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1.  Lysine-specific demethylase 1-mediated demethylation of histone H3 lysine 9 contributes to interleukin 1β-induced microsomal prostaglandin E synthase 1 expression in human osteoarthritic chondrocytes 
Arthritis Research & Therapy  2014;16(3):R113.
Microsomal prostaglandin E synthase 1 (mPGES-1) catalyzes the terminal step in the biosynthesis of PGE2, a critical mediator in the pathophysiology of osteoarthritis (OA). Histone methylation plays an important role in epigenetic gene regulation. In this study, we investigated the roles of histone H3 lysine 9 (H3K9) methylation in interleukin 1β (IL-1β)-induced mPGES-1 expression in human chondrocytes.
Chondrocytes were stimulated with IL-1β, and the expression of mPGES-1 mRNA was evaluated using real-time RT-PCR. H3K9 methylation and the recruitment of the histone demethylase lysine-specific demethylase 1 (LSD1) to the mPGES-1 promoter were evaluated using chromatin immunoprecipitation assays. The role of LSD1 was further evaluated using the pharmacological inhibitors tranylcypromine and pargyline and small interfering RNA (siRNA)-mediated gene silencing. The LSD1 level in cartilage was determined by RT-PCR and immunohistochemistry.
The induction of mPGES-1 expression by IL-1β correlated with decreased levels of mono- and dimethylated H3K9 at the mPGES-1 promoter. These changes were concomitant with the recruitment of the histone demethylase LSD1. Treatment with tranylcypromine and pargyline, which are potent inhibitors of LSD1, prevented IL-1β-induced H3K9 demethylation at the mPGES-1 promoter and expression of mPGES-1. Consistently, LSD1 gene silencing with siRNA prevented IL-1β-induced H3K9 demethylation and mPGES-1 expression, suggesting that LSD1 mediates IL-1β-induced mPGES-1 expression via H3K9 demethylation. We show that the level of LSD1 was elevated in OA compared to normal cartilage.
These results indicate that H3K9 demethylation by LSD1 contributes to IL-1β-induced mPGES-1 expression and suggest that this pathway could be a potential target for pharmacological intervention in the treatment of OA and possibly other arthritic conditions.
PMCID: PMC4060543  PMID: 24886859
2.  NFAT3 and TGF-β/SMAD3 regulate the expression of miR-140 in osteoarthritis 
Arthritis Research & Therapy  2013;15(6):R197.
MicroRNAs (miRNAs) down-regulate their target genes. The intronic miR-140, present in the WW domain containing E3 ubiquitin protein ligase 2 (WWP2) gene, decreases the expression of genes that play detrimental roles in osteoarthritis (OA). As the expression level of miR-140 is significantly decreased in human OA chondrocytes, we investigated its regulation in those cells.
Gene expression in human chondrocytes was determined by quantitative polymerase chain reaction (qPCR) and gene silencing was done in OA chondrocytes by transient transfection with specific small interfering RNAs (siRNAs). Binding sites of the miR-140 regulatory sequence (rsmiR-140) were identified by mutagenesis and chromatin immunoprecipitation (ChIP) in OA chondrocytes. The effects of translocation on OA chondrocytes were determined by immunocytochemistry and qPCR.
In contrast to miR-140, the expression of WWP2 was similar in both normal and OA cells, suggesting that miR-140 has an additional level of regulation. rsmiR-140 showed activity and predicted binding sites for nuclear matrix transcription factor 4 (NMP4), myc-associated zinc (MAZ), nuclear factor of activated T-cells (NFAT), and mothers against decapentaplegic homolog 3 (SMAD3). Silencing NFAT3 (P ≤0.01) and SMAD3 (P ≤0.05) differentially regulated miR-140 independently of WWP2. Silencing NFAT5 decreased both miR-140 and WWP2 (P ≤0.003 and P ≤0.05, respectively). NFAT3 activation increased and transforming growth factor-β (TGF-β) decreased rsmiR-140 activity. Mutagenesis of rsmiR-140 and ChIP assays identified binding sites at which NFAT3 (activator) and SMAD3 (repressor) directly regulated miR-140. TGF-β interfered with NFAT3 translocation, and subsequently with miR-140 expression.
