Cultured Rabbit Articular Chondrocytes Research Articles

Effect of oleanolic acid on the activity, secretion and gene expression of matrix metalloproteinase-3 in articular chondrocytes in vitro and the production of matrix metalloproteinase-3 in vivo.

In the present study, we tried to examine whether oleanolic acid regulates the activity, secretion and gene expression of matrix metalloproteinase-3 (MMP-3) in primary cultured rabbit articular chondrocytes, as well as the production of MMP-3 in the knee joint of rat to evaluate the potential chondroprotective effect of oleanolic acid. Rabbit articular chondrocytes were cultured in a monolayer, and reverse transcription-polymerase chain reaction (RT-PCR) was used to measure interleukin-1β (IL-1β)-induced gene expression of MMP-3, MMP-1, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), ADAMTS-5 and type II collagen. In rabbit articular chondrocytes, the effects of oleanolic acid on IL-1β-induced secretion and proteolytic activity of MMP-3 were investigated using western blot analysis and casein zymography, respectively. The effect of oleanolic acid on in vivo MMP-3 protein production was also examined, after intra-articular injection to the knee joint of rat. The results were as follows: (1) oleanolic acid inhibited the gene expression of MMP-3, MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5, but increased the gene expression of type II collagen; (2) oleanolic acid reduced the secretion and proteolytic activity of MMP-3; (3) oleanolic acid suppressed the production of MMP-3 protein in vivo. These results suggest that oleanolic acid can regulate the activity, secretion and gene expression of MMP-3, by directly acting on articular chondrocytes.

Betulin suppressed interleukin-1β-induced gene expression, secretion and proteolytic activity of matrix metalloproteinase in cultured articular chondrocytes and production of matrix metalloproteinase in the knee joint of rat.

We investigated whether betulin affects the gene expression, secretion and proteolytic activity of matrix metalloproteinase-3 (MMP-3) in primary cultured rabbit articular chondrocytes, as well as in vivo production of MMP-3 in the rat knee joint to evaluate the potential chondroprotective effect of betulin. Rabbit articular chondrocytes were cultured and reverse transcription-polymerase chain reaction (RT-PCR) was used to measure interleukin-1β (IL-1β)-induced gene expression of MMP-3, MMP-1, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), ADAMTS-5 and type II collagen. Effect of betulin on IL-1β-induced secretion and proteolytic activity of MMP-3 was investigated using western blot analysis and casein zymography, respectively. Effect of betulin on MMP-3 protein production was also examined in vivo. The results were as follows: (1) betulin inhibited the gene expression of MMP-3, MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5, but increased the gene expression of type II collagen; (2) betulin inhibited the secretion and proteolytic activity of MMP-3; (3) betulin suppressed the production of MMP-3 protein in vivo. These results suggest that betulin can regulate the gene expression, secretion, and proteolytic activity of MMP-3, by directly acting on articular chondrocytes.

Apigenin Regulates Interleukin-1β-Induced Production of Matrix Metalloproteinase Both in the Knee Joint of Rat and in Primary Cultured Articular Chondrocytes

We examined whether apigenin affects the gene expression, secretion and activity of matrix metalloproteinase-3 (MMP-3) in primary cultured rabbit articular chondrocytes, as well as in vivo production of MMP-3 in the knee joint of rat to evaluate the potential chondroprotective effects of apigenin. Rabbit articular chondrocytes were cultured in a monolayer, and reverse transcription - polymerase chain reaction (RT-PCR) was used to measure interleukin-1β (IL-1β)-induced expression of MMP-3, MMP-1, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), and ADAMTS-5. In rabbit articular chondrocytes, the effects of apigenin on IL-1β-induced secretion and proteolytic activity of MMP-3 were investigated using western blot analysis and casein zymography, respectively. The effect of apigenin on MMP-3 protein production was also examined in vivo. In rabbit articular chondrocytes, apigenin inhibited the gene expression of MMP-3, MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5. Furthermore, apigenin inhibited the secretion and proteolytic activity of MMP-3 in vitro, and inhibited production of MMP-3 protein in vivo. These results suggest that apigenin can regulate the gene expression, secretion, and activity of MMP-3, by directly acting on articular chondrocytes.

Effects of prunetin on the proteolytic activity, secretion and gene expression of MMP-3 in vitro and production of MMP-3 in vivo.

