N. Zayed

Histone deacetylase inhibitors suppress interleukin-1β-induced nitric oxide and prostaglandin E2 production in human chondrocytes

OBJECTIVE Overproduction of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) plays an important role in the pathogenesis of osteoarthritis (OA). In the present study, we determined the effect of trichostatin A (TSA) and butyric acid (BA), two histone deacetylase (HDAC) inhibitors, on NO and PGE(2) synthesis, inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 expression, and nuclear factor (NF)-kappaB DNA-binding activity, in interleukin-1beta (IL-1)-stimulated human OA chondrocytes, and on IL-1-induced proteoglycan degradation in cartilage explants. METHODS Chondrocytes were stimulated with IL-1 in the absence or presence of increasing concentrations of TSA or BA. The production of NO and PGE(2) was evaluated using Griess reagent and an enzyme immunoassay, respectively. The expression of iNOS and COX-2 proteins and mRNAs was evaluated using Western blotting and real-time reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. Proteoglycan degradation was measured with dimethymethylene blue assay. Electrophoretic mobility shift assay (EMSA) was utilized to analyze the DNA-binding activity of NF-kappaB. RESULTS HDAC inhibition with TSA or BA resulted in a dose-dependent inhibition of IL-1-induced NO and PGE(2) production. IL-17- and tumor necrosis factor-alpha (TNF-alpha)-induced NO and PGE(2) production was also inhibited by TSA and BA. This inhibition correlated with the suppression of iNOS and COX-2 protein and mRNA expression. TSA and BA also prevented IL-1-induced proteoglycan release from cartilage explants. Finally, we demonstrate that the DNA-binding activity of NF-kappaB, was induced by IL-1, but was not affected by treatment with HDAC inhibitors. CONCLUSIONS These data indicate that HDAC inhibitors suppressed IL-1-induced NO and PGE(2) synthesis, iNOS and COX-2 expression, as well as proteoglycan degradation. The suppressive effect of HDAC inhibitors is not due to impaired DNA-binding activity of NF-kappaB. These findings also suggest that HDAC inhibitors may be of potential therapeutic value in the treatment of OA.

Inhibition of interleukin‐1β–induced matrix metalloproteinases 1 and 13 production in human osteoarthritic chondrocytes by prostaglandin D2

OBJECTIVE 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. METHODS 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. RESULTS 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. CONCLUSION 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.

Human articular chondrocytes express 15-lipoxygenase-1 and -2: potential role in osteoarthritis

Introduction15-Lipoxygenases and their metabolites have been shown to exhibit anti-inflammatory and immunomodulatory properties, but little is known regarding their expression and function in chondrocytes. The objective of this study was to evaluate the expression of 15-lipoxygenase-1 and -2 in human articular chondrocytes, and to investigate the effects of their metabolites 13(S)-hydroxy octadecadienoic and 15(S)-hydroxyeicosatetraenoic acids on IL-1β-induced matrix metalloproteinase (MMP)-1 and MMP-13 expression.MethodsThe expression levels of 15-lipoxygenase-1 and -2 were analyzed by reverse transcription PCR and Western blotting in chondrocytes, and by immunohistochemistry in cartilage. Chondrocytes or cartilage explants were stimulated with IL-1β in the absence or presence of 13(S)-hydroxy octadecadienoic and 15(S)-hydroxyeicosatetraenoic acids, and the levels of MMP-1 and MMP-13 protein production and type II collagen cleavage were evaluated using immunoassays. The role of peroxisome proliferator-activated receptor (PPAR)γ was evaluated using transient transfection experiments and the PPARγ antagonist GW9662.ResultsArticular chondrocytes express 15-lipoxygenase-1 and -2 at the mRNA and protein levels. 13(S)-hydroxy octadecadienoic and 15(S)-hydroxyeicosatetraenoic acids dose dependently decreased IL-1β-induced MMP-1 and MMP-13 protein and mRNA expression as well as type II collagen cleavage. The effect on MMP-1 and MMP-13 expression does not require de novo protein synthesis. 13(S)-hydroxy octadecadienoic and 15(S)-hydroxyeicosatetraenoic acids activated endogenous PPARγ, and GW9662 prevented their suppressive effect on MMP-1 and MMP-13 production, suggesting the involvement of PPARγ in these effects.ConclusionsThis study is the first to demonstrate the expression of 15-lipoxygenase-1 and -2 in articular chondrocytes. Their respective metabolites, namely 13(S)-hydroxy octadecadienoic and 15(S)-hydroxyeicosatetraenoic acids, suppressed IL-1β-induced MMP-1 and MMP-13 expression in a PPARγ-dependent pathway. These data suggest that 15-lipoxygenases may have chondroprotective properties by reducing MMP-1 and MMP-13 expression.

