CCAAT/enhancer-binding proteins mediate the repression of transcription of cartilage-derived retinoic acid-sensitive protein induced by IL-1β

Introduction

IL-1β is one of the major proinflammatory cytokines involved in arthritis joints. IL-1β promotes the arachidonic acid cascade, resulting in production of prostaglandin. In addition, IL-1β represses the expression of matrix proteins in cartilage, such as type II, type IX and type XI collagen and cartilage-derived retinoic acid-sensitive protein (CD-RAP), leading to degradation of the cartilage structures.

Objective

To investigate the transcriptional mechanism by which CD-RAP expression is repressed by IL-1β.

Methods and results

Deletion constructs of the CD-RAP promoter were transfected into rat chondrocytes and incubated in the absence or presence of IL-1β. The results revealed an IL-1β-responsive element located between -2138 and -2068 bp. As this element contains the CCAAT/enhancer-binding protein (C/EBP) motif, the function of C/EBP-β was examined. IL-1β stimulated the expression of C/EBP-β. The direct binding of C/EBP-β to the C/EBP motif was confirmed by electrophoretic mobility shift assay. Expression of the -2251 bp CD-RAP promoter construct was downregulated by cotransfection with C/EBP-β expression vectors in a dose-dependent manner. Mutation of the C/EBP motif within the -2251 bp construct abolished the inhibitory response to IL-1β. These results suggest that C/EBP-β is a critical factor mediating IL-1β-induced repression of CD-RAP transcription. C/EBP-β expression vectors were also found to downregulate the reporter construct containing the promoter and enhancer of the type II collagen gene. The IL-1β-induced repression of CD-RAP and type II collagen genes via stimulation of C/EBP-β were confirmed in chondrocytes from normal human articular cartilage. Finally, the enhancer factor, Sox9, known to be downregulated by IL-1, was shown to bind adjacent to the C/EBP site competing with C/EBP binding.

Discussion

C/EBP-β is known to mediate the arachidonic acid cascade induced by IL-1β. These results suggest that C/EBP-β play an important role in the distinct effects of IL-1β : the promotion of inflammatory reaction and the repression of cartilage-specific proteins in joint disease. Expression of matrix proteins are influenced by availability of both positive and negative transacting factors.