This is the first study to provide evidence of a regulatory mechanism of miR-140 independent of WWP2, and new and differential roles for NFAT3 and SMAD3 in the OA process in the regulation of miR-140 transcription. Such knowledge could advance therapeutic strategies targeting OA.
PMCID: PMC3978709  PMID: 24257415
4.  Ramipril attenuates lipid peroxidation and cardiac fibrosis in an experimental model of rheumatoid arthritis 
Arthritis Research & Therapy  2012;14(5):R223.
Recent studies revealed that co-morbidity and mortality due to cardiovascular disease are increased in patients with rheumatoid arthritis (RA) but little is known about factors involved in these manifestations. This study aimed at characterizing the impact of arthritis on oxidative stress status and tissue fibrosis in the heart of rats with adjuvant-induced arthritis (AIA).
AIA was induced with complete Freund's adjuvant in female Lewis rats. Animals were treated by oral administration of vehicle or angiotensin-converting enzyme inhibitor ramipril (10 mg/kg/day) for 28 days, beginning 1 day after arthritis induction. Isolated adult cardiomyocytes were exposed to 10 μM 4-hydroxynonenal (HNE) for 24 hours in the presence or absence of 10 μM ramipril.
Compared to controls, AIA rats showed significant 55 and 30% increase of 4-HNE/protein adducts in serum and left ventricular (LV) tissues, respectively. Cardiac mitochondrial NADP+-isocitrate dehydrogenase (mNADP-ICDH) activity decreased by 25% in AIA rats without any changes in its protein and mRNA expression. The loss of mNADP-ICDH activity was correlated with enhanced accumulation of HNE/mNADP-ICDH adducts as well as with decrease of glutathione and NADPH. Angiotensin II type 1 receptor (AT1R) expression and tissue fibrosis were induced in LV tissues from AIA rats. In isolated cardiomyocytes, HNE significantly decreased mNADP-ICDH activity and enhanced type I collagen and connective tissue growth factor expression. The oral administration of ramipril significantly reduced HNE and AT1R levels and restored mNADP-ICDH activity and redox status in LV tissues of AIA rats. The protective effects of this drug were also evident from the decrease in arthritis scoring and inflammatory markers.
Collectively, our findings disclosed that AIA induced oxidative stress and fibrosis in the heart. The fact that ramipril attenuates inflammation, oxidative stress and tissue fibrosis may provide a novel strategy to prevent heart diseases in RA.
PMCID: PMC3580534  PMID: 23079082
5.  Egr-1 contributes to IL-1-mediated down-regulation of peroxisome proliferator-activated receptor γ expression in human osteoarthritic chondrocytes 
Peroxisome proliferator-activated receptor (PPAR)γ has been shown to exhibit anti-inflammatory and anti-catabolic properties and to be protective in animal models of osteoarthritis (OA). We have previously shown that interleukin-1β (IL-1) down-regulates PPARγ expression in human OA chondrocytes. However, the mechanisms underlying this effect have not been well characterized. The PPARγ promoter harbors an overlapping Egr-1/specificity protein 1 (Sp1) binding site. In this study, our objective was to define the roles of Egr-1 and Sp1 in IL-1-mediated down-regulation of PPARγ expression.
Chondrocytes were stimulated with IL-1 and the expression levels of Egr-1 and Sp1 mRNAs and proteins were evaluated using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting, respectively. The role of de novo protein synthesis was evaluated using the protein synthesis inhibitor cycloheximide (CHX). The recruitment of Sp1 and Egr-1 to the PPARγ promoter was evaluated using chromatin immunoprecipitation (ChIP) assays. The PPARγ promoter activity was analyzed in transient transfection experiments. The roles of Egr-1 and Sp1 were further evaluated using small interfering RNA (siRNA) approaches. The level of Egr-1 in cartilage was determined using immunohistochemistry.