We investigated whether prunetin affects the proteolytic activity, secretion, and gene expression of matrix metalloproteinase-3 (MMP-3) in primary cultured rabbit articular chondrocytes, as well as in vivo production of MMP-3 in the rat knee joint to evaluate the potential chondroprotective eff ect of prunetin. Rabbit articular chondrocytes were cultured in a monolayer, and reverse transcription-polymerase chain reaction (RT-PCR) was used to measure interleukin-1β (IL-1β)-induced expression of MMP-3, MMP-1, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), and ADAMTS-5. In rabbit articular chondrocytes, the effects of prunetin on IL-1β-induced secretion and proteolytic activity of MMP-3 were investigated using western blot analysis and casein zymography, respectively. The eff ect of prunetin on MMP-3 protein production was also examined in vivo. The results were as follows: (1) prunetin inhibited the gene expression of MMP-3, MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5; (2) prunetin inhibited the secretion and proteolytic activity of MMP-3; (3) prunetin suppressed the production of MMP-3 protein in vivo. These results suggest that prunetin can regulate the gene expression, secretion, and proteolytic activity of MMP-3, by directly acting on articular chondrocytes.

Chondroprotective Effects of Wogonin in Experimental Models of Osteoarthritis in vitro and in vivo.

We evaluated the chondroprotective effects of wogonin by investigating its effects on the gene expression and production of matrix metalloproteinase-3 (MMP-3) in primary cultured rabbit articular chondrocytes, as well as on production of MMP-3 in the rat knee. Rabbit articular chondrocytes were cultured in a monolayer, and RT-PCR was used to measure interleukin-1β (IL-1β)-induced expression of MMP-3, MMP-1, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), and type II collagen. In rabbit articular chondrocytes, the effects of wogonin on IL-1β-induced production and proteolytic activity of MMP-3 were investigated using western blot analysis and casein zymography, respectively. The effect of wogonin on MMP-3 protein production was also examined in vivo. In rabbit articular chondrocytes, wogonin inhibited the expression of MMP-3, MMP-1, MMP-13, and ADAMTS-4, but increased expression of type II collagen. Furthermore, wogonin inhibited the production and proteolytic activity of MMP-3 in vitro, and inhibited production of MMP-3 protein in vivo. These results suggest that wogonin can regulate the gene expression and production of MMP-3, by directly acting on articular chondrocytes.

Luteolin Inhibits the Activity, Secretion and Gene Expression of MMP-3 in Cultured Articular Chondrocytes and Production of MMP-3 in the Rat Knee.

We investigated whether luteolin affects the gene expression, secretion and activity of matrix metalloproteinase-3 (MMP-3) in primary cultured rabbit articular chondrocytes, as well as production of MMP-3 in the rat knee to evaluate the potential chondro-protective effects of luteolin. Rabbit articular chondrocytes were cultured in a monolayer and IL-1β-induced gene expression levels of MMP-3, MMP-1, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), ADAMTS-5 and type II collagen were measured by reverse transcription - polymerase chain reaction (RT-PCR). Effects of luteolin on interleukin-1β (IL-1β)-induced secretion and enzyme activity of MMP-3 in rabbit articular chondrocytes were investigated by western blot analysis and casein zymography, respectively. The effect of luteolin on MMP-3 protein production was also examined in vivo. The results were as follows: (1) luteolin inhibited the gene expression levels of MMP-3, MMP-1, MMP-13, ADAMTS-4 and ADAMTS-5. However, it increased the gene expression level of collagen in rabbit articular chondrocytes; (2) luteolin inhibited the secretion and activity of MMP-3; (3) luteolin inhibited in vivo production of MMP-3 protein. These results suggest that luteolin can regulate the gene expression, secretion and activity of MMP-3, by directly acting on articular chondrocytes.

3D dynamic culture of rabbit articular chondrocytes encapsulated in alginate gel beads using spinner flasks for cartilage tissue regeneration.

