title: Aggrecan degradation in human articular cartilage explants is mediated by both ADAMTS-4 and ADAMTS-5.
abstract: OBJECTIVE
  Recent published studies have shown that cartilage from ADAMTS-5-knockout mice, but not ADAMTS-4- or ADAMTS-1-knockout mice, is significantly protected from degradation. The present study was undertaken to evaluate the respective roles of these enzymes in human cartilage breakdown, using a small interfering RNA (siRNA) approach to assess the effects of inhibition of each enzyme in normal and osteoarthritic (OA) explants.
  
  
  METHODS
  The activities of siRNA specifically targeting ADAMTS-1, -4, and -5 were assessed by transfection into primary human chondrocytes and cultured human cartilage explants. At 24 hours, a cytokine stimulus was applied to normal, but not OA, samples to initiate a catabolic response. At designated times, total RNA was isolated and gene expression was measured by quantitative real-time reverse transcription-polymerase chain reaction. Aggrecan release and aggrecanase-generated neoepitope formation were determined by dye binding analysis and Western blotting, respectively.
  
  
  RESULTS
  Human chondrocytes and explants were efficiently transfected with siRNA that specifically decreased the expression of each targeted gene. Suppression of ADAMTS-4 and ADAMTS-5, individually or in combination, attenuated the degradation of aggrecan in cytokine-stimulated normal cartilage. A reduction in aggrecan degradation was also observed following siRNA-mediated knockdown of either gene in unstimulated OA cartilage. In contrast, knockdown of ADAMTS-1 failed to inhibit aggrecan loss.
  
  
  CONCLUSIONS
  Despite the apparent dominant role of ADAMTS-5 in genetically modified mice, our data suggest that both ADAMTS-4 and ADAMTS-5 contribute to the structural damage that characterizes human OA.
authors.affiliation: Pfizer Global Research and Development, St Louis, MO 63017, USA.
authors.forename: Ruo-Hua
authors.initials: RH
authors.lastname: Song
authors.forename: Micky D
authors.initials: MD
authors.lastname: Tortorella
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Malfait
authors.forename: James T
authors.initials: JT
authors.lastname: Alston
authors.forename: Zhiyong
authors.initials: Z
authors.lastname: Yang
authors.forename: Elizabeth C
authors.initials: EC
authors.lastname: Arner
authors.forename: David W
authors.initials: DW
authors.lastname: Griggs
mesh_terms: D051722:ADAM Proteins / Q000235:genetics / Q000378:metabolism*; D000071097:ADAMTS1 Protein; D000071121:ADAMTS4 Protein; D000071116:ADAMTS5 Protein; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D053674:Aggrecans / Q000378:metabolism*; D002358:Cartilage, Articular / Q000166:cytology / Q000378:metabolism* / Q000473:pathology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism / Q000473:pathology; D005786:Gene Expression Regulation; D020868:Gene Silencing; D006801:Humans; D008297:Male; D008875:Middle Aged; D010003:Osteoarthritis / Q000378:metabolism* / Q000473:pathology; D011348:Procollagen N-Endopeptidase / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D053674:Aggrecans; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D051722:ADAM Proteins; D000071097:ADAMTS1 Protein; C103859:ADAMTS1 protein, human; D000071116:ADAMTS5 Protein; C121532:ADAMTS5 protein, human; D011348:Procollagen N-Endopeptidase; D000071121:ADAMTS4 Protein; C000603918:ADAMTS4 protein, human; C000603919:Adamts4 protein, mouse