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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Objective

Prostaglandin E2 (PGE2) is one of the main catabolic factors involved in osteoarthritis (OA), and metalloproteinases (MMPs) are crucial for cartilage degradation. PGE2 synthesis under inflammatory conditions is catalyzed by cyclooxygenase 2 and microsomal PGE synthase 1 (mPGES-1), whereas NAD+-dependent 15-hydroxy–PG dehydrogenase (15-PGDH) is the key enzyme implicated in PGE2 catabolism. The present study was undertaken to investigate the contribution of visfatin, an adipose tissue–derived hormone, to the pathophysiology of OA, by examining its role in PGE2 synthesis and matrix degradation.

Methods

The synthesis of visfatin by human chondrocytes from OA patients, with and without stimulation with interleukin-1β (IL-1β) and the role of visfatin in PGE2 synthesis were analyzed by real-time reverse transcriptase–polymerase chain reaction (RT-PCR) and immunoblotting. The effects of visfatin (1–10 μg/ml) on mPGES-1 and 15-PGDH synthesis, on the subsequent release of PGE2, and on MMP-3, MMP-13, ADAMTS-4, ADAMTS-5, and PG synthesis by primary immature mouse articular chondrocytes were examined by quantitative RT-PCR, immunoblotting, and enzyme-linked immunosorbent assay. Finally, small interfering RNA (siRNA) was used to assess the influence of visfatin on IL-1β–induced release of PGE2 in immature mouse articular chondrocytes.

Results

Human OA chondrocytes produced visfatin, and visfatin synthesis was increased by IL-1β treatment. Visfatin, like IL-1β, triggered excessive release of PGE2, due to increased mPGES-1 synthesis and decreased 15-PGDH synthesis. Visfatin knockout with siRNA reduced IL-1β–induced PGE2 overrelease. Visfatin triggered ADAMTS-4 and ADAMTS-5 expression and MMP-3 and MMP-13 synthesis and release, and reduced synthesis of high molecular weight PG by immature mouse articular chondrocytes.

Conclusion

The findings of this study indicate that visfatin has a catabolic function in cartilage and may have an important role in the pathophysiology of OA.

Osteoarthritis (OA) causes pain and dysfunction and is the leading cause of disability in elderly people in industrialized countries (1). It results in breakdown of articular cartilage with concomitant changes in the underlying bone, development of osteophytes, and some degree of synovial inflammation (2). The extracellular matrix of cartilage is destroyed and the phenotype of chondrocytes altered due to changes in their pattern of gene expression. They lose their differentiated phenotype and undergo focal cell death and degeneration (3). Several epidemiologic studies have shown a positive association between obesity and hip and knee OA, highlighting the key role of mechanical loading on cartilage metabolism (4). An association between obesity and OA in non–weight-bearing joints, such as the hand, has also been described (5, 6). Moreover, the loss of body fat seems to be more important than the loss of body weight in improving the symptoms of OA (7). The increased fat mass in obesity may therefore alter the metabolism of articular tissues such as cartilage.

Obesity is a chronic metabolic disease that is becoming increasingly common in industrialized countries (8). Fat cells secrete a variety of proteins with the functional and structural properties of cytokines; these are termed “adipokines.” Adiponectin, leptin, and resistin are the most abundant adipokines produced by adipose tissue, and production of leptin and resistin is increased in obese individuals (8). Adipokines have autocrine, paracrine, and endocrine effects and may be an important link between the immune response and metabolism, predisposing individuals to increased risk of disease (9, 10). For example, the increased level of circulating leptin in obese patients is positively correlated with increased concentrations of interleukin-1 (IL-1) receptor antagonist, IL-6, and tumor necrosis factor α (TNFα) in serum, and with type 2 diabetes and cardiovascular disease (11, 12). Interestingly, plasma levels of leptin, adiponectin, and resistin are increased in arthropathies, such as rheumatoid arthritis (RA) (13, 14) and OA (15). Presle and colleagues demonstrated that adipokines were expressed by tissue from various OA-affected joints and that levels of adipokines in serum did not correspond to levels in synovial fluid (15). Therefore, it appears that the joint cavity is the site at which each adipokine's expression is individually regulated.

One recently described adipokine is visfatin. It is secreted by mature adipocytes, and its plasma concentration markedly increases in parallel with the amount of visceral fat. Visfatin exerts insulin-mimetic effects in vivo and in vitro (16). This 52-kd protein binds to and activates insulin receptor, with a similar binding equilibrium dissociation constant. Mutation of the extracellular α-subunit of insulin receptor abrogates binding of insulin, but not of visfatin. Therefore, visfatin activates insulin receptor in a manner distinct from its activation by insulin, but the two proteins trigger the same signaling pathways in adipocytes (16).

Visfatin was previously identified as a secreted growth factor for early B lymphocytes (pre–B cell colony-enhancing factor [PBEF]). Several tissues produce visfatin/PBEF, including skeletal muscle, liver, and bone marrow (17). It is pleiotropic and regulates both inflammatory and immune responses. Its synthesis is regulated by cytokines such as TNFα, IL-1β, and IL-6, by lipopolysaccharide (LPS), and by dexamethasone (18–20). Visfatin is overproduced in colorectal cancer and is increased in acute lung injury, where it is a potential biomarker (21). It is produced by LPS-induced neutrophils and inhibits their apoptosis via a caspase 3– and caspase 8–mediated mechanism (18). Moreover, visfatin was recently found in foam cell macrophages within unstable atherosclerotic lesions, where it was involved in plaque destabilization (22). Finally, plasma concentrations of visfatin are increased in patients with RA (13). However, the effect of visfatin on cartilage physiology remains completely unknown.