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

IntroductionProstaglandin 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.MethodsThe 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.ResultsL-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.ConclusionsThis 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.

Valproic Acid Suppresses Interleukin-1ß-induced Microsomal Prostaglandin E2 Synthase-1 Expression in Chondrocytes Through Upregulation of NAB1

Objective. Microsomal prostaglandin E2 synthase-1 (mPGES-1) catalyzes the terminal step in the biosynthesis of PGE2. Early growth response factor-1 (Egr-1) is a key transcription factor in the regulation of mPGES-1, and its activity is negatively regulated by the corepressor NGF1-A-binding protein-1 (NAB1). We examined the effects of valproic acid (VA), a histone deacetylase inhibitor, on interleukin 1ß (IL-1ß)-induced mPGES-1 expression in human chondrocytes, and evaluated the roles of Egr-1 and NAB1 in these effects. Methods. Chondrocytes were stimulated with IL-1 in the absence or presence of VA, and the level of mPGES-1 protein and mRNA expression were evaluated using Western blotting and real-time reverse-transcription polymerase chain reaction (PCR), respectively. mPGES-1 promoter activity was analyzed in transient transfection experiments. Egr-1 and NAB1 recruitment to the mPGES-1 promoter was evaluated using chromatin immunoprecipitation assays. Small interfering RNA (siRNA) approaches were used to silence NAB1 expression. Results. VA dose-dependently suppressed IL-1-induced mPGES-1 protein and mRNA expression as well as its promoter activation. Treatment with VA did not alter IL-1-induced Egr-1 expression, or its recruitment to the mPGES-1 promoter, but prevented its transcriptional activity. The suppressive effect of VA requires de novo protein synthesis. VA induced the expression of NAB1, and its recruitment to the mPGES-1 promoter, suggesting that NAB1 may mediate the suppressive effect of VA. Indeed, NAB1 silencing with siRNA blocked VA-mediated suppression of IL-1-induced mPGES-1 expression. Conclusion. VA inhibited IL-1-induced mPGES-1 expression in chondrocytes. The suppressive effect of VA was not due to reduced expression or recruitment of Egr-1 to the mPGES-1 promoter and involved upregulation of NAB1.

Prostaglandin D2 enhances interleukin -1beta- induced cyclooxygenase-2 expression in osteoarthritic cartilage

Methods Chondrocytes were stimulated with IL-1 in the presence or absence of PGD2, and expression of COX-2 protein was evaluated by western-blotting. Messenger RNA (mRNA) expression was analyzed by real-time reverse transcription-polymerase chain reaction. 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.

Valproic acid suppresses interleukin-1β-induced microsomal prostaglandin E2 Synthase-1 expression in chondrocytes

Microsomal prostaglandin E2 Synthase (mPGES)-1 catalyzes the terminal step in the biosynthesis of PGE2. Early growth response factor-1 (Egr-1) is a key transcription factor in the regulation of mPGES-1. In the present study we examined the effects of valproic acid (VA), a histone deacetylase (HDAC) inhibitor, on interleukin (IL)-1β-induced mPGES-1-expression in human chondrocytes.

Expression of PPARα, β, and γ in the Hartley guinea pig model of primary osteoarthritis

