title: Depletion of SIRT6 causes cellular senescence, DNA damage, and telomere dysfunction in human chondrocytes.
abstract: OBJECTIVE
  SIRT6, a member of the sirtuin family of nicotinamide adenine dinucleotide (NAD(+))-dependent protein deacetylases, has been implicated as a key factor in aging-related diseases. However, the role of SIRT6 in chondrocytes has not been fully explored. The purpose of this study was to examine the role of SIRT6 in human chondrocytes by inhibiting SIRT6 in vitro.
  
  
  METHODS
  First, the localization of SIRT6 and proliferation cell nuclear antigen (PCNA) in human cartilages was examined by immunohistochemistry. Next, SIRT6 was depleted by RNA interference (RNAi), and the effect of SIRT6 depletion on changes in gene expression, protein levels, proliferation, and senescence in human chondrocytes was assessed. Furthermore, to detect DNA damage and telomere dysfunction, γH2AX foci and telomere dysfunction-induced foci (TIFs) were examined using immunofluorescence microscopy. The protein levels of two mediators for DNA damage induced-senescence, p16 and p21, were examined by western blotting.
  
  
  RESULTS
  Immunohistochemical analysis showed SIRT6 was preferentially expressed in the superficial zone chondrocytes and PCNA-positive cluster-forming chondrocytes in the osteoarthritic cartilage tissue samples. Real-time PCR analysis showed that matrix metalloproteinase 1 (MMP-1) and MMP-13 mRNA were significantly increased by SIRT6 inhibition. Moreover, SIRT6 inhibition significantly reduced proliferation and increased senescence associated β-galactosidase (SA-β-Gal)-positive chondrocytes; it also led to increased p16 levels. Immunofluorescence microscopy showed that γH2AX foci and TIFs were increased by SIRT6 inhibition.
  
  
  CONCLUSIONS
  Depletion of SIRT6 in human chondrocytes caused increased DNA damage and telomere dysfunction, and subsequent premature senescence. These findings suggest that SIRT6 plays an important role in the regulation of senescence of human chondrocytes.
authors.affiliation: Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan. Electronic address: kant_kantona@yahoo.co.jp.
authors.forename: K
authors.initials: K
authors.lastname: Nagai
authors.affiliation: Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan. Electronic address: matsushi@kobe-u.ac.jp.
authors.forename: T
authors.initials: T
authors.lastname: Matsushita
authors.affiliation: Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan. Electronic address: tokiomatsuzaki@yahoo.co.jp.
authors.forename: T
authors.initials: T
authors.lastname: Matsuzaki
authors.affiliation: Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan. Electronic address: kojitakayama1978@gmail.com.
authors.forename: K
authors.initials: K
authors.lastname: Takayama
authors.affiliation: Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan. Electronic address: matsun@m4.dion.ne.jp.
authors.forename: T
authors.initials: T
authors.lastname: Matsumoto
authors.affiliation: Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan. Electronic address: kurodar@med.kobe-u.ac.jp.
authors.forename: R
authors.initials: R
authors.lastname: Kuroda
authors.affiliation: Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan. Electronic address: kurosaka@med.kobe-u.ac.jp.
authors.forename: M
authors.initials: M
authors.lastname: Kurosaka
mesh_terms: D049109:Cell Proliferation; D002478:Cells, Cultured; D016922:Cellular Senescence; D019902:Chondrocytes / Q000378:metabolism / Q000473:pathology*; D004249:DNA Damage; D006657:Histones / Q000378:metabolism; D006801:Humans; D020781:Matrix Metalloproteinase 1 / Q000235:genetics / Q000378:metabolism; D053509:Matrix Metalloproteinase 13 / Q000235:genetics / Q000378:metabolism; D020370:Osteoarthritis, Knee / Q000473:pathology; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D037761:Sirtuins / Q000172:deficiency* / Q000235:genetics; D016615:Telomere; D015854:Up-Regulation; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: C468783:H2AX protein, human; D006657:Histones; D012333:RNA, Messenger; D001616:beta-Galactosidase; D053509:Matrix Metalloproteinase 13; D020781:Matrix Metalloproteinase 1; C516592:SIRT6 protein, human; D037761:Sirtuins
keywords: Chondrocyte; DNA damage; SIRT6; Senescence; Sirtuin; Telomere dysfunction