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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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Related in: MedlinePlus

LiCl treatment determines a DNA damage response and increases expression of markers of chondrocyte differentiation.4A, LiCl mediated increased GSK3β phosphorylation leads to a DNA damage response (DDR) (a representative example of one out of several experiments). The DDR includes markers of DNA damage (double strand breaks evidenced as γH2AX), increased expression of GADD45β and p21. 4B, Cumulative densitometric analysis following either 5mM LiCl (LiCl) or 10μM SB216763 (SB) treatment (“n” of experiments detailed within brackets for LiCl and SB216763) of the “fold change increase” in comparison of not stimulated samples (NS) of γH2AX (7 patients for LiCl and 5 for SB216763), GADD45β (6,2), p21 (7,3). IKKα (8,4) was also evaluated. 4C, 16 hours LiCl treatment significantly increased gene expression of IKKα (n = 3) and of its target gene MMP-10 (n = 3). 4D, Cumulative correlation analysis of the fold changes protein expression values, considering all the samples independently of time and stimulus, indicated the strong association between the hypertrophy marker GADD45β and both γH2AX (left graph: Spearman r value = 0.48, p = 0.0066, n = 25) and IKKα (right graph: Spearman r value = 0.37, p = 0.024, n = 30). *P < 0.05; **P < 0.01;***P < 0.001.
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pone.0143865.g004: LiCl treatment determines a DNA damage response and increases expression of markers of chondrocyte differentiation.4A, LiCl mediated increased GSK3β phosphorylation leads to a DNA damage response (DDR) (a representative example of one out of several experiments). The DDR includes markers of DNA damage (double strand breaks evidenced as γH2AX), increased expression of GADD45β and p21. 4B, Cumulative densitometric analysis following either 5mM LiCl (LiCl) or 10μM SB216763 (SB) treatment (“n” of experiments detailed within brackets for LiCl and SB216763) of the “fold change increase” in comparison of not stimulated samples (NS) of γH2AX (7 patients for LiCl and 5 for SB216763), GADD45β (6,2), p21 (7,3). IKKα (8,4) was also evaluated. 4C, 16 hours LiCl treatment significantly increased gene expression of IKKα (n = 3) and of its target gene MMP-10 (n = 3). 4D, Cumulative correlation analysis of the fold changes protein expression values, considering all the samples independently of time and stimulus, indicated the strong association between the hypertrophy marker GADD45β and both γH2AX (left graph: Spearman r value = 0.48, p = 0.0066, n = 25) and IKKα (right graph: Spearman r value = 0.37, p = 0.024, n = 30). *P < 0.05; **P < 0.01;***P < 0.001.

Mentions: As expected, LiCl or SB216763 were effective in increasing the extent of phosphorylated GSK3β. Noteworthy, the treatment also induced a slight increase of total GSK3β expression, that also appeared to change in non stimulated cells at 16 and 24 hours, reflecting cell cycle progression. We then performed western blot analysis to investigate whether the LiCl induced increased S phase could be dependent on an activated intra S checkpoint following DNA damage [26]. As shown in the representative case in Fig 4A, LiCl leads to a DNA damage response (DDR), with increased expression of γH2AX, GADD45β and p21. Fig 4B and S4 File show the cumulative densitometric analysis of the level of these proteins following either 5mM LiCl or 10μM SB216763 treatment. γH2AX level was increased at 8 hours by both LiCl and SB216763 and at 24 hours by LiCl. Moreover, γH2AX increase was an effective stimulus for the early induction of GADD45β, significantly increased by SB216763 at 8 hours and by LiCl at 24 hours. GADD45β dependence on γH2AX was further confirmed by the strong correlation between the two proteins (Fig 4D, left graph and S4 File). GADD45β, in turns, led to an increased expression of p21, a marker of senescence [27], significantly induced at 16 hours by both LiCl and SB216763. The effects of GSK3β inactivation on cell growth prompted us to investigate regulation of IKKα, involved in chondrocyte proliferation [28]. Fig 4B shows the cumulative results of 8 (LiCl) or 4 (SB216763) independent experiments and indicates that LiCl induces a modest, yet significant increase of IKKα protein expression at both 8 and 16 hours. In keeping with these findings, at 16 hours stimulation, 5mM LiCl but not 10 μM SB216763 significantly increases IKKα mRNA expression (Fig 4C and S4 File), and of its target gene MMP-10 (Fig 4C and S4 File). IKKα was also found to correlate with GADD45β expression (Fig 4D, right graph and S4 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)

LiCl treatment determines a DNA damage response and increases expression of markers of chondrocyte differentiation.4A, LiCl mediated increased GSK3β phosphorylation leads to a DNA damage response (DDR) (a representative example of one out of several experiments). The DDR includes markers of DNA damage (double strand breaks evidenced as γH2AX), increased expression of GADD45β and p21. 4B, Cumulative densitometric analysis following either 5mM LiCl (LiCl) or 10μM SB216763 (SB) treatment (“n” of experiments detailed within brackets for LiCl and SB216763) of the “fold change increase” in comparison of not stimulated samples (NS) of γH2AX (7 patients for LiCl and 5 for SB216763), GADD45β (6,2), p21 (7,3). IKKα (8,4) was also evaluated. 4C, 16 hours LiCl treatment significantly increased gene expression of IKKα (n = 3) and of its target gene MMP-10 (n = 3). 4D, Cumulative correlation analysis of the fold changes protein expression values, considering all the samples independently of time and stimulus, indicated the strong association between the hypertrophy marker GADD45β and both γH2AX (left graph: Spearman r value = 0.48, p = 0.0066, n = 25) and IKKα (right graph: Spearman r value = 0.37, p = 0.024, n = 30). *P < 0.05; **P < 0.01;***P < 0.001.
© Copyright Policy
Related In: Results  -  Collection

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pone.0143865.g004: LiCl treatment determines a DNA damage response and increases expression of markers of chondrocyte differentiation.4A, LiCl mediated increased GSK3β phosphorylation leads to a DNA damage response (DDR) (a representative example of one out of several experiments). The DDR includes markers of DNA damage (double strand breaks evidenced as γH2AX), increased expression of GADD45β and p21. 4B, Cumulative densitometric analysis following either 5mM LiCl