prostaglandin-d2 and Osteoarthritis

We previously reported that synovial mast cells (MCs) from patients with rheumatoid arthritis (RA) produced TNF-α in response to immune complexes via FcγRI and FcγRIIA. However, the specific functions of synovial MCs in RA remain unclear. This study aimed to elucidate those functions. Synovial tissues and fluid were obtained from RA and osteoarthritis (OA) patients undergoing joint replacement surgery. Synovium-derived, cultured MCs were generated by culturing dispersed synovial cells with stem cell factor. We performed microarray-based screening of mRNA and microRNA (miRNA), followed by quantitative RT-PCR-based verification. Synovial MCs from RA patients showed significantly higher prostaglandin systhetase (PTGS)1 and PTGS2 expression compared with OA patients' MCs, and they produced significantly more prostaglandin D

Topics: Aged; Aged, 80 and over; Arthritis, Rheumatoid; Cells, Cultured; Cyclooxygenase 1; Cyclooxygenase 2; Dinoprostone; Female; Fibroblasts; Gene Expression Profiling; Gene Expression Regulation; Histamine Release; Humans; Immunity, Innate; Interleukin-8; Lymphocytes; Male; Mast Cells; MicroRNAs; Middle Aged; Osteoarthritis; Prostaglandin D2; Receptors, IgG; RNA, Messenger; Signal Transduction; Synovial Fluid; Synovial Membrane

Lipocalin-type prostaglandin D synthase (L-PGDS) catalyzes the formation of prostaglandin D. Collectively, these findings suggest a protective role of L-PGDS in OA, and therefore enhancing levels of L-PGDS may constitute a promising therapeutic strategy.

Topics: ADAMTS5 Protein; Animals; Arthritis, Experimental; Bone and Bones; Cartilage, Articular; Chondrocytes; Interleukin-1alpha; Intramolecular Oxidoreductases; Lipocalins; Matrix Metalloproteinase 13; Menisci, Tibial; Mice; Mice, Knockout; Osteoarthritis; Prostaglandin D2; Proteoglycans; Stifle; X-Ray Microtomography

Osteoarthritis (OA) is the most common musculoskeletal disorder among the elderly. It is characterized by progressive cartilage degradation, synovial inflammation, subchondral bone remodeling and pain. Lipocalin prostaglandin D synthase (L-PGDS) is responsible for the biosynthesis of PGD

Topics: ADAMTS5 Protein; Aggrecans; Aging; Animals; Behavior, Animal; Cartilage, Articular; Cell Count; Collagen Type II; Femur; Immunohistochemistry; Intramolecular Oxidoreductases; Lipocalins; Locomotion; Matrix Metalloproteinase 13; Mice; Mice, Knockout; Open Field Test; Osteoarthritis; Prostaglandin D2; Synovitis; Tibia; X-Ray Microtomography

Synovial fibrosis is a pathological process that is observed in several musculoskeletal disorders and characterized by the excessive deposition of extracellular matrix, as well as cell migration and proliferation. Despite the fact that glucocorticoids are widely employed in the treatment of rheumatic pathologies such as osteoarthritis (OA) and rheumatoid arthritis, the mechanisms by which glucocorticoids act in the joint and their impacts on pro-fibrotic pathways are still unclear.. Human OA synovial fibroblasts were obtained from knee and hip joints. Cells were treated with prednisolone (1 mM) or transforming growth factor-beta 1 (TGF-β1) (10 ng/ml) for 1 and 7 days for quantification of RNA and protein expression (by real-time quantitative reverse transcription-PCR and western blot, respectively), 72 h for immunocytochemistry analysis, and 48 h for proliferation (by BrdU assay) and migration (by wound assay) studies. In addition, cells were preincubated with prednisolone and/or the peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist 15-deoxy-Δ-12,14-prostaglandin J2 (15d-PGJ2) for 6 h before adding TGF-β1. pSmad1/5, pSmad2 and β-catenin levels were analyzed by Western blot. The activin receptor-like kinase-5 (ALK-5) inhibitor (SB-431542) was employed for the mechanistic assays.. Prednisolone showed a predominant anti-fibrotic impact on fibroblast-like synoviocytes as it attenuated the spontaneous and TGF-β-induced gene expression of pro-fibrotic markers. Prednisolone also reduced α-sma protein and type III collagen levels, as well as cell proliferation and migration after TGF-β stimulation. However, prednisolone did not downregulate the gene expression of all the pro-fibrotic markers tested and did not restore the reduced PPAR-γ levels after TGF-β stimulation. Interestingly, anti-fibrotic actions of the glucocorticoid were reinforced in the presence of the PPAR-γ agonist 15d-PGJ2. Combined pretreatment modulated Smad2/3 levels and, similar to the ALK-5 inhibitor, blocked β-catenin accumulation elicited by TGF-β.. Prednisolone, along with 15d-PGJ2, modulates pro-fibrotic pathways activated by TGF-β in synovial fibroblasts at least partially through the inhibition of ALK5/Smad2 signaling and subsequent β-catenin accumulation. These findings shed light on the potential therapeutic effects of glucocorticoids treatment combined with a PPAR-γ agonist against synovial fibrosis, although future studies are warranted to further evaluate this concern.

