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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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In vitro pharmacological inhibition of GSK3β determines ROS production, oxidative damage, stress dependent growth inhibition and activation of an intra-S checkpoint.2A, Compared to control (NS), 5 mM LiCl and 10 μM SB216763 increases ROS production and mitochondria activation at 4 hours treatment. Merged and separated signals of Hoechst 33258 nuclear counterstaining, DCHF-DA and Mitotracker Orange CMTMRos mitochondrial staining. Right: high magnification detail of a LiCl treated cell. Right Graph: at 16 hours, LiCl (black columns) but not SB216763 (grey columns) induced a significant increased (n = 5) accumulation of 8-oxo-dG compared to controls (white columns) on the basis of a flow cytometry analysis. 2B, LiCl (black columns) and SB216763 (grey columns): longitudinal assessment of the effects of GSK3β inhibition on cell growth versus the control (white columns). Upper graphs: counts normalized versus the 8 hours count; lower graph: percentage count reduction due to either LiCl or SB216763 at each time point. 2C, longitudinal assessment of the effects of siRNA mediated GSK3β silencing (squared columns) on cell growth versus the control non targeting siRNA (white columns). Right graph: Population doublings reduction following either 5 mM LiCl or 10 μM SB216763 stimulation as percentage of each unstimulated control at 8, 16 and 24 hours of both siCTL and siGSK3β treated cells 2D, Left: LiCl determines a significant increased () percentage of cells in the S phase at 24 hours, as evidenced by DNA staining (Sytox green, n = 9 different experiments with LiCl and 4 with SB216763). Right: a representative example with cell cycle analysis of control (left) versus 5mM LiCl (right) treated cells at each time point. *P < 0.05; **P < 0.01;***P < 0.001.
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pone.0143865.g002: In vitro pharmacological inhibition of GSK3β determines ROS production, oxidative damage, stress dependent growth inhibition and activation of an intra-S checkpoint.2A, Compared to control (NS), 5 mM LiCl and 10 μM SB216763 increases ROS production and mitochondria activation at 4 hours treatment. Merged and separated signals of Hoechst 33258 nuclear counterstaining, DCHF-DA and Mitotracker Orange CMTMRos mitochondrial staining. Right: high magnification detail of a LiCl treated cell. Right Graph: at 16 hours, LiCl (black columns) but not SB216763 (grey columns) induced a significant increased (n = 5) accumulation of 8-oxo-dG compared to controls (white columns) on the basis of a flow cytometry analysis. 2B, LiCl (black columns) and SB216763 (grey columns): longitudinal assessment of the effects of GSK3β inhibition on cell growth versus the control (white columns). Upper graphs: counts normalized versus the 8 hours count; lower graph: percentage count reduction due to either LiCl or SB216763 at each time point. 2C, longitudinal assessment of the effects of siRNA mediated GSK3β silencing (squared columns) on cell growth versus the control non targeting siRNA (white columns). Right graph: Population doublings reduction following either 5 mM LiCl or 10 μM SB216763 stimulation as percentage of each unstimulated control at 8, 16 and 24 hours of both siCTL and siGSK3β treated cells 2D, Left: LiCl determines a significant increased () percentage of cells in the S phase at 24 hours, as evidenced by DNA staining (Sytox green, n = 9 different experiments with LiCl and 4 with SB216763). Right: a representative example with cell cycle analysis of control (left) versus 5mM LiCl (right) treated cells at each time point. *P < 0.05; **P < 0.01;***P < 0.001.

Mentions: In monolayer cultures at log phase, GSK3β inactivation with either LiCl or SB216763 determined increased ROS production in activated mitochondria as detected by combining the green DCHF-DA ROS probe with the red Mitotracker Orange CMTMRos mitochondrial probe, that yielded an orange staining (Fig 2A). Noteworthy, besides the increased DCHF-DA signal, the increased Mitotracker CMTMRos signal is a confirmation that the treatment with both the GSK3β inhibitors induces ROS production, since Mitotracker signal is increased by these species [16]. Confocal microscopy analysis revealed interesting morphological features: in most chondrocytes the overlapped staining had a perinuclear pattern, ROS also accumulated in the nucleus (see high magnification image in Fig 2A) and some characteristic nuclear mitotracker stained spots became evident in treated cells (right images in Fig 2A).


