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J Biol Chem, Vol. 275, Issue 15, 10738-10744, April 14, 2000
From the Departments of 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 ~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 °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.
SOX9 Enhances Aggrecan Gene Promoter/Enhancer Activity and Is
Up-regulated by Retinoic Acid in a Cartilage-derived Cell Line,
TC6*
,,
,, and**
Molecular
Pharmacology, Medical Research Institute, and Orthopaedic
Surgery, Tokyo Medical and Dental University, Tokyo 101, Japan, the
§ Center for Molecular and Cellular Biology, University of
Queensland, Brisbane, Queensland 4072, Australia, and the
¶ Laboratory of Developmental Biology, NIDCR, National Institutes
of Health, Bethesda, Maryland 20892-4370
*
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.
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