CCAAT/enhancer binding protein beta mediates expression of matrix metalloproteinase 13 in human articular chondrocytes in inflammatory arthritis.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
C/EBPbeta directly binds to the MMP-13 promoter region and stimulates the expression of MMP-13 in chondrocytes in inflammatory arthritis.
Hayashida
Mitsumasa
M
Kyushu University, Fukuoka, Japan.
Okazaki
Ken
K
Fukushi
Junichi
J
Sakamoto
Akio
A
Iwamoto
Yukihide
Y
D000368:Aged; D000369:Aged, 80 and over; D001172:Arthritis, Rheumatoid / Q000378:metabolism* / Q000473:pathology; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000378:metabolism*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D053583:Interleukin-1beta / Q000494:pharmacology; D007719:Knee Joint / Q000378:metabolism* / Q000473:pathology; D053509:Matrix Metalloproteinase 13 / Q000378:metabolism*; D008875:Middle Aged; D020370:Osteoarthritis, Knee / Q000378:metabolism* / Q000473:pathology; D011401:Promoter Regions, Genetic; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D022782:CCAAT-Enhancer-Binding Protein-beta; D053583:Interleukin-1beta; D014409:Tumor Necrosis Factor-alpha; D053509:Matrix Metalloproteinase 13