Am. Freyria et al., 2-DIMENSIONAL ELECTROPHORESIS OF INTRACELLULAR AND SECRETED PROTEIN SYNTHESIZED BY FETAL BOVINE CHONDROCYTES IN HIGH-DENSITY CULTURE, Electrophoresis, 16(7), 1995, pp. 1268-1272
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).