher Full Text

28. Altman RD: Criteria for the classification of osteoarthritis of the knee and hip.

    Scand J Rheumatol Suppl 1987, 65:31-39. PubMed Abstract | Publisher Full Text

29. Afif H, Benderdour M, Mfuna-Endam L, Martel-Pelletier J, Pelletier JP, Duval N, Fahmi H: Peroxisome proliferator-activated receptor gamma1 expression is diminished in human osteoarthritic cartilage and is downregulated by interleukin-1beta in articular chondrocytes.

    Arthritis Res Ther 2007, 9:R31. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text

30. O'Sullivan S, Mueller MJ, Dahlen SE, Kumlin M: Analyses of prostaglandin D₂ metabolites in urine: comparison between enzyme immunoassay and negative ion chemical ionisation gas chromatography-mass spectrometry.

    Prostaglandins Other Lipid Mediat 1999, 57:149-165. PubMed Abstract | Publisher Full Text

31. Geng Y, Valbracht J, Lotz M: Selective activation of the mitogen-activated protein kinase subgroups c-Jun NH2 terminal kinase and p38 by IL-1 and TNF in human articular chondrocytes.

    J Clin Invest 1996, 98:2425-2430. PubMed Abstract | Publisher Full Text | PubMed Central Full Text

32. Ding GJ, Fischer PA, Boltz RC, Schmidt JA, Colaianne JJ, Gough A, Rubin RA, Miller DK: Characterization and quantitation of NF-kappaB nuclear translocation induced by interleukin-1 and tumor necrosis factor-alpha. Development and use of a high capacity fluorescence cytometric system.

    J Biol Chem 1998, 273:28897-28905. PubMed Abstract | Publisher Full Text

33. Mengshol JA, Vincenti MP, Coon CI, Barchowsky A, Brinckerhoff CE: Interleukin-1 induction of collagenase 3 (matrix metalloproteinase 13) gene expression in chondrocytes requires p38, c-Jun N-terminal kinase, and nuclear factor kappaB: differential regulation of collagenase 1 and collagenase 3.

    Arthritis Rheum 2000, 43:801-811. PubMed Abstract | Publisher Full Text

34. Mendes AF, Caramona MM, Carvalho AP, Lopes MC: Role of mitogen-activated protein kinases and tyrosine kinases on IL-1-Induced NF-kappaB activation and iNOS expression in bovine articular chondrocytes.

    Nitric Oxide 2002, 6:35-44. PubMed Abstract | Publisher Full Text

35. Fan Z, Bau B, Yang H, Aigner T: IL-1beta induction of IL-6 and LIF in normal articular human chondrocytes involves the ERK, p38 and NFkappaB signaling pathways.

    Cytokine 2004, 28:17-24. PubMed Abstract | Publisher Full Text

36. Fiuza UM, Arias AM: Cell and molecular biology of Notch.

    J Endocrinol 2007, 194:459-474. PubMed Abstract | Publisher Full Text

37. Fujimori K, Kadoyama K, Urade Y: Protein kinase C activates human lipocalin-type prostaglandin D synthase gene expression through de-repression of notch-HES signaling and enhancement of AP-2 beta function in brain-derived TE671 cells.

    J Biol Chem 2005, 280:18452-18461. PubMed Abstract | Publisher Full Text

38. Kagitani-Shimono K, Mohri I, Oda H, Ozono K, Suzuki K, Urade Y, Taniike M: Lipocalin-type prostaglandin D synthase (beta-trace) is upregulated in the alphaB-crystallin-positive oligodendrocytes and astrocytes in the chronic multiple sclerosis.

    Neuropathol Appl Neurobiol 2006, 32:64-73. PubMed Abstract | Publisher Full Text

39. Hirawa N, Uehara Y, Ikeda T, Gomi T, Hamano K, Totsuka Y, Yamakado M, Takagi M, Eguchi N, Oda H, Seiki K, Nakajima H, Urade Y: Urinary prostaglandin D synthase (beta-trace) excretion increases in the early stage of diabetes mellitus.

    Nephron 2001, 87:321-327. PubMed Abstract | Publisher Full Text

40. Hirawa N, Uehara Y, Yamakado M, Toya Y, Gomi T, Ikeda T, Eguchi Y, Takagi M, Oda H, Seiki K, Urade Y, Umemura S: Lipocalin-type prostaglandin d synthase in essential hypertension.

    Hypertension 2002, 39:449-454. PubMed Abstract | Publisher Full Text

41. Mohri I, Taniike M, Okazaki I, Kagitani-Shimono K, Aritake K, Kanekiyo T, Yagi T, Takikita S, Kim HS, Urade Y, Suzuki K: Lipocalin-type prostaglandin D synthase is up-regulated in oligodendrocytes in lysosomal storage diseases and binds gangliosides.

    J Neurochem 2006, 97:641-651. PubMed Abstract | Publisher Full Text

42. Pelletier JP, Lascau-Coman V, Jovanovic D, Fernandes JC, Manning P, Connor JR, Currie MG, Martel-Pelletier J: Selective inhibition of inducible nitric oxide synthase in experimental osteoarthritis is associated with reduction in tissue levels of catabolic factors.

    J Rheumatol 1999, 26:2002-2014. PubMed Abstract

43. Tetlow LC, Adlam DJ, Woolley DE: Matrix metalloproteinase and proinflammatory cytokine production by chondrocytes of human osteoarthritic cartilage: associations with degenerative changes.

