NUCLEAR FACTOR-BINDING TO AN AP-1 SITE IS ASSOCIATED WITH THE ACTIVATION OF PRO-ALPHA-1(I)-COLLAGEN GENE IN DEDIFFERENTIATING CHONDROCYTES

Isolated chondrocytes grown on plastic gradually lose their differenti ated 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 dediffer entiating human and murine fetal chondrocytes. Nuclear extracts from d edifferentiated 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 alpha1(I) collagen gene. T his binding activity was not present in freshly isolated human or muri ne chondrocytes, which produced type II, but not type I, collagen mRNA in culture. Thus the binding activity was induced simultaneously with alpha1(I)-collagen-gene expression during dedifferentiation. The spec ific interaction was sensitive to dephosphorylation of the nuclear ext ract and to chemical modification of reduced cysteine residues. The AP -1 site we studied had previously been shown to be a positive transcri ptional contributor in the first intron to the expression of the alpha 1(I) collagen gene. In transient transfections into dedifferentiating chondrocytes, an alpha1(I) collagen expression plasmid carrying a muta ted AP-1 site in the first intron resulted in three-times-lower report er gene RNA levels than a plasmid carrying the respective functional A P-1 site. These data suggest that the AP-1 sequence and its respective trans-acting factor's may play a role in the transcriptional regulati on of the alpha1(I) collagen gene during dedifferentiation of chondroc ytes.