title: Interleukin-1 Acts via the JNK-2 Signaling Pathway to Induce Aggrecan Degradation by Human Chondrocytes.
abstract: OBJECTIVE
  Aggrecan enables articular cartilage to bear load and resist compression. Aggrecan loss occurs early in osteoarthritis and rheumatoid arthritis and can be induced by inflammatory cytokines such as interleukin-1 (IL-1). IL-1 induces cleavage of specific aggrecans characteristic of the ADAMTS proteinases. The aim of this study was to identify the intracellular signaling pathways by which IL-1 causes aggrecan degradation by human chondrocytes and to investigate how aggrecanase activity is controlled by chondrocytes.
  
  
  METHODS
  We developed a cell-based assay combining small interfering RNA (siRNA)-induced knockdown with aggrecan degradation assays. Human articular chondrocytes were overlaid with bovine aggrecan after transfection with siRNAs against molecules of the IL-1 signaling pathway. After IL-1 stimulation, released aggrecan fragments were detected with AGEG and ARGS neoepitope antibodies. Aggrecanase activity and tissue inhibitor of metalloproteinases 3 levels were measured by enzyme-linked immunosorbent assay. Low-density lipoprotein receptor-related protein 1 (LRP-1) shedding was analyzed by Western blotting.
  
  
  RESULTS
  ADAMTS-5 is a major aggrecanase in human chondrocytes, regulating aggrecan degradation in response to IL-1. The tumor necrosis factor receptor-associated 6 (TRAF-6)/transforming growth factor β-activated kinase 1 (TAK-1)/MKK-4 signaling axis is essential for IL-1-induced aggrecan degradation, while NF-κB is not. Of the 3 MAPKs (ERK, p38, and JNK), only JNK-2 showed a significant role in aggrecan degradation. Chondrocytes constitutively secreted aggrecanase, which was continuously endocytosed by LRP-1, keeping the extracellular level of aggrecanase low. IL-1 induced aggrecanase activity in the medium in a JNK-2-dependent manner, possibly by reducing aggrecanase endocytosis, because IL-1 caused JNK-2-dependent shedding of LRP-1.
  
  
  CONCLUSIONS
  The signaling axis TRAF-6/TAK-1/MKK-4/JNK-2 mediates IL-1-induced aggrecanolysis. The level of aggrecanase is controlled by its endocytosis, which may be reduced upon IL-1 stimulation because of LRP-1 shedding.
authors.affiliation: Kennedy Institute of Rheumatology and University of Oxford, Oxford, UK.
authors.forename: Heba M
authors.initials: HM
authors.lastname: Ismail
authors.affiliation: Kennedy Institute of Rheumatology and University of Oxford, Oxford, UK.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Yamamoto
authors.affiliation: Kennedy Institute of Rheumatology and University of Oxford, Oxford, UK.
authors.forename: Tonia L
authors.initials: TL
authors.lastname: Vincent
authors.affiliation: Kennedy Institute of Rheumatology and University of Oxford, Oxford, UK.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Nagase
authors.affiliation: Kennedy Institute of Rheumatology and University of Oxford, Oxford, UK.
authors.forename: Linda
authors.initials: L
authors.lastname: Troeberg
authors.affiliation: Kennedy Institute of Rheumatology and University of Oxford, Oxford, UK.
authors.forename: Jeremy
authors.initials: J
authors.lastname: Saklatvala
mesh_terms: D051722:ADAM Proteins / Q000502:physiology; D000071116:ADAMTS5 Protein; D053674:Aggrecans / Q000378:metabolism*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology / Q000502:physiology*; D026503:Low Density Lipoprotein Receptor-Related Protein-1 / Q000378:metabolism; D048670:MAP Kinase Kinase 4 / Q000502:physiology; D048688:MAP Kinase Kinase 7 / Q000502:physiology*; D020930:MAP Kinase Kinase Kinases / Q000502:physiology; D034741:RNA, Small Interfering / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology*; D048029:TNF Receptor-Associated Factor 6 / Q000502:physiology; D000099151:MAP Kinase Kinase Kinase 7
chemical_list: D051722:ADAM Proteins; D000071116:ADAMTS5 Protein; D053674:Aggrecans; D007375:Interleukin-1; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D048670:MAP Kinase Kinase 4; D048688:MAP Kinase Kinase 7; D020930:MAP Kinase Kinase Kinases; D034741:RNA, Small Interfering; D048029:TNF Receptor-Associated Factor 6; D000099151:MAP Kinase Kinase Kinase 7; C475476:LRP1 protein, human; C482559:MAP2K4 protein, human; C121532:ADAMTS5 protein, human