Cell-based therapy using chondrocytes for cartilage repair suffers from chondrocyte dedifferentiation. In the present study, the effects of an integrated three-dimensional and dynamic culture on rabbit articular chondrocytes were investigated. Cells (passages 1 and 4) were encapsulated in alginate gel beads and cultured in spinner flasks in chondrogenic and chondrocyte growth media. Subcutaneous implantation of the cell-laden beads was performed to evaluate the ectopic chondrogenesis. It was found that cells remained viable after 35 days in the three-dimensional dynamic culture. Passage 1 cells demonstrated a proliferative growth in both media. Passage 4 cells showed a gradual reduction in DNA content in growth medium, which was attenuated in chondrogenic medium. Deposition of glycosaminoglycans (GAG) was found in all cultures. While passage 1 cells generally produced higher amounts of GAG than passage 4 cells, GAG/DNA became similar on day 35 for both cells in growth media. Interestingly, GAG/DNA in growth medium was greater than that in chondrogenic medium for both cells. Based on GAG quantification and gene expression analysis, encapsulated passage 1 cells cultured in growth medium displayed the best ectopic chondrogenesis. Taken together, the three-dimensional and dynamic culture for chondrocytes holds great potential in cartilage regeneration.

Co-culture of chondrocytes and bone marrow mesenchymal stem cells in vitro enhances the expression of cartilaginous extracellular matrix components

Chondrocytes and bone marrow mesenchymal stem cells (BMSCs) are frequently used as seed cells in cartilage tissue engineering. In the present study, we determined if the co-culture of rabbit articular chondrocytes and BMSCs in vitro promotes the expression of cartilaginous extracellular matrix and, if so, what is the optimal ratio of the two cell types. Cultures of rabbit articular chondrocytes and BMSCs were expanded in vitro and then cultured individually or at a chondrocyte:BMSC ratio of 4:1, 2:1, 1:1, 1:2, 1:4 for 21 days and cultured in DMEM/F12. BMSCs were cultured in chondrogenic induction medium. Quantitative real-time RT-PCR and Western blot were used to evaluate gene expression. In the co-cultures, type II collagen and aggrecan expression increased on days 14 and 21. At the mRNA level, the expression of type II collagen and aggrecan on day 21 was much higher in the 4:1, 2:1, and 1:1 groups than in either the articular chondrocyte group or the induced BMSC group, and the best ratio of co-culture groups seems to be 2:1. Also on day 21, the expression of type II collagen and aggrecan proteins in the 2:1 group was much higher than in all other groups. The results demonstrate that the co-culture of rabbit chondrocytes and rabbit BMSCs at defined ratios can promote the expression of cartilaginous extracellular matrix. The optimal cell ratio appears to be 2:1 (chondrocytes:BMSCs). This approach has potential applications in cartilage tissue engineering since it provides a protocol for maintaining and promoting seed-cell differentiation and function.

Co-culture of chondrocytes and bone marrow mesenchymal stem cells in vitro enhances the expression of cartilaginous extracellular matrix components

Chondrocytes and bone marrow mesenchymal stem cells (BMSCs) are frequently used as seed cells in cartilage tissue engineering. In the present study, we determined if the co-culture of rabbit articular chondrocytes and BMSCs in vitro promotes the expression of cartilaginous extracellular matrix and, if so, what is the optimal ratio of the two cell types. Cultures of rabbit articular chondrocytes and BMSCs were expanded in vitro and then cultured individually or at a chondrocyte:BMSC ratio of 4:1, 2:1, 1:1, 1:2, 1:4 for 21 days and cultured in DMEM/F12. BMSCs were cultured in chondrogenic induction medium. Quantitative real-time RT-PCR and Western blot were used to evaluate gene expression. In the co-cultures, type II collagen and aggrecan expression increased on days 14 and 21. At the mRNA level, the expression of type II collagen and aggrecan on day 21 was much higher in the 4:1, 2:1, and 1:1 groups than in either the articular chondrocyte group or the induced BMSC group, and the best ratio of co-culture groups seems to be 2:1. Also on day 21, the expression of type II collagen and aggrecan proteins in the 2:1 group was much higher than in all other groups. The results demonstrate that the co-culture of rabbit chondrocytes and rabbit BMSCs at defined ratios can promote the expression of cartilaginous extracellular matrix. The optimal cell ratio appears to be 2:1 (chondrocytes:BMSCs). This approach has potential applications in cartilage tissue engineering since it provides a protocol for maintaining and promoting seed-cell differentiation and function.

