title: Fibronectin fragment-induced expression of matrix metalloproteinases is mediated by MyD88-dependent TLR-2 signaling pathway in human chondrocytes.
abstract: BACKGROUND
  Fibronectin fragments (FN-fs) are increased in the cartilage of patients with osteoarthritis (OA) and have a potent chondrolytic effect. However, little is known about the cellular receptors and signaling mechanisms that are mediated by FN-fs. We investigated whether the 29-kDa amino-terminal fibronectin fragment (29-kDa FN-f) regulates cartilage catabolism via the Toll-like receptor (TLR)-2 signaling pathway in human chondrocytes.
  
  
  METHODS
  Small interfering RNA was used to knock down TLR-2 and myeloid differentiation factor 88 (MyD88). TLR-2 was overexpressed in chondrocytes transfected with a TLR-2 expression plasmid. The expression levels of matrix metalloproteinase (MMP)-1, MMP-3, and MMP-13 were analyzed using quantitative real-time reverse transcription polymerase chain reactions, immunoblotting, or enzyme-linked immunosorbent assay. The effect of TLR-2 on 29-kDa FN-f-mediated signaling pathways was investigated by immunoblotting.
  
  
  RESULTS
  TLR-2, TLR-3, TLR-4, and TLR-5 mRNA were significantly overexpressed in OA cartilage compared with normal cartilage, whereas no significant difference of TLR-1 mRNA expression was found. 29-kDa FN-f significantly increased TLR-2 expression in human chondrocytes in a dose- and time-dependent manner. Knockdown of TLR-2 or MyD88, the latter a downstream adaptor of TLR-2, significantly inhibited 29-kDa FN-f-induced MMP production at the mRNA and protein levels. Conversely, TLR-2 overexpression led to enhanced MMP production by 29-kDa FN-f. In addition, TLR-2 knockdown apparently inhibited 29-kDa FN-f-mediated activation of phosphorylated nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha, and p38, but not of c-Jun N-terminal kinase or extracellular signal-regulated kinase. Exposure to synovial fluid (SF) from affected joints of patients with OA elevated MMP-1, MMP-3, and MMP-13 expression markedly in primary chondrocytes without reducing cell viability. However, TLR-2 knockdown in chondrocytes significantly suppressed SF-induced MMP induction.
  
  
  CONCLUSIONS
  Our data demonstrate that the MyD88-dependent TLR-2 signaling pathway may be responsible for 29-kDa FN-f-mediated cartilage catabolic responses. Our results will enhance understanding of cartilage catabolic mechanisms driven by cartilage degradation products, including FN-f. The modulation of TLR-2 signaling activated by damage-associated molecular patterns, including 29-kDa FN-f, is a potential therapeutic strategy for the prevention of cartilage degradation in OA.
authors.affiliation: Division of rheumatology, Department of Internal Medicine, Hallym University Sacred Heart Hospital, 896, Pyungchon, Anyang, Kyunggi, 431-070, Korea. wazzup@hallym.ac.kr.|Institute for Skeletal Aging, Hallym University, Chunchon, 200-702, Korea. wazzup@hallym.ac.kr.
authors.forename: Hyun Sook
authors.initials: HS
authors.lastname: Hwang
authors.affiliation: Division of rheumatology, Department of Internal Medicine, Hallym University Sacred Heart Hospital, 896, Pyungchon, Anyang, Kyunggi, 431-070, Korea. sujin153@naver.com.
authors.forename: Su Jin
authors.initials: SJ
authors.lastname: Park
authors.affiliation: Division of rheumatology, Department of Internal Medicine, Hallym University Sacred Heart Hospital, 896, Pyungchon, Anyang, Kyunggi, 431-070, Korea. jjaymedi@gmail.com.|Institute for Skeletal Aging, Hallym University, Chunchon, 200-702, Korea. jjaymedi@gmail.com.
authors.forename: Eun Jeong
authors.initials: EJ
authors.lastname: Cheon
authors.affiliation: Division of rheumatology, Department of Internal Medicine, Hallym University Sacred Heart Hospital, 896, Pyungchon, Anyang, Kyunggi, 431-070, Korea. cride02@naver.com.|Institute for Skeletal Aging, Hallym University, Chunchon, 200-702, Korea. cride02@naver.com.
authors.forename: Mi Hyun
authors.initials: MH
authors.lastname: Lee
authors.affiliation: Division of rheumatology, Department of Internal Medicine, Hallym University Sacred Heart Hospital, 896, Pyungchon, Anyang, Kyunggi, 431-070, Korea. kimha@hallym.ac.kr.|Institute for Skeletal Aging, Hallym University, Chunchon, 200-702, Korea. kimha@hallym.ac.kr.
authors.forename: Hyun Ah
authors.initials: HA
authors.lastname: Kim
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D005353:Fibronectins / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008297:Male; D020782:Matrix Metalloproteinases / Q000096:biosynthesis*; D008875:Middle Aged; D053594:Myeloid Differentiation Factor 88 / Q000096:biosynthesis*; D010446:Peptide Fragments / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D051195:Toll-Like Receptor 2 / Q000096:biosynthesis*
chemical_list: D005353:Fibronectins; C507779:MYD88 protein, human; D053594:Myeloid Differentiation Factor 88; D010446:Peptide Fragments; C495342:TLR2 protein, human; D051195:Toll-Like Receptor 2; D020782:Matrix Metalloproteinases