Visfatin is also called Nampt because of its nicotinamide phosphoribosyltransferase (NAmPRTase) activity. Visfatin/Nampt has been implicated in the synthesis of NAD, an essential cofactor for cell metabolism. Rongvaux and colleagues showed that during polyclonal immune responses, visfatin/PBEF production is increased in lymphocytes, and could stimulate their proliferation (23). Visfatin is present in both the cytoplasm and the nucleus in rat and mouse cell lines, where its concentration depends upon the cell cycle phase (24). Finally, Nampt appears to be a longevity gene which adds stress-resistant life to human smooth muscle cells (25). Therefore, visfatin is a multifunctional protein whose intracellular and extracellular effects are dissimilar.

Cartilage degradation in OA is due in part to increased release of catabolic mediators, such as IL-1β and prostaglandin E2 (PGE2) (2, 26). IL-1β concentrations are markedly elevated in the synovial fluid of patients with RA (27) and OA (28). PGE2 is a prostanoid derived from arachidonic acid, and its production in the setting of inflammation depends on the coordinated activities of cyclooxygenase 2 and microsomal PGE synthase 1 (mPGES-1). Collagen-induced arthritis has been shown to be significantly less severe, and cartilage better preserved, in mice lacking mPGES-1 than in control mice (29). The synthesis of mPGES-1 by chondrocytes from OA patients is also increased by IL-1β (30). In vivo, PGE2 is rapidly converted to an inactive metabolite by 15-hydroxy–PG dehydrogenase (15-PGDH) (31). Synthesis of 15-PGDH is subnormal in various diseases, including cancer (32) and inflammatory bowel disease (33). We recently reported that 15-PGDH is synthesized by mouse cartilage (34), but its regulation in this tissue has not been characterized.

Increased release of these catabolic mediators in OA cartilage triggers the expression of matrix metalloproteinases (MMPs) and ADAMTS (also called aggrecanases) (35). MMPs are zinc-dependent endopeptidases that can break down all kinds of extracellular matrix proteins and are probably involved in both normal turnover and breakdown under disease conditions. MMP-13 (collagenase 3) and MMP-3 (stromelysin 1) are implicated in degradation of the main constituents of cartilage matrix, type II collagen and aggrecans, whereas ADAMTS-4 and ADAMTS-5 are aggrecanases most efficient at degrading aggrecans (35). Their production is increased in experimental models of OA and in articular cartilage from OA patients (36, 37).

In the present study we investigated the role of visfatin in cartilage metabolism, focusing on inflammation and matrix degradation, and its possible implication in OA. We found that visfatin is produced by human OA chondrocytes and that this synthesis is increased by IL-1β. Visfatin also triggers PGE2 synthesis by increasing production of mPGES-1 and reducing expression of 15-PGDH. Moreover, it increases the synthesis and release of MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5 by chondrocytes and decreases aggrecan production. Finally, inhibition of visfatin synthesis reduces IL-1β–induced PGE2 synthesis.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Materials.

All reagents were purchased from Sigma-Aldrich (St. Quentin Fallavier, France), unless noted otherwise. Recombinant human IL-1β was from PeproTech (Tebu- Bio, Le Perray-en-Yvelines, France). Recombinant mouse and human visfatin (produced in Escherichia coli with residual LPS contamination <100 pg/ml in accordance with the recommendations of the manufacturer) was from Alexis Biochemicals (Paris, France).

Primary cultures of human cells.

Human cartilage samples were obtained from patients undergoing joint replacement surgery for OA at Saint Antoine Hospital (Paris, France). Informed consent was obtained from each patient prior to surgery. The diagnosis of OA was based on clinical and radiographic evaluations according to the criteria of the American College of Rheumatology (formerly, the American Rheumatism Association) (38). Cartilage samples were collected from 7 patients (6 women and 1 man; mean ± SD age 70.8 ± 8.9 years), all of whom were overweight (mean ± SD body mass index 31.1 ± 10.25 kg/m2). Our institutional ethics committee approved the study protocol.

Human articular chondrocytes were isolated by enzymatic digestion of cartilage from tibial plateaus according to a previously described procedure (39). After 4 days of culture, cells were starved in serum-free Dulbecco's modified Eagle's medium (DMEM) (4.5 mg/liter glucose) supplemented with 100 IU/ml penicillin, 100 μg/ml streptomycin, and 4 mML-glutamine containing 0.3% bovine serum albumin (BSA; Euromedex, Strasbourg, France), for 24 hours. Morphologic features, along with the levels of type II collagen and aggrecan expression, were consistent with a chondrocyte phenotype, rather fibroblast-like cells.

Primary cultures of mouse cells.