Down-regulation of PPARγ expression by IL-1 requires de novo protein synthesis and was concomitant with the induction of the transcription factor Egr-1. Treatment with IL-1 induced Egr-1 recruitment and reduced Sp1 occupancy at the PPARγ promoter. Overexpression of Egr-1 potentiated, whereas overexpression of Sp1 alleviated, the suppressive effect of IL-1 on the PPARγ promoter, suggesting that Egr-1 may mediate the suppressive effect of IL-1. Consistently, Egr-1 silencing prevented IL-1-mediated down-regulation of PPARγ expression. We also showed that the level of Egr-1 expression was elevated in OA cartilage compared to normal cartilage.
Our results indicate that induction and recruitment of Egr-1 contributed to the suppressive effect of IL-1 on PPARγ expression. They also suggest that modulation of Egr-1 levels in the joint may have therapeutic potential in OA.
PMCID: PMC3446440  PMID: 22455954
8.  mPGES-1 null mice are resistant to bleomycin-induced skin fibrosis 
Microsomal prostaglandin E2 synthase-1 (mPGES-1) is an inducible enzyme that acts downstream of cyclooxygenase (COX) to specifically catalyze the conversion of prostaglandin (PG) H2 to PGE2. mPGES-1 plays a key role in inflammation, pain and arthritis; however, the role of mPGES-1 in fibrogenesis is largely unknown. Herein, we examine the role of mPGES-1 in a mouse model of skin scleroderma using mice deficient in mPGES-1.
Wild type (WT) and mPGES-1 null mice were subjected to the bleomycin model of cutaneous skin scleroderma. mPGES-1 expressions in scleroderma fibroblasts and in fibroblasts derived from bleomycin-exposed mice were assessed by Western blot analysis. Degree of fibrosis, dermal thickness, inflammation, collagen content and the number of α-smooth muscle actin (α-SMA)-positive cells were determined by histological analyses. The quantity of the collagen-specific amino acid hydroxyproline was also measured.
Compared to normal skin fibroblasts, mPGES-1 protein expression was elevated in systemic sclerosis (SSc) fibroblasts and in bleomycin-exposed mice. Compared to WT mice, mPGES-1-null mice were resistant to bleomycin-induced inflammation, cutaneous thickening, collagen production and myofibroblast formation.
mPGES-1 expression is required for bleomycin-induced skin fibrogenesis. Inhibition of mPGES-1 may be a viable method to alleviate the development of cutaneous sclerosis and is a potential therapeutic target to control the onset of fibrogenesis.
PMCID: PMC3546456  PMID: 21266028
10.  Perturbation of adhesion molecule-mediated chondrocyte-matrix interactions by 4-hydroxynonenal binding: implication in osteoarthritis pathogenesis 
Arthritis Research & Therapy  2010;12(5):R201.
Objectives were to investigate whether interactions between human osteoarthritic chondrocytes and 4-hydroxynonenal (HNE)-modified type II collagen (Col II) affect cell phenotype and functions and to determine the protective role of carnosine (CAR) treatment in preventing these effects.
Human Col II was treated with HNE at different molar ratios (MR) (1:20 to 1:200; Col II:HNE). Articular chondrocytes were seeded in HNE/Col II adduct-coated plates and incubated for 48 hours. Cell morphology was studied by phase-contrast and confocal microscopy. Adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and α1β1 integrin at protein and mRNA levels were quantified by Western blotting, flow cytometry and real-time reverse transcription-polymerase chain reaction. Cell death, caspases activity, prostaglandin E2 (PGE2), metalloproteinase-13 (MMP-13), mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) were assessed by commercial kits. Col II, cyclooxygenase-2 (COX-2), MAPK, NF-κB-p65 levels were analyzed by Western blotting. The formation of α1β1 integrin-focal adhesion kinase (FAK) complex was revealed by immunoprecipitation.
Col II modification by HNE at MR approximately 1:20, strongly induced ICAM-1, α1β1 integrin and MMP-13 expression as well as extracellular signal-regulated kinases 1 and 2 (ERK1/2) and NF-κB-p65 phosphorylation without impacting cell adhesion and viability or Col II expression. However, Col II modification with HNE at MR approximately 1:200, altered chondrocyte adhesion by evoking cell death and caspase-3 activity. It inhibited α1β1 integrin and Col II expression as well as ERK1/2 and NF-κB-p65 phosphorylation, but, in contrast, markedly elicited PGE2 release, COX-2 expression and p38 MAPK phosphorylation. Immunoprecipitation assay revealed the involvement of FAK in cell-matrix interactions through the formation of α1β1 integrin-FAK complex. Moreover, the modification of Col II by HNE at a 1:20 or approximately 1:200 MR affects parameters of the cell shape. All these effects were prevented by CAR, an HNE-trapping drug.
