Interleukin-1␤ (IL-1␤) is a pleiotropic cytokine that was shown to inhibit the biosynthesis of articular cartilage components. Here we demonstrate that IL-1␤ inhibits the production of newly synthesized collagens in proliferating rabbit articular chondrocytes and that this effect is accompanied by a decrease in the steady-state levels of type II collagen mRNA. IL-1␤ down-regulates COL2A1 gene transcription through a ؊41/؊33 bp sequence that binds a multimeric complex including Sp1 and Sp3 transcription factors. Specificity of IL-1␤ effects on COL2A1 promoter activity was demonstrated in experiments in which transfection of a wild type ؊50/؉1 sequence of COL2A1 promoter as a decoy oligonucleotide abolished the IL-1␤ inhibition of a ؊63/؉47 COL2A1-mediated transcription. By contrast, transfection of the related oligonucleotide harboring a targeted mutation in the ؊41/؊33 sequence did not modify the negative effect the cytokine. Because we demonstrated previously that Sp1 was a strong activator of COL2A1 gene expression via the ؊63/؉1 promoter region, whereas Sp3 overexpression blocked Sp1-induced promoter activity and inhibited COL2A1 gene transcription, we conclude that IL-1␤ down-regulation of that gene, as we found previously for transforming growth factor-␤1, is mediated by an increase in the Sp3/Sp1 ratio. Moreover, IL-1␤ increased steady-state levels of Sp1 and Sp3 mRNAs, whereas it enhanced Sp3 protein expression and inhibited Sp1 protein biosynthesis. Nevertheless, IL-1␤ decreased the binding activity of both Sp1 and Sp3 to the 63-bp short COL2A1 promoter, suggesting that the cytokine exerts a post-transcriptional regulatory mechanism on Sp1 and Sp3 gene expressions. Altogether, these data indicate that modulation of Sp3/Sp1 ratio in cartilage could be a potential target to prevent or limit the tissue degradation.Articular cartilage is a highly specialized tissue composed of a complex extracellular matrix of proteoglycans, collagens, and noncollagenous glycoproteins. Cartilage collagens include type II as the major form and types VI, IX, and XI as minor components (1). Type II collagen is an homotrimer composed of ␣1(II) chains encoded by the COL2A1 gene. Previous studies have delineated minimal sequences in the first intron of human, mouse, and rat COL2A1 genes which are sufficient to direct chondrocyte-specific expression in cultured chondrocytes and transgenic mice (2-5). Several binding sites of the intronic enhancer sequences were shown to interact with transcription factors that form chondrocyte-specific complexes, such as SOX9, L-SOX5, and SOX6 (6, 7), and also with factors having less tissue-specific expression, such as Sp1, Sp3, and C-KROX (5, 8). Indeed, promoter sequences are also implicated through interaction with the intronic enhancer sequence, for tissuespecific expression during in vivo and in vitro chondrogenesis (7, 9, 10). In a 266-bp promoter of the human COL2A1 gene mediating enhanced transcription activity, we identified several binding sites for Sp1, Sp3, and C-KROX (5, 8, 11). Sp1 w...

Abstract. Heparin blocks the phorbol ester-induced progression of nontransformed cells through the Go/G, phase

Objective. To determine the effects of hypoxia and reoxygenation on the metabolism of chondrocytes and their response to interleukin-1␤ (IL-1␤). The study included activation of hypoxia-inducible factor 1 (HIF-1), NF-B, and activator protein 1 (AP-1) transcription factors, expression of matrix components and metalloproteases and transforming growth factor ␤ (TGF␤) and TGF␤ 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-1␤. 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-1␤-induced DNA binding of NF-B and AP-1 was significantly higher in hypoxic and reoxygenated cultures than in normoxia. Greater activation of the MAPKs was also observed with IL-1␤ treatment in hypoxia compared with normoxia. Steady-state levels of type II collagen and aggrecan core protein messenger RNA (mRNA) were decreased by IL-1␤ in all instances. Matrix metalloprotease 1 (MMP-1) and MMP-3 mRNA were increased by IL-1␤ 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-1␤ in normoxia or hypoxia, whereas it was enhanced in reoxygenated cultures. MMP-9 mRNA was dramatically decreased by IL-1␤ 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-1␤ in reoxygenated cultures. Stimulation of TGF␤1 expression by IL-1␤ was observed only in normal atmospheric conditions. One of the more striking findings of the study was the greater stimulating effect of IL-1␤ on NO production observed in hypoxia, which was much higher than in normoxia, whereas the reverse was observed for IL-1␤-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-1␤. In hypoxic conditions, which mimic the in vivo condition of cartilage, the effects of IL-1␤ 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-1␤-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 bio...

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% 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 level of c-jun protein, an element of AP-1 complex, was increased by exposure of the chondrocytes to IL-1beta after 2, 6, and 24 h. Addition of rhein at 10(-5) M for 24 h did not reduce the c-jun protein amount. 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 (MMPI) 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 proinflammatory genes by chondrocytes. In addition, the drug can reduce the procatabolic effect of the cytokine, by reducing the MMPI synthesis, and enhance the synthesis of matrix components, such as type II collagen and aggrecan. These results may explain the antiosteoarthritic properties of rhein and its disease-modifying effects on OA cartilage, in spite of absence of inhibition at prostaglandin level.

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.