To determine the function of CCAAT/enhancer binding protein beta (C/EBPbeta) in the expression of matrix metalloproteinase 13 (MMP-13) in chondrocytes in inflammatory arthritis.
Cartilage obtained from patients with rheumatoid arthritis and osteoarthritis was immunostained for expression of C/EBPbeta or MMP-13. Interleukin-1beta- or tumor necrosis factor alpha (TNFalpha)-stimulated chondrocytes were subjected to Western blotting and real-time reverse transcriptase-polymerase chain reaction (RT-PCR). MMP-13 promoter assays were conducted, and the C/EBPbeta response element was characterized by deletion and mutation analysis. C-28/I2 cells were treated with TNFalpha and subjected to chromatin immunoprecipitation (ChIP) assays. Finally, C/EBPbeta-liver-enriched activator protein (LAP) was overexpressed in C-28/I2 cells or cartilage tissues, and MMP-13 expression was analyzed.
C/EBPbeta and MMP-13 expression was colocalized in chondrocytes in arthritic cartilage. MMP-13 promoter activity was stimulated by C/EBPbeta overexpression in a dose-dependent manner. Luciferase assays revealed that a -981-bp promoter had the greatest activity, while deletion to -936 bp strongly diminished promoter activity. Luciferase activity was repressed to basal levels by mutations in potential C/EBP binding sites. The stimulatory effects of C/EBPbeta overexpression were diminished by mutation. ChIP assays revealed that TNFalpha treatment enhanced the binding of C/EBPbeta to the MMP-13 promoter. When C/EBPbeta-LAP was overexpressed in C-28/I2 cells, endogenous MMP-13 expression was stimulated up to 32-fold as detected by real-time RT-PCR. Furthermore, following adenoviral overexpression of C/EBPbeta-LAP in organ culture of articular cartilage, stimulation of MMP-13 was also detected by immunohistochemistry.
C/EBPbeta directly binds to the MMP-13 promoter region and stimulates the expression of MMP-13 in chondrocytes in inflammatory arthritis.