All experiments were performed according to protocols approved by the French/European Ethics Committee. Immature mouse articular chondrocytes were isolated by enzymatic digestion of articular cartilage from 6-day-old newborn animals from one Swiss mouse litter, according to a previously described procedure (40). After 6–7 days of culture, the cells were placed in serum-free DMEM (1 mg/liter of glucose) supplemented with penicillin, streptomycin, and L-glutamine containing 1% BSA, for 24 hours.

Transfection of small interfering RNA (siRNA).

Two siRNA directed against mouse visfatin (siRNA1 and siRNA2) were designed and purchased from Ambion Cenix (Austin, TX). The reported results were obtained with siRNA1 and confirmed with siRNA2. The sequences specific for mouse visfatin were as follows: siRNA1 forward 5′-GGCACCACUAAUCAUCAGAtt-3′, reverse 5′-UCUGAUGAUUAGUGGUGCCtc-3′; siRNA2 forward 5′-GCACAGUACCAUAACGGCUtt-3′, reverse 5′-AGCCGUUAUGGUACUGUGCtc-3′.

Mouse chondrocytes were cultured as described above. Confluent cells were removed with trypsin, and 6 × 105 chondrocytes were seeded in 6-cm tissue culture plates and grown for 24 hours, to 70–80% confluence. Normal growth medium containing 10% fetal bovine serum was changed prior to siRNA transfection. Transfections were performed as described for the RNAi Starter Kit (Qiagen, Courtaboeuf, France). Cells were incubated for 18 hours with siRNA and transfection reagent, rinsed twice with phosphate buffered saline (PBS), and placed in DMEM (1 mg/liter of glucose) supplemented with penicillin, streptomycin, and L-glutamine containing 1% BSA, with or without IL-1β (10 ng/ml) for 6 hours or 24 hours. Transfection of siRNA against MAPK-1, a ubiquitously produced mouse cell protein, was used as a positive control. A nonsilencing siRNA that has no homology with any known mammalian gene (RNAi Starter Kit) and scrambled siRNA (Ambion) were used as negative controls.

Cell viability analysis.

Immature mouse articular chondrocytes were plated at 2.5 × 106 cells/well in 96-well plates and treated with visfatin (in various concentrations up to 10 μg/ml) for 6 hours. Cell viability was evaluated by TACS MTT Assay, in accordance with the instructions of the manufacturer (R&D Systems, Lille, France).

PGE2 and MMP-3 assays.

PGE2 in the medium was measured with an enzyme immunoassay kit from Cayman Chemical SPI-BIO (Massy, France), and total mouse MMP-3 was measured with an enzyme-linked immunosorbent assay (ELISA) kit from R&D Systems. The limits of detection were 9 pg/ml for PGE2 and 5 pg/ml for MMP-3. The PGE2 and MMP-3 concentrations were analyzed in duplicate at serial dilutions and were read against standard curves.

RNA extraction, reverse transcription, and real-time quantitative reverse transcriptase–polymerase chain reaction (RT-PCR).

Messenger RNA (mRNA) for visfatin, aggrecan, MMP, ADAMTS, PGES, and 15-PGDH was quantified using the iCycler iQ Real Time PCR kit (Bio-Rad, Marnes-la-coquette, France) and the QuantiTect SYBR PCR kit (Qiagen), as previously described (40). Levels of mRNA were normalized to those of murine hypoxanthine guanine phosphoribosyltransferase or human GAPDH. Probe sequences, amplicon lengths, and conditions were as previously described (34), except as follows: GAPDH forward 5′-CCATCACCATCTTCCA-3′, reverse 5′-CCTTCTCCATGGTGGT-3′ (58°C); MMP-3 forward 5′-ATGAAAATGAAGGGTCTTCCGG-3′, reverse 5′-GCAGAAGCTCCATACCAGCA-3′ (58°C, 108 bp); MMP-13 forward 5′-TGATGGCACTGCTGACATCAT-3′, reverse 5′-TGTAGCCTTTGGAACTGCTT-3′ (58°C, 173 bp); ADAMTS-4 forward 5′-GGCAAGGACTATGACGC-3′, reverse 5′-TCAGCCCAAGGTGAGTG-3′ (60°C, 155 bp); ADAMTS-5 forward 5′-TCAGCCACCATCACAGAA-3′, reverse 5′-CCAGGGCACACCGAGTA-3′ (60°C, 161 bp); aggrecan forward 5′-CAGAGTTAGTGGAGGGTGTGA-3′, reverse 5′-AGACCCTGGGAAGTTTGT-3′ (60°C, 152 bp). Mouse and human visfatin and the MAPK-1 primers were from Qiagen.

Protein extraction and Western blotting.

Cell lysates were prepared and Western blotting was carried out as described previously (30), with anti-mouse mPGES-1 polyclonal antibody (Cayman Chemical SPI-BIO), anti-mouse and anti-human visfatin polyclonal antibodies (Alexis Biochemicals), anti-human MMP-3 and MMP-13 polyclonal antibodies (Santa Cruz Biotechnology, Tebu-Bio; Le Perray en Yvelines, France), and anti-mouse β-actin monoclonal antibody (Sigma-Aldrich). Recombinant human or mouse visfatin and cell extracts containing overproduced mPGES-1 were used as positive controls. For densitometry analysis, we used MultiGauge version 3.0 software (Fujifilm, Courbevoie, France).

35SO4 incorporation into PGs.

Chondrocytes were incubated in serum-free, sulfate-free DMEM plus 1.5 μCi/ml Namath imageSO4 (75 MBq/ml; Amersham, Little Chalfont, UK) for 20 hours in the presence or absence of IL-1β or visfatin. Controls were prepared without any additive compound. Radiolabeled PGs were prepared as previously described (41). Aliquots were applied to a Sepharose 2B column (Amersham, Uppsala, Sweden), and PGs were eluted with 0.5M sodium acetate (pH 6.8) as previously described (41). For each column, radioactivity eluted at Kav 0.1–0.2 (high molecular weight [HMW] aggregated PG) was measured and expressed as a percent of total radioactivity eluted on the column (void volume to total volume). In each experiment, 2 similarly treated flasks were each analyzed in duplicate.

Statistical analysis.

Each result is expressed as the fold induction compared with control (set at 1). All data are reported as the mean ± SD unless stated otherwise. Statistical analysis was performed with the Welch corrected t-test to compare mean values between 2 groups, and with one-way analysis of variance with Bonferroni post hoc correction to compare mean values among >2 groups, using GraphPad Prism software (GraphPad Software, San Diego, CA). P values less than or equal to 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Synthesis of visfatin in human articular chondrocytes from OA patients, and its induction by IL-1β.

Primary cultures of human chondrocytes from the knees of OA patients were used to assess the synthesis of visfatin by human cartilage. We used quantitative real-time RT-PCR analysis and immunoblotting to investigate the influence of IL-1β on the transcription and translation of the visfatin gene. Visfatin mRNA and protein were constitutively produced in the chondrocytes from OA patients (Figures 1A and 1B). IL-1β (10 ng/ml) stimulated visfatin mRNA production at 6 hours (6-fold increases) (P < 0.05) (Figure 1A) and visfatin protein expression from 6 hours up to 24 hours (Figure 1B).

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Figure 1. Expression and regulation of visfatin in human chondrocytes from osteoarthritis (OA) patients by interleukin-1β (IL-1β), and role of visfatin in prostaglandin E2 (PGE2) release. A and B, Primary cultures of human chondrocytes from OA patients were starved and then stimulated with 10 ng/ml IL-1β for 6 hours or 24 hours. Visfatin synthesis was analyzed by reverse transcriptase–polymerase chain reaction (A) and immunoblotting (B). Values in A are the fold increase compared with control (cont; C) (set at 1) and are the mean and SD of 3 independent experiments with 1 well/condition, analyzed in duplicate. Blots in B are representative of 4–5 independent experiments. The densitometric quantification of visfatin was normalized to β-actin and expressed as arbitrary units (AU; control set at 1); values in the graph in B are the mean and SD of 4–5 independent experiments. C, Primary cultures of human chondrocytes from OA patients were left untreated or treated with 1–10 μg/ml human visfatin for 6 hours. The amount of PGE2 released into the medium (pg/ml) was measured by enzyme immunoassay. Values are the fold increase in PGE2 release compared with control (untreated chondrocytes) (set at 1) and are the mean and SD of 3 independent experiments with 2 wells/condition, analyzed in duplicate. ∗ = P < 0.05 versus control.

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Visfatin-induced synthesis of PGE2 by human articular chondrocytes from OA patients.

Because PGE2 is a pivotal catabolic mediator of OA, we tested its effects on PGE2 synthesis by human chondrocytes from OA cartilage. Visfatin administered for 6 hours at 1, 2.5, 5, or 10 μg/ml triggered a dose-dependent increase in PGE2 release (increases of 8.4-fold [P not significant], 10.2-fold [P < 0.05], 14.31-fold [P < 0.05], and 27.85-fold [P < 0.05], respectively, compared with controls [mean ± SD 1,249 ± 1,515 pg/ml]) (Figure 1C).

Increased PGE2 synthesis by immature mouse articular chondrocytes stimulated with visfatin.

Since only limited amounts of human cartilage were available, we used primary cultures of immature mouse chondrocytes for more extensive studies. Visfatin mRNA and protein were constitutively produced by immature mouse articular chondrocytes, and their synthesis was increased by IL-1β in a time-dependent manner (0–24 hours) and a dose-dependent manner (0–10 ng/ml) (data not shown).

In order to characterize the role of visfatin in PGE2 synthesis in cartilage, we stimulated immature mouse articular chondrocytes with visfatin. Visfatin administered for 6 hours at 2.5, 5, or 10 μg/ml triggered a dose-dependent increase in PGE2 release (1.5-fold [P < 0.001], 1.8-fold [P < 0.01], and 3-fold [P < 0.01], respectively, compared with controls [mean ± SD 46.9 ± 8.36 pg/ml]) (Figure 2A).