Our novel findings indicate that HNE-binding to Col II results in multiple abnormalities of chondrocyte phenotype and function, suggesting its contribution in osteoarthritis development. CAR was shown to be an efficient HNE-snaring agent capable of counteracting these outcomes.
PMCID: PMC2991038  PMID: 20977750
11.  Regulation of microsomal prostaglandin E2 synthase-1 and 5-lipoxygenase-activating protein/5-lipoxygenase by 4-hydroxynonenal in human osteoarthritic chondrocytes 
This study aimed to investigate whether hydroxynonenal (HNE) depletion is responsible for the switch from cyclooxygenase-2 (COX-2) and microsomal prostaglandin E2 synthase-1 (mPGES-1) to 5-lipoxygenase-activating protein (FLAP) and 5-lipoxygenase (5-LOX).
For COX-2 and mPGES-1 studies, human osteoarthritic chondrocytes were stimulated at different incubation times (up to 24 hours) with a single or repetitive addition of 10 μM HNE to the cultures at 2-hour intervals, up to 14 hours. For 5-LOX and FLAP studies, cells were treated with a single addition of 10 μM HNE for 24 hours, 48 hours, and 72 hours in the presence or absence of naproxen (a nonspecific COX-2 inhibitor) or antibody anti-transforming growth factor-beta 1 (TGF-β1). The protein levels of COX-2, mPGES-1 and early growth response factor-1 (Egr-1) transcription factor were evaluated by western blot, and those of prostaglandin E2 (PGE2), leukotriene B4 (LTB4) and TGF-β1 were determined with commercial kits. The levels of mPGES-1, FLAP and 5-LOX mRNA were measured by real-time RT-PCR. Transient transfection was performed to determine promoter activities of mPGES-1 and 5-LOX.
Single addition of 10 μM HNE to cultured chondrocytes induced PGE2 release as well as COX-2 and mPGES-1 expression at the protein and mRNA levels, with a plateau reached respectively at 8 and 16 hours of incubation, followed by a subsequent decline. However, repeated treatments with HNE prevented the decline of COX-2 and mPGES-1 expression that occurred with a single aldehyde addition. HNE induced mPGES-1 promoter activity, possibly through transcription factor Egr-1 activation. After 48 hours, when COX-2 expression decreased, the LTB4 level rose through 5-LOX and FLAP upregulation. The addition of naproxen to cultured chondrocytes revealed that FLAP and 5-LOX regulation by HNE required PGE2 production. Furthermore, our data showed that HNE significantly induced TGF-β1 production. The addition of anti-TGF-β1 antibody reduced HNE-induced 5-LOX and FLAP expression by 40%, indicating the partial involvement of a TGF-β1-dependent mechanism.
Our data demonstrate that the shunt to the FLAP and 5-LOX pathway in HNE-induced human osteoarthritic chondrocytes is attributed to COX-2 and mPGES-1 inhibition, probably due to HNE depletion. PGE2 and TGF-β1 are suggested to be involved in this regulation.
PMCID: PMC2875653  PMID: 20144213
12.  Human articular chondrocytes express 15-lipoxygenase-1 and -2: potential role in osteoarthritis 
15-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.
The 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.
Articular 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.
This 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.
PMCID: PMC2688191  PMID: 19296842
13.  Increased expression of lipocalin-type prostaglandin D2 synthase in osteoarthritic cartilage 
Arthritis Research & Therapy  2008;10(6):R146.
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.
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.
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.
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.
PMCID: PMC2656251  PMID: 19094210
14.  4-Hydroxynonenal induces apoptosis in human osteoarthritic chondrocytes: the protective role of glutathione-S-transferase 
Arthritis Research & Therapy  2008;10(5):R107.
