Ajulemic acid, a synthetic cannabinoid acid, induces an antiinflammatory profile of eicosanoids in human synovial cells

AIMS: To better understand mechanisms whereby Ajulemic acid (AjA), a synthetic antiinflammatory cannabinoid, promotes resolution of acute and chronic inflammation in animal models, we investigated its influence on cyclooxygenase 2 (COX2) expression and eicosanoid production in human fibroblast-like synovial cells (FLS). MAIN METHODS: FLS isolated from tissue obtained at joint replacement surgery or cultured from synovial fluid were treated for 60 min with AjA (10-30 microM), then stimulated with tumor necrosis factor alpha (TNFalpha). COX2 mRNA was measured by hybridization/colorimetric assay of whole cell lysates collected 4 h after stimulation. To determine effects on arachidonic acid release, FLS were incubated with (14)C-arachidonic acid for 20 h then treated with AjA (8-32 microM). Arachidonic acid release was measured by scintillation counting. Prostaglandins (PG) were measured by enzyme linked immunosorbent assay (ELISA) in cell supernatants collected 4 and 24 h after stimulation. KEY FINDINGS: AjA increased the steady state levels of COX2 mRNA in and arachidonic acid release from FLS. Treatment of FLS with AjA increased 15-deoxy-delta(12,14)-PGJ(2) (15d-PGJ(2)) production in a concentration dependent manner, but did not affect PGE(2) production significantly. SIGNIFICANCE: The capacity of AjA to increase selectively and markedly 15d-PGJ(2), an eicosanoid which facilitates resolution of inflammation, suggests that AjA may have value as a therapeutic agent for the treatment of rheumatoid arthritis (RA) and other diseases characterized by acute and chronic inflammation.     

15-deoxy-Δ(12,14) -prostaglandin-J2 and ciglitazone inhibit TNF-α-induced matrix metalloproteinase 13 production via the antagonism of NF-κB activation in human synovial fibroblasts

Collagenase-3 (matrix metalloproteinase, MMP-13) plays an important role in the degradation of cartilage in pathologic conditions. MMP-13 is elevated in joint tissues in both rheumatoid arthritis (RA) and osteoarthritis (OA). In addition, inflammation-stimulated synovial fibroblasts are able to release MMP-13 and other cytokines in these diseases. The peroxisome proliferator-activated receptor-γ (PPARγ) ligands are recently considered as new anti-inflammatory compounds and these ligands were reported to ameliorate inflammatory arthritis. The aim of this study is to evaluate the mechanisms how PPARγ ligands inhibit the inflammatory response in synovial fibroblasts. Two PPARγ ligands, cyclopentenone prostaglandin 15-deoxy-Δ(12,14) -prostaglandin-J2 (15d-PGJ2) and synthetic thiazolidinedione compound ciglitazone were examined in this study. Here we found that 15d-PGJ2 and ciglitazone markedly inhibited TNF-α-induced MMP-13 production in human synovial fibroblasts. In addition, activation of nuclear factor κB (NF-κB) is strongly associated with MMP-13 induction by TNF-α and the activation of NF-κB was determined by Western blot, reporter assay, and immunofluorescence. It was found that 15d-PGJ2 markedly attenuated the translocation of NF-κB by direct inhibition of the activation of IKK via a PPARγ-independent manner. Ciglitazone also inhibits TNF-α-induced MMP-13 expression by suppressing NF-κB activation mainly via the modulation of p38-MAPK. Collectively, our data demonstrate that 15d-PGJ2 and ciglitazone attenuated TNF-α-induced MMP-13 expression in synovial fibroblasts primarily through the modulation of NF-κB signaling pathways. These compounds may have therapeutic application in inflammatory arthritis.     

