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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.

Guidotti S, Minguzzi M, Platano D, Cattini L, Trisolino G, Mariani E, Borzì RM - PLoS ONE (2015)

Bottom Line: 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).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.Conversely, GSK3β inactivation, although preserving chondrocyte survival, results in functional impairment via induction of hypertrophy and senescence.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio di Immunoreumatologia e Rigenerazione Tessutale, Istituto Ortopedico Rizzoli, Bologna, Italy.

ABSTRACT

Introduction: 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.

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Effects of GSK3β inhibition on senescence markers and scatter properties.SA-β Galactosidase activity and PAS staining were quantitatively evaluated by image analysis: cells were automatically detected by SyBr Green nuclear counterstaining and the percentage of cells above a given staining intensity threshold was determined. 3A, SA-β Galactosidase activity. 5mM LiCl increases the percentage of SA-β Gal positive cells already at 8 hours of treatment (n = 6 different experiments with LiCl and 4 with SB216763). Right images: representative pictures of non stimulated (NS) and LiCl or SB216763 treated cells (10x original magnification). Hypertrophic cells also show the strongest level of SA-β Gal activity. 3B, PAS staining. A significant increase in PAS staining was observed in chondrocytes treated with 5mM LiCl (n = 5) or 10μM SB216763 (n = 4) at 24 hours. Right image: representative pictures showing that hypertrophic cells also has the strongest level of PAS staining (10x original magnification). 3C, SA-β Galactosidase activity is increased by GSK3β silencing after 24 hours. 3D, Cell cycle phase distribution of Forward Scatter (left graph, a parameter related to cell size) and Side scatter (right graph, a parameter related to cell granularity) of different experiments (n = 8 with LiCl and 4 with SB216763), with values normalized to that of each control G1 phase cells. 5mM LiCl treatment determines an increase of scatter values at G1, S and G2M phase. *P < 0.05; **P < 0.01;***P < 0.001.
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pone.0143865.g003: Effects of GSK3β inhibition on senescence markers and scatter properties.SA-β Galactosidase activity and PAS staining were quantitatively evaluated by image analysis: cells were automatically detected by SyBr Green nuclear counterstaining and the percentage of cells above a given staining intensity threshold was determined. 3A, SA-β Galactosidase activity. 5mM LiCl increases the percentage of SA-β Gal positive cells already at 8 hours of treatment (n = 6 different experiments with LiCl and 4 with SB216763). Right images: representative pictures of non stimulated (NS) and LiCl or SB216763 treated cells (10x original magnification). Hypertrophic cells also show the strongest level of SA-β Gal activity. 3B, PAS staining. A significant increase in PAS staining was observed in chondrocytes treated with 5mM LiCl (n = 5) or 10μM SB216763 (n = 4) at 24 hours. Right image: representative pictures showing that hypertrophic cells also has the strongest level of PAS staining (10x original magnification). 3C, SA-β Galactosidase activity is increased by GSK3β silencing after 24 hours. 3D, Cell cycle phase distribution of Forward Scatter (left graph, a parameter related to cell size) and Side scatter (right graph, a parameter related to cell granularity) of different experiments (n = 8 with LiCl and 4 with SB216763), with values normalized to that of each control G1 phase cells. 5mM LiCl treatment determines an increase of scatter values at G1, S and G2M phase. *P < 0.05; **P < 0.01;***P < 0.001.

Mentions: GSK3β inactivation induced chondrocyte senescence as assessed by the significant increase of SA-β Gal activity [17] after 5mM LiCl. Noteworthy, the cells with stronger SA-β Gal staining were larger and with a “hypertrophic” phenotype. A quantitative analysis of the increased percentage of senescent/hypertrophic cells was undertaken at 8, 16 and 24 hours and indicated a significant increase already at 8 hours (Fig 3A and S3 File). GSK3β inactivation by either LiCl or SB216763 also led to glycogen accumulation: the number of PAS positive cells was significantly higher at 24 hours for both 5mM LiCl and 10μM SB216763 (Fig 3B and S3 File). Compared to SB216763, 5mM LiCl was a more effective stimulus for glycogenesis, since treated cells had a significantly higher PAS staining already at 16 hours. Interestingly, at 24 hours LiCl treatment, hypertrophic chondrocytes showed both stronger level of SA-β Gal and of PAS staining. A flow cytometric analysis combining cell cycle information and light scattering properties confirmed that already at 8 hours stimulation at each cell cycle phase, LiCl treatment led to the accumulation of chondrocytes larger than control and richer of intracellular structures that can reflect the light, as evidenced by their increased forward and side scatter [25], respectively (Fig 3C and S3 File). We also obtained a statistically significant higher level of SA-β Gal staining in siGSK3β compared to siCTL chondrocytes, while in siGSK3β cells, the addition of either LiCl or SB216763 did not increase further the level of senescence (Fig 3C and S3 File).


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.

