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.
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.
复制标题
视黄酸对高密度培养中胎牛软骨细胞蛋白质合成的影响：下调葡萄糖调节蛋白、GRP-78 和 II 型胶原。
作者：
A. Freyria;M. Ronzière;M. Boutillon;Daniel Herbage
在高密度培养（0.6 × 10(6)个细胞/cm2）中，研究了0.1-10微米维甲酸（RA）对胎牛软骨细胞的影响。在不含抗坏血酸的培养基中培养5天后，对照软骨细胞呈典型的圆形，并合成了II、IX、XI和III型胶原。通过[35S]蛋氨酸标记，在培养第2-5天RA处理后，细胞呈现成纤维细胞样形状，总蛋白（48%）和胃蛋白酶抗性蛋白（60%）的合成减少。添加RA后没有I型胶原的表达，但通过SDS/PAGE后相应带的直接放射自显影测量，诱导了软骨特异性胶原（II、IX和XI）合成的定量变化。主要变化是II型胶原合成，细胞层含量减少80%，培养基含量减少89%；对IX型和XI型胶原合成的抑制作用分别为25%和31%。通过与计算机成像系统相结合的二维电泳确定RA诱导的细胞内蛋白的修饰。RA处理后，其中一个较丰富的蛋白（pI 4.8; 78 kDa）的合成减少了75%。该蛋白经微测序鉴定为葡萄糖调节蛋白78 （grp78）。据报道，它可以结合变性胶原蛋白和突变的I型前胶原蛋白分子，并作为胶原蛋白分子的分子伴侣。这里观察到的grp78和II型胶原蛋白的平行下调是否对应于这两种蛋白的协调调节，还有待证明。
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.