Effect of peroxisome proliferator activated receptor (PPAR)gamma agonists on prostaglandins cascade in joint cells

In response to inflammatory cytokines, chondrocytes and synovial fibroblasts produce high amounts of prostaglandins (PG) which self-perpetuate locally the inflammatory reaction. Prostaglandins act primarily through membrane receptors coupled to G proteins but also bind to nuclear Peroxisome Proliferator-Activated Receptors (PPARs). Amongst fatty acids, the cyclopentenone metabolite of PGD2, 15-deoxy-Delta12,14PGJ2 (15d-PGJ2), was shown to be a potent ligand of the PPARgamma isotype prone to inhibit the production of inflammatory mediators. As the stimulated synthesis of PGE2 originates from the preferential coupling of inducible enzymes, cyclooxygenase-2 (COX-2) and membrane PGE synthase-1 (mPGES-1), we investigated the potency of 15d-PGJ2 to regulate prostaglandins synthesis in rat chondrocytes stimulated with interleukin-1beta (IL-1beta). We demonstrated that 15d-PGJ2, but not the high-affinity PPARgamma ligand rosiglitazone, decreased almost completely PGE2 synthesis and mPGES-1 expression. The inhibitory potency of 15d-PGJ2 was unaffected by changes in PPARgamma expression and resulted from inhibition of NF-kappaB nuclear binding and IkappaBalpha sparing, secondary to reduced phosphorylation of IKKbeta. Consistently with 15d-PGJ2 being a putative endogenous regulator of the inflammatory reaction if synthesized in sufficient amounts, the present data confirm the variable PPARgamma-dependency of its effects in joint cells while underlining possible species and cell types specificities.     

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

We have previously shown that nuclear factor-kappaB inhibition by adenovirus expressing mutated IkappaB-alpha or by proteasome inhibitor increases human articular chondrocytes sensibility to apoptosis. Moreover, the nuclear factor-kappaB inhibitor BAY11-7085, a potent anti-inflammatory drug in rat adjuvant arthritis, is itself a proapoptotic agent for chondrocytes. In this work, we show that BAY 11-7085 but not the proteasome inhibitor MG-132 induced a rapid and sustained phosphorylation of extracellular signal-regulated kinases (ERK1/2) in human articular chondrocytes. The level of ERK1/2 phosphorylation correlated with BAY 11-7085 concentration and chondrocyte apoptosis. 15-Deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2) and its precursor prostaglandin (PG) D2 but not PGE2 and PGF2alpha rescued chondrocytes from BAY 11-7085-induced apoptosis. 15d-PGJ2 markedly inhibited BAY 11-7085-induced phosphorylation of ERK1/2. BAY 11-7085 also induced ERK1/2 phosphorylation and apoptosis in human synovial fibroblasts, and these reactions were down-regulated by 15d-PGJ2. Further analysis in synovial fibroblasts showed that only molecules that suppressed BAY 11-7085-induced phosphorylation of ERK1/2 (i.e. 15d-PGJ2, PGD2, and to a lesser extent, MEK1/2 inhibitor UO126, but not prostaglandins E2 and F2alpha or peroxisome proliferator-activated receptor-gamma agonist ciglitazone) were able protect cells from apoptosis. These results suggested that the antiapoptotic effect of 15d-PGJ2 on chondrocytes and synovial fibroblasts might involve inhibition of ERK1/2 phosphorylation.     

15-deoxy-delta12,14-prostaglandin J2 regulates the functional state and the survival of microglial cells through multiple molecular mechanisms

We have previously reported that rat primary microglial cultures express the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and that several functions associated with the activation of these cells, including nitric oxide (NO) and tumor necrosis factor-alpha synthesis, are down-regulated by 15-deoxy-delta12,14-prostaglandin J2 (15d-PGJ2) and ciglitazone, two specific PPAR-gamma agonists. Here we demonstrate that microglial cells not only express a functionally active PPAR-gamma, but also synthesize large amounts of 15d-PGJ2 upon stimulation with lipopolysaccharide (LPS). In addition, we show that, although 15d-PGJ2 and ciglitazone were equally effective in reducing microglial activation when used at 1-5 microm concentrations, 15d-PGJ2, but not of ciglitazone, reduced PGE2 production at low concentration (0.1 microm) and induced a time-dependent microglial impairment and apoptosis at high concentration (10 microm). Interestingly, the inhibition of PGE2 production was achieved mainly through the inhibition of cycloxygenase-2 enzymatic activity, as the expression of this enzyme and that of the microsomal isoform of PGE synthase remained unaltered. These findings suggest that 15d-PGJ2 affects the functional state and the survival of activated microglia through mechanisms only in part dependent on PPAR-gamma and that the concentration of 15d-PGJ2 is crucial in determining the particular microglial function affected.     

