Dynamic compression counteracts IL-1beta induced iNOS and COX-2 activity by human chondrocytes cultured in agarose constructs

*NO and PGE2 are inflammatory mediators derived from the inducible iNOS and COX enzymes and are potentially important pharmacological targets in OA. Both mechanical loading and IL-1beta will influence the release of *NO and PGE2. Accordingly, the current study examines the effect of dynamic compression on *NO and PGE2 release by human chondrocytes cultured in agarose constructs in the presence and absence of selective iNOS and COX-2 inhibitors. The current data demonstrate that IL-1beta induced nitrite and PGE2 release and inhibited [3H]-thymidine and 35SO4 incorporation. Inhibitor experiments indicate that 1400W and NS-398 either partially reversed or abolished IL-1beta induced nitrite and PGE2 release. IL-1beta induced inhibition of cell proliferation and proteoglycan synthesis was partially reversed with 1400W but was not influenced by NS-398. For the dynamic loading experiments, 1400W and NS-398 either reduced or abolished the compression-induced inhibition of *NO and PGE2 release in the presence of IL-1beta. The IL-1beta induced inhibition of cell proliferation was not influenced by 1400W or NS-398 whereas strain-induced stimulation of proteoglycan synthesis in the presence of IL-1beta was enhanced by 1400W. The data obtained using human chondrocytes demonstrate that IL-1beta induced *NO and PGE2 release via an iNOS-driven-COX-2 inter-dependent pathway. This response could be reversed by dynamic compression. These data indicate interactions exist between the NOS and COX pathways, a finding which will provide new insights in the development of pharmacological or biophysical treatments for cartilage disorders such as OA.     

Tetracycline up-regulates COX-2 expression and prostaglandin E2 production independent of its effect on nitric oxide

Tetracyclines (doxycycline and minocycline) augmented (one- to twofold) the PGE2 production in human osteoarthritis-affected cartilage (in the presence or absence of cytokines and endotoxin) in ex vivo conditions. Similarly, bovine chondrocytes stimulated with LPS showed (one- to fivefold) an increase in PGE2 accumulation in the presence of doxycycline. This effect was observed at drug concentrations that did not affect nitric oxide (NO) production. In murine macrophages (RAW 264.7) stimulated with LPS, tetracyclines inhibited NO release and increased PGE2 production. Tetracycline(s) and L-N-monomethylarginine (L-NMMA) (NO synthase inhibitor) showed an additive effect on inhibition of NO and PGE2 accumulation, thereby uncoupling the effects of tetracyclines on NO and PGE2 production. The enhancement of PGE2 production in RAW 264.7 cells by tetracyclines was accompanied by the accumulation of both cyclooxygenase (COX)-2 mRNA and cytosolic COX-2 protein. In contrast to tetracyclines, L-NMMA at low concentrations (< or = 100 microM) inhibited the spontaneous release of No in osteoarthritis-affected explants and LPS-stimulated macrophages but had no significant effect on the PGE2 production. At higher concentrations, L-NMMA (500 microM) inhibited NO release but augmented PGE2 production. This study indicates a novel mechanism of action of tetracyclines to augment the expression of COX-2 and PGE2 production, an effect that is independent of endogenous concentration of NO.     

Regulation of cytosolic COX-2 and prostaglandin E2 production by nitric oxide in activated murine macrophages

Murine macrophages (RAW 264.7) when stimulated with LPS show 90% distribution of cyclooxygenase-2 (COX-2) in the nuclear fraction and approximately 10% in the cytosolic fraction. Further analysis of this cytosolic fraction at 100,000 x g indicates that the COX-2 is distributed both in the 100,000 x g soluble fraction and membrane fraction. Stimulation of RAW 264.7 cells with LPS in the presence of inducible nitric oxide synthase inhibitor L-NMMA at concentrations that inhibit nitrite accumulation by /=85% with higher concentrations of L-NMMA shows 1) up-regulation of PGE2 production, 2) accumulation of COX-2 protein in the 100,000 x g soluble and membrane fractions of the cytosolic fraction, and 3) with no significant effects on the accumulation of COX-2 mRNA. These experiments suggest that low concentrations of nitric oxide (10-15% of the total) attenuate PGE2 production in response to LPS in RAW 264.7 cells. This inhibition is, in part, due to decreased expression of cytosolic COX-2 protein.     