Background Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily. Three isoforms have been identified: PPARa, PPARb-δ and PPARg. Several in vitro and in vivo studies suggest that PPARg may have protective roles in osteoarthritis (OA). So far, little is known about the pattern of PPAR expression during the progression of OA and cartilage degradation. Aim To investigate the expression of PPARa, b, and g in cartilage over the course of OA in the spontaneous Hartley guinea pig model. Methods Hartley guinea pigs were sacrificed at 2 (control group), 4, 8, and 12 (n =6p er group) month-old of age. Cartilage was obtained from the central portion of the medial tibial plateau. Cartilage degradation was evaluated histologically using the Osteoarthritis Research Society International (OARSI) guidelines. The expression of PPARa, b and g was analyzed by immunohistochemistry. The non-parametric Spearman test was used for the correlation analysis between the protein expression levels and histological scores. Results PPARa, b and g, were detected in medial tibial plateaus from control animals. There was no significant change in the levels of PPARa and PPARb over the course of OA. In contrast, PPARg expression decreased during the progression of OA. Correlation analysis revealed a negative correlation between PPARg levels and histological score of OA. Conclusion Expression of PPARg in cartilage decreased during the course of OA. These data suggest that loss of PPARg expression in cartilage may contribute to the pathogenesis of OA.

Egr-1 mediates the suppressive effect of IL-1 on PPARγ expression in human OA chondrocytes

Background Peroxisome proliferator-activated receptor gamma (PPARg) is a ligand activated transcription factor and member the nuclear hormone receptor superfamily. Several lines of evidence indicate that PPARg have protective effects in osteoarthritis (OA). Indeed, PPARg has been shown to down-regulate several inflammatory and catabolic responses in articular cartilage and chondrocytes and to be protective in animal models of OA. Aim We have previously shown that IL-1 down-regulated PPARg expression in OA chondrocytes. In the present study we will investigate the mechanisms underlying this effect of IL-1. Methods Chondrocytes were stimulated with IL-1, and the level of PPARg and Egr-1 protein and mRNA were evaluated using Western blotting and real-time reverse-transcription polymerase chain reaction, respectively. The PPARg promoter activity was analyzed in transient transfection experiments. Egr-1 recruitment to the PPARg promoter was evaluated using chromatin immunoprecipitation (ChIP) assays. Small interfering RNA (siRNA) approaches were used to silence Egr-1 expression. Results We demonstrated that the suppressive effect of IL-1 on PPARg expression requires de novo protein synthesis and was concomitant with the induction of the transcription factor Egr-1. ChIP analyses revealed that IL-1 induced Egr-1 recruitment at the PPARg promoter. IL-1 inhibited the activity of PPARg promoter and overexpression of Egr-1 potentiated the inhibitory effect of IL-1, suggesting that Egr-1 may mediate the suppressive effect of IL-1. Finally, Egr-1 silencing with small interfering RNA blocked IL-1-mediated down-regulation of PPARg expression. Conclusion These results indicate that Egr-1 contributes to IL-1mediated down-regulation of PPARg expression in OA chondrocytes and suggest that this pathway could be a potential target for pharmacologic intervention in the treatment of OA and possibly other arthritic diseases.

199 INTERLEUKIN-1-INDUCED COX-2 AND INOS EXPRESSION IN OA HUMAN CHONDROCYTES IS ASSOCIATED WITH HISTONE H3K4 METHYLATION

Purpose: Increased expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 plays a key role in the pathogenesis of osteoarthritis (OA). Methylation of lysine 4 on histone H3 (H3K4) was shown to be of fundamental importance in the regulation of gene expression. In the present study, we investigated the role of H3K4 methylation in interleukin-1β (IL-1)-induced COX-2 and iNOS expression in human OA chondrocytes Methods: Chondrocytes were stimulated with IL-1 for various time periods and the expression of iNOS and COX-2 mRNAs and proteins were evaluated using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting, respectively. H3K4 methylation at the iNOS and COX-2 promoters was evaluated using chromatin immunoprecipitation (ChIP) assays. The role of histone methylation was further evaluated using the methyltransferase inhibitor, 5’-deoxy-5’(methylthio) adenosine (MTA). Results: IL-1 induced iNOS and COX-2 mRNA and protein in a doseand time-dependent manner. The induction of iNOS and COX-2 expression by IL-1 was associated with H3K4 diand trimethylation at the iNOS and COX-2 promoters, whereas the levels of H3K4 monomethylation remained unchanged. Treatment with MTA inhibited IL-1-induced H3K4 methylation as well as IL-1-induced iNOS and COX-2 expression. Conclusions: These results indicate that H3K4 methylation contributes to IL-1-induced iNOS and COX-2 expression and suggest that this pathway could be a potential target for pharmacological intervention in the treatment of OA.