15-Deoxy-Delta(12,14)-prostaglandin J2 inhibits the expression of proinflammatory genes in human blood monocytes via a PPAR-gamma-independent mechanism

The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) has been implicated in inhibition of the expression of proinflammatory cytokines and inducible enzymes such as cyclooxygenase-2 (COX-2). Using real-time RT-PCR the present study investigates the impact of two PPAR-gamma agonists, 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) and ciglitazone, on the expression of several proinflammatory genes in lipopolysaccharide (LPS)-stimulated human blood monocytes. Stimulation of cells with LPS resulted in a profound induction of the expression of COX-2, interleukin (IL)-1, IL-6, tumor necrosis factor (TNF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Treatment of cells with 15d-PGJ(2) (10 microM) was associated with a nearly complete inhibition of the expression of all genes that remained unaltered in the presence of the PPAR-gamma antagonist bisphenol A diglycidyl ether (BADGE; 100 microM). By contrast, treatment of cells with another potent PPAR-gamma agonist, ciglitazone (50 microM), and the PPAR-alpha agonist WY-14,643 (100 microM) did not suppress LPS-induced expression of the investigated genes. Stimulation of monocytes with LPS resulted in an 88% inhibition of PPAR-gamma mRNA expression that was fully restored by 15d-PGJ(2) but only to a partial extent by ciglitazone and WY-14,643. Again, BADGE did not alter the effect of 15d-PGJ(2). Collectively, our results show that alterations of gene expression by 15d-PGJ(2) in LPS-stimulated human blood monocytes are mediated by PPAR-gamma-independent mechanisms. Moreover, it is concluded that both inhibition of proinflammatory gene expression and restoration of LPS-induced decrease of PPAR-gamma expression may contribute to the biological action of 15d-PGJ(2).     

Peroxisome proliferator-activated receptor-gamma1 is dephosphorylated and degraded during BAY 11-7085-induced synovial fibroblast apoptosis

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.     

LPS induces fibroblast-like synoviocytes RSC-364 cells to pyroptosis through NF-κB mediated dual signalling pathway

Journal of Molecular Histology - Rheumatoid arthritis (RA) is a chronic, progressive, and systemic inflammatory joint disease characterized by synovial inflammation and joint damage. Abnormal...     

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

Microsomal prostaglandin E synthase (mPGES)-1 is a newly identified inducible enzyme of the arachidonic acid cascade with a key function in prostaglandin (PG)E2 synthesis. We investigated the kinetics of inducible cyclo-oxygenase (COX)-2 and mPGES-1 expression with respect to the production of 6-keto-PGF1alpha and PGE2 in rat chondrocytes stimulated with 10 ng/ml IL-1beta, and compared their modulation by peroxisome-proliferator-activated receptor (PPAR)gamma agonists. Real-time PCR analysis showed that IL-1beta induced COX-2 expression maximally (37-fold) at 12 hours and mPGES-1 expression maximally (68-fold) at 24 hours. Levels of 6-keto-PGF1alpha and PGE2 peaked 24 hours after stimulation with IL-1beta; the induction of PGE2 was greater (11-fold versus 70-fold, respectively). The cyclopentenone 15-deoxy-Delta12,14prostaglandin J2 (15d-PGJ2) decreased prostaglandin synthesis in a dose-dependent manner (0.1 to 10 microM), with more potency on PGE2 level than on 6-keto-PGF1alpha level (-90% versus -66% at 10 microM). A high dose of 15d-PGJ2 partly decreased COX-2 expression but decreased mPGES-1 expression almost completely at both the mRNA and protein levels. Rosiglitazone was poorly effective on these parameters even at 10 microM. Inhibitory effects of 10 microM 15d-PGJ2 were neither reduced by PPARgamma blockade with GW-9662 nor enhanced by PPARgamma overexpression, supporting a PPARgamma-independent mechanism. EMSA and TransAM analyses demonstrated that mutated IkappaBalpha almost completely suppressed the stimulating effect of IL-1beta on mPGES-1 expression and PGE2 production, whereas 15d-PGJ2 inhibited NF-kappaB transactivation. These data demonstrate the following in IL-1-stimulated rat chondrocytes: first, mPGES-1 is rate limiting for PGE2 synthesis; second, activation of the prostaglandin cascade requires NF-kappaB activation; third, 15d-PGJ2 strongly inhibits the synthesis of prostaglandins, in contrast with rosiglitazone; fourth, inhibition by 15d-PGJ2 occurs independently of PPARgamma through inhibition of the NF-kappaB pathway; fifth, mPGES-1 is the main target of 15d-PGJ2.     

