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: 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 GSK3? inactivation in vivo in OA knee cartilage and the molecular events downstream GSK3? inactivation in vitro to assess their contribution to cell senescence and hypertrophy.In vivo level of phosphorylated GSK3? was analyzed in cartilage and oxidative damage was assessed by 8-oxo-deoxyguanosine staining. The in vitro effects of GSK3? 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 (?H2AX, GADD45? and p21), flow cytometric analysis of cell cycle and light scattering properties, quantitative assessment of senescence associated ? galactosidase activity, and PAS staining.In vivo chondrocytes from obese OA patients showed higher levels of phosphorylated GSK3?, oxidative damage and expression of GADD45? and p21, in comparison with chondrocytes of nonobese OA patients. LiCl mediated GSK3? 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 ?H2AX, GADD45? and p21. Moreover, LiCl boosted 8-oxo-dG staining, expression of IKK? and MMP-10.In articular chondrocytes, GSK3? activity is required for the maintenance of proliferative potential and phenotype. Conversely, GSK3? inactivation, although preserving chondrocyte survival, results in functional impairment via induction of hypertrophy and senescence. Indeed, GSK3? inactivation is responsible for ROS production, triggering oxidative stress and DNA damage response.

SUBMITTER: Guidotti S 

PROVIDER: S-EPMC4664288 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature