Differential effects of interleukin-1 and transforming growth factor beta on the synthesis of small proteoglycans by rabbit articular chondrocytes cultured in alginate beads as compared to monolayers.

Small proteoglycans (PGs) are supposed to play great roles in the assembly of cartilage matrix but the influence of cytokines and growth factors on their synthesis by articular chondrocytes is largely unknown. We investigated whether IL-1 and TGFbeta1 influence the production of small leucine-rich proteoglycans by chondrocytes cultured in a three-dimensional gel, as compared to the common monolayer system. Rabbit articular chondrocytes were cultured in alginate beads for 14 days or as monolayers for 7 days. The effect of 2 ng/ml IL-1beta or TGFbeta1 during the last two days in culture was determined, after [35S]methionine labeling over the last 24 h. Cell-associated and further-removed matrix compartments were separated by centrifugation after sodium citrate/EDTA treatment of alginate beads whereas medium and cell-layer fractions were isolated from monolayer cultures. Total newly synthesized PGs were first isolated by anion-exchange chromatography and the small PGs were further separated from aggrecans by gel-filtration (Sepharose CL-4B) and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Addition of TGFbeta1 resulted in an overall rise in neosynthesized small PG content in both culture systems. However, TGFbeta1 significantly increased to the same extent the percentage of small PGs laid down in the cell-associated and the further-removed matrix compartments of the beads culture (+60%) whereas it augmented the content of small PGs in the medium (+40%) and reduced that of the cell fraction (-35%) in the monolayer culture. By adding IL-1, the amount of total newly synthesized small PGs was decreased in monolayers while it increased in alginate beads. IL-1 was also shown to change the relative distribution of these molecules in the monolayer system in contrast to the alginate beads culture where the proportions were not significantly altered. Electrophoretic analysis of the 35S-labeled small PGs-containing fractions confirmed these effects at the level of the 45-50 kDa-related core proteins. This study demonstrates that TGFbeta and IL-1 differently influence small PG synthesis of rabbit articular chondrocytes depending on whether they are cultured in alginate beads or in monolayers. Moreover, the regulation of small PG expression appears to be different from that of high-molecular weight aggrecans. As these small molecules are playing major roles in matrix assembly and growth factor regulation, the data may have great relevance to the pathogenesis of osteoarthritis and repair of articular cartilage lesions.

Expression of decorin and biglycan by rabbit articular chondrocytes. Effects of cytokines and phenotypic modulation.

In this study, the levels of mRNAs coding for aggrecan, decorin and biglycan in rabbit articular chondrocytes were investigated, using both monolayer and 3D-alginate cultures treated with TGF-beta 1 and IL-1 beta. The cells were shown to express higher amounts of proteoglycan messages, specially the aggrecan, in gels than in monolayers. TGF-beta 1 increased aggrecan mRNA in both systems, whereas biglycan message was elevated only in alginate. It markedly decreased decorin expression in monolayer, either in primary or passaged cultures. In contrast, IL-1 beta had a weak inhibitory effect on both decorin and biglycan expression. Subculturing induced a dramatic decrease of aggrecan mRNA, while that of decorin augmented. Biglycan expression transiently increased after two passages, whereas it declined in further subcultures. Passaged chondrocytes transferred to alginate re-expressed high levels of aggrecan, decorin and biglycan. The data point to the influence of morphology, proliferative state and environment of the articular chondrocytes on their biosynthetic responses to cytokines. Although these immature cells do not fully reflect the adult chondrocytes present in the cartilage, this study may help in understanding the behaviour of these cells in osteoarticular diseases, where the surrounding extracellular matrix is profoundly altered.

Differential expression of membrane-anchored proteoglycans in rabbit articular chondrocytes cultured in monolayers and in alginate beads. Effect of transforming growth factor-beta 1.

Cell-surface proteoglycans (PGs) were extracted with Triton X-100 from rabbit articular chondrocytes cultured in monolayers and in alginate beads. They were first purified on DEAE-Trisacryl columns and the proportion of hydrophobic PGs was determined by both Octyl-Sepharose chromatography and partitioning in Triton X-114. These two methods revealed that the proportion of hydrophobic PGs was higher in monolayer culture system as compared to alginate beads (24 and 15%, respectively). Characterization of the PGs by Sepharose CL 6B gel filtration followed by electrophoresis indicated that the PGs isolated from monolayers were composed of three chondroitin sulfate (CS) PGs (core proteins of 180, 100 and 50 kDa) and a heparan sulfate (HS) PG (core protein of 60 kDa). In the alginate system. CSPGs with core proteins of 180, 45 and 32 kDa were observed, but no HSPG was present. In parallel, the effect of TGF-beta on the distribution of membrane-associated PGs was studied. The results showed that the synthesis of cell-surface PGs was stimulated by TGF-beta in monolayers whereas it was inhibited in alginate beads, but the amount of hydrophobic PGs was not altered by the growth factor. These data clearly indicate that TGF-beta induces a differential expression of the PG families present at the cell surface. Taken together, the results reveal the complex regulation of cell-surface PG distribution, which obviously depends on the culture method used and suggest that rabbit articular chondrocytes may differentially respond to extracellular ligands according to their morphological state and environment.

[Effect of diacerhein (ART 50) on the matrix synthesis and collagenase secretion by cultured joint chondrocytes in rabbits]. / Effet de la diacerhéine (ART 50) sur la synthèse de la matrice et la sécrétion de collagénase par des chondrocytes articulaires de lapin en culture.

Cultured rabbit joint chondrocytes were exposed to diacerhein (DAR : ART 50, Negma, 10(-6) to 10(-4) M), which has proved effective and safe when given for two months for the treatment of osteoarthritis. Experiments were performed with and without 500 pg/ml human recombinant interleukin-1 to determine whether diacerhein antagonizes the effects of this monokine. Glycosaminoglycan production was measured by 35S-sulfate incorporation followed by cetylpyridinium precipitation, collagen production by 3H-proline labeling and bacterial collagenase digestion, and collagenase production by determination of the amount of 3H-collagen that underwent degradation. Incubation of chondrocytes with diacerhein for 24 hours was not associated with substantial changes in glycosaminoglycan or collagen production but substantially antagonized interleukin-1-mediated enhancement of collagenase production. With longer incubation periods (6 days) with the 10(-6) M concentration of diacerhein, production of glycosaminoglycans and collagen increased. Incubation with both diacerhein and interleukin-1 for six days partly antagonized the cytokine's inhibitory effect on glycosaminoglycan and collagen production. During these experiments, the medium's ability to break down collagen was consistently reduced by diacerhein, even in the presence of interleukin-1. These data demonstrate that diacerhein can reduce or even abolish interleukin-1-mediated enhancement of collagenase production by joint chondrocytes. This effect may lead to less erosion of cartilage in degenerative joint diseases.