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.
Demoor-Fossard
M
M
Laboratoire de Biochimie du Tissu Conjonctif, Faculté de Médecine, Caen, France.
Boittin
M
M
Redini
F
F
Pujol
J P
JP
D000464:Alginates / Q000378:metabolism; D000818:Animals; D002358:Cartilage, Articular / Q000166:cytology*; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D020723:Glucuronic Acid; D006603:Hexuronic Acids; D007375:Interleukin-1 / Q000378:metabolism* / Q000494:pharmacology; D008297:Male; D011506:Proteins / Q000032:analysis; D011509:Proteoglycans / Q000096:biosynthesis* / Q000187:drug effects; D011817:Rabbits; D013462:Sulfur Radioisotopes; D016212:Transforming Growth Factor beta / Q000378:metabolism* / Q000494:pharmacology
D000464:Alginates; D006603:Hexuronic Acids; D007375:Interleukin-1; D011506:Proteins; D011509:Proteoglycans; D013462:Sulfur Radioisotopes; D016212:Transforming Growth Factor beta; D020723:Glucuronic Acid