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Papers In Press, published online ahead of print June 18, 2002
J. Biol. Chem, 10.1074/jbc.M202815200
Submitted on March 22, 2002
Revised on June 18, 2002
Accepted on June 18, 2002
Orthopaedic Surgery, Washington University School of Medicine, St. Louis, MO 63110
Corresponding Author: hochg{at}msnotes.wustl.edu
Cartilage-derived retinoic acid-sensitive protein (CD-RAP) is a secreted protein expressed by chondrocytes: the expression is repressed by interleukin 1b (IL-1b). To investigate the transcriptional mechanism, by which CD-RAP expression is suppressed by IL-1b, deletion constructs of the mouse CD-RAP promoter were transfected into rat chondrocytes treated with or without IL-1b. The results revealed an IL-1b-responsive element located between –2138 and –2068 bp. As this element contains CAAT/enhancer-binding protein (C/EBP) motif, the function of C/EBPb and C/EBPd was examined. IL-1b stimulated the expression of C/EBPb and d and the direct binding of C/EBPb to the C/EBP motif was confirmed. The –2251 bp CD-RAP promoter activity was down-regulated by co-transfection with C/EBP expression vectors. Mutation of the C/EBP motif abolished the inhibitory response to IL-1b. Additionally, C/EBP expression vectors were found to down-regulate the construct containing the promoter and enhancer of the type II collagen gene. Lastly, the enhancer factor, Sox9, was shown to bind adjacent to the C/EBP site competing with C/EBP binding. Taken together, these results suggest that C/EBPb and d may play an important role in the IL-1b-induced repression of cartilage-specific proteins and that expression of matrix proteins will be influenced by availability of positive and negative trans-acting factors.
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