Optimum Combination of Insulin-Transferrin-Selenium and Fetal Bovine Serum for Culture of Rabbit Articular Chondrocytes in Three-Dimensional Alginate Scaffolds

Fetal bovine serum (FBS) has been reported to affect chondrocyte biosynthesis in monolayer culture. Insulin-Transferrin-Selenium (ITS) was investigated as a partial replacement for FBS during in vitro culture of rabbit articular chondrocytes in three-dimensional alginate scaffold. Chondrocyte-seeded alginate hydrogels were cultured in Dulbecco's modified Eagle's medium plus 10% FBS, 1% ITS plus 2% FBS, 1% ITS plus 4% FBS, or 1% ITS plus 8% FBS. At designed time point, the Chondrocyte-seeded alginate hydrogels were harvested and evaluated with histological staining, immunohistochemistry, and quantitative gene expression analysis. Viable cell density and cell division were also evaluated. Chondrocytes biosynthesis and cell division in 1% ITS with 2% FBS medium were similar to that in medium added with 10% FBS. For a total culture of 3 weeks, phenotypic gene expression in chondrocyte-seeded hydrogels was maintained at high levels in medium with 1% ITS plus 2% FBS, while it was decreased to varying degrees in the other groups. In conclusion, with 1% ITS, medium with 2% FBS could promote chondrocyte biosynthesis and cell division, and prevented cell dedifferentiation in three-dimensional alginate scaffolds.

Natural chondroitin sulphates increase aggregation of proteoglycan complexes and decrease adamts-5 expression in interleukin 1β-treated chondrocytes

Objective:To assess the effect of natural chondroitin sulphate (CS) on the ability of neosynthesised sulphated proteoglycans (PGs) to aggregate in cultured chondrocytes treated with interleukin (IL)1β.Methods:Primary cultured rabbit articular chondrocytes...

Effects of growth factors on heparin‐carrying polystyrene‐coated atelocollagen scaffold for articular cartilage tissue engineering

The specific aim of our investigation is to study the potential use of a collagen/heparin-carrying polystyrene (HCPS) composite extracellular matrix for articular cartilage tissue engineering. Here, we created a high-performance extracellular matrix (HpECM) scaffold to build an optimal extracellular environment using an HCPS we originally developed, and an atelocollagen honeycomb-shaped-scaffold (ACHMS-scaffold) with a membrane seal. This scaffold was coated with HCPS to enable aggregation of heparin-binding growth factors such as FGF-2 and TGF-beta1 within the scaffold. Three-dimensional culture of rabbit articular chondrocytes within the HpECM-scaffold and subsequent preparation of a tissue-engineered cartilage were investigated. The results showed remarkably higher cell proliferative activity within the HpECM-pretreated-FGF-2 scaffold and the sustenance of phenotype within the HpECM-pretreated-TGF-beta1 scaffold. It was thought that both FGF-2 and TGF-beta1 were stably immobilized in the HpEMC-scaffold since HCPS generated an extracellular environment similar to that of heparan sulfate proteoglycan within the scaffold. These results suggest that an ACHMS-scaffold immobilized with HCPS can be a HpECM for cartilage regeneration to retain the heparin-binding growth factors within the scaffolds.

The Phylogenetically Conserved Molluscan Chitinase-like Protein 1 (Cg-Clp1), Homologue of Human HC-gp39, Stimulates Proliferation and Regulates Synthesis of Extracellular Matrix Components of Mammalian Chondrocytes

Members of chitinase-like proteins (CLPs) have attracted much attention because of their ability to promote cell proliferation in insects (imaginal disc growth factors) and mammals (YKL-40). To gain insights into the molecular processes underlying the physiological control of growth and development in Lophotrochozoa, we report here the cloning and biochemical characterization of the first Lophotrochozoan CLP from the oyster Crassostrea gigas (Cg-Clp1). Gene expression profiles monitored by real time quantitative reverse transcription-PCR in different adult tissues and during development support the involvement of this protein in the control of growth and development in C. gigas. Recombinant Cg-Clp1 demonstrates a strong affinity for chitin but no chitinolytic activity, as was described for the HC-gp39 mammalian homolog. Furthermore, transient expression of Cg-Clp1 in primary cultures of rabbit articular chondrocytes as well as the use of both purified recombinant protein and conditioned medium from Cg-Clp1-expressing rabbit articular chondrocytes established that Cg-Clp1 stimulates cell proliferation and regulates extracellular matrix component synthesis, showing for the first time a possible involvement of a CLP on type II collagen synthesis regulation. These observations together with the fact that Cg-Clp1 gene organization strongly resembles that of its mammalian homologues argue for an early evolutionary origin and a high conservation of this class of proteins at both the structural and functional levels.