Topics: Adult; Aged; beta Catenin; Cells, Cultured; Female; Fibroblasts; Fibrosis; Humans; Male; Middle Aged; Osteoarthritis; PPAR gamma; Prednisolone; Prostaglandin D2; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

The role of PGD2 has been recognized in allergy, innate immunity and inflammation. Western blot analysis identified 21 kDa lipocalin (L)-prostaglandin D2 (PGD2) synthase (S) in human osteoarthritis (OA)-affected cartilage, whose expression was increased by IL-1β and TNFα. Similarly, PGD2 was spontaneously released by human OA-affected cartilage (and upregulated by IL-β) in ex vivo conditions and could be inhibited by indomethacin. Addition of PGD2 to human OA-affected cartilage significantly increased accumulation of PGE2, PGF1α, PGF2α, TXB2, but inhibited LTB4 and nitric oxide (NO) accumulation. Similarly, PGD2 (but not 13,14-dihydro-15-keto PGD2) augmented IL-1β induced PGE2 but inhibited IL-β induced nitric oxide (NO) in human chondrocytes. Celecoxib (10 μM) inhibits COX-1 mediated PGD2, and nitric oxide synthase (NOS) mediated NO in human OA-affected cartilage. Furthermore, celecoxib (1 μM) counter balances (IL-1β induced+PGD2 modulated) levels of NO and PGE2 in human OA-affected cartilage and chondrocytes to basal levels. These results show concentration-dependent, pro- and anti-inflammatory activity of PGD2 in human chondrocytes and cartilage, which can be neutralized by celecoxib. In view of the broad prostaglandin dependent and independent mechanism of action of celecoxib, these observations further reaffirm the broader role of celecoxib as a "Disease Modifying Drug" for human Osteoarthritis.

Topics: Celecoxib; Cells, Cultured; Chondrocytes; Dinoprost; Dinoprostone; Humans; Inflammation Mediators; Interleukin-1beta; Knee; Leukotriene B4; Nitric Oxide; Osteoarthritis; Prostaglandin D2; Prostaglandins F; Pyrazoles; Sulfonamides; Thromboxane B2

Mechanical overloading of articular cartilage producing hydrostatic stress, tensile strain, and fluid flow results in irreversible cartilage erosion and osteoarthritis (OA). Application of high fluid shear to chondrocytes recapitulates the earmarks of OA as evidenced by the induction of proinflammatory cytokines and prostaglandins, which are capable of inducing the expression of matrix-degrading enzymes. Matrix metalloproteinase-9 (MMP-9) synthesis is detected at early but not late stages of OA. However, the underlying mechanism(s) of the MMP-9 temporal regulation remains unknown. Using the T/C-28a2 chondrocyte cell line as a model system, we demonstrated that high fluid shear induces a marked increase in MMP-9 expression at short shear exposure times (3-6 h), which falls below basal levels after prolonged shear exposure (12-48 h). High fluid shear stress induced the rapid and sustained synthesis of IL-1β, activating PI3K, ERK1/2, and JNK, which are in turn responsible for MMP-9 expression. Prolonged shear exposure (>12 h) induced 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)) synthesis, which exerted an antagonistic effect on IL-1β-mediated PI3K-, ERK1/2-, and JNK-dependent NF-κB activation, thereby suppressing MMP-9 expression in human chondrocytes. Reconstructing the signaling network that regulates shear-mediated MMP-9 expression in human chondrocytes may provide insights for developing strategies to treat arthritic disorders.

