Rhein, the metabolite of diacerhein, reduces the proliferation of osteoarthritic chondrocytes and synoviocytes without inducing apoptosis.

OBJECTIVE: The aim of this study was to determine the effects of pharmacologically relevant concentrations of rhein (1,8-dihydroxy-3-carboxyanthraquinone) on the cell proliferation rate of human chondrocytes and synoviocytes. METHODS: Cultures of human osteoarthritic synoviocytes and chondrocytes were incubated with 10(-6), 10(-5), and 10(-4) M rhein. [3H]thymidine incorporation was used to determine rhein proliferative effects after incubation periods of 24 h, 48 h, and 1 week. The cytotoxicity of the drug was assayed with a nonradioactive assay kit. Nuclear extracts were used to detect variations in cell-cycle proteins (p21, p27, and cyclin D1) by Western blotting. The effect of rhein on apoptosis was investigated by measurement of caspase-3/7 activity and DNA fragmentation. RESULTS: Rhein was found to downregulate the proliferation rate of both chondrocytes and synoviocytes, two-fold for 10(-5) M rhein and five- to six-fold for 10(-4) M rhein. No cytotoxicity of the drug was observed. Rhein (10(-4) M) decreased caspase-3/7 activity and did not induce DNA fragmentation. Western blots showed that 10(-4) M rhein increased the expression of p21 and/or p27, but not that of cyclin D1. CONCLUSIONS: Rhein has previously been shown to reduce the interleukin (IL)-1beta deleterious effects on osteoarthritis (OA) cartilage through inhibition of the expression of degrading enzymes. Here, rhein was also found to inhibit proliferation of both synoviocytes and chondrocytes, suggesting that the drug may decrease the development of the inflammatory synovial tissue that accompanies joint pathologies. Both its anti-catabolic and anti-proliferative effects may explain its beneficial effect in the treatment of joint diseases.

Differentiation potential of human muscle-derived cells towards chondrogenic phenotype in alginate beads culture.

OBJECTIVE: The aim of this study was to evaluate the differentiation potential of two populations of muscle-derived cells (CD56- and CD56+) towards chondrogenic phenotype in alginate beads culture and to compare the effect of transforming growth factor beta 1 (TGFbeta1) on the differentiation process in these populations. METHODS: Muscle CD56- and CD56+ cells were cultured in alginate beads, in a chondrogenic medium, containing or not TGFbeta1 (10 ng/ml). Cultures were maintained for 3, 7, 14 or 21 days in a humidified culture incubator. At harvest, one culture of each set was fixed for alcian blue staining and aggrecan detection. The steady-state level of matrix macromolecules mRNA was assessed by real-time polymerase chain reaction (PCR). Protein detection was performed by western-blot analysis. The binding activity of nuclear extracts to Cbfa1 DNA sequence was also evaluated by electrophoretic mobility shift assays (EMSA). RESULTS: Chondrogenic differentiation of both CD56+ and CD56- muscle-derived cells was improved in alginate scaffold, even without growth factor, as suggested by increased chondrogenesis markers expression during the culture. Furthermore, TGFbeta1 enhanced the differentiation process and allowed to maintain a high expression of markers of mature chondrocytes. Of importance, the combination of alginate and TGFbeta1 treatment resulted in a further down-regulation of collagen type I and type X, as well as Cbfa1 both expression and binding activity. CONCLUSIONS: Thus, alginate scaffold and chondrogenic medium are sufficient to lead both populations CD56+ and CD56- towards chondrogenic differentiation. Moreover, TGFbeta1 enhances this process and allows to maintain the chondrogenic phenotype by inhibiting terminal differentiation, particularly for CD56- cells.

17Beta-oestradiol up-regulates the expression of a functional UDP-glucose dehydrogenase in articular chondrocytes: comparison with effects of cytokines and growth factors.

OBJECTIVES: To investigate the mechanisms by which cytokines and 17beta-oestradiol (17beta-E2) modulate gene expression and activity of uridine diphosphoglucose dehydrogenase (UGDH), a key enzyme of GAG synthesis in articular chondrocytes. METHODS: Rabbit articular chondrocytes (RAC) from 3-week-old animals were incubated for 24 h with TGF-beta, insulin like growth factor-I (IGF-I), IL-1beta, IL-6 and 17beta-E2. GAG synthesis was measured by [35S]-sulphate labelling and the expression of the UGDH gene was estimated by both real-time polymerase chain reaction and western blotting, whereas the enzyme activity was assayed by a spectrophotometric procedure. In addition, the transcriptional activity of several UGDH gene promoter constructs was determined in RAC transiently transfected with wild-type or deleted human oestrogen receptor-alpha gene (hER alpha66 or hER alpha46, respectively). RESULTS: 17Beta-E2 and its receptor hER alpha66 enhanced GAG neosynthesis in rabbit articular chondrocytes, as did TGF-beta1 whereas IL-1beta decreased this synthesis. 17Beta-E2 was found to exert positive regulatory effects at mRNA, protein and UGDH activity levels. In addition, the receptor hER alpha66, but not hER alpha46, increased the transcriptional activity of the UGDH gene. In contrast, no clear correlation between transcription, translation and activity of the UGDH was found under the effects of the cytokines studied. However, TGF-beta enhanced the enzyme activity, whereas IL-1beta, IL-6 and IGF-I were without significant effect. CONCLUSIONS: 17Beta-E2 enhanced GAG synthesis in chondrocytes via up-regulation of the UGDH gene expression and enzyme activity. These data provide insights into the molecular mechanisms involved in the regulation of the UGDH gene and offer new approaches to investigate its potential alteration in joint diseases.