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Figure 2. Involvement of visfatin in prostaglandin E2 (PGE2) synthesis by immature mouse articular chondrocytes. Chondrocytes were left untreated or incubated with 2.5–10 μg/ml visfatin for 6 hours, or with 1 ng/ml interleukin-1β (IL-1β) with or without 1–5 μg/ml visfatin for 24 hours. The PGE2 concentration in the medium was measured, and total RNA was extracted and levels of microsomal PGE synthase 1 (mPGES-1) and 15-hydroxy–PG dehydrogenase (15-PGDH) mRNA assayed. A, Effect of visfatin on PGE2 release by chondrocytes. The amount of PGE2 released into the medium (pg/ml) was measured by enzyme immunoassay. B and C, Effect of visfatin on levels of mPGES-1 mRNA (B) and 15-PGDH mRNA (C) in chondrocytes, determined by real-time polymerase chain reaction. D, Additive effects of visfatin and IL-1β on PGE2 release by chondrocytes. Values are the fold increase compared with control (set at 1) and are the mean and SD of 2 independent experiments with 2 wells/condition (A and D) or 1 well/condition (B and C), analyzed in duplicate. ∗ = P < 0.05 versus control; ∗∗ = P < 0.01 versus control; ∗∗∗ = P < 0.001 versus control; # = P < 0.05 versus stimulation with IL-1β alone. HPRT = hypoxanthine guanine phosphoribosyltransferase.

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We studied the transcription of the mPGES-1 and 15-PGDH genes in immature mouse articular chondrocytes to determine the effects of visfatin on the enzymes involved in PGE2 synthesis and catabolism. Incubation with visfatin at 5 μg/ml or 10 μg/ml for 6 hours triggered increased synthesis of mPGES-1 mRNA (6.9-fold and 13.8-fold, respectively) (both P < 0.01) (Figure 2B). Transcription of the 15-PGDH gene was dramatically reduced by visfatin at 2.5, 5, and 10 μg/ml (75%, 85%, and 90%, respectively; all P < 0.05) (Figure 2C).

Immature mouse articular chondrocytes were incubated with both IL-1β and visfatin to determine whether visfatin acted synergistically with IL-1β (Figure 2D). Chondrocytes incubated with 1 ng/ml IL-1β released 15 times more PGE2 than controls (P < 0.001), while chondrocytes incubated for 24 hours with visfatin at 1, 2.5, or 5 μg/ml also exhibited increased PGE2 release (2.4-fold, 3.8-fold, and 14-fold, respectively, compared with controls [mean ± SD 33.75 ± 4 pg/ml]). This increase was significant (P < 0.001) with the 5-μg/ml concentration. Incubation for 24 hours with 1 ng/ml IL-1β plus 1, 2.5, or 5 μg/ml visfatin had an additive effect on PGE2 release (increases of 1.23-fold, 2.03-fold, and 3.35-fold, respectively, compared with incubation with IL-1β alone) (all P < 0.05).

Because the recombinant visfatin protein is produced by E coli, we performed experiments to confirm that PGE2 release was not triggered by any residual LPS contamination. LPS (1 ng/ml) triggered the release of 29.89 ± 13.22 pg/ml PGE2 by chondrocytes (mean ± SD; n = 4), compared with 27.6 ± 3.06 pg/ml in controls (P not significant). Moreover, the MTT assay for cell viability revealed that visfatin in concentrations of up to 10 μg/ml did not trigger chondrocyte death or proliferation (data not shown).

Implication of visfatin in IL-1β–induced synthesis of PGE2 and mPGES-1 by immature mouse articular chondrocytes.

Because IL-1β is essential for PGE2 synthesis and IL-1β increased visfatin production by chondrocytes, we tested whether visfatin mediated the action of IL-1β on cartilage metabolism. We used an siRNA-based strategy to assess the role of visfatin in IL-1β–induced PGE2 release. Transfection with predesigned siRNA against visfatin significantly suppressed visfatin expression at the mRNA and protein levels (decreases of 70% and 35%, respectively, compared with control; n = 4) (P < 0.05), demonstrating efficient gene silencing by this approach.

Treatment with 10 ng/ml IL-1β for 24 hours induced a 10-fold increase (P < 0.001) in PGE2 release by immature mouse articular chondrocytes (mean ± SD PGE2 release by controls 80 ± 12 pg/ml). Visfatin siRNA did not reduce constitutive PGE2 release by cells incubated without IL-1β. However, PGE2 release was decreased by 33% (P < 0.001) when visfatin siRNA was transfected before IL-1β stimulation (Figure 3A).

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Figure 3. Involvement of visfatin in IL-1β–induced PGE2 release by immature mouse articular chondrocytes. Immature mouse articular chondrocytes were transfected for 18 hours with small interfering RNA (siRNA) targeting the visfatin gene. The cells were then starved and incubated with 10 ng/ml IL-1β. A, Effect of visfatin siRNA transfection on PGE2 release by chondrocytes stimulated with IL-1β for 24 hours. The amount of PGE2 released into the medium (pg/ml) was measured by enzyme immunoassay. B and C, Effect of visfatin siRNA transfection on mPGES-1 mRNA levels analyzed by real-time polymerase chain reaction (RT-PCR) (B) and protein synthesis analyzed by immunoblotting (C), in chondrocytes stimulated with IL-1β for 6 hours (B) or 24 hours (C). D, Effect of IL-1β (6-hour treatment) and visfatin siRNA on 15-PGDH synthesis by chondrocytes, analyzed by real-time RT-PCR. Values in A, B, and D are the mean and SD of 4 independent experiments with 1 well/condition, analyzed in duplicate. Blots in C are representative of 3 independent experiments. ∗∗ = P < 0.01; ∗∗∗ = P < 0.001, versus stimulation with IL-1β alone. See Figure 2 for other definitions.

