CCAAT/ENHANCER binding protein β mediates expression of matrix metalloproteinase 13 in human articular chondrocytes in inflammatory arthritis

Authors


Abstract

Objective

To determine the function of CCAAT/enhancer binding protein β (C/EBPβ) in the expression of matrix metalloproteinase 13 (MMP-13) in chondrocytes in inflammatory arthritis.

Methods

Cartilage obtained from patients with rheumatoid arthritis and osteoarthritis was immunostained for expression of C/EBPβ or MMP-13. Interleukin-1β– or tumor necrosis factor α (TNFα)–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/EBPβ response element was characterized by deletion and mutation analysis. C-28/I2 cells were treated with TNFα and subjected to chromatin immunoprecipitation (ChIP) assays. Finally, C/EBPβ–liver-enriched activator protein (LAP) was overexpressed in C-28/I2 cells or cartilage tissues, and MMP-13 expression was analyzed.

Results

C/EBPβ and MMP-13 expression was colocalized in chondrocytes in arthritic cartilage. MMP-13 promoter activity was stimulated by C/EBPβ 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/EBPβ overexpression were diminished by mutation. ChIP assays revealed that TNFα treatment enhanced the binding of C/EBPβ to the MMP-13 promoter. When C/EBPβ-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/EBPβ-LAP in organ culture of articular cartilage, stimulation of MMP-13 was also detected by immunohistochemistry.

Conclusion

C/EBPβ directly binds to the MMP-13 promoter region and stimulates the expression of MMP-13 in chondrocytes in inflammatory arthritis.

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