Interleukin-1beta up-regulation of Smad7 via NF-kappaB activation in human chondrocytes.

OBJECTIVE: We have previously shown that interleukin-1beta (IL-1beta) impairs transforming growth factor beta (TGFbeta) signaling through TGFbeta receptor type II (TGFbetaRII) down-regulation and Smad7 up-regulation. This mechanism could account for the reduced responsiveness of osteoarthritic chondrocytes to TGFbeta and the cartilage breakdown linked to this disease. The aim of this study was to investigate the molecular mechanism underlying the IL-1beta-induced stimulation of Smad7 in human articular chondrocytes. METHODS: Human articular chondrocytes were treated with IL-1beta in the presence of TGFbeta1, pyrrolidine dithiocarbamate (a repressor of the NF-kappaB pathway), or cycloheximide. Then, steady-state messenger RNA and protein levels were estimated by real-time reverse transcription-polymerase chain reaction and immunocytology. In addition, transient transfections of p65 expression vector or p65-targeted short hairpin RNA were performed to define the effect of NF-kappaB on Smad7 expression. RESULTS: TGFbetaRII overexpression restored the TGFbeta response of human articular chondrocytes. However, this effect was transient, implying that a secondary mechanism was responsible for the alteration of the TGFbeta response with long-term exposure to IL-1beta. Moreover, IL-1beta caused a late induction of the inhibitory Smad7. This effect was direct, since it did not require de novo synthesis. In addition, we established, by experiments with gain/loss of function, that the up-regulation of Smad7 by IL-1beta is mediated through the NF-kappaB pathway, especially the p65 subunit. CONCLUSION: These findings clarify the regulatory process of IL-1beta on Smad7 expression. Understanding the molecular basis of IL-1beta induction of Smad7 and the reduction of chondrocyte responsiveness to TGFbeta provides new insights into the molecular mechanisms of osteoarthritis and may facilitate the identification of novel approaches for its treatment.

Chondroitin sulfate modulation of matrix and inflammatory gene expression in IL-1beta-stimulated chondrocytes--study in hypoxic alginate bead cultures.

OBJECTIVE: To determine the effect of avian chondroitin sulfate (CS) on interleukin-1beta (IL-1beta)-induced expression of genes related to catabolic, anabolic and inflammatory aspects in chondrocytes cultured in hypoxic alginate beads. DESIGN: Articular chondrocytes from bovine metacarpal joint were isolated and cultured in alginate beads, using low oxygen atmosphere (5% O2). After 1-week exposure to CS (1, 10 and 100microg/ml), they were treated by recIL-1beta (10ng/ml) for 24 or 48h, in the presence of CS. RNA was extracted and used to determine, by quantitative reverse transcription-polymerase chain reaction, the steady-state levels of mRNAs encoding several genes related to anabolic, catabolic and inflammatory aspects. Glycosaminoglycan (GAG) synthesis was also assayed by 35S-sulfate incorporation. RESULTS: CS decreased IL-1beta-induced expression of matrix metalloproteases-1, -3 and -13 and aggrecanases-1 and -2. It slightly enhanced the aggrecan core protein mRNA and the GAG synthesis. Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA levels were found to be reduced by CS treatment. However, no CS-induced decrease of NO was observed in IL-1beta-treated chondrocytes, whereas prostaglandin E2 production was diminished in correlation with the COX-2 mRNA amounts. Furthermore, CS was capable of counteracting IL-1beta-depressed expression of transforming growth factor-beta (TGF-beta) receptors. CONCLUSIONS: CS can repress expression of genes encoding proteolytic enzymes involved in cartilage degradation. It also inhibits IL-1beta-induced expression of the pro-inflammatory genes iNOS and COX-2 and restores TGF-beta receptors I and II (TGF-betaRI and RII) mRNA levels. These data suggest that CS may exert both chondroprotective and anti-inflammatory limited effects on articular cartilage that could have long-term beneficial action on the osteoarthritic process.

NFkappaB mediates IL-1beta-induced down-regulation of TbetaRII through the modulation of Sp3 expression.