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We then assessed the effect of visfatin on mPGES-1 synthesis induced by IL-1β. IL-1β administered at 10 ng/ml for 6 hours or 24 hours increased both mPGES-1 mRNA (2.8-fold) (P < 0.001) (Figure 3B) and mPGES-1 protein (Figure 3C). The increase in mPGES-1 mRNA induced by IL-1β was partially reversed when visfatin siRNA was transfected (38% less than the increase observed with IL-1β alone) (P < 0.01) (Figure 3B). Similarly, immature mouse articular chondrocytes transfected with visfatin siRNA produced less IL-1β–induced mPGES-1 protein (Figure 3C). Neither the transcription nor the translation of the cytosolic constitutive isoform of PGES was significantly altered in cells, with or without visfatin siRNA or IL-1β treatment (data not shown).

Messenger RNA for 15-PGDH was constitutively produced in immature mouse articular chondrocytes. Treatment with IL-1β (10 ng/ml) dramatically decreased (by 95%) the 15-PGDH mRNA concentration (P < 0.001). This effect was reversed to a small extent when visfatin siRNA was transfected prior to the IL-1β treatment (15-PGDH mRNA level 1.38-fold higher than that obtained with IL-1β alone (P not significant) (Figure 3D).

Visfatin-induced reduction of aggrecan mRNA levels and the pool of HMW proteoglycans.

In order to characterize the role of visfatin in chondrocyte matrix synthesis, we analyzed the ratio of type II collagen to type I collagen and the synthesis of aggrecan in response to increasing concentrations of visfatin. Treatment with visfatin for 6 hours did not modify the type II collagen mRNA:type I collagen mRNA ratio (data not shown), but did trigger a decrease in aggrecan mRNA levels (reductions of 40% [P < 0.05], 55% [P < 0.01], and 50% [P < 0.05], respectively, with visfatin concentrations of 2.5, 5, and 10 μg/ml) (Figure 4A).

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Figure 4. Decreased aggrecan and high molecular weight (HMW) PG synthesis by immature mouse articular chondrocytes treated with visfatin. A, Effect of visfatin on aggrecan mRNA levels. The effect of visfatin on aggrecan synthesis was assessed by real-time reverse transcriptase–polymerase chain reaction. Values are the mean and SD of 3 independent experiments with 1 well/condition, analyzed in duplicate. B, Sepharose 2B elution profile of 35SO4-sulfated PGs synthesized and secreted by immature mouse articular chondrocytes. Chondrocytes were left untreated or stimulated with 10 ng/ml IL-1β or 5 μg/ml visfatin in the presence of radiolabeled sulfate. Sulfated PGs were extracted with protease inhibitors, and aliquots were applied to a Sepharose 2B column. Eluates were collected, and radioactivity was measured by scintillation counting. Under each experimental condition, HMW radioactive material (grey area) eluted at Kav <0.2 was measured. Individual symbols represent the radioactivity of each eluate. Results are representative of 2 independent experiments, analyzed in duplicate. V0 = void volume; Vt = total volume (ml). C, Quantification of radioactivity eluted at Kav <0.2 (HMW PGs) and expressed as the percentage of total radioactivity eluted on the column (V0 to Vt). In each experiment, 2 similarly treated flasks were analyzed. Values are the mean and SD of 2 experiments. ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001, versus control. See Figure 2 for other definitions.

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Immature mouse articular chondrocytes were then left untreated or treated for 20 hours with 10 ng/ml IL-1β or 5 μg/ml visfatin, and PG synthesis after 35SO4 incorporation was analyzed by chromatography on a Sepharose 2B column (Figure 4B). Under basal conditions, >25 ± 0.6% of sulfated PGs (mean ± SD) were eluted as HMW aggregated complexes (Kav < 0.2). As expected, IL-1β decreased the HMW aggregated pool (to 30% of that seen under basal conditions) (P < 0.001). Interestingly, visfatin triggered a decrease in HMW aggregated PGs in the same range as that observed with IL-1β (decrease to 31% of the level seen under basal conditions) (P < 0.05) (Figure 4C).

Increased MMP-3 and MMP-13 synthesis and release and ADAMTS-4 and ADAMTS-5 expression by immature mouse articular chondrocytes incubated with visfatin.

Since the matrix breakdown that occurs in OA is due to MMPs (especially MMP-3 and MMP-13) and to ADAMTS-4 and ADAMTS-5, we assessed the influence of visfatin on the synthesis of these enzymes by chondrocytes. Immature mouse articular chondrocytes incubated for 6 hours with 2.5, 5, or 10 μg/ml visfatin contained significantly more MMP-3 and MMP-13 mRNA than controls (in which mRNA for both MMP-3 and MMP-13 was undetectable). Compared with levels obtained with 2.5 μg/ml visfatin, MMP-3 mRNA levels were increased 3-fold (P < 0.05) and 5.4-fold (P < 0.01) and MMP-13 mRNA levels were increased 2.3-fold (P < 0.01) and 6.2-fold (P < 0.01) with addition of visfatin at 5 μg/ml and 10 μg/ml, respectively (Figure 5A). Using immunoblotting and ELISA, we determined the effects of visfatin on MMP-3 and MMP-13 release by chondrocytes by measuring the translation of these enzymes in chondrocytes incubated with visfatin for 24 hours. Visfatin (1, 2.5, or 5 μg/ml) triggered the release of MMP-3 and MMP-13 protein (not detectable with control). The concentration of MMP-3 in the medium of cells incubated with 5 μg/ml visfatin was 10 times greater (P < 0.05) than that of cells incubated with 1 μg/ml visfatin (mean ± SD 59 ± 37.6 ng/ml) (Figure 5B).