Topics: Cartilage, Articular; Cells, Cultured; Chondrocytes; Gene Expression Regulation; Humans; Interleukin-1beta; Matrix Metalloproteinase 9; Models, Biological; Osteoarthritis; Prostaglandin D2; Shear Strength; Signal Transduction; Stress, Mechanical; Time Factors

Injury to articular cartilage is often associated with an inflammatory reaction and frequently results in the development of post-traumatic osteoarthritis (post-traumatic OA). Cell death, inflammation and loss of proteoglycans participate in these mechanisms with p38MAPK being one of the pivotal signaling kinases. Therefore, the interaction of trauma and of the pro-inflammatory cytokine IL-1β was investigated in an in vitro tissue model of human osteoarthritic cartilage. Trauma was induced by impacting cartilage explants with a drop-tower system and its effect was measured in terms of cell survival, gene expression and the release of mediators. In addition, the effect of concomitant IL‑1β stimulation and p38MAPK inhibition by SB203580 was investigated. We found a significant decrease in chondrocyte viability after trauma, but no additional effect of IL-1β stimulation. SB203580 had a tendency to improve cell survival suggesting a role for p38 signaling in cell viability after impact in an inflammatory environment. We showed that various mediators are released in response to trauma with or without IL-1β stimulation, differing in composition and time response. Trauma resulted in an increased release of IL-6, whereas TNF-α and IL-1β release was unaffected. Prostaglandin (PG) and NO synthesis pathways were both affected by trauma and/or IL-1β. We demonstrate for the first time an elevated release of prostaglandin D2 (PGD2) by human articular cartilage in response to a single mechanical impact. The up-regulation of mediators was time-dependent, with a more early increase of PGD2 compared to prostaglandin E2 (PGE2) and a late induction of NO by co-stimulation with IL-1β between 6 and 24 h.

Topics: Aged; Aged, 80 and over; Cartilage, Articular; Cell Survival; Chondrocytes; Cytokines; Enzyme Inhibitors; Gene Expression Regulation; Humans; Imidazoles; Inflammation Mediators; Interleukin-1beta; Middle Aged; Nitric Oxide Synthase Type II; Osteoarthritis; p38 Mitogen-Activated Protein Kinases; Prostaglandin D2; Pyridines; Signal Transduction; Up-Regulation

To investigate the effects of prostaglandin D2 (PGD2) on interleukin-1beta (IL-1beta)-induced matrix metalloproteinase 1 (MMP-1) and MMP-13 expression in human chondrocytes and the signaling pathways involved in these effects.. Chondrocytes were stimulated with IL-1 in the presence or absence of PGD2, and expression of MMP-1 and MMP-13 proteins was evaluated by enzyme-linked immunosorbent assay. Messenger RNA (mRNA) expression and promoter activity were analyzed by real-time reverse transcription-polymerase chain reaction 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/protein kinase A (PKA) pathway was determined using cAMP-elevating agents and PKA inhibitors.. PGD2 decreased in a dose-dependent manner IL-1-induced MMP-1 and MMP-13 protein and mRNA expression as well as their promoter activation. DP1 and CRTH2 were expressed and functional in chondrocytes. The effect of PGD2 was mimicked by BW245C, a selective agonist of DP1, but not by 13,14-dihydro-15-keto-PGD2, a selective agonist of CRTH2. 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 H89 reversed the inhibitory effect of PGD2, suggesting that the effect of PGD2 is mediated by the cAMP/PKA pathway.. PGD2 inhibits IL-1-induced production of MMP-1 and MMP-13 by chondrocytes through the DP1/cAMP/PKA signaling pathway. These data also suggest that modulation of PGD2 levels in the joint may have therapeutic potential in the prevention of cartilage degradation.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Aged; Antibodies; Carbazoles; Cells, Cultured; Chondrocytes; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme-Linked Immunosorbent Assay; Humans; Interleukin-1beta; Isoquinolines; Matrix Metalloproteinase 1; Matrix Metalloproteinase 13; Osteoarthritis; Prostaglandin D2; Pyrroles; Receptors, Immunologic; Receptors, Leukotriene B4; Receptors, Prostaglandin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Sulfonamides; Th2 Cells