Activation of the peroxisome proliferator–activated receptor α pathway potentiates interleukin‐1 receptor antagonist production in cytokine‐treated chondrocytes

To determine whether peroxisome proliferator-activated receptor alpha (PPARalpha) agonists protect chondrocytes against the effects of interleukin-1beta (IL-1beta). PPARalpha expression and function in cultured rabbit articular chondrocytes were studied by Northern blotting, electrophoretic mobility shift assay, and transient expression of a luciferase reporter construct bearing the human IL-1 receptor antagonist (Il-1Ra) gene promoter. Chondrocytes were incubated in vitro with IL-1beta alone or in combination with CloFibrate (CloF) or other PPAR ligands. Proteoglycans were evaluated by 35S-sulfate incorporation, matrix metalloproteinase (MMP) levels were assessed by zymography and enzyme-linked immunosorbent assay (ELISA), and MMP messenger RNA (mRNA) levels were measured by Northern blotting and real-time reverse transcriptase-polymerase chain reaction. IL-1beta and IL-1Ra soluble contents were measured by ELISA. CloF counteracted IL-1beta-induced 35S-proteoglycan degradation, gelatinolytic activity, and MMP-1, -3, and -13 mRNA expression. CloF also maximized IL-1beta-induced endogenous production of soluble IL-1Ra (sIL-1Ra). This stimulating effect on IL-1Ra gene expression was shown, by transient expression assay, to be transcriptional. Inhibition of sIL-1Ra expression by a specific small interfering RNA suppressed the effect of CloF on IL-1beta-induced MMP expression. The stimulatory effect of CloF was enhanced by cotransfection with wild-type PPARalpha and abolished by a dominant-negative PPARalpha mutant. Fenofibrate and WY-14643 displayed a similar stimulating effect on the IL-1Ra promoter, while rosiglitazone did not. Two PPAR response elements, an NF-kappaB-binding site, and a CCAAT/enhancer binding protein-binding site were identified in the IL-1Ra promoter. All 4 sites were necessary for mediation of the effects of CloF. Our findings support the notion that there is a PPARalpha-dependent mechanism that inhibits IL-1beta function in chondrocytes, which operates via an increase in sIL-1Ra production.

Chondrocyte response to growth factors is modulated by p38 mitogen-activated protein kinase inhibition

Inhibitors of p38 mitogen-activated protein kinase (MAPK) diminish inflammatory arthritis in experimental animals. This may be effected by diminishing the production of inflammatory mediators, but this kinase is also part of the IL-1 signal pathway in articular chondrocytes. We determined the effect of p38 MAPK inhibition on proliferative and synthetic responses of lapine chondrocytes, cartilage, and synovial fibroblasts under basal and IL-1-activated conditions.Basal and growth factor-stimulated proliferation and proteoglycan synthesis were determined in primary cultures of rabbit articular chondrocytes, first-passage synovial fibroblasts, and cartilage organ cultures. Studies were performed with or without p38 MAPK inhibitors, in IL-1-activated and control cultures. Media nitric oxide and prostaglandin E2 were assayed.p38 MAPK inhibitors blunt chondrocyte and cartilage proteoglycan synthesis in response to transforming growth factor beta; responses to insulin-like growth factor 1 (IGF-1) and fetal calf serum (FCS) are unaffected. p38 MAPK inhibitors significantly reverse inhibition of cartilage organ culture proteoglycan synthesis by IL-1. p38 MAPK inhibition potentiated basal, IGF-1-stimulated and FCS-stimulated chondrocyte proliferation, and reversed IL-1 inhibition of IGF-1-stimulated and FCS-stimulated DNA synthesis. Decreases in nitric oxide but not prostaglandin E2 synthesis in IL-1-activated chondrocytes treated with p38 MAPK inhibitors are partly responsible for this restoration of response. Synovial fibroblast proliferation is minimally affected by p38 MAPK inhibition.p38 MAPK activity modulates chondrocyte proliferation under basal and IL-1-activated conditions. Inhibition of p38 MAPK enhances the ability of growth factors to overcome the inhibitory actions of IL-1 on proliferation, and thus could facilitate restoration and repair of diseased and damaged cartilage.

