Lentiviral shRNA knock-down of ADAMTS-5 and -9 restores matrix deposition in 3D chondrocyte culture.
Aggrecan is one of the two major constituents of articular cartilage, and during diseases such as osteoarthritis (OA) it is subject to degradation by proteolytic enzymes. The primary proteases responsible for aggrecan cleavage are the aggrecanases, identified as members of the ADAMTS family of proteases, which are upregulated in response to inflammatory stimuli. It is uncertain which of the six aggrecanases (ADAMTS-1, -4, -5, -8, -9 and -15) are primarily responsible for the degradation of aggrecan in human cartilage. Here we show that four of the six aggrecanases are expressed in immortalized chondrocyte cell-lines and can be upregulated in response to inflammatory cytokines. Using RNA interference, we demonstrate robust knock-down of ADAMTS-5 and -9 expression in these cells and, by culturing them on three-dimensional (3D) scaffolds, show that reduction in expression of ADAMTS-5 enzyme results in an increase in matrix deposition. These data suggest that the quality of tissue-engineered cartilage matrix might be improved by targeted depletion of aggrecanase expression. Moreover, this work also provides further evidence that ADAMTS-5 may be a therapeutic target in the treatment of arthritic disease.
Coughlan
Teresa C
TC
Department of Infection and Immunity, University of Sheffield Medical School, UK. teresa.c.coughlan@nottingham.ac.uk
Crawford
Aileen
A
Goldring
Mary B
MB
Hatton
Paul V
PV
Barker
Michael D
MD
D051722:ADAM Proteins / Q000235:genetics / Q000378:metabolism*; D000071116:ADAMTS5 Protein; D000071119:ADAMTS9 Protein; D002460:Cell Line; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D019902:Chondrocytes / Q000201:enzymology*; D005109:Extracellular Matrix / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D055785:Gene Knockdown Techniques; D006025:Glycosaminoglycans / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D054457:Tissue Scaffolds
D006025:Glycosaminoglycans; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D051722:ADAM Proteins; D000071116:ADAMTS5 Protein; C121532:ADAMTS5 protein, human; D000071119:ADAMTS9 Protein; C412869:ADAMTS9 protein, human