    Arthritis Rheum 2001, 44:585-594. PubMed Abstract | Publisher Full Text

44. Towle CA, Hung HH, Bonassar LJ, Treadwell BV, Mangham DC: Detection of interleukin-1 in the cartilage of patients with osteoarthritis: a possible autocrine/paracrine role in pathogenesis.

    Osteoarthritis Cartilage 1997, 5:293-300. PubMed Abstract | Publisher Full Text

45. Melchiorri C, Meliconi R, Frizziero L, Silvestri T, Pulsatelli L, Mazzetti I, Borzi RM, Uguccioni M, Facchini A: Enhanced and coordinated in vivo expression of inflammatory cytokines and nitric oxide synthase by chondrocytes from patients with osteoarthritis.

    Arthritis Rheum 1998, 41:2165-2174. PubMed Abstract | Publisher Full Text

46. Moos V, Fickert S, Muller B, Weber U, Sieper J: Immunohistological analysis of cytokine expression in human osteoarthritic and healthy cartilage.

    J Rheumatol 1999, 26:870-879. PubMed Abstract

47. Kapoor M, Kojima F, Yang L, Crofford LJ: Sequential induction of pro- and anti-inflammatory prostaglandins and peroxisome proliferators-activated receptor-gamma during normal wound healing: a time course study.

    Prostaglandins Leukot Essent Fatty Acids 2007, 76:103-112. PubMed Abstract | Publisher Full Text | PubMed Central Full Text

48. Cipollone F, Fazia M, Iezzi A, Ciabattoni G, Pini B, Cuccurullo C, Ucchino S, Spigonardo F, De Luca M, Prontera C, Chiarelli F, Cuccurullo F, Mezzetti A: Balance between PGD synthase and PGE synthase is a major determinant of atherosclerotic plaque instability in humans.

    Arterioscler Thromb Vasc Biol 2004, 24:1259-1265. PubMed Abstract | Publisher Full Text

49. Fournier T, Fadok V, Henson PM: Tumor necrosis factor-alpha inversely regulates prostaglandin D₂ and prostaglandin E₂ production in murine macrophages. Synergistic action of cyclic AMP on cyclooxygenase-2 expression and prostaglandin E₂ synthesis.

    J Biol Chem 1997, 272:31065-31072. PubMed Abstract | Publisher Full Text

50. Matsumoto H, Naraba H, Murakami M, Kudo I, Yamaki K, Ueno A, Oh-ishi S: Concordant induction of prostaglandin E₂ synthase with cyclooxygenase-2 leads to preferred production of prostaglandin E₂ over thromboxane and prostaglandin D₂ in lipopolysaccharide-stimulated rat peritoneal macrophages.

    Biochem Biophys Res Commun 1997, 230:110-114. PubMed Abstract | Publisher Full Text

51. Jung SM, Schumacher HR, Kim H, Kim M, Lee SH, Pessler F: Reduction of urate crystal-induced inflammation by root extracts from traditional oriental medicinal plants: elevation of prostaglandin D₂ levels.

    Arthritis Res Ther 2007, 9:R64. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text

52. Stewart MD, Li J, Wong J: Relationship between histone H3 lysine 9 methylation, transcription repression, and heterochromatin protein 1 recruitment.

    Mol Cell Biol 2005, 25:2525-2538. PubMed Abstract | Publisher Full Text | PubMed Central Full Text

53. Kobayashi T, Notoya K, Naito T, Unno S, Nakamura A, Martel-Pelletier J, Pelletier JP: Pioglitazone, a peroxisome proliferator-activated receptor gamma agonist, reduces the progression of experimental osteoarthritis in guinea pigs.

    Arthritis Rheum 2005, 52:479-487. PubMed Abstract | Publisher Full Text

54. Boileau C, Martel-Pelletier J, Fahmi H, Mineau F, Boily M, Pelletier JP: The peroxisome proliferator-activated receptor gamma agonist pioglitazone reduces the development of cartilage lesions in an experimental dog model of osteoarthritis: in vivo protective effects mediated through the inhibition of key signaling and catabolic pathways.

    Arthritis Rheum 2007, 56:2288-2298. PubMed Abstract | Publisher Full Text

55. Fujimori K, Fujitani Y, Kadoyama K, Kumanogoh H, Ishikawa K, Urade Y: Regulation of lipocalin-type prostaglandin D synthase gene expression by Hes-1 through E-box and interleukin-1 beta via two NF-kappa B elements in rat leptomeningeal cells.

    J Biol Chem 2003, 278:6018-6026. PubMed Abstract | Publisher Full Text

56. Miyagi M, Miwa Y, Takahashi-Yanaga F, Morimoto S, Sasaguri T: Activator protein-1 mediates shear stress-induced prostaglandin d synthase gene expression in vascular endothelial cells.

    Arterioscler Thromb Vasc Biol 2005, 25:970-975. PubMed Abstract | Publisher Full Text

57. Joo M, Kwon M, Sadikot RT, Kingsley PJ, Marnett LJ, Blackwell TS, Peebles RS Jr, Urade Y, Christman JW: Induction and function of lipocalin prostaglandin D synthase in host immunity.

    J Immunol 2007, 179:2565-2575. PubMed Abstract | Publisher Full Text

Have something to say? Post a comment on this article!