Cyclooxygenase-Independent NSAID Action

Nonsteroidal anti-inflammatory drugs (NSAIDs) alleviate the pain and inflammation of arthritis through inhibition of cyclooxygenase (COX) and a consequent reduction in prostaglandin production. NSAIDs also affect cartilage production by articular chondrocytes and protect chondrocytes from dedifferentiation and apoptosis, processes implicated in the pathophysiology of arthritis. Yoon et al . investigated the mechanisms by which NSAIDs inhibit dedifferentiation and apoptosis in response to nitric oxide (NO), which activates multiple signaling pathways with differing effects on chondrocyte apoptosis and dedifferentiation. The authors used sodium nitroprusside to stimulate NO production and to trigger dedifferentiation and apoptosis in primary cultures of rabbit articular chondrocytes and three-dimensional cultures of rabbit cartilage explants. Various NSAIDs inhibited dedifferentiation and apoptosis under both conditions. The NSAID indomethacin enhanced activation of extracellular signal-regulated protein kinases (ERK1 and 2) as assessed by Western analysis, which would be expected to promote NO-mediated dedifferentiation but inhibit NO-mediated apoptosis. Indomethacin inhibited NO-mediated activation of p38 kinase, as determined by an in vitro kinase assay, which would also be expected to promote NO-mediated dedifferentiation and inhibit NO-mediated apoptosis. Indomethacin also blocked NO-mediated inhibition of protein kinase C (PKC) α and ζ, effects that would be expected to inhibit both NO-mediated dedifferentiation and apoptosis. Pharmacological inhibition of COX-2, as well as treatment with prostaglandin E 2 , indicated that NSAID inhibition of NO-mediated apoptosis and dedifferentiation was independent of effects on COX-2 activity and prostaglandin synthesis. Thus, NSAIDs appear to inhibit chondrocyte apoptosis and dedifferentiation through multiple signaling pathways distinct from their inhibition of COX pathway-dependent pain and inflammation. J.-B. Yoon, S.-J. Kim, S.-G. Hwang, S. Chang, S.-S. Kang, Non-steroidal anti-inflammatory drugs inhibit nitric oxide-induced apoptosis and dedifferentiation of articular chondrocytes independent of cyclooxygenase activity. J. Biol. Chem . 278 , 15319-15325 (2003). [Abstract] [Full Text]

Induction of heme oxygenase-1 inhibits IL-1b nitrite oxide production in articular chondrocytes

IL-1b is one of the main cytokines leading to the degradation of articular cartilage. One mechanism through which this cytokine exerts its effects is by articular chondrocytes being induced to produce nitrite oxide (NO). These findings are supported by clinical studies revealing a significant increase of NO production in the synovial fluid of patients with rheumatoid arthritis and osteoarthritis. Modulating IL-1b-induced NO in articular chondrocytes appears to be an interesting challenge for reducing or inhibiting the cartilage destruction. Heme oxygenase-1 (HO-1) is an inducible enzyme catalyzing the degradation of heme. The beneficial effects of HO-1 have been described for several diseases. This enzyme confers protection mainly through its antioxidant and anti-inflammatory functions. The objectives of this work were to investigate the expression and activity of HO-1 in IL-1b-treated articular chondrocytes, and the corresponding molecular mechanism. We used high-density primary cultures of rabbit articular chondrocytes. HO-1 expression was evaluated by Western blotting. HO-1 activity on IL-1b-induced NO was evaluated with use of the Griess reaction. Transient transfection of and Western blotting for IkBa, NF-κB, inducible nitrite oxide synthase and HO-1 elucidated the molecular mechanism of the HO-1 activity on IL-1b-induced NO. We found evidence, for the first time, of the inducible expression of HO-1 in articular chondrocytes. Overproduction or overexpression of HO-1 is able to decrease dramatically in a dose-dependent manner the IL-1b-induced NO release in the culture medium from articular chondrocyte culture. The inhibition of NO production occurs at the transcriptional level through an NF-κB-dependent pathway. These results demonstrate the critical role of HO-1 in inhibiting the IL-1b-induced NO. In vivo experiments are under investigation.

