EFFECT OF RETINOIC ACID ON PROTEIN-SYNTHESIS BY FETAL BOVINE CHONDROCYTES IN HIGH-DENSITY CULTURE - DOWN-REGULATION OF THE GLUCOSE-REGULATED PROTEIN, GRP-78, AND TYPE-II COLLAGEN

The effect of 0.1-10 mu M retinoic acid (RA) on foetal bovine chondroc ytes was investigated in high-density cultures (0.6 x 10(6) cells/cm(2 )). After 5 days of culture in ascorbate-free medium, control chondroc ytes presented a typical rounded shape and synthesized type II, IX, XI and III collagens. After RA treatment on days 2-5 of culture, the cel ls exhibited a fibroblast-like shape and decreased synthesis of total protein (48%) and pepsin-resistant proteins (60%) as determined by [S- 35]methionine labelling. Addition of RA was not followed by the expres sion of type I collagen, but induced quantitative changes in the synth esis of cartilage-specific collagens (II, IX and XI) as measured by di rect autoradiography of the corresponding bands after SDS/PAGE. The ma in change was in type II collagen synthesis, with a 80% decrease in th e cell-layer fraction and a 89% decrease in culture-medium fraction; i nhibition of type IX and XI collagen synthesis was limited to 25 and 3 1% respectively. Modifications to intracellular proteins induced by RA were determined by using two-dimensional electrophoresis associated w ith a computerized imaging system. Synthesis of one of the more abunda nt proteins (pI 4.8; 78 kDa) was decreased by 75% after RA treatment. This protein was characterized by micro-sequencing as the glucose-regu lated protein 78 (GRP 78). It was reported previously to bind denature d collagen and mutated type I procollagen molecule and to function as a molecular chaperone for collagen molecules. It remains to demonstrat e whether the parallel down-regulation of GRP 78 and type II collagen observed here corresponds to a co-ordinate regulation of these two pro teins.