We previously showed that interleukin-1beta (IL-1beta) down-regulation of type II TGFbeta receptor (TbetaRII) involves NFkappaB pathway and requires de novo synthesis of a yet unknown protein. Here, we demonstrate that this effect is mediated through Sp1 site located at position -25 of human TbetaRII promoter. Inhibition of transcription factors binding (decoy oligonucleotides or mithramycin) abolished IL-1beta effect. EMSA and ChIP revealed that this treatment induced Sp3 binding to cis-sequence whereby IL-1beta exerts its transcriptional effects whereas it decreased that of Sp1. Moreover, although the cytokine did not modulate Sp1 expression, it increased that of Sp3 via NFkappaB pathway. Experiments of gain and loss of function clearly showed that Sp3 inhibited TbetaRII expression whereas its silencing abolished IL-1beta effect. In addition, both Sp1 and Sp3 were found to interact with NFkappaB, which therefore may indirectly interact with TbetaRII pro moter. Altogether, these data suggest that IL-1beta decreases TbetaRII expression by inducing Sp3 via NFkappaB and its binding on core promote at the expense of Sp1, which could explain the loss of cell responsiveness in certain conditions. These findings bring new insights in the knowledge of the interference between two antagonistic transduction pathways involved in multiple physiopathological processes.

[Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation: a review of 28 cases]. / Greffe de chondrocytes autologues dans le traitement des pertes de substance condylienne du genou: bilan de 28 implantations.

PURPOSE OF THE STUDY: The knee has little capacity for spontaneous regeneration of deep cartilage defects. In 1999, the French Society of Arthroscopy initiated a multicentric clinical trial on autologous chondrocyte transplantation using the technique described by Brittberg and Peterson. The protocol of this prospective study was validated by the ethics committee and all patients provided the informed consent for participation. MATERIAL AND METHODS: Patients underwent surgery in seven hospitals: 28 patients (7 female, 21 male, mean age 28 years, age range 18-45 years). The underlying condition was: osteochondritis (n=14), isolated posttraumatic chondropathy (n=8), chondropathy plus ACL tear (n=6). All patients presented deep condylar cartilage defects (ICRS grades 3 and 4). Mean surface area involved after debridement was 490 mm2 (range 150-1050 mm2). Patients were reviewed two years at least after transplantation for functional assessment and an MRI performed 2 to 3 years after transplantation. Control arthroscopy was also performed in 13 patients with biopsy for histology and immunohistochemistry for 10. RESULTS: Twenty-six patients were reviewed with more than two years follow-up (mean 2 years 9 months). There were no general complications; three patients presented a partial avulsion of the autograft treated arthroscopically and one arthrolysis was performed at six months. Function was improved in all patients but four, but pain persisted in one patient. The mean ICRS score improved from 41 points (19-55 points) to 74 points (54-86 points), for an 80% gain. Follow-up MRI was available for 16 knees: the graft was hypertrophied in 11, at level in 3 and insufficient in 2; marginal integration was good in 10 knees and fair in 6. Items of marginal and subchondral integration had a very high positive predictive value for good clinical outcome. The arthroscopic score was nearly normal (range 8-11) in 8 knees and abnormal (range 4-7) in 5. The Knutsen histological groups according to richness of hyaline cartilage were: group 1 (>60%) (n=1); group 2 (>40%) (n=3), group 3 (<40%) n(=4) and group 4 (bone or fibrous tissue) (n=1). The function scores (r=0.80) and the MRI scores (r=0.76) were correlated with the arthroscopy scores. There was no correlation between the histological findings but the sample size was too small for meaningful analysis. DISCUSSION: The clinical results demonstrate an improvement in more than 80% of knees, findings similar to earlier reports. The arthroscopic and histological results were equivalent to those reported by Knutsen, but less satisfactory than those reported by Bentley or Peterson. Cell injections under a periosteal patch constitute the first generation of autologous chondrocyte grafts. Resorbable matrices loaded with chondrocytes before implantation are under development and have provided promising early results.

Interleukin-1beta impairment of transforming growth factor beta1 signaling by down-regulation of transforming growth factor beta receptor type II and up-regulation of Smad7 in human articular chondrocytes.