Guidotti S, Minguzzi M, Platano D, Cattini L, Trisolino G, Mariani E, Borzì RM - PLoS ONE (2015)

Effects of GSK3β inhibition on senescence markers and scatter properties.SA-β Galactosidase activity and PAS staining were quantitatively evaluated by image analysis: cells were automatically detected by SyBr Green nuclear counterstaining and the percentage of cells above a given staining intensity threshold was determined. 3A, SA-β Galactosidase activity. 5mM LiCl increases the percentage of SA-β Gal positive cells already at 8 hours of treatment (n = 6 different experiments with LiCl and 4 with SB216763). Right images: representative pictures of non stimulated (NS) and LiCl or SB216763 treated cells (10x original magnification). Hypertrophic cells also show the strongest level of SA-β Gal activity. 3B, PAS staining. A significant increase in PAS staining was observed in chondrocytes treated with 5mM LiCl (n = 5) or 10μM SB216763 (n = 4) at 24 hours. Right image: representative pictures showing that hypertrophic cells also has the strongest level of PAS staining (10x original magnification). 3C, SA-β Galactosidase activity is increased by GSK3β silencing after 24 hours. 3D, Cell cycle phase distribution of Forward Scatter (left graph, a parameter related to cell size) and Side scatter (right graph, a parameter related to cell granularity) of different experiments (n = 8 with LiCl and 4 with SB216763), with values normalized to that of each control G1 phase cells. 5mM LiCl treatment determines an increase of scatter values at G1, S and G2M phase. *P < 0.05; **P < 0.01;***P < 0.001.
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Related In: Results  -  Collection

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pone.0143865.g003: Effects of GSK3β inhibition on senescence markers and scatter properties.SA-β Galactosidase activity and PAS staining were quantitatively evaluated by image analysis: cells were automatically detected by SyBr Green nuclear counterstaining and the percentage of cells above a given staining intensity threshold was determined. 3A, SA-β Galactosidase activity. 5mM LiCl increases the percentage of SA-β Gal positive cells already at 8 hours of treatment (n = 6 different experiments with LiCl and 4 with SB216763). Right images: representative pictures of non stimulated (NS) and LiCl or SB216763 treated cells (10x original magnification). Hypertrophic cells also show the strongest level of SA-β Gal activity. 3B, PAS staining. A significant increase in PAS staining was observed in chondrocytes treated with 5mM LiCl (n = 5) or 10μM SB216763 (n = 4) at 24 hours. Right image: representative pictures showing that hypertrophic cells also has the strongest level of PAS staining (10x original magnification). 3C, SA-β Galactosidase activity is increased by GSK3β silencing after 24 hours. 3D, Cell cycle phase distribution of Forward Scatter (left graph, a parameter related to cell size) and Side scatter (right graph, a parameter related to cell granularity) of different experiments (n = 8 with LiCl and 4 with SB216763), with values normalized to that of each control G1 phase cells. 5mM LiCl treatment determines an increase of scatter values at G1, S and G2M phase. *P < 0.05; **P < 0.01;***P < 0.001.
Mentions: GSK3β inactivation induced chondrocyte senescence as assessed by the significant increase of SA-β Gal activity [17] after 5mM LiCl. Noteworthy, the cells with stronger SA-β Gal staining were larger and with a “hypertrophic” phenotype. A quantitative analysis of the increased percentage of senescent/hypertrophic cells was undertaken at 8, 16 and 24 hours and indicated a significant increase already at 8 hours (Fig 3A and S3 File). GSK3β inactivation by either LiCl or SB216763 also led to glycogen accumulation: the number of PAS positive cells was significantly higher at 24 hours for both 5mM LiCl and 10μM SB216763 (Fig 3B and S3 File). Compared to SB216763, 5mM LiCl was a more effective stimulus for glycogenesis, since treated cells had a significantly higher PAS staining already at 16 hours. Interestingly, at 24 hours LiCl treatment, hypertrophic chondrocytes showed both stronger level of SA-β Gal and of PAS staining. A flow cytometric analysis combining cell cycle information and light scattering properties confirmed that already at 8 hours stimulation at each cell cycle phase, LiCl treatment led to the accumulation of chondrocytes larger than control and richer of intracellular structures that can reflect the light, as evidenced by their increased forward and side scatter [25], respectively (Fig 3C and S3 File). We also obtained a statistically significant higher level of SA-β Gal staining in siGSK3β compared to siCTL chondrocytes, while in siGSK3β cells, the addition of either LiCl or SB216763 did not increase further the level of senescence (Fig 3C and S3 File).

Bottom Line: 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).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.Conversely, GSK3β inactivation, although preserving chondrocyte survival, results in functional impairment via induction of hypertrophy and senescence.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio di Immunoreumatologia e Rigenerazione Tessutale, Istituto Ortopedico Rizzoli, Bologna, Italy.

ABSTRACT

Introduction: 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.

Show MeSH
Related in: MedlinePlus