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)

In vitro pharmacological inhibition of GSK3β determines ROS production, oxidative damage, stress dependent growth inhibition and activation of an intra-S checkpoint.2A, Compared to control (NS), 5 mM LiCl and 10 μM SB216763 increases ROS production and mitochondria activation at 4 hours treatment. Merged and separated signals of Hoechst 33258 nuclear counterstaining, DCHF-DA and Mitotracker Orange CMTMRos mitochondrial staining. Right: high magnification detail of a LiCl treated cell. Right Graph: at 16 hours, LiCl (black columns) but not SB216763 (grey columns) induced a significant increased (n = 5) accumulation of 8-oxo-dG compared to controls (white columns) on the basis of a flow cytometry analysis. 2B, LiCl (black columns) and SB216763 (grey columns): longitudinal assessment of the effects of GSK3β inhibition on cell growth versus the control (white columns). Upper graphs: counts normalized versus the 8 hours count; lower graph: percentage count reduction due to either LiCl or SB216763 at each time point. 2C, longitudinal assessment of the effects of siRNA mediated GSK3β silencing (squared columns) on cell growth versus the control non targeting siRNA (white columns). Right graph: Population doublings reduction following either 5 mM LiCl or 10 μM SB216763 stimulation as percentage of each unstimulated control at 8, 16 and 24 hours of both siCTL and siGSK3β treated cells 2D, Left: LiCl determines a significant increased () percentage of cells in the S phase at 24 hours, as evidenced by DNA staining (Sytox green, n = 9 different experiments with LiCl and 4 with SB216763). Right: a representative example with cell cycle analysis of control (left) versus 5mM LiCl (right) treated cells at each time point. *P < 0.05; **P < 0.01;***P < 0.001.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4664288&req=5

pone.0143865.g002: In vitro pharmacological inhibition of GSK3β determines ROS production, oxidative damage, stress dependent growth inhibition and activation of an intra-S checkpoint.2A, Compared to control (NS), 5 mM LiCl and 10 μM SB216763 increases ROS production and mitochondria activation at 4 hours treatment. Merged and separated signals of Hoechst 33258 nuclear counterstaining, DCHF-DA and Mitotracker Orange CMTMRos mitochondrial staining. Right: high magnification detail of a LiCl treated cell. Right Graph: at 16 hours, LiCl (black columns) but not SB216763 (grey columns) induced a significant increased (n = 5) accumulation of 8-oxo-dG compared to controls (white columns) on the basis of a flow cytometry analysis. 2B, LiCl (black columns) and SB216763 (grey columns): longitudinal assessment of the effects of GSK3β inhibition on cell growth versus the control (white columns). Upper graphs: counts normalized versus the 8 hours count; lower graph: percentage count reduction due to either LiCl or SB216763 at each time point. 2C, longitudinal assessment of the effects of siRNA mediated GSK3β silencing (squared columns) on cell growth versus the control non targeting siRNA (white columns). Right graph: Population doublings reduction following either 5 mM LiCl or 10 μM SB216763 stimulation as percentage of each unstimulated control at 8, 16 and 24 hours of both siCTL and siGSK3β treated cells 2D, Left: LiCl determines a significant increased () percentage of cells in the S phase at 24 hours, as evidenced by DNA staining (Sytox green, n = 9 different experiments with LiCl and 4 with SB216763). Right: a representative example with cell cycle analysis of control (left) versus 5mM LiCl (right) treated cells at each time point. *P < 0.05; **P < 0.01;***P < 0.001.
Mentions: In monolayer cultures at log phase, GSK3β inactivation with either LiCl or SB216763 determined increased ROS production in activated mitochondria as detected by combining the green DCHF-DA ROS probe with the red Mitotracker Orange CMTMRos mitochondrial probe, that yielded an orange staining (Fig 2A). Noteworthy, besides the increased DCHF-DA signal, the increased Mitotracker CMTMRos signal is a confirmation that the treatment with both the GSK3β inhibitors induces ROS production, since Mitotracker signal is increased by these species [16]. Confocal microscopy analysis revealed interesting morphological features: in most chondrocytes the overlapped staining had a perinuclear pattern, ROS also accumulated in the nucleus (see high magnification image in Fig 2A) and some characteristic nuclear mitotracker stained spots became evident in treated cells (right images in Fig 2A).

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