OBJECTIVE: Extracellular matrix deposition is tightly controlled by a network of regulatory cytokines. Among them, interleukin-1beta (IL-1beta) and transforming growth factor beta1 (TGFbeta1) have been shown to play antagonistic roles in tissue homeostasis. The purpose of this study was to determine the influence of IL-1beta on TGFbeta receptor type II (TGFbetaRII) regulation and TGFbeta1 responsiveness in human articular chondrocytes. METHODS: TGFbeta1-induced gene expression was analyzed through plasminogen activator inhibitor 1 and p3TP-Lux induction. Receptor-activated Smad (R-Smad) phosphorylation, TGFbeta receptors, and Smad expression were determined by Western blotting and real-time reverse transcription-polymerase chain reaction techniques. Signaling pathways were investigated using specific inhibitors, messenger RNA (mRNA) silencing, and expression vectors. RESULTS: IL-1beta down-regulated TGFbetaRII expression at both the protein and mRNA levels and led to inhibition of the TGFbeta1-induced gene expression and Smad2/3 phosphorylation. Moreover, IL-1beta strongly stimulated the expression of inhibitory Smad7. TGFbetaRII overexpression abolished the loss of TGFbeta1 responsiveness induced by IL-1beta. The decrease in TGFbetaRII required de novo protein synthesis and involved both the NF-kappaB and JNK pathways. CONCLUSION: We demonstrate that IL-1beta impairs TGFbeta1 signaling through down-regulation of TGFbetaRII, which is mediated by the p65/NF-kappaB and activator protein 1/JNK pathways, and secondarily through the up-regulation of Smad7. These findings show that there is cross-talk in the signaling of 2 regulatory cytokines involved in inflammation.

Rhein, a diacerhein-derived metabolite, modulates the expression of matrix degrading enzymes and the cell proliferation of articular chondrocytes by inhibiting ERK and JNK-AP-1 dependent pathways.

OBJECTIVE: To determine the effects of rhein on the expression of matrix metalloproteinases (MMP-1, -3, 13) and ADAMTs 4, 5 (a disintegrin and metalloproteinase with thrombospondin type-I repeat)/aggrecanases-1, -2 in interleukin-1-stimulated bovine articular chondrocytes, and to investigate the signalling pathways involved in the effects of the drug on gene expression and cell proliferation. METHODS: Bovine chondrocytes were treated with 10(-4) M rhein for 18 h, followed by 10 ng/ml IL-1Beta for 30 min (cytoplasmic extracts) or 24 h (RNA extraction and EMSA). mRNA was assessed by RT-PCR for the expression of MMPs and aggrecanases, and the phosphorylation of MAP kinases was studied by Western blotting. NF-kappaB and AP-1 DNA binding were determined by gel retardation assay. The effects of inhibitors of these signalling pathways were compared to those of rhein. The proliferation of human chondrocytes and synoviocytes treated with the drug was also investigated. RESULTS: IL-1Beta-induced stimulation of the MMPs and aggrecanase-1 was markedly inhibited by rhein. The drug reduced IL-1Beta-induced NF-kappaB and AP-1 DNA binding, as well as the phosphorylation of ERK and JNK. Similar effects were produced by the specific inhibitors of these signalling pathways. In addition, rhein reduced the proliferation of both human chondrocytes and synoviocytes. CONCLUSION: Our data indicate that rhein may reduce the deleterious effects of IL-1Beta on osteoarthritic cartilage through its effects on the ERK- and JNK-dependent pathways. Both its anti-catabolic and anti-proliferative properties may explain its value in the treatment of joint diseases.

Inhibition of interleukin-1beta-induced activation of MEK/ERK pathway and DNA binding of NF-kappaB and AP-1: potential mechanism for Diacerein effects in osteoarthritis.

In the present report we have shown that bovine articular chondrocytes cultured in low oxygen tension, i.e. in conditions mimicking their hypoxic in vivo environment, respond to IL-1beta (10 ng/ml) by an increased DNA binding activity of NF-kappaB and AP-l transcription factors. Incubation of the cells with 10(-5) M Rhein, the active metabolite of Diacerhein, for 24 h was found to reduce this activity particularly in the case of AP-1. Mitogen activated kinases (ERK-1 and ERK-2) were activated by exposure of the chondrocytes to a 1 h treatment with IL-1beta. This effect was greater in hypoxia (3% O2) than in normoxia (21% O2). Rhein was capable of reducing the IL-1beta-stimulated ERK1/ERK2 pathway whatever the tension of oxygen present in the environment. The mRNA steady-state levels of collagen type II (COL2A1) and aggrecan core protein were found to be significantly increased by a 24-h treatment with 10(-5) M Rhein. This stimulating effect was also observed in the presence of IL-1beta, suggesting that the drug could prevent or reduce the IL-1beta-induced inhibition of extra cellular matrix synthesis. IL-1-induced collagenase (MMP1) expression was significantly decreased by Rhein under the same conditions. In conclusion, Rhein can effectively inhibit the IL-1-activated MAPK pathway and the binding of NF-kappaB and AP-1 transcription factors, two key factors involved in the expression of several pro-inflammatory genes by chondrocytes. In addition, the drug can reduce the procatabolic effect of the cytokine, by reducing the MMP1 synthesis, and enhance the synthesis of matrix components, such as type II collagen and aggrecan. These results may explain the anti-osteoarthritic properties of Rhein and its disease-modifying effects on OA cartilage, in spite of the absence of inhibition at prostaglandin level.

Effect of interleukin-1beta on transforming growth factor-beta and bone morphogenetic protein-2 expression in human periodontal ligament and alveolar bone cells in culture: modulation by avocado and soybean unsaponifiables.