Differentiation status-dependent regulation of cyclooxygenase-2 expression and prostaglandin E2 production by epidermal growth factor via mitogen-activated protein kinase in articular chondrocytes

Although large amounts of epidermal growth factor (EGF) are found in the synovial fluids of arthritic cartilage, the role of EGF in arthritis is not clearly understood. This study investigated the effect of EGF on differentiation and on inflammatory responses such as cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) (PGE(2)) production in articular chondrocytes. EGF caused a loss of differentiated chondrocyte phenotype as demonstrated by inhibition of type II collagen expression and proteoglycan synthesis. EGF also induced COX-2 expression and PGE(2) production. EGF-induced dedifferentiation was caused by EGF receptor-mediated activation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) but not p38 kinase, whereas the activation of both ERK1/2 and p38 kinase was necessary for COX-2 expression and PGE(2) production. Neither the inhibition of COX-2 expression and PGE(2) production nor the addition of exogenous PGE(2) affected EGF-induced dedifferentiation. However, COX-2 expression and PGE(2) production were significantly enhanced in chondrocytes that were dedifferentiated by serial subculture, and EGF also potentiated COX-2 expression and PGE(2) production, although these cells were less sensitive to EGF. Dedifferentiation-induced COX-2 expression and PGE(2) production were mediated by ERK1/2 and p38 kinase signaling. Our results indicate that EGF in articular chondrocytes stimulates COX-2 expression and PGE(2) production via ERK and p38 kinase signaling in association with differentiation status.     

Dynamic compression inhibits fibronectin fragment induced iNOS and COX-2 expression in chondrocyte/agarose constructs

Mechanical loading and the fibronectin fragments (FN-fs) are known to stimulate the anabolic and catabolic processes in articular cartilage, possible through pathways mediated by ^·NO. This study examined the combined effects of dynamic compression and the NH₂-hep I or COOH-hep II FN-fs on the expression levels of iNOS and COX-2 and production of ^·NO and PGE₂ release. Both types of fragments induced iNOS and COX-2 expression and stimulated the production of ^·NO release. This response was inhibited by dynamic compression. Inhibitor experiments indicated that both dynamic compression and the iNOS inhibitor were important in restoring cell proliferation and proteoglycan synthesis in the presence of the FN-fs. This is the first study which demonstrates a downregulation of the FN-f-induced iNOS and COX-2 expression by dynamic compression. The combination of mechanical and pharmacological interventions makes this study a powerful tool to examine further the interactions of biomechanics and cell signalling in osteoarthritis.     

Catalase potentiates interleukin-1beta-induced expression of nitric oxide synthase in rat vascular smooth muscle cells

The role of reactive oxygen species (ROS) in regulating the expression of the inducible nitric oxide synthase (iNOS) was studied in rat aortic vascular smooth muscle cells (VSMC). We hypothesized that ROS regulate iNOS expression through the mitogen-activated protein kinases ERK and p38(MAPK). We found that interleukin-1beta (IL-1beta) stimulated the production of hydrogen peroxide (H2O2) which could be inhibited by loading the cells with the H2O2-scavenging enzyme catalase. Inhibition of the upstream ERK1,2 activator MEK1,2 with U0126 prevented IL-1beta-stimulated iNOS expression, while the p38MAPK inhibitor SB03580 potentiated iNOS expression. Loading the cells with catalase enhanced ERK activation and iNOS expression but had no effect on p38MAPK activation or PDGF-induced ERK activation. These data indicated that H2O2 negatively regulates iNOS expression through ERK inhibition independently of p38MAPK. The present results outline a novel role for H2O2 in suppressing signaling pathways leading to gene expression such as iNOS in VSMC in response to cytokines.     