4-Hydroxynonenal (HNE) is one of the most abundant and reactive aldehydes of lipid peroxidation products and exerts various effects on intracellular and extracellular signalling cascades. We have previously shown that HNE at low concentrations could be considered as an important mediator of catabolic and inflammatory processes in osteoarthritis (OA). In the present study, we focused on characterizing the signalling cascade induced by high HNE concentration involved in cell death in human OA chondrocytes.
Markers of apoptosis were quantified with commercial kits. Protein levels were evaluated by Western blotting. Glutathione (GSH) and ATP levels were measured with commercial kits. Glucose uptake was assessed by 2-deoxy-D-[3H]-glucose. The role of GSH-S-transferase A4-4 (GSTA4-4) in controlling HNE-induced chondrocyte apoptosis was investigated by chondrocyte transfection with small interfering RNA (siRNA) or with the expression vector of GSTA4-4.
Our data showed that HNE at concentrations of up to 10 μM did not alter cell viability but was cytotoxic at concentrations of greater than or equal to 20 μM. HNE-induced chondrocyte death exhibited several classical hallmarks of apoptosis, including caspase activation, cytochrome c and apoptosis-induced factor release from mitochondria, poly (ADP-ribose) polymerase cleavage, Bcl-2 downregulation, Bax upregulation, and DNA fragmentation. Our study of signalling pathways revealed that HNE suppressed pro-survival Akt kinase activity but, in contrast, induced Fas/CD95 and p53 expression in chondrocytes. All of these effects were inhibited by an antioxidant, N-acetyl-cysteine. Analysis of cellular energy and redox status showed that HNE induced ATP, NADPH, and GSH depletion and inhibited glucose uptake and citric acid cycle activity. GSTA4-4 ablation by the siRNA method augmented HNE cytotoxicity, but, conversely, its overexpression efficiently protected chondrocytes from HNE-induced cell death.
Our study provides novel insights into the potential mechanisms of cell death in OA cartilage and suggests the potential role of HNE in OA pathophysiology. GSTA4-4 expression is critically important for cellular defence against oxidative stress-induced cell death in OA cartilage, possibly by HNE elimination.
PMCID: PMC2592788  PMID: 18782442
15.  Activation of proteinase-activated receptor 2 in human osteoarthritic cartilage upregulates catabolic and proinflammatory pathways capable of inducing cartilage degradation: a basic science study 
Proteinase-activated receptors (PARs) belong to a family of G protein-coupled receptors. PARs are activated by a serine-dependent cleavage generating a tethered activating ligand. PAR-2 was shown to be involved in inflammatory pathways. We investigated the in situ levels and modulation of PAR-2 in human normal and osteoarthritis (OA) cartilage/chondrocytes. Furthermore, we evaluated the role of PAR-2 on the synthesis of the major catabolic factors in OA cartilage, including metalloproteinase (MMP)-1 and MMP-13 and the inflammatory mediator cyclooxygenase 2 (COX-2), as well as the PAR-2-activated signalling pathways in OA chondrocytes. PAR-2 expression was determined using real-time reverse transcription-polymerase chain reaction and protein levels by immunohistochemistry in normal and OA cartilage. Protein modulation was investigated in OA cartilage explants treated with a specific PAR-2-activating peptide (PAR-2-AP), SLIGKV-NH2 (1 to 400 μM), interleukin 1 beta (IL-1β) (100 pg/mL), tumor necrosis factor-alpha (TNF-α) (5 ng/mL), transforming growth factor-beta-1 (TGF-β1) (10 ng/mL), or the signalling pathway inhibitors of p38 (SB202190), MEK1/2 (mitogen-activated protein kinase kinase) (PD98059), and nuclear factor-kappa B (NF-κB) (SN50), and PAR-2 levels were determined by immunohistochemistry. Signalling pathways were analyzed on OA chondrocytes by Western blot using specific phospho-antibodies against extracellular signal-regulated kinase 1/2 (Erk1/2), p38, JNK (c-jun N-terminal kinase), and NF-κB in the presence or absence of the PAR-2-AP and/or IL-1β. PAR-2-induced MMP and COX-2 levels in cartilage were determined by immunohistochemistry. PAR-2 is produced by human chondrocytes and is significantly upregulated in OA compared with normal chondrocytes (p < 0.04 and p < 0.03, respectively). The receptor levels were significantly upregulated by IL-1β (p < 0.006) and TNF-α (p < 0.002) as well as by the PAR-2-AP at 10, 100, and 400 μM (p < 0.02) and were downregulated by the inhibition of p38. After 48 hours of incubation, PAR-2 activation significantly induced MMP-1 and COX-2 starting at 10 μM (both p < 0.005) and MMP-13 at 100 μM (p < 0.02) as well as the phosphorylation of Erk1/2 and p38 within 5 minutes of incubation (p < 0.03). Though not statistically significant, IL-1β produced an additional effect on the activation of Erk1/2 and p38. This study documents, for the first time, functional consequences of PAR-2 activation in human OA cartilage, identifies p38 as the major signalling pathway regulating its synthesis, and demonstrates that specific PAR-2 activation induces Erk1/2 and p38 in OA chondrocytes. These results suggest PAR-2 as a potential new therapeutic target for the treatment of OA.