BACKGROUND: In periodontal disease, interleukin-1beta (IL-1beta) is responsible for the matrix breakdown through excessive production of degrading enzymes by periodontal ligament fibroblasts and osteoblasts. Transforming growth factor-beta (TGF-beta) plays an important role in tissue regeneration as one of the factors capable of counteracting IL-1beta effects. In this study, we investigated the in vitro effect of avocado and soya unsaponifiables (ASU) on the expression of TGF-beta1, TGF-beta2, and bone morphogenetic protein-2 (BMP-2) by human periodontal ligament (HPL) and human alveolar bone (HAB) cells in the presence of IL-1beta. METHODS: HPL and HAB cells were incubated for 48 hours with ASU (10 microg/ml) in the presence or absence of IL-1beta (10 ng/ml). The steady-state levels of TGF-beta1, TGF-beta2, and BMP-2 mRNAs were determined by Northern blot or reverse transcription-polymerase chain reaction (RT-PCR). The amounts of TGF-beta1 and TGF-beta2 proteins were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: The data indicated that IL-1beta strongly decreases the expression of TGF-beta1 and TGF-beta2 by HPL cells. ASU were capable of opposing the cytokine effect. In HAB cells, TGF-beta1 and BMP-2 mRNA levels were downregulated by the cytokine. ASU were found to reverse the IL-1beta-inhibiting effect. In contrast, the cytokine stimulated the production of TGF-beta2 in alveolar bone cells, with no significant effect of ASU. CONCLUSIONS: The results indicate that the IL-1beta-driven erosive effect in periodontitis could be enhanced by a decreased expression of members of the TGF-beta family. The ASU stimulation of TGF-beta1, TGF-beta2, and BMP-2 expression may explain their promoting effects in the treatment of periodontal disorders, at least partly. These findings support the hypothesis that ASU could exert a preventive action on the deleterious effects exerted by IL-1beta in periodontal diseases.

Comparative effects of IL-1beta and hydrogen peroxide (H2O2) on catabolic and anabolic gene expression in juvenile bovine chondrocytes.

OBJECTIVE: To compare the effects of hydrogen peroxide (H(2)O(2)) to those of interleukin-1beta (IL-1beta) on gene expression in juvenile bovine articular chondrocytes (BAC). The study analyses the activation of nuclear factor-kappa B (NF-kappaB) and activator protein-1 (AP-1) transcription factors, and the mRNA steady-state levels of the type II collagen, aggrecan core protein matrix, metalloproteinases (MMP-1, -3), and transforming growth factor-beta1 (TGF-beta1) genes. METHODS: Confluent BAC cultures were treated for 3 and 24h with IL-1beta and/or different concentrations of H(2)O(2) (Protocol 1). Following initial treatment, a part of the cells was further subjected to another 24h with medium, in the presence of IL-1beta, to determine the effect of the cytokine on H(2)O(2) pre-treated cells (Protocol 2). Total RNA and nuclear protein extractions were performed to study mRNA steady-state levels (real-time polymerase chain reaction) and AP-1/NF-kappaB DNA binding (Electrophoretic Mobility Shift Assays), respectively. RESULTS: IL-1beta enhanced both AP-1 and NF-kappaB binding, whereas H(2)O(2) only activated AP-1. H(2)O(2) pre-treatment decreased the IL-1beta activation of NF-kappaB. Both H(2)O(2) and IL-1beta down-regulated type II collagen and aggrecan expression and increased that of MMP-1 and -3. When cells were pre-treated with H(2)O(2), followed by IL-1beta, the effects were the same as those observed with H(2)O(2) alone. However, although H(2)O(2) and IL-1beta were capable of increasing TGF-beta1 expression separately, subsequent incubation with both factors led to a partial or total abolition of TGF-beta1 up-regulation. CONCLUSION: The different regulation of NF-kappaB and AP-1 by H(2)O(2) and IL-1beta underlines the distinct roles played by the two transcription factors in the regulation of gene expression. H(2)O(2) and IL-1beta exert similar effects on matrix, MMPs and TGF-beta1 gene expression. However, the association of H(2)O(2) and IL-1beta does not cause synergic effect, and rather leads, in some cases, to an opposite effect. These data provide further insights into the respective roles of reactive oxygen species and cytokine in the pathophysiology of joint diseases.

Immunohistochemical analysis of transforming growth factor beta isoforms and their receptors in human cartilage from normal and osteoarthritic femoral heads.

