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Two‐dimensional electrophoresis of intracellular and secreted protein synthesized by fetal bovine chondrocytes in high‐density culture

In order to study the mechanisms involved in the differentiation/dedifferentiation of chondrocytes, fetal bovine chondrocytes in high‐density cultures were treated with retinoic acid, an agent known to modify the chondrocyte phenotype (10 μmol/L between day 2 to day 5 of culture). The synthesis of intracellular and secreted proteins was studied by two‐dimensional electrophoresis in cell lysates and culture media after labeling with [35S]methionine for the last 14 h of culture. The proteins expressed in control and retinoic acid‐treated cells were identified by microsequencing after “in‐gel” tryptic digestion of the spot or by immunodetection with specific antibodies after two‐dimensional gel blotting. Intracellular protein modifications included one of 56.9 kDa and with an isoelectric point (pI) of 5.8 whose synthesis was previously reported to be up‐regulated by 75%. Microsequencing of two internal peptides did not reveal a known protein. Changes to the chondrocyte phenotype were also recorded in the culture medium, as a decrease in type II collagen synthesis and expression of the small proteoglycan, decorin. Several new spots were also observed after treatment with retinoic acid, including a large, diffuse spot, not yet characterized, with a mean molecular mass of 39 kDa and a pI of 4.5–5.0. Under our experimental conditions, retinoic acid induces morphological changes of the chondrocytes and dramatic changes in the synthesis of several intracellular and secreted proteins that predate the synthesis of collagen type I (the classical marker of chondrocyte dedifferentiation).