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Figure 5. Involvement of visfatin in synthesis of matrix metalloproteinase 3 (MMP-3), MMP-13, ADAMTS-4, and ADAMTS-5 by immature mouse articular chondrocytes. Immature mouse articular chondrocytes were left untreated or incubated with visfatin at 2.5–10 μg/ml or 1–5 μg/ml. RNA was extracted after 6-hour incubation and assays for MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5 mRNA levels performed; media from cells incubated for 24 hours were assayed for MMP-3 and MMP-13 levels. A and C, Effect of visfatin on the synthesis of mRNA for MMP-3 and MMP-13 (A) and ADAMTS-4 and ADAMTS-5 (C). Levels of mRNA were assessed by real-time reverse transcriptase–polymerase chain reaction. Values are the mean and SD of 2 independent experiments with 2 wells/condition, analyzed in duplicate. B, Effect of visfatin on MMP-3 and MMP-13 release from chondrocytes into the medium. Immunoblotting was performed, and the amount of MMP-3 released into the medium (ng/ml) was measured by enzyme-linked immunosorbent assay. Blots are representative of 3 independent experiments; values in the graph are the mean and SD of 2 independent experiments with 2 wells/conditions, analyzed in duplicate. Values are the fold increase compared with 2.5 μg/ml visfatin (A), 1 μg/ml visfatin (B), or untreated controls (C). ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001, versus 2.5 μg/ml visfatin (A), 1 μg/ml visfatin (B), or controls (C). HPRT = hypoxanthine guanine phosphoribosyltransferase; ND = not detectable.

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We then tested MMP-3 and MMP-13 expression in chondrocytes treated with visfatin siRNA, with or without addition of 10 ng/ml IL-1β. The results demonstrated that, in contrast to mPGES-1 expression, visfatin was not implicated in IL-1β–induced MMP-3 and MMP-13 expression (data not shown).

Finally, the effect of visfatin on ADAMTS-4 and ADAMTS-5 expression was investigated. Visfatin stimulated ADAMTS-4 and ADAMTS-5 mRNA expression in immature mouse articular chondrocytes, in a dose-dependent manner (compared with control, increases in ADAMTS-4 mRNA of 1.96-fold [P < 0.05], 2.72-fold [P < 0.001], and 5.02-fold [P < 0.001], and increases in ADAMTS-5 mRNA of 1.54-fold [P not significant], 2.52-fold [P < 0.05], and 4.28-fold [P < 0.01] with visfatin concentrations of 2.5 μg/ml, 5 μg/ml, and 10 μg/ml, respectively) (Figure 5C).

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Visfatin is a newly identified adipokine that is produced by adipose tissue, bone marrow, and skeletal muscle (16). It was originally discovered as a growth factor for B lymphocyte precursors, and is alternatively called PBEF (17). Visfatin/PBEF exerts actions in pathophysiologic processes such as colorectal cancer, acute lung injury, and atherosclerosis. In the present work, we studied its extracellular role in cartilage homeostasis. Our findings reveal that visfatin is produced by chondrocytes from patients with OA, and that its production is increased by IL-1β. Moreover, visfatin is a potent inducer of PGE2 release in both human and immature mouse articular chondrocytes, as a result of increased mPGES-1 and decreased 15-PGDH synthesis. Finally, visfatin triggers the synthesis and release of MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5 by chondrocytes and reduces HMW aggregated PGs. This demonstration of the potent proinflammatory and prodegradative effects of visfatin suggests that it also contributes to the progression of OA.

Interestingly, blocking of visfatin synthesis reduces the effect of IL-1β on PGE2 release in chondrocytes. Our working hypothesis of the interactive effect of IL-1β and visfatin on PGE2 synthesis in cartilage is illustrated in Figure 6. Increased mPGES-1 synthesis and the subsequent release of PGE2 by chondrocytes stimulated with IL-1β has been well described (30). We showed in this study that IL-1β induced a dramatic decrease in 15-PGDH levels, enhancing this effect. IL-1β stimulated the synthesis of visfatin by chondrocytes, and visfatin increased the production of mPGES-1 and reduced that of 15-PGDH, leading to the release of excess PGE2 from articular chondrocytes. Therefore, after IL-1β–triggered release of visfatin into the medium, an autocrine/paracrine loop would allow visfatin to act on the chondrocytes themselves via its receptor, and to modify the production of mPGES-1 and 15-PGDH in the same way that IL-1β does. Interestingly, a similar mechanism was not found with regard to MMP-3 and MMP-13 expression. The receptors for visfatin are insulin receptor and insulin-like growth factor 1 (IGF-1) receptor. Insulin receptor has not yet been found in chondrocytes, and affinity of the IGF-1 receptor for visfatin is too low to trigger an intracellular signal (16). We are presently working to identify the signaling pathway of visfatin in chondrocytes.