OBJECTIVE: Osteoarthritis (OA) is characterized by erosion of cartilage and formation of osteophytes. Since transforming growth factor beta (TGF-beta) is known to be involved in chondrogenesis and osteogenesis, we studied by immunochemistry the expression of TGF-beta isoform types 1, 2, and 3 and their receptor types I and II in slightly and strongly altered areas of human OA cartilage and in osteophytes. METHODS: Specimens were collected from femoral heads at the time of hip arthroplasty, selecting osteophytic regions and areas of slight or severe degradation according to the Mankin score. Cryostat sections were prepared and submitted to immunohistochemistry using appropriate antibodies to TGF-beta(1-3) and TGF-beta receptors I and II. RESULTS: TGF-beta1 expression was shown to be depressed in strongly degraded cartilage, compared to normal and slightly altered areas. TGF-beta2 was barely detectable in all samples studied. In osteophytes, a marked overexpression of TGF-beta1 and -beta3 was observed. An important decrease in TGF-beta receptor II was found in fibrillated cartilage areas. CONCLUSIONS: The three major isoforms of TGF-beta are expressed in human OA cartilage, albeit the TGF-beta2 level is very low. Their expression patterns and the ratio of receptors I and II varies according to the degree of OA severity. The decrease in TGF-beta1 production and marked downregulation of receptor II in fibrillated cartilage may lead to reduced chondrocyte responsiveness to TGF-beta and contribute to the irreversibility of the disease. Overexpression of TGF-beta1 and -beta3 in osteophytes suggests that the two isoforms are involved in the formation of these structures.

Effect of hypoxia and reoxygenation on gene expression and response to interleukin-1 in cultured articular chondrocytes.

OBJECTIVE: To determine the effects of hypoxia and reoxygenation on the metabolism of chondrocytes and their response to interleukin-1beta (IL-1beta). The study included activation of hypoxia-inducible factor 1 (HIF-1), NF-kappaB, and activator protein 1 (AP-1) transcription factors, expression of matrix components and metalloproteases and transforming growth factor beta (TGFbeta) and TGFbeta receptors, and production of nitric oxide (NO) and prostaglandin E(2) (PGE(2)). METHODS: Bovine articular chondrocytes (BACs) were cultured to confluency in either 5% O(2) (hypoxia) or 21% O(2) (normoxia) in media supplemented with 10% fetal calf serum (FCS). BACs were preincubated for 18 hours in media with 1% FCS only and then incubated for 24 hours in the presence of IL-1beta. For reoxygenation experiments, cells were treated in the same way in 5% O(2), except that cultures were transferred to normal atmospheric conditions and used after 4 hours for RNA extraction or after 30 minutes for cytoplasmic or nuclear protein extraction. RESULTS: In hypoxic and reoxygenated chondrocytes, we observed strong DNA binding of HIF-1. IL-1beta-induced DNA binding of NF-kappaB and AP-1 was significantly higher in hypoxic and reoxygenated cultures than in normoxia. Greater activation of the MAPKs was also observed with IL-1beta treatment in hypoxia compared with normoxia. Steady-state levels of type II collagen and aggrecan core protein messenger RNA (mRNA) were decreased by IL-1beta in all instances. Matrix metalloprotease 1 (MMP-1) and MMP-3 mRNA were increased by IL-1beta in normoxia and hypoxia, whereas only MMP-3 mRNA was enhanced in reoxygenated cultures. The MMP-2 mRNA level was not significantly affected by IL-1beta in normoxia or hypoxia, whereas it was enhanced in reoxygenated cultures. MMP-9 mRNA was dramatically decreased by IL-1beta only in low oxygen tension. Tissue inhibitor of metalloproteinases 1 (TIMP-1) message was significantly enhanced by the cytokine in most instances, whereas TIMP-2 message was markedly decreased by IL-1beta in reoxygenated cultures. Stimulation of TGFbeta1 expression by IL-1beta was observed only in normal atmospheric conditions. One of the more striking findings of the study was the greater stimulating effect of IL-1beta on NO production observed in hypoxia, which was much higher than in normoxia, whereas the reverse was observed for IL-1beta-induced PGE(2) production. CONCLUSION: Oxygen level and reoxygenation stress significantly modulate gene expression and the response of articular chondrocytes to cytokines such as IL-1beta. In hypoxic conditions, which mimic the in vivo condition of cartilage, the effects of IL-1beta on both synthesis and degradative processes are significantly different from those in normoxia, conditions that are unlikely encountered by chondrocytes in a normal state. In low oxygen tension, high IL-1beta-induced NO production is associated with a significant decrease in PGE(2) synthesis. These data should influence our concept of the role of oxygen in the pathophysiology of joint disease and may help define the best conditions in which to develop bioartificial cartilage.

Articular chondrocytes cultured in hypoxia: their response to interleukin-1beta and rhein, the active metabolite of diacerhein.

