2-DIMENSIONAL ELECTROPHORESIS OF INTRACELLULAR AND SECRETED PROTEIN SYNTHESIZED BY FETAL BOVINE CHONDROCYTES IN HIGH-DENSITY CULTURE

In o rder to stu dy the mechanisms involved in the differentiation! de differentiation of chondrocytes, fetal bovine chondrocytes in high-den sity cultures were treated with retinoic acid, an agent known to modif y the chondrocyte phenotype (10 mu mol/L between day 2 to day 5 of cul ture). The synthesis of intracellular and secreted proteins was studie d by two-dimensional electrophoresis in cell lysates and culture media after labeling with [S-35]methionine for the last 14 h of culture. Th e proteins expressed in control and retinoic acid-treated cells were i dentified by microsequencing after ''in-gel'' tryptic digestion of the spot or by immunodetection with specific antibodies after two-dimensi onal gel blotting. Intracellular protein modifications included one of 56.9 kDa and with an isoelectric point (pI) of 5.8 whose synthesis wa s previously reported to be up-regulated by 75%. Microsequencing of tw o internal peptides did not reveal a known protein. Changes to the cho ndrocyte phenotype were also recorded in the culture medium, as a decr ease in type II collagen synthesis and expression of the small proteog lycan, decorin. Several new spots were also observed after treatment w ith retinoic acid, including a large, diffuse spot, not yet characteri zed, with a mean molecular mass of 39 kDa and a pI of 4.5-5.0. Under o ur experimental conditions, retinoic acid induces morphological change s of the chondrocytes and dramatic changes in the synthesis of several intracellular and secreted proteins that predate the synthesis of col lagen type I (the classical marker of chondrocyte dedifferentiation).