Rho-kinase mediates spinal nitric oxide formation by prostaglandin E2 via EP3 subtype

Prostaglandin E2 (PGE2), the principal pro-inflammatory prostanoid, is known to play versatile roles in pain transmission via four PGE receptor subtypes, EP1-EP4. We recently demonstrated that continuous production of nitric oxide (NO) by neuronal NO synthase (nNOS) following phosphorylation of myristoylated alanine-rich C-kinase substrate (MARCKS) and NMDA receptor NR2B subunits is essential for neuropathic pain. These phosphorylation and nNOS activity visualized by NADPH-diaphorase histochemistry were blocked by indomethacin, a PG synthesis inhibitor. To clarify the interaction between cyclooxygenase and nNOS pathways in the spinal cord, we examined the effect of EP subtype-selective agonists on NO production. NO formation was stimulated in the spinal superficial layer by EP1, EP3, and EP4 agonists. While the EP1- and the EP4-stimulated NO formation was markedly blocked by MK-801, an NMDA receptor antagonist, the EP3-stimulated one was completely inhibited by H-1152, a Rho-kinase inhibitor. Phosphorylation of MARCKS and NADPH-diaphorase activity stimulated by the EP3 agonist were also blocked by H-1152. These results suggest that PGE2 stimulates NO formation by Rho-kinase via EP3, a mechanism(s) different from EP1 and EP4.     

Hyaluronan fragments activate nitric oxide synthase and the production of nitric oxide by articular chondrocytes

Chondrocyte CD44 receptors anchor hyaluronan to the cell surface, enabling the assembly and retention of proteoglycan aggregates in the pericellular matrix. Hyaluronan-CD44 interactions also provide signaling important for maintaining cartilage homeostasis. Disruption of chondrocyte-hyaluronan contact alters CD44 occupancy, initiating alternative signaling cascades. Treatment with hyaluronan oligosaccharides is one approach to uncouple CD44 receptors from its native ligand, hyaluronan. In bovine articular chondrocytes, treatment with hyaluronan oligosaccharides or purified hyaluronan hexasaccharides induced the production of nitric oxide that mirrored nitric oxide production following interleukin-1 treatment. In contrast, 120 and 1,260 kDa hyaluronan did not induce production of nitric oxide. Human chondrocytes responded similarly to treatment with hyaluronan or hyaluronan oligosaccharides. Nitric oxide production from chondrocytes was mediated by activation of the inducible nitric oxide synthase, as confirmed by mRNA expression and inhibition of nitric oxide production by diphenyleneiodonium. Co-treatment of chondrocytes with hyaluronan oligosaccharides and interleukin-1 did not demonstrate additive effects. Blocking interleukin-1 receptors with an antagonist did not abolish the production of nitric oxide induced by treatment with hyaluronan oligosaccharides. Moreover, only COS-7 following transfection with a pCD44, not the CD44-null parental cells, responded to treatment with hyaluronan oligosaccharides by releasing nitric oxide. This study demonstrates a novel signaling potential by hyaluronan fragments, in lieu of endogenous hyaluronan-chondrocyte interactions, resulting in the activation of inducible nitric oxide synthase.     

Sphingosylphosphorylcholine acts in an anti-inflammatory manner in renal mesangial cells by reducing interleukin-1beta-induced prostaglandin E2 formation

Sphingosylphosphorylcholine (SPC) is a bioactive lipid that binds to G protein-coupled-receptors and activates various signaling cascades. Here, we show that in renal mesangial cells, SPC not only activates various protein kinase cascades but also activates Smad proteins, which are classical members of the transforming growth factor-beta (TGFbeta) signaling pathway. Consequently, SPC is able to mimic TGFbeta-mediated cell responses, such as an anti-inflammatory and a profibrotic response. Interleukin-1beta-stimulated prostaglandin E(2) formation is dose-dependently suppressed by SPC, which is paralleled by reduced secretory phospholipase A(2) (sPLA(2)) protein expression and activity. This effect is due to a reduction of sPLA(2) mRNA expression caused by inhibited sPLA(2) promoter activity. Furthermore, SPC upregulates the profibrotic connective tissue growth factor (CTGF) protein and mRNA expression. Blocking TGFbeta signaling by a TGFbeta receptor kinase inhibitor causes an inhibition of SPC-stimulated Smad activation and reverses both the negative effect of SPC on sPLA(2) expression and the positive effect on CTGF expression. In summary, our data show that SPC, by mimicking TGFbeta, leads to a suppression of proinflammatory mediator production and stimulates a profibrotic cell response that is often the end point of an anti-inflammatory reaction. Thus, targeting SPC receptors may represent a novel therapeutic strategy to cope with inflammatory diseases.     