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) plays a central role in whole body metabolism by regulating adipocyte differentiation and energy storage. Recently, however, PPAR-gamma has also been demonstrated to affect proliferation, differentiation, and apoptosis of different cell types. As we have previously shown that BAY 11-7085-induced synovial fibroblast apoptosis is prevented by PPAR-gamma agonist 15d-PGJ2; the expression of PPAR-gamma in these cells was studied. Both PPAR-gamma1 and PPAR-gamma2 isoforms were cloned from synovial fibroblast RNA, but only PPAR-gamma1 was detected by Western blot, showing constitutive nuclear expression. Within minutes of BAY 11-7085 treatment, a PPAR-gamma1-specific band was shifted into a form of higher mobility, suggesting dephosphorylation, as confirmed by phosphatase treatment of cell extracts. Of interest, BAY 11-7085-induced PPAR-gamma1 dephosphorylation was followed by PARP and caspase-8 cleavage as well as by PPAR-gamma1 protein degradation. PPAR-gamma1 dephosphorylation was followed by the loss of PPAR-DNA binding activity ubiquitously present in synovial fibroblast nuclear extracts. Unlike the phosphorylated form, dephosphorylated PPAR-gamma1 was found in insoluble membrane cell fraction and was not ubiquitinated before degradation. PPAR-gamma1 dephosphorylation coincided with ERK1/2 phosphorylation that accompanies BAY 11-7085-induced synovial fibroblasts apoptosis. 15d-PGJ2, PGD2, and partially UO126, down-regulated ERK1/2 phosphorylation, protected cells from BAY 11-7085-induced apoptosis, and reversed both PPAR-gamma dephosphorylation and degradation. Furthermore, PPAR-gamma antagonist BADGE induced PPAR-gamma1 degradation, ERK1/2 phosphorylation, and synovial fibroblasts apoptosis. The results presented suggest an anti-apoptotic role for PPAR-gamma1 in synovial fibroblasts. Since apoptotic marker PARP is cleaved after PPAR-gamma1 dephosphorylation but before PPAR-gamma1 degradation, dephosphorylation event might be enough to mediate BAY 11-7085-induced apoptosis in synovial fibroblasts.

Topics: Apoptosis; Cell Line; Cell Nucleus; Fibroblasts; Gene Expression; Humans; Nitriles; Osteoarthritis; Phosphorylation; PPAR gamma; Prostaglandin D2; Sulfones; Synovial Membrane; Transfection

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily and have a dominant regulatory role in adipocyte and monocyte differentiation. PPAR-gamma agonists are also negative regulators of macrophage activation and have modulatory effects on tumorigenesis. In this study we demonstrate that synovial tissue localized expression of PPAR-gamma in patients with rheumatoid arthritis (RA). We detected markedly enhanced expression of PPAR-gamma in macrophages, as well as modestly enhanced expression in the synovial lining layer, fibroblasts, and endothelial cells. Activation of the PPAR-gamma by 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) and the synthetic PPAR-gamma ligand (troglitazone) induced RA synoviocyte apoptosis in vitro. Moreover, intraperitoneal administration of these PPAR-gamma ligands ameliorated adjuvant-induced arthritis with suppression of pannus formation and mononuclear cell infiltration in female Lewis rats. Anti-inflammatory effects of 15d-PGJ(2) were more potent than troglitazone. These findings suggest that PPAR-gamma may be an important immunoinflammatory mediator and its ligands, especially 15d-PGJ(2), may be useful in the treatment of RA.

Topics: Animals; Apoptosis; Arthritis; Arthritis, Experimental; Arthritis, Rheumatoid; Cells, Cultured; Chromans; Female; Humans; Ligands; Osteoarthritis; Prostaglandin D2; Rats; Rats, Inbred Lew; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Synovial Membrane; Thiazoles; Thiazolidinediones; Tissue Distribution; Transcription Factors; Troglitazone