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CCAAT/enhancer binding protein β regulates expression of matrix metalloproteinase-3 in arthritis
  1. Hidetoshi Tsushima,
  2. Ken Okazaki,
  3. Mitsumasa Hayashida,
  4. Takahiro Ushijima,
  5. Yukihide Iwamoto
  1. Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
  1. Correspondence to Dr Ken Okazaki, Department of Orthopaedic Surgery, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812–8582, Japan; okazaki{at}ortho.med.kyushu-u.ac.jp

Abstract

Objectives To investigate whether CCAAT/enhancer binding protein β (C/EBPβ) mediates the expression of matrix metalloproteinase-3 (MMP-3) and aggrecanases in arthritis.

Methods Localisation of C/EBPβ and MMP-3 in synovium and cartilage from patients with rheumatoid arthritis and osteoarthritis was determined by immunohistochemistry. Cell lines SW982, C28/I2 and human fibroblast-like synoviocytes stimulated by interleukin 1β (IL-1β) were subjected to western blotting and quantitative PCR. Overexpression of C/EBPβ by adenovirus was performed in cells and organ culture of normal cartilage. Knockdown of C/EBPβ by small interference RNA was performed in cells. Activity of the human MMP-3 and aggrecanase-2 ADAMTS-5 (a disintegrin and metalloproteinase with thrombospondin motifs) promoters was analysed by a luciferase assay. To determine whether C/EBPβ directly binds to the MMP-3 or ADAMTS-5 promoter,a chromatin immunoprecipitation assay was performed.

Results Immunohistochemistry showed that C/EBPβ and MMP-3 were co-localised in arthritic synovium and cartilage. Western blots revealed increased C/EBPβ expression in cells treated with IL-1β. Expression of MMP-3, MMP-13 and ADAMTS-5 mRNA was significantly increased by the overexpression of C/EBPβ. C/EBPβ stimulated MMP-3 expression and induced matrix degradation in cartilage explants. C/EBPβ knockdown reduced MMP-3 and ADAMTS-5 expression. C/EBPβ stimulated the 2011 bp MMP-3 promoter and the 1768 bp ADAMTS-5 promoter in a dose-dependent manner. Deletion and mutation analysis of the MMP-3 promoter showed that the C/EBPβ core responsive element was located between −108 bp and −100 bp. The chromatin immunoprecipitation assay showed that C/EBPβ was directly bound to MMP-3 and ADAMTS-5 promoters.

Conclusions These data demonstrate that C/EBPβ is involved in expression of MMP-3 and ADAMTS-5 in arthritic synovium and cartilage.

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Footnotes

  • Funding This work was supported in part by a Grant-in-Aid for Scientific Research (No. 19591758) from the Japan Society for the Promotion of Science and Research Grant for Intractable Diseases from the Ministry of Health and Welfare of Japan.

  • Ethical approval All studies were carried out with the approval of the University Institutional Ethics Board and in accordance with the tenets of the Declaration of Helsinki.

  • Competing interests None.

  • Provenance and peer review Not commissioned; internally peer reviewed.

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