No abstract

Decorin, a small leucine-rich proteoglycan may play an important role in the attempt of cartilage repair initiated by chondrocytes in early stages of osteoarthritis, through its ability to bind collagen fibrils and growth factors such as transforming growth factor-␤ (TGF-␤). We previously demonstrated that TGF-␤ decreased decorin mRNA steady state levels in articular chondrocytes (Demoor, M., Ré dini, F., Boittin, M., and Pujol, J.-P. . DNase I footprinting analysis delineated a negative TGF-␤-responsive region between ؊140 and ؊111 bp in the decorin proximal promoter. Gel retardation assays demonstrated that TGF-␤ modulates decorin gene expression through transcription factors, the nature and mode of action of which depend on the differentiation state of the chondrocytes; two DNA-protein complexes were formed in the region ؊144/؊127 bp with nuclear extracts from primary chondrocytes, whereas a higher mobility complex was observed in the ؊127/؊111 bp region for dedifferentiated cells. Antibodies against vitamin D and retinoic acid receptors used in supershift experiments showed that these nuclear receptors are involved in the regulation of decorin gene expression in articular chondrocytes.

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.

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.