title: Lithium Chloride Dependent Glycogen Synthase Kinase 3 Inactivation Links Oxidative DNA Damage, Hypertrophy and Senescence in Human Articular Chondrocytes and Reproduces Chondrocyte Phenotype of Obese Osteoarthritis Patients.
abstract: BACKGROUND
  Recent evidence suggests that GSK3 activity is chondroprotective in osteoarthritis (OA), but at the same time, its inactivation has been proposed as an anti-inflammatory therapeutic option. Here we evaluated the extent of GSK3β inactivation in vivo in OA knee cartilage and the molecular events downstream GSK3β inactivation in vitro to assess their contribution to cell senescence and hypertrophy.
  
  
  METHODS
  In vivo level of phosphorylated GSK3β was analyzed in cartilage and oxidative damage was assessed by 8-oxo-deoxyguanosine staining. The in vitro effects of GSK3β inactivation (using either LiCl or SB216763) were evaluated on proliferating primary human chondrocytes by combined confocal microscopy analysis of Mitotracker staining and reactive oxygen species (ROS) production (2',7'-dichlorofluorescin diacetate staining). Downstream effects on DNA damage and senescence were investigated by western blot (γH2AX, GADD45β and p21), flow cytometric analysis of cell cycle and light scattering properties, quantitative assessment of senescence associated β galactosidase activity, and PAS staining.
  
  
  RESULTS
  In vivo chondrocytes from obese OA patients showed higher levels of phosphorylated GSK3β, oxidative damage and expression of GADD45β and p21, in comparison with chondrocytes of nonobese OA patients. LiCl mediated GSK3β inactivation in vitro resulted in increased mitochondrial ROS production, responsible for reduced cell proliferation, S phase transient arrest, and increase in cell senescence, size and granularity. Collectively, western blot data supported the occurrence of a DNA damage response leading to cellular senescence with increase in γH2AX, GADD45β and p21. Moreover, LiCl boosted 8-oxo-dG staining, expression of IKKα and MMP-10.
  
  
  CONCLUSIONS
  In articular chondrocytes, GSK3β activity is required for the maintenance of proliferative potential and phenotype. Conversely, GSK3β inactivation, although preserving chondrocyte survival, results in functional impairment via induction of hypertrophy and senescence. Indeed, GSK3β inactivation is responsible for ROS production, triggering oxidative stress and DNA damage response.
authors.affiliation: Laboratorio di Immunoreumatologia e Rigenerazione Tessutale, Istituto Ortopedico Rizzoli, Bologna, Italy.|Dipartimento di Scienze Mediche e Chirurgiche-DIMEC, Università di Bologna, Bologna, Italy.
authors.forename: Serena
authors.initials: S
authors.lastname: Guidotti
authors.affiliation: Laboratorio di Immunoreumatologia e Rigenerazione Tessutale, Istituto Ortopedico Rizzoli, Bologna, Italy.|Dipartimento di Scienze Mediche e Chirurgiche-DIMEC, Università di Bologna, Bologna, Italy.
authors.forename: Manuela
authors.initials: M
authors.lastname: Minguzzi
authors.affiliation: Laboratorio di Immunoreumatologia e Rigenerazione Tessutale, Istituto Ortopedico Rizzoli, Bologna, Italy.|Dipartimento di Scienze Mediche e Chirurgiche-DIMEC, Università di Bologna, Bologna, Italy.|Dipartimento di Scienze Biomediche e Neuromotorie-DIBINEM, Università di Bologna, Bologna, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Platano
authors.affiliation: Laboratorio di Immunoreumatologia e Rigenerazione Tessutale, Istituto Ortopedico Rizzoli, Bologna, Italy.|Dipartimento RIT, Laboratorio RAMSES, Istituto Ortopedico Rizzoli, Bologna, Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Cattini
authors.affiliation: Chirurgia ricostruttiva articolare dell'anca e del ginocchio, Istituto Ortopedico Rizzoli, Bologna, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Trisolino
authors.affiliation: Laboratorio di Immunoreumatologia e Rigenerazione Tessutale, Istituto Ortopedico Rizzoli, Bologna, Italy.|Dipartimento di Scienze Mediche e Chirurgiche-DIMEC, Università di Bologna, Bologna, Italy.|Dipartimento RIT, Laboratorio RAMSES, Istituto Ortopedico Rizzoli, Bologna, Italy.
authors.forename: Erminia
authors.initials: E
authors.lastname: Mariani
authors.affiliation: Laboratorio di Immunoreumatologia e Rigenerazione Tessutale, Istituto Ortopedico Rizzoli, Bologna, Italy.|Dipartimento RIT, Laboratorio RAMSES, Istituto Ortopedico Rizzoli, Bologna, Italy.
authors.forename: Rosa Maria
authors.initials: RM
authors.lastname: Borzì
mesh_terms: D049109:Cell Proliferation / Q000187:drug effects; D048429:Cell Size / Q000187:drug effects; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000187:drug effects; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects* / Q000473:pathology; D004249:DNA Damage; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D018021:Lithium Chloride / Q000494:pharmacology*; D008301:Maleimides / Q000494:pharmacology; D009765:Obesity / Q000201:enzymology / Q000473:pathology*; D020370:Osteoarthritis, Knee / Q000201:enzymology / Q000473:pathology*; D018384:Oxidative Stress / Q000187:drug effects; D010766:Phosphorylation / Q000187:drug effects
chemical_list: D007211:Indoles; D008301:Maleimides; C417521:SB 216763; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3; D018021:Lithium Chloride