In the present report, we show that bovine articular chondrocytes cultured in low oxygen tension, i.e. in conditions mimicking their hypoxic in vivo environment, respond to IL-1beta (10 ng/ml) by an increased DNA binding activity of NF-kappaB and AP-1 transcription factors. Incubation of the cells with 10(-5) M rhein for 24 h was found to reduce this activity, particularly in the case of AP-1. Mitogen activated kinases (ERK-1 and ERK-2) were activated by exposure of the chondrocytes to 1-h treatment with IL-1beta. This effect was greater in hypoxia (3% O(2)) than in normoxia (21% O(2)). Rhein was capable of reducing the IL-1beta-stimulated ERK1/ERK2 pathway whatever the tension of oxygen present in the environment. The mRNA steady-state levels of collagen type II (COL2A1) and aggrecan core protein were found to be significantly increased by a 24-h treatment with 10(-5) M rhein. This stimulating effect was also observed in the presence of IL-1beta, suggesting that the drug could prevent or reduce the IL-1beta-induced inhibition of extracellular matrix synthesis. IL-1-induced collagenase (MMP1) expression was significantly decreased by rhein in the same conditions. In conclusion, rhein can effectively inhibit the IL-1-activated MAPK pathway and the binding of NF-kappaB and AP-1 transcription factors, two key factors involved in the expression of several pro-inflammatory genes by chondrocytes. In addition, the drug can reduce the procatabolic effect of the cytokine, by reducing the MMP1 synthesis, and enhance the synthesis of matrix components, such as type II collagen and aggrecan. These results may explain the anti-osteoarthritic properties of rhein and its disease-modifying effects on OA cartilage, in spite of absence of inhibition at prostaglandin level.

The role of reactive oxygen species in homeostasis and degradation of cartilage.

OBJECTIVES: The metabolism of cells in articular joint tissues in normal and pathological conditions is subject to a complex environmental control. In addition to soluble mediators such as cytokines and growth factors, as well as mechanical stimuli, reactive oxygen species (ROS) emerge as major factors in this regulation. ROS production has been found to increase in joint diseases, such as osteoarthritis and rheumatoid arthritis, but their role in joint diseases initiation and progression remains questionable. METHOD: This review is focused on the role of ROS, mainly nitric oxide, peroxynitrite and superoxide anion radicals, in the signaling mechanisms implied in the main cellular functions, including synthesis and degradation of matrix components. The direct effects of ROS on cartilage matrix components as well as their inflammatory and immunomodulatory effects are also considered. RESULTS: Some intracellular signaling pathways are redox sensitive and ROS are involved in the regulation of the production of some biochemical factors involved in cartilage degradation and joint inflammation. Further, ROS may cause damage to all matrix components, either by a direct attack or indirectly by reducing matrix components synthesis, by inducing apoptosis or by activating latent metalloproteinases. Finally, we have highlighted the uncoupling effect of ROS on tissue remodeling and synovium inflammation, suggesting that antioxidant therapy could be helpful to treat structural changes but not to relieve symptoms. CONCLUSIONS: This review of the literature supports the concept that ROS are not only deleterious agents involved in cartilage degradation, but that they also act as integral factors of intracellular signaling mechanisms. Further investigation is required to support the concept of antioxidant therapy in the management of joint diseases.

Mediation of interleukin-1beta-induced transforming growth factor beta1 expression by activator protein 4 transcription factor in primary cultures of bovine articular chondrocytes: possible cooperation with activator protein 1.

OBJECTIVE: Interleukin-1 (IL-1) and transforming growth factor beta1 (TGFbeta1) play major roles in osteoarticular diseases, exerting opposite effects on both the catabolism and anabolism of cartilage matrix. Previous findings suggest that IL-1 and TGFbeta1 could function in a feedback interaction. However, the effect exerted by IL-1 on expression of TGFbeta by articular chondrocytes is, so far, poorly understood. The present study was carried out to determine the influence of IL-1beta on the expression of TGFbeta1 by bovine articular chondrocytes (BACs) in primary culture. METHODS: BAC primary cultures were treated with IL-1beta, and TGFbeta1 messenger RNA (mRNA) steady-state levels and protein expression were measured by real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Transient transfection of TGFbeta1 gene promoter constructs was performed to delineate the DNA sequences that mediate the IL-1beta effect. Electrophoretic mobility shift assays (EMSAs) and supershift analysis were used to characterize the transcription factors binding to these sequences. RESULTS: Cultured BACs responded to IL-1beta exposure by exhibiting an increase of TGFbeta1 expression at both the mRNA and protein levels. The effect was found to be mediated by a major 80-bp sequence located between -732 and -652 upstream of the transcription initiation site. EMSA and supershift analysis revealed that the transcription factors activator protein 4 (AP-4) and AP-1 specifically bound to the -720/-696 part of this sequence under IL-1beta treatment. Overexpression of AP-4 in the BAC cultures resulted in stimulation of the transcriptional activity of the -732/+11 TGFbeta1 promoter construct through the same IL-1beta-responsive element. CONCLUSION: IL-1beta induces an increase of TGFbeta1 in articular chondrocytes through activation of AP-4 and AP-1 binding to the TGFbeta1 gene promoter. These findings may help us understand the role of IL-1beta in the disease process. Notwithstanding its deleterious effect on cartilage, IL-1 could initiate the repair response displayed by injured cartilage in the early stages of osteoarthritis through its ability to enhance TGFbeta1 expression by local chondrocytes.