Dynamic compression inhibits the synthesis of nitric oxide and PGE(2) by IL-1beta-stimulated chondrocytes cultured in agarose constructs

Both mechanical loading and interleukin-1beta (IL-1beta) are known to regulate metabolic processes in articular cartilage through pathways mediated by nitric oxide ((*)NO) and PGE(2). This study uses a well-characterized model system involving isolated chondrocytes cultured in agarose constructs to test the hypothesis that dynamic compression alters the synthesis of (*)NO and PGE(2) by IL-1beta-stimulated articular chondrocytes. The data presented demonstrate for the first time that dynamic compression counteracts the effects of IL-1beta on articular chondrocytes by suppressing both (*)NO and PGE(2) synthesis. Inhibitor experiments indicated that the dynamic compression-induced inhibition of PGE(2) synthesis and stimulation of proteoglycan synthesis were (*)NO mediated, while compression-induced stimulation of cell proliferation was (*)NO independent. The inhibition of (*)NO and PGE(2) by dynamic compression is a finding of major significance that could contribute to the development of novel strategies for the treatment of cartilage-degenerative disorders.     

Green tea polyphenol epigallocatechin-3-gallate inhibits the IL-1 beta-induced activity and expression of cyclooxygenase-2 and nitric oxide synthase-2 in human chondrocytes

We have previously shown that green tea polyphenols inhibit the onset and severity of collagen II-induced arthritis in mice. In the present study, we report the pharmacological effects of green tea polyphenol epigallocatechin-3-gallate (EGCG), on interleukin-1 beta (IL-1 beta)-induced expression and activity of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in human chondrocytes derived from osteoarthritis (OA) cartilage. Stimulation of human chondrocytes with IL-1 beta (5 ng/ml) for 24 h resulted in significantly enhanced production of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) when compared to untreated controls (p <.001). Pretreament of human chondrocytes with EGCG showed a dose-dependent inhibition in the production of NO and PGE(2) by 48% and 24%, respectively, and correlated with the inhibition of iNOS and COX-2 activities (p <.005). In addition, IL-1 beta-induced expression of iNOS and COX-2 was also markedly inhibited in human chondrocytes pretreated with EGCG (p <.001). Parallel to these findings, EGCG also inhibited the IL-1 beta-induced LDH release in chondrocytes cultures. Overall, the study suggests that EGCG affords protection against IL-1 beta-induced production of catabolic mediators NO and PGE(2) in human chondrocytes by regulating the expression and catalytic activity of their respective enzymes. Furthermore, our results also indicate that ECGC may be of potential therapeutic value for inhibiting cartilage resorption in arthritic joints.     

Induction of COX-2 expression by nitric oxide in rheumatoid synovial cells

Prostaglandins formed by cyclooxygenase (COX) enzymes are important mediators of inflammation. The contribution of inducible COX-2 in the rheumatoid synovium is well documented. In this study, we evaluated the contribution of nitric oxide (NO) to COX-2 expression in rheumatoid synovial cells. Exposure of rheumatoid synovial cells to a NO donor, SNAP, induced COX-2 protein expression in a dose-dependent manner. RT-PCR analysis also demonstrated that COX-2 mRNA was induced in SNAP-treated synovial cells. Dexamethasone at therapeutic concentrations markedly inhibited this NO-mediated COX-2 expression in synovial cells. In contrast to its effect on COX-2 expression, SNAP did not affect the constitutive expression of COX-1 in rheumatoid synovial cells. Our findings suggest that NO is an important modulator of COX-2 expression and that glucocorticoids exert their anti-inflammatory action in rheumatoid synovium, at least in part, by suppression of COX-2 induction.     