Evidence that rosiglitazone acts as an antidegradative agent on cartilage cells in vitro

Recent studies have documented the role of the natural ligand (15d-PGJ2) of peroxisome proliferator-activated receptor gamma (PPARγ) as an anti-inflammatory agent in IL-1-treated cartilage cells and synovial fibroblasts in culture. Similar but less pronounced effects have been reported with pharmacological concentrations of synthetic PPARγ ligands, such as thiazolidinediones. These anti-IL-1 effects were attributed to the transcriptional inhibition of NF-κB and/or AP-1-dependent genes, while PPARγ implication remained questionable. We studied the effects of rosiglitazone, a thiazolidinedione with high affinity for PPARγ, in IL-1-treated rabbit articular chondrocytes (ARC) in culture. When used at 10 μM, rosiglitazone inhibited IL-1-induced nitric oxide production, COX-2 mRNA, decreased the degradative effect of IL-1 on 35S-sulfated proteoglycans and the 55 kDa gelatinolytic activity secreted by ARC, and downregulated MMP-1 mRNA. By contrast, when used at 0.1–1 μM, rosiglitazone decreased proteoglycan degradation and MMP-1 gene expression, whereas it did not modify chondrocyte nitric oxide production nor COX-2 mRNA expression. Transient transfection of ARC with MMP-1-Luc showed that IL-1 stimulation was inhibited in a dose-dependent manner by rosiglitazone from 0.1 μM to 1 μM, indicating that rosiglitazone was acting at the transcriptional level. The electrophoretic mobility shift assay showed that IL-1-induced NF-κB binding activity was not changed by rosiglitazone, while IL-1-induced AP-1 binding activity was reduced. We analysed rosiglitazone's effect on MMP-1 promoter activation in cells transiently cotransfected with MMP-1-Luc vector. We showed that the inhibitory effect of rosiglitazone was significantly more pronounced in cells cotransfected with wild-type PPARγ than without, while this effect was completely suppressed by cotransfecting cells with the dominant-negative PPARγ. Altogether, these data show for the first time that rosiglitazone has a selective inhibitory effect on IL-1-induced MMP-1 in chondrocytes, which involves a mechanism whereby PPARγ and AP-1 are implicated.

Design of a chimeric promoter induced by pro-inflammatory mediators in articular chondrocytes

We have designed a chimeric promoter that can be stimulated by various pro-inflammatory mediators and so drive the expression of therapeutic genes under inflammatory conditions. The promoter has two parts, the [−247/+20] fragment of the human type IIA secreted phospholipase A2 gene promoter, which is stimulated by the pro-inflammatory cytokine interleukin-1β (IL-1β), and a double peroxisome proliferator-activated receptor response element that is activated by some eicosanoids and by non-steroidal anti-inflammatory drugs (NSAIDs). Transfection experiments using rabbit articular chondrocytes in primary culture showed that this chimeric promoter produced a low basal activity and was induced by NSAIDs, WY-14643, IL-1β, and 15-deoxy Δ 12,14 prostaglandin J2. The latter two compounds stimulated the promoter synergistically.

Induction of osteogenic protein-1 expression by interleukin-1beta in cultured rabbit articular chondrocytes.

To elucidate the effects of interleukin-1beta (IL-1beta) on osteogenic protein-1 (OP-1) gene expression in a polylayer culture of rabbit articular chondrocytes, we measured rabbit OP-1 mRNA using quantitative TaqMan reverse transcriptase-polymerase chain reaction (RT-PCR) techniques. Rabbit articular chondrocytes were isolated and cultured in minimum essential medium eagle alpha modification containing 10% fetal bovine serum for 7 days. IL-1beta was then added and cultures were continued for 48 or 96 hours. OP-1 gene expression was detected in cell cultures both with and without addition of IL-1beta. However, the level of expression was very low in the control group. OP-1 gene expression was significantly increased about 450- to 800-fold in IL-1beta-treated groups (0.1, 1, and 10 ng/ml) versus the control group. Evaluation of serial changes in OP-1 expression after addition of IL-1beta (10 ng/ml) revealed that OP-1 gene expression increased rapidly after addition of IL-1beta, reaching a peak at 48 hours, and then decreasing. Simultaneous assay of CD44 expression demonstrated a rapid increase, similar to that of OP-1 expression, following addition of IL-1beta: this was followed by a more gradual increase. Assay of hyaluronan synthase-2 (HAS-2) expression following addition of IL-1beta showed an increase after OP-1 expression had already reached a peak. Our results demonstrate that OP-1 expression is induced by IL-1beta and suggest that this expression, like that of HAS-2, may play a role as a protective mechanism against inflammatory cytokines.