SOX9 enhances aggrecan gene promoter/enhancer activity and is up-regulatedby retinoic acid in a cartilage-derived cell line, TC6

SOX9 is a transcription factor that plays a key role in chondrogenesis, Agg recan is one of the major structural components in cartilage; however, the molecular mechanism of aggrecan gene regulation has not yet been fully eluc idated, TC6 is a clonal chondrocytic cell line derived from articular carti lage, The purpose of this study was to examine whether SOX9 modulates aggre can gene expression and to further identify molecules that regulate Sox9 ex pression in TC6 cells. SOX9 overexpression in TC6 cells enhanced by similar to 3-fold the transcriptional activity of the AgCAT-8 construct containing S-kilobase (kb) promoter/first exon/first intron fragments of the aggrecan gene. SOX9 enhancement of aggrecan promoter activity was lost when we dele ted a 4.5-kb fragment from the 3'-end of the 8-kb fragment corresponding to the region including the first intron, In TC6 cells, SOX9 enhanced the tra nscriptional activity of a reporter construct containing the Sry/Sox consen sus sequence >10-fold. SOX9 enhancement of aggrecan gene promoter activity and SOX9 transactivation through the Sry/Sox consensus sequence were not ob served in osteoblastic osteosarcoma cells (ROS17/2.8), indicating the depen dence on the cellular background. Northern blot analysis indicated that TC6 cells constitutively express Sox9 mRNA at relatively low levels. To examin e regulation of Sox9 gene expression, we investigated the effects of calcio tropic hormones and cytokines, Among these, retinoic acid (RA) specifically enhanced Sox9 mRNA expression in TC6 cells. The basal levels of Sox9 expre ssion and its enhancement by RA were observed similarly at both permissive (33 degrees C) and nonpermissive (39 degrees C) temperatures. Furthermore, RA treatment enhanced the transcriptional activity of a reporter construct containing the Sry/Sox consensus sequence in TC6 cells. Moreover, RA treatm ent also enhanced the transcriptional activity of another reporter construc t containing the enhancer region of the type II procollagen gene in TC6 cel ls. These observations indicate that SOX9 enhances aggrecan promoter activi ty and that its expression is up-regulated by RA in TC6 cells.