The induction of cell death in human osteoarthritis chondrocytes by nitric oxide is related to the production of prostaglandin E2 via the induction of cyclooxygenase-2

There is increasing evidence suggesting that chondrocyte death may contribute to the progression of osteoarthritis (OA). This study focused on the characterization of signaling cascade during NO-induced cell death in human OA chondrocytes. The NO generator, sodium nitroprusside (SNP), promoted chondrocyte death in association with DNA fragmentation, caspase-3 activation, and down-regulation of Bcl-2. Both caspase-3 inhibitor Z-Asp(OCH3)-Glu(OCH3)-Val-Asp(OCH3)-CH2F and caspase-9 inhibitor Z-Leu-Glu(OCH3)-His-Asp(OCH3)-CH2F prevented the chondrocyte death. Blocking the mitogen-activated protein kinase pathway by the mitogen-activated protein kinase kinase 1/2 inhibitor PD98059 or p38 kinase inhibitor SB202190 also inhibited the SNP-mediated cell death, suggesting possible requirements of both extracellular signal-related protein kinase 1/2 and p38 kinase for the NO-induced cell death. Furthermore, the selective inhibition of cyclooxygenase (COX)-2 by NS-398 or the inhibition of COX-1/COX-2 by indomethacin blocked the SNP-induced cell death. The chondrocyte death induced by SNP was associated with an overexpression of COX-2 protein (as determined by Western blotting) and an increase in PGE2 release. PD98059 and SB202190, but neither Z-DEVD FMK nor Z-LEHD FMK completely inhibited the SNP-mediated PGE2 production. Analysis of interactions between PGE2 and the cell death showed that PGE2 enhanced the SNP-mediated cell death, whereas PGE2 alone did not induce the chondrocyte death. These data indicate that NO-induced chondrocyte death signaling includes PGE2 production via COX-2 induction and suggest that both extracellular signal-related protein kinase 1/2 and p38 kinase pathways are upstream signaling of the PGE2 production. The results also demonstrate that exogenous PGE2 may sensitize human OA chondrocytes to the cell death induced by NO.     

Influence of macrophage products on the release of plasminogen activator, collagenase, beta-glucuronidase and prostaglandin E2 by articular chondrocytes.

We describe the effects of products of mononuclear phagocytes on the secretory activity of chondrocytes. The primary confluent cultures of rabbit articular chondrocytes were exposed to standard medium alone or enriched with conditioned medium obtained from cultures of rabbit peritoneal macrophages, the mouse macrophage cell line P388D1 or human blood mononuclear cells. Four markers of release were assessed, the neutral proteinases plasminogen activator and collagenase, the acid hydrolase beta-glucuronidase and prostaglandin E2, and the kinetics of their changes were monitored. Chondrocytes that were cultured in standard medium secreted large amounts of plasminogen activator, some beta-glucuronidase, but no collagenase, and released only minor amounts of prostaglandin E2. The addition of conditioned medium from rabbit macrophages induced a rapid release of large quantities of prostaglandin E2 and an abundant secretion of collagenase, while abolishing or strongly decreasing plasminogen activator secretion. In addition, beta-glucuronidase secretion was markedly enhanced. The decrease in secretion of plasminogen activator appeared to reflect a diminished production, since no evidence was found for the generation of inhibitors or for an accelerated extracellular breakdown of the enzyme. Conditioned media of the mouse and human mononuclear cells influenced the secretory activities of rabbit articular chondrocytes in a similar way, suggesting that the factor (or factors) acting on chondrocytes is produced by a variety of macrophages, and that its action is not species-restricted. The time course and concentration-dependence of the effects observed indicate that the secretion of plasminogen activator and collagenase are influenced in a strictly reciprocal fashion by the macrophage products. The release of prostaglandin E2 paralleled that of collagenase.     