PMCID: PMC2246240  PMID: 18031579
16.  Chondroitin and glucosamine sulfate in combination decrease the pro-resorptive properties of human osteoarthritis subchondral bone osteoblasts: a basic science study 
Early in the pathological process of osteoarthritis (OA), subchondral bone remodelling, which is related to altered osteoblast metabolism, takes place. In the present study, we explored in human OA subchondral bone whether chondroitin sulfate (CS), glucosamine sulfate (GS), or both together affect the major bone biomarkers, osteoprotegerin (OPG), receptor activator of nuclear factor-kappa B ligand (RANKL), and the pro-resorptive activity of OA osteoblasts. The effect of CS (200 μg/mL), GS (50 and 200 μg/mL), or both together on human OA subchondral bone osteoblasts, in the presence or absence of 1,25(OH)2D3 (vitamin D3) (50 nM), was determined on the bone biomarkers alkaline phosphatase and osteocalcin, on the expression (mRNA) and production (enzyme-linked immunosorbent assay) of bone remodelling factors OPG and RANKL, and on the pro-resorptive activity of these cells. For the latter experiments, human OA osteoblasts were incubated with differentiated peripheral blood mononuclear cells on a sub-micron synthetic calcium phosphate thin film. Data showed that CS and GS affected neither basal nor vitamin D3-induced alkaline phosphatase or osteocalcin release. Interestingly, OPG expression and production under basal conditions or vitamin D3 treatment were upregulated by CS and by both CS and GS incubated together. Under basal conditions, RANKL expression was significantly reduced by CS and by both drugs incubated together. Under vitamin D3, these drugs also showed a decrease in RANKL level, which, however, did not reach statistical significance. Importantly, under basal conditions, CS and both compounds combined significantly upregulated the expression ratio of OPG/RANKL. Vitamin D3 decreased this ratio, and GS further decreased it. Both drugs reduced the resorption activity, and statistical significance was reached for GS and when CS and GS were incubated together. Our data indicate that CS and GS do not overly affect cell integrity or bone biomarkers. Yet CS and both compounds together increase the expression ratio of OPG/RANKL, suggesting a positive effect on OA subchondral bone structural changes. This was confirmed by the decreased resorptive activity for the combination of CS and GS. These data are of major significance and may help to explain how these two drugs exert a positive effect on OA pathophysiology.