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Figure 6. Hypothetical role of visfatin in synthesis of PGE2, matrix metalloproteinase 3 (MMP-3), MMP-13, ADAMTS-4, and ADAMTS-5 by articular chondrocytes. In this model, expression of visfatin (mRNA and protein) in chondrocytes is increased by IL-1β (after binding to its receptor [IL-1R]). Visfatin then exerts action on the chondrocytes, in an autocrine manner. Extracellular visfatin, after binding to its receptor (which remains to be identified), increases mPGES-1 mRNA levels and reduces 15-PGDH mRNA levels, and subsequently triggers PGE2 release. In addition, extracellular visfatin triggers the synthesis and release of MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5 by chondrocytes. Therefore, visfatin appears to act as a mediator of inflammation and cartilage breakdown. See Figure 2 for other definitions.

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New concepts to explain the pathophysiology of OA have recently emerged. While OA is usually considered to be cartilage driven, other tissues, such as bone, muscle, and juxtaarticular adipose tissue, may also be involved. Therefore, OA could be a systemic disorder in which altered lipid homeostasis is a predisposing factor (42). Recent studies on the link between obesity and OA reveal potential roles of adipokines, such as leptin, adiponectin, and resistin, in cartilage degradation (15, 43, 44). Presle and colleagues recently reported that concentrations of adipokines in the serum do not reflect adipokine concentrations in the joint space and suggested that OA is associated with a specific local dysregulation of adipokines (15). The present study shows that human OA chondrocytes produce visfatin and that its production is increased by IL-1β, enforcing this hypothesis. Moreover, the visfatin gene is activated by hypoxia in a human breast cancer cell line, involving functional hypoxia-responsive element sites located within the proximal promoter region (45). Visfatin could be a critical factor in cartilage degradation, given that mature cartilage is an avascular tissue.

Visfatin is a highly conserved protein, and the death of mice lacking visfatin during embryogenesis (16) highlights its physiologic importance. Visfatin has separate extracellular and intracellular roles, and its implication in pathophysiology remains unclear. Two extracellular functions of visfatin have been identified. First, it acts as an insulin analog in glucose homeostasis (16). Second, it may be a mediator of late-stage inflammation: even though it does not have a signal peptide for its secretion and is unlike other known cytokines (46), its synthesis is increased in severely infected tissue (18, 46). Our data show that visfatin levels in chondrocytes increase in response to IL-1β and act in an autocrine/paracrine manner to trigger PGE2 synthesis. The human visfatin gene has various regulatory elements in the 5′-upstream region, such as binding sites for NF-κB and activator protein 1, the two major signaling pathways activated by IL-1β (46). Moreover, visfatin is an antiapoptotic mediator in neutrophils in both experimental inflammation and clinical sepsis (18). Apoptotic death of articular chondrocytes has been implicated in the pathogenesis of OA. PGE2 may sensitize chondrocytes to the cell death induced by nitric oxide (47) and can directly trigger apoptosis of bovine chondrocytes via cAMP signaling (48). Further experiments are needed to decipher the role of visfatin in chondrocyte apoptosis.

Visfatin has key intracellular roles, as evidenced by the implication of its NAmPRTase activity in cell proliferation and differentiation, demonstrated in B cells (23). Therefore, visfatin may act on chondrocyte differentiation in immature mouse articular chondrocytes. However, we did not observe any modification of the main marker of chondrocyte differentiation, i.e., the ratio of type II collagen mRNA to type I collagen mRNA. Thus, although we cannot definitively rule out a role of visfatin in the chondrocyte differentiation process, our results do not support such a role.

Several lines of evidence demonstrate that adipokines, released by white adipose tissue, participate in a wide variety of physiologic and pathophysiologic processes, including immunity and inflammation. Therefore, adipokines seem to link metabolic disorders to inflammatory and/or autoimmune conditions (49). It has been suggested that changes in lipid metabolism are involved in the diverse physiologic changes in generalized OA (50). However, there has been little reported experimental evidence to support this. We believe that the present results are the first demonstration of a catabolic role of visfatin, a newly discovered adipokine, in OA. Further studies are needed for more precise elucidation of the proinflammatory activities of visfatin in osteoarthritic joints and the contribution of each tissue to its synthesis. Broader knowledge of the deleterious action of visfatin in OA may lead to its emergence as a novel target for therapy.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Dr. Berenbaum had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study design. Berenbaum, Jacques.

Acquisition of data. Gosset, Salvat, Sautet, Pigenet, Tahiri.

Analysis and interpretation of data. Gosset.

Manuscript preparation. Gosset, Berenbaum, Jacques.

Statistical analysis. Gosset.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

We thank Dr. J.-M. Dayer for critically reviewing the manuscript and making valuable suggestions, and Dr. M. Corvol for designing the study of proteoglycan degradation. We are grateful to Dr. G. Nourissat for technical assistance.

REFERENCES

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  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES
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