Temporal expression patterns of distinct cytokines and M1/M2 macrophage polarization regulate rheumatoid arthritis progression

Molecular Biology Reports - Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of synovial joints and often associated with chronic pain. Chronic joint...     

Apoptosis as a target for gene therapy in rheumatoid arthritis

Rheumatoid arthritis (RA) is characterized by chronic inflammation of the synovial joints resulting from hyperplasia of synovial fibroblasts and infiltration of lymphocytes, macrophages and plasma cells, all of which manifest signs of activation. All these cells proliferate abnormally, invade bone and cartilage, produce an elevated amount of pro-inflammatory cytokines, metalloproteinases and trigger osteoclast formation and activation. Some of the pathophysiological consequences of the disease may be explained by the inadequate apoptosis, which may promote the survival of autoreactive T cells, macrophages or synovial fibroblasts. Although RA does not result from single genetic mutations, elucidation of the molecular mechanisms implicated in joint destruction has revealed novel targets for gene therapy. Gene transfer strategies include inhibition of pro-inflammatory cytokines, blockade of cartilage-degrading metalloproteinases, inhibition of synovial cell activation and manipulation of the Th1-Th2 cytokine balance. Recent findings have iluminated the idea that induction of apoptosis in the rheumatoid joint can be also used to gain therapeutic advantage in the disease. In the present review we will discuss different strategies used for gene transfer in RA and chronic inflammation. Particularly, we will high-light the importance of programmed cell death as a novel target for gene therapy using endogenous biological mediators, such as galectin-1, a beta-galactoside-binding protein that induces apoptosis of activated T cells and immature thymocytes.     

7beta-Hydroxy-epiandrosterone-mediated regulation of the prostaglandin synthesis pathway in human peripheral blood monocytes

7alpha-Hydroxy-DHEA, 7beta-hydroxy-DHEA and 7beta-hydroxy-EpiA are native metabolites of dehydroepiandrosterone (DHEA) and epiandrosterone (EpiA). Since numerous steroids are reported to interfere with inflammatory and immune processes, our objective was to test the effects of these hydroxysteroids on prostaglandin (PG) production and related enzyme gene expression. Human peripheral blood monocytes were cultured for 4 and 24 h in the presence of each of the steroids (1-100 nM), with and without addition of TNF-alpha (10 ng/mL). Levels of PGE(2), PGD(2) and 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) were measured in the incubation medium, and cell content of cyclooxygenase (COX-2), and PGE and PGD synthases (m-PGES1, H-PGDS, L-PGDS), and peroxisome proliferator activated receptor (PPAR-gamma) was assessed by quantitative RT-PCR and Western blots. Addition of TNF-alpha resulted in elevated PG production and increased COX-2 and m-PGES1 levels. Among the three steroids tested, only 7beta-hydroxy-EpiA decreased COX-2, m-PGES1 and PPAR-gamma expression while markedly decreasing PGE(2) and increasing 15d-PGJ(2) production. These results suggest that 7beta-hydroxy-EpiA is a native trigger of cellular protection through simultaneous activation of 15d-PGJ(2) and depression of PGE(2) synthesis, and that these effects may be mediated by activation of a putative receptor, specific for 7beta-hydroxy-EpiA.     