PMCID: PMC2246236  PMID: 17996099
17.  Peroxisome proliferator-activated receptor γ1 expression is diminished in human osteoarthritic cartilage and is downregulated by interleukin-1β in articular chondrocytes 
Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor involved in the regulation of many cellular processes. We and others have previously shown that PPARγ activators display anti-inflammatory and chondroprotective properties in vitro and improve the clinical course and histopathological features in an experimental animal model of osteoarthritis (OA). However, the expression and regulation of PPARγ expression in cartilage are poorly defined. This study was undertaken to investigate the quantitative expression and distribution of PPARγ in normal and OA cartilage and to evaluate the effect of IL-1β, a prominent cytokine in OA, on PPARγ expression in cultured chondrocytes. Immunohistochemical analysis revealed that the levels of PPARγ protein expression were significantly lower in OA cartilage than in normal cartilage. Using real-time RT-PCR, we demonstrated that PPARγ1 mRNA levels were about 10-fold higher than PPARγ2 mRNA levels, and that only PPARγ1 was differentially expressed: its levels in OA cartilage was 2.4-fold lower than in normal cartilage (p < 0.001). IL-1 treatment of OA chondrocytes downregulated PPARγ1 expression in a dose- and time-dependent manner. This effect probably occurred at the transcriptional level, because IL-1 decreases both PPARγ1 mRNA expression and PPARγ1 promoter activity. TNF-α, IL-17, and prostaglandin E2 (PGE2), which are involved in the pathogenesis of OA, also downregulated PPARγ1 expression. Specific inhibitors of the mitogen-activated protein kinases (MAPKs) p38 (SB203580) and c-Jun N-terminal kinase (SP600125), but not of extracellular signal-regulated kinase (PD98059), prevented IL-1-induced downregulation of PPARγ1 expression. Similarly, inhibitors of NF-κB signaling (pyrrolidine dithiocarbamate, MG-132, and SN-50) abolished the suppressive effect of IL-1. Thus, our study demonstrated that PPARγ1 is downregulated in OA cartilage. The pro-inflammatory cytokine IL-1 may be responsible for this downregulation via a mechanism involving activation of the MAPKs (p38 and JNK) and NF-κB signaling pathways. The IL-1-induced downregulation of PPARγ expression might be a new and additional important process by which IL-1 promotes articular inflammation and cartilage degradation.
PMCID: PMC1906809  PMID: 17386086
18.  Alterations of metabolic activity in human osteoarthritic osteoblasts by lipid peroxidation end product 4-hydroxynonenal 
4-Hydroxynonenal (HNE), a lipid peroxidation end product, is produced abundantly in osteoarthritic (OA) articular tissues, but its role in bone metabolism is ill-defined. In this study, we tested the hypothesis that alterations in OA osteoblast metabolism are attributed, in part, to increased levels of HNE. Our data showed that HNE/protein adduct levels were higher in OA osteoblasts compared to normal and when OA osteoblasts were treated with H2O2. Investigating osteoblast markers, we found that HNE increased osteocalcin and type I collagen synthesis but inhibited alkaline phosphatase activity. We next examined the effects of HNE on the signaling pathways controlling cyclooxygenase-2 (COX-2) and interleukin-6 (IL-6) expression in view of their putative role in OA pathophysiology. HNE dose-dependently decreased basal and tumour necrosis factor-α (TNF-α)-induced IL-6 expression while inducing COX-2 expression and prostaglandin E2 (PGE2) release. In a similar pattern, HNE induces changes in osteoblast markers as well as PGE2 and IL-6 release in normal osteoblasts. Upon examination of signaling pathways involved in PGE2 and IL-6 production, we found that HNE-induced PGE2 release was abrogated by SB202190, a p38 mitogen-activated protein kinase (MAPK) inhibitor. Overexpression of p38 MAPK enhanced HNE-induced PGE2 release. In this connection, HNE markedly increased the phosphorylation of p38 MAPK, JNK2, and transcription factors (CREB-1, ATF-2) with a concomitant increase in the DNA-binding activity of CRE/ATF. Transfection experiments with a human COX-2 promoter construct revealed that the CRE element (-58/-53 bp) was essential for HNE-induced COX-2 promoter activity. However, HNE inhibited the phosphorylation of IκBα and subsequently the DNA-binding activity of nuclear factor-κB. Overexpression of IKKα increased TNF-α-induced IL-6 production. This induction was inhibited when TNF-α was combined with HNE. These findings suggest that HNE may exert multiple effects on human OA osteoblasts by selective activation of signal transduction pathways and alteration of osteoblastic phenotype expression and pro-inflammatory mediator production.
PMCID: PMC1794501  PMID: 17042956

Results 1-18 (18)