Reactive oxygen species downregulate the expression of pro-inflammatory genes by human chondrocytes.

OBJECTIVES: To determine the regulatory effects of reactive oxygen species (ROS) on the expression by human osteoarthritic chondrocytes of interleukin (IL)-1beta, -6 and -8, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) gene in response to interleukin (IL)-1beta or lipopolysaccharide (LPS). METHODS: Human chondrocytes in monolayer culture were incubated for 3 h with ROS generating molecules such as S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 100 microM), 3-morpholinosydnonimine (SIN-1, 100 microM), with chemically synthesised peroxynitrite (ONOO-, 10 microM) or hydrogen peroxide (H2O2, 100 microM). After treatment by ROS, chondrocytes were washed and then cultured for the next 24 h with or without lipopolysaccharide LPS (10 microg/ml) or IL-1beta (1.10(-11) M). IL-1beta, IL-6, IL-8, iNOS and COX-2 gene expression was analysed by real time and quantitative RT PCR. IL-6, IL-8 and prostaglandin (PG) E2 productions were assayed by specific immunoassays. Nitrite was measured in the culture supernatants by the Griess procedure. RESULTS: LPS and IL-1beta stimulated IL-1beta, IL-6, IL-8, iNOS and COX-2 gene expression. SNAP significantly downregulated LPS induced overall gene expressions, whereas SIN-1 had no effect. ONOO- inhibited iNOS and COX-2 gene expression but not that of the cytokine genes. When chondrocytes were incubated with IL-1beta, SIN-1 and ONOO dramatically decreased all gene expressions while SNAP was inefficient. H2O2 treatment inhibited both LPS and IL-1beta induced gene expressions. CONCLUSIONS: These data provide an evidence that ROS may have anti-inflammatory properties by depressing inflammatory gene expression. Further, we demonstrate that ROS effects are dependent on the nature of radical species and the signalling pathway that is activated. These findings should be taken into consideration for the management of antioxidant therapy in treatment of inflammatory joint diseases.

Regulation by reactive oxygen species of interleukin-1beta, nitric oxide and prostaglandin E(2) production by human chondrocytes.

OBJECTIVES: To determine the effects of two drugs, N-monomethyl-L-arginine (L-NMMA) and N-acetylcysteine (NAC), on interleukin-1beta (IL-1beta), nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production by human chondrocytes. The effect of aceclofenac (ACECLO), a non-steroidal antiinflammatory drug (NSAID), was also examined. METHODS: Human chondrocytes were enzymatically isolated from osteoarthritic knee cartilage and then maintained in culture in suspension for 48h in the absence or in the presence of lipopolysaccharide (LPS) (10 microg/ml), L-NMMA (0.5mM), NAC (1mM) or ACECLO (6.10(-6)M). IL-1beta and PGE(2) productions were quantified by specific immunoassays. Nitrite was measured in the culture supernatants by a spectrophotometric method based upon the Griess reaction. Cyclooxygenase-2 (COX-2), inducible NO synthase (iNOS) and IL-1beta gene expressions were quantified by transcription of mRNA followed by real time and quantitative polymerase chain reaction. COX-2 protein expression was analysed by Western blot. RESULTS: LPS markedly increased the expression of IL-1beta, iNOS and COX-2 genes. In parallel, NO(2) and PGE(2) amounts found in the culture supernatants were significantly enhanced whereas IL-1beta was immunologically undetectable. The addition of L-NMMA (0.5mM) fully blocked LPS-induced NO production but greatly increased PGE(2) production, suggesting a negative effect of NO on PGE(2) synthesis. Inversely, NO production was stimulated by NAC while PGE(2) production was not affected. Interestingly, NAC increased the IL-1beta and iNOS mRNA levels but did not significantly modify COX-2 mRNA expression. L-NMMA did not significantly affect the expression of IL-1beta, iNOS and COX-2. The amount of COX-2 protein did not change in the presence of the antioxidants. Finally, ACECLO fully blocked the production of PGE(2) by chondrocytes without affecting the levels of COX-2 mRNA. CONCLUSIONS: The stimulation of IL-1beta, NO and PGE(2) production by LPS is differentially controlled by reactive oxygen species (ROS). In fact, L-NMMA and NAC have different mechanisms of action on the regulation of NO and PGE(2) productions. L-NMMA fully inhibits NO but increases PGE(2) production whereas NAC up-regulates NO but does not modify PGE(2) synthesis. The stimulating effect of L-NMMA on PGE(2) production is not controlled at the transcriptional level. These findings suggest that antioxidant therapy could have different effects according to the oxygen radical species targeted.