Effect of retinoic acid on protein synthesis by foetal 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 microM retinoic acid (RA) on foetal bovine chondrocytes was investigated in high-density cultures (0.6 x 10(6) cells/cm2). After 5 days of culture in ascorbate-free medium, control chondrocytes presented a typical rounded shape and synthesized type II, IX, XI and III collagens. After RA treatment on days 2-5 of culture, the cells exhibited a fibroblast-like shape and decreased synthesis of total protein (48%) and pepsinresistant proteins (60%) as determined by [35S]methionine labelling. Addition of RA was not followed by the expression of type I collagen, but induced quantitative changes in the synthesis of cartilage-specific collagens (II, IX and XI) as measured by direct autoradiography of the corresponding bands after SDS/PAGE. The main change was in type II collagen synthesis, with a 80% decrease in the cell-layer fraction and a 89% decrease in culture-medium fraction; inhibition of type IX and XI collagen synthesis was limited to 25 and 31% respectively. Modifications to intracellular proteins induced by RA were determined by using two-dimensional electrophoresis associated with a computerized imaging system. Synthesis of one of the more abundant proteins (pI 4.8; 78 kDa) was decreased by 75% after RA treatment. This protein was characterized by micro-sequencing as the glucose-regulated protein 78 (GRP 78). It was reported previously to bind denatured collagen and mutated type I procollagen molecule and to function as a molecular chaperone for collagen molecules. It remains to demonstrate whether the parallel down-regulation of GRP 78 and type II collagen observed here corresponds to a co-ordinate regulation of these two proteins.
Freyria
A M
AM
Institut de Biologie et Chimie des Protéines, CNRS UPR 412, Lyon France.
Ronzière
M C
MC
Boutillon
M M
MM
Herbage
D
D
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000096:biosynthesis*; D002358:Cartilage, Articular / Q000166:cytology / Q000187:drug effects / Q000196:embryology / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis*; D015536:Down-Regulation; D015180:Electrophoresis, Gel, Two-Dimensional; D000091342:Endoplasmic Reticulum Chaperone BiP; D005333:Fetus / Q000166:cytology; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D017421:Sequence Analysis; D014212:Tretinoin / Q000494:pharmacology*
D002352:Carrier Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D010446:Peptide Fragments; D014212:Tretinoin; D003094:Collagen