Identification in human osteoarthritic chondrocytes of proteins binding to the novel regulatory site AGRE in the human matrix metalloprotease 13 proximal promoter

Zhiyong Fan, Ginette Tardif, Christelle Boileau, Joseph P. Bidwell, Changshan Geng, David Hum, Alexander Watson, Jean Pierre Pelletier, Martin Lavigne, Johanne Martel-Pelletier

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Abstract

Objective. Matrix metalloprotease 13 (MMP-13) plays a major role in osteoarthritic (OA) processes. We previously identified the AG-rich element (AGRE) regulatory site (GAAAAGAAAAAG) in the proximal promoter of this gene. Electrophoretic mobility shift assays (EMSAs) done with nuclear extracts from OA chondrocytes showed the presence of 2 AGRE protein-binding complexes, the formation of which depended on the pathophysiologic state (high or low) of the cells; the low OA (L-OA) chondrocytes have low MMP-13 basal levels and high interleukin-1β (IL-1β) inducibility, and the high OA (H-OA) chondrocytes have high MMP-13 basal levels and low IL-1β inducibility. In this study, we sought to determine the importance of individual AGRE bases in promoter activity and to identify AGRE binding proteins from L-OA and H-OA chondrocyte complexes. Methods. Promoter activity was determined following transient transfection into human OA chondrocytes. AGRE binding proteins were identified by mass spectroscopy. Results. Individual mutations of the AGRE site differentially modulated promoter activity, indicating that the intact AGRE site is required for optimal MMP-13 expression. Damage-specific DNA binding protein 1 (DDB-1) was identified in the L-OA chondrocyte-binding complex. EMSA experiments performed with the mutation of the left AGRE site (GTGCTGAAAAAG) and nuclear extracts of L-OA chondrocytes reproduced the pattern seen in the H-OA chondrocytes. Mass spectroscopy identified p130cas as one of the proteins in this complex. Supershift experiments showed the presence of p130cas and nuclear matrix transcription factor 4 (NMP-4) in the wild-type AGRE/H-OA chondrocyte complex. Conclusion. These data suggest that the binding of p130cas and NMP-4 to the ACRE site regulates MMP-13 expression and may trigger the change in human chondrocytes from the L-OA state to the H-OA state.

Original languageEnglish (US)
Pages (from-to)2471-2480
Number of pages10
JournalArthritis and Rheumatism
Volume54
Issue number8
DOIs
StatePublished - Aug 1 2006

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ASJC Scopus subject areas

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

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