Journal of Biological Chemistry
Volume 275, Issue 15, 14 April 2000, Pages 10738-10744
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GENES: STRUCTURE AND REGULATION
SOX9 Enhances Aggrecan Gene Promoter/Enhancer Activity and Is Up-regulated by Retinoic Acid in a Cartilage-derived Cell Line, TC6*

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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 regulateSox9 expression in TC6 cells. SOX9 overexpression in TC6 cells enhanced by ∼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 expressSox9 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 °C) and nonpermissive (39 °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.

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This work was supported by Grants-in-aid 11877357, 11152209, 10044246, 10877223, and 0930734 from the Japanese Ministry of Education; grants from Core Research for Evolutional Science and Technology of the Japan Science and Technology Corp.; Grant 96100205 from the Research for the Future Program of the Japan Society for the Promotion of Science; and grants from the Traffic Medicine Foundation, the Interdisciplinary Cancer Research Foundation, the Inamori Foundation, the Cell Fate Modulation Research Unit, and National Space Development Agency of Japan (to M. N.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.