Määttä A, Glumoff V, Paakkonen P, Liska D, Penttinen R P, Elima K
Department of Medical Biochemistry, University of Turku, Finland.
Biochem J. 1993 Sep 1;294 ( Pt 2)(Pt 2):365-71. doi: 10.1042/bj2940365.
Isolated chondrocytes grown on plastic gradually lose their differentiated phenotype upon subculturing. This dedifferentiation is manifested by an altered production of extracellular-matrix molecules (ECM): e.g., the cartilage specific type II collagen is replaced by types I and III. We have studied the regulation of ECM gene expression in dedifferentiating human and murine fetal chondrocytes. Nuclear extracts from dedifferentiated cells, human fetal fibroblasts and 3T3 cells contained a protein that bound in an electrophoretic mobility shift assay to an AP-1 site in the first intron of the human alpha 1(I) collagen gene. This binding activity was not present in freshly isolated human or murine chondrocytes, which produced type II, but not type I, collagen mRNA in culture. Thus the binding activity was induced simultaneously with alpha 1(I)-collagen-gene expression during dedifferentiation. The specific interaction was sensitive to dephosphorylation of the nuclear extract and to chemical modification of reduced cysteine residues. The AP-1 site we studied had previously been shown to be a positive transcriptional contributor in the first intron to the expression of the alpha 1(I) collagen gene. In transient transfections into dedifferentiating chondrocytes, an alpha 1(I) collagen expression plasmid carrying a mutated AP-1 site in the first intron resulted in three-times-lower reporter gene RNA levels than a plasmid carrying the respective functional AP-1 site. These data suggest that the AP-1 sequence and its respective trans-acting factors may play a role in the transcriptional regulation of the alpha 1(I) collagen gene during dedifferentiation of chondrocytes.
在塑料培养皿上生长的分离软骨细胞在传代培养后会逐渐丧失其分化表型。这种去分化表现为细胞外基质分子（ECM）产生的改变：例如，软骨特异性的II型胶原蛋白被I型和III型胶原蛋白所取代。我们研究了人及小鼠胎儿软骨细胞去分化过程中ECM基因表达的调控。去分化细胞、人胎儿成纤维细胞和3T3细胞的核提取物中含有一种蛋白质，在电泳迁移率变动分析中，该蛋白质可与人α1（I）胶原蛋白基因第一个内含子中的AP-1位点结合。新鲜分离的人或小鼠软骨细胞中不存在这种结合活性，这些软骨细胞在培养中产生II型而非I型胶原蛋白mRNA。因此，在去分化过程中，这种结合活性与α1（I）胶原蛋白基因的表达同时被诱导。这种特异性相互作用对核提取物的去磷酸化以及还原型半胱氨酸残基的化学修饰敏感。我们研究的AP-1位点先前已被证明是α1（I）胶原蛋白基因第一个内含子中表达的正向转录贡献因子。在对去分化软骨细胞进行的瞬时转染中，第一个内含子中携带突变AP-1位点的α1（I）胶原蛋白表达质粒导致报告基因RNA水平比携带相应功能性AP-1位点的质粒低三倍。这些数据表明，AP-1序列及其相应的反式作用因子可能在软骨细胞去分化过程中α1（I）胶原蛋白基因的转录调控中发挥作用。