Autocrine prostaglandin E2 signaling promotes promonocytic leukemia cell survival via COX-2 expression and MAPK pathway

The COX-2/PGE₂ pathway has been implicated in the occurrence and progression of cancer. The underlying mechanisms facilitating the production of COX-2 and its mediator, PGE₂, in cancer survival remain unknown. Herein, we investigated PGE₂-induced COX-2 expression and signaling in HL-60 cells following menadione treatment. Treatment with PGE₂ activated anti-apoptotic proteins such as Bcl-2 and Bcl-xL while reducing pro-apoptotic proteins, thereby enhancing cell survival. PGE₂ not only induced COX-2 expression, but also prevented casapse-3, PARP, and lamin B cleavage. Silencing and inhibition of COX-2 with siRNA transfection or treatment with indomethacin led to a pronounced reduction of the extracellular levels of PGE₂, and restored the menadione-induced cell death. In addition, pretreatment of cells with the MEK inhibitor PD98059 and the PKA inhibitor H89 abrogated the PGE₂-induced expression of COX-2, suggesting involvement of the MAPK and PKA pathways. These results demonstrate that PGE₂ signaling acts in an autocrine manner, and specific inhibition of PGE₂ will provide a novel approach for the treatment of leukemia. [BMB Reports 2015; 48(2): 109-114]     

Induction of COX-2 enzyme and down-regulation of COX-1 expression by lipopolysaccharide (LPS) control prostaglandin E2 production in astrocytes

Pathological conditions and pro-inflammatory stimuli in the brain induce cyclooxygenase-2 (COX-2), a key enzyme in arachidonic acid metabolism mediating the production of prostanoids that, among other actions, have strong vasoactive properties. Although low basal cerebral COX-2 expression has been reported, COX-2 is strongly induced by pro-inflammatory challenges, whereas COX-1 is constitutively expressed. However, the contribution of these enzymes in prostanoid formation varies depending on the stimuli and cell type. Astrocyte feet surround cerebral microvessels and release molecules that can trigger vascular responses. Here, we investigate the regulation of COX-2 induction and its role in prostanoid generation after a pro-inflammatory challenge with the bacterial lipopolysaccharide (LPS) in astroglia. Intracerebral administration of LPS in rodents induced strong COX-2 expression mainly in astroglia and microglia, whereas COX-1 expression was predominant in microglia and did not increase. In cultured astrocytes, LPS strongly induced COX-2 and microsomal prostaglandin-E(2) (PGE(2)) synthase-1, mediated by the MyD88-dependent NFκB pathway and influenced by mitogen-activated protein kinase pathways. Studies in COX-deficient cells and using COX inhibitors demonstrated that COX-2 mediated the high production of PGE(2) and, to a lesser extent, other prostanoids after LPS. In contrast, LPS down-regulated COX-1 in an MyD88-dependent fashion, and COX-1 deficiency increased PGE(2) production after LPS. The results show that astrocytes respond to LPS by a COX-2-dependent production of prostanoids, mainly vasoactive PGE(2), and suggest that the coordinated down-regulation of COX-1 facilitates PGE(2) production after TLR-4 activation. These effects might induce cerebral blood flow responses to brain inflammation.     

The effects of interleukin-1 on the expression of thrombospondin and fibronectin by rabbit articular chondrocytes

Studies to evaluate the effects of recombinant interleukin-1 beta (IL-1) on the expression of matrix proteins by rabbit articular chondrocytes were conducted. Chondrocytes expressed high levels of message for thrombospondin (Tsp) and fibronectin (Fn). RNA slot-blot analysis demonstrated that treatment of the cultures with IL-1 (100 ng/ml) for 24 h caused a 70% suppression of their steady-state Tsp mRNA levels whereas those of Fn were not affected. Steady-state mRNA levels for the intracellular protein, actin, were not modulated by treatment with IL-1. The suppression of Tsp mRNA levels by IL-1 (100 ng/ml) was maximal by 4 h and was concentration dependent; half-maximal suppression was estimated to require 0.12 ng/ml IL-1. Cycloheximide treatment enhanced Tsp mRNA levels, but did not modulate IL-1 suppression of Tsp mRNA. Using pulse-labeling and immunoprecipitation techniques, we found that IL-1 suppression of Tsp mRNA levels was reflected in a coordinate inhibition of Tsp protein synthesis. Chondrocyte synthesis of Fn was not affected by IL-1. These data suggest that IL-1 specifically regulates chondrocyte expression of Tsp at least in part by decreasing the amount of Tsp mRNA available for translation.