Abstract
SOX9 is a transcription factor that plays a key role in chondrogenesis. Aggrecan is one of the major structural components in cartilage; however, the molecular mechanism of aggrecan gene regulation has not yet been fully elucidated. TC6 is a clonal chondrocytic cell line derived from articular cartilage. The purpose of this study was to examine whether SOX9 modulates aggrecan gene expression and to further identify molecules that regulate Sox9 expression in TC6 cells. SOX9 overexpression in TC6 cells enhanced by approximately 3-fold the transcriptional activity of the AgCAT-8 construct containing 8-kilobase (kb) promoter/first exon/first intron fragments of the aggrecan gene. SOX9 enhancement of aggrecan promoter activity was lost when we deleted 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 transcriptional activity of a reporter construct containing the Sry/Sox consensus sequence >10-fold. SOX9 enhancement of aggrecan gene promoter activity and SOX9 transactivation through the Sry/Sox consensus sequence were not observed in osteoblastic osteosarcoma cells (ROS17/2.8), indicating the dependence on the cellular background. Northern blot analysis indicated that TC6 cells constitutively express Sox9 mRNA at relatively low levels. To examine regulation of Sox9 gene expression, we investigated the effects of calciotropic hormones and cytokines. Among these, retinoic acid (RA) specifically enhanced Sox9 mRNA expression in TC6 cells. The basal levels of Sox9 expression 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 treatment also enhanced the transcriptional activity of another reporter construct containing the enhancer region of the type II procollagen gene in TC6 cells. These observations indicate that SOX9 enhances aggrecan promoter activity and that its expression is up-regulated by RA in TC6 cells.
Highlights
SOX9 is a transcription factor that plays a key role in chondrogenesis
SOX9 Enhances the Transcriptional Activity of the Aggrecan Gene Promoter/Enhancer in TC6 Cells—To examine whether SOX9 regulates the transcriptional activity of the aggrecan gene, a Chloramphenicol Acetyltransferase (CAT) reporter gene construct (AgCAT-8) containing an
SOX9 enhancement was no longer observed when we deleted a 4.5-kb intron fragment from the 3Ј-end of the 8-kb fragment corresponding to the region including the first intron
Summary
SOX9 is a transcription factor that plays a key role in chondrogenesis. Aggrecan is one of the major structural components in cartilage; the molecular mechanism of aggrecan gene regulation has not yet been fully elucidated. In TC6 cells, SOX9 enhanced the transcriptional activity of a reporter construct containing the Sry/Sox consensus sequence >10-fold. RA treatment enhanced the transcriptional activity of another reporter construct containing the enhancer region of the type II procollagen gene in TC6 cells These observations indicate that SOX9 enhances aggrecan promoter activity and that its expression is up-regulated by RA in TC6 cells. SOX9Ϫ/Ϫ cells are excluded from all cartilaginous tissues during embryonic development and are present in juxtaposed mesenchyme that does not express chondrocyte-specific markers such as procollagen types II, IX, and XI and aggrecan [11] These observations indicate that SOX9 plays a key role in chondrogenesis and skeletogenesis. We found that SOX9 expression is enhanced by RA1 in TC6 cells
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