A potential role of 15-deoxy-delta(12,14)-prostaglandin J2 for induction of human articular chondrocyte apoptosis in arthritis

The cyclopentenone prostaglandin (PG) J2 is formed within the cyclopentenone ring of the endogenous prostaglandin PG D2 by a nonenzymatic reaction. The PG J family is involved in mediating various biological effects including the regulation of cell cycle progression and inflammatory responses. Here we demonstrate the potential role of 15-deoxy-Delta(12,14)-prostaglandin J2 (15d-PG J2) in human articular chondrocyte apoptosis. 15d-PG J2 was released by human articular chondrocytes and found in joint synovial fluids taken from osteoarthritis or rheumatoid arthritis patients. Proinflammatory cytokines such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) up-regulated chondrocyte release of 15d-PG J2. PG D2 synthase mRNA expression was up-regulated by IL-1beta, TNF-alpha, or nitric oxide. 15d-PG J2 induced apoptosis of chondrocytes from osteoarthritis or rheumatoid arthritis patients as well as control nonarthritic subjects in a time- and dose-dependent manner and in a peroxisome proliferator-activated receptor gamma-dependent manner. Peroxisome proliferator-activated receptor gamma expression was up-regulated by IL-1beta and TNF-alpha. Inhibition of NF-kappaB, and the activation of p38 MAPK were also found to be involved in 15d-PG J2-induced chondrocyte apoptosis. Such signal pathways led to the activation of the downstream pro-apoptotic molecule p53 and caspase cascades. Together, these results suggest that 15d-PGJ2 may play an important role in the pathogenesis of arthritic joint destruction via a regulation of chondrocyte apoptosis.     

PPAR-gamma inhibits ANG II-induced cell growth via SHIP2 and 4E-BP1

The present study evaluated the effects of peroxisome proliferator-activated receptor (PPAR)-gamma activators on ANG II-induced signaling pathways and cell growth. Vascular smooth muscle cells (VSMC) derived from rat mesenteric arteries were treated with ANG II, with/without the AT1 receptor blocker valsartan or the AT2 receptor blocker PD-123319, after pretreatment for 24 h with the PPAR-gamma activators 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2) or rosiglitazone. Both 15d-PGJ2 and rosiglitazone decreased ANG II-induced DNA synthesis. Rosiglitazone treatment increased nuclear PPAR-gamma expression and activity in VSMC. However, rosiglitazone did not alter expression of PPAR-alpha/beta, ERK 1/2, Akt, or ANG II receptors. 15d-PGJ2 and rosiglitazone decreased ERK 1/2 and Akt peak activity, both of which were induced by ANG II via the AT1 receptor. Rosiglitazone inhibited ANG II-enhanced phosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), as well as Src homology (SH) 2-containing inositol phosphatase 2 (SHIP2). PPAR-gamma activation reduced ANG II-induced growth associated with inhibition of ERK 1/2, Akt, 4E-BP1, and SHIP2. Modulation of these pathways by PPAR-gamma activators may contribute to regression of vascular remodeling in hypertension.     

Methionine aminopeptidase-2 blockade reduces chronic collagen-induced arthritis: potential role for angiogenesis inhibition

The enzyme methionine aminopeptidase-2 (MetAP-2) is thought to play an important function in human endothelial cell proliferation, and as such provides a valuable target in both inflammation and cancer. Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with increased synovial vascularity, and hence is a potential therapeutic target for angiogenesis inhibitors. We examined the use of PPI-2458, a selective non-reversible inhibitor of MetAP-2, in disease models of RA, namely acute and chronic collagen-induced arthritis (CIA) in mice. Whilst acute CIA is a monophasic disease, CIA induced with murine collagen type II manifests as a chronic relapsing arthritis and mimics more closely the disease course of RA. Our study showed PPI-2458 was able to reduce clinical signs of arthritis in both acute and chronic CIA models. This reduction in arthritis was paralleled by decreased joint inflammation and destruction. Detailed mechanism of action studies demonstrated that PPI-2458 inhibited human endothelial cell proliferation and angiogenesis in vitro, without affecting production of inflammatory cytokines. Furthermore, we also investigated release of inflammatory cytokines and chemokines from human RA synovial cell cultures, and observed no effect of PPI-2458 on spontaneous expression of cytokines and chemokines, or indeed on the angiogenic molecule vascular endothelial growth factor (VEGF). These results highlight MetAP-2 as a good candidate for therapeutic intervention in RA.