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

Mitsumasa Hayashida, Ken Okazaki, Jun-Ichi Fukushi, Akio Sakamoto, Yukihide Iwamoto

Research output: Contribution to journalArticle

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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 over-expression 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.

Original languageEnglish
Pages (from-to)708-716
Number of pages9
JournalArthritis and rheumatism
Volume60
Issue number3
DOIs
Publication statusPublished - Mar 1 2009

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CCAAT-Enhancer-Binding Proteins
Matrix Metalloproteinase 13
Chondrocytes
Arthritis
Joints
Cartilage
Tumor Necrosis Factor-alpha
Chromatin Immunoprecipitation
Luciferases
Reverse Transcriptase Polymerase Chain Reaction
Real-Time Polymerase Chain Reaction
Liver
Mutation
Proteins
Sequence Deletion
Organ Culture Techniques
Response Elements
Articular Cartilage
Interleukin-1
Genetic Promoter Regions

All Science Journal Classification (ASJC) codes

  • Immunology
  • Immunology and Allergy
  • Rheumatology
  • Pharmacology (medical)

Cite this

CCAAT/enhancer binding protein β mediates expression of matrix metalloproteinase 13 in human articular chondrocytes in inflammatory arthritis. / Hayashida, Mitsumasa; Okazaki, Ken; Fukushi, Jun-Ichi; Sakamoto, Akio; Iwamoto, Yukihide.

In: Arthritis and rheumatism, Vol. 60, No. 3, 01.03.2009, p. 708-716.

Research output: Contribution to journalArticle

Hayashida, Mitsumasa ; Okazaki, Ken ; Fukushi, Jun-Ichi ; Sakamoto, Akio ; Iwamoto, Yukihide. / CCAAT/enhancer binding protein β mediates expression of matrix metalloproteinase 13 in human articular chondrocytes in inflammatory arthritis. In: Arthritis and rheumatism. 2009 ; Vol. 60, No. 3. pp. 708-716.
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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 over-expression 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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T1 - CCAAT/enhancer binding protein β mediates expression of matrix metalloproteinase 13 in human articular chondrocytes in inflammatory arthritis

AU - Hayashida, Mitsumasa

AU - Okazaki, Ken

AU - Fukushi, Jun-Ichi

AU - Sakamoto, Akio

AU - Iwamoto, Yukihide

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N2 - 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 over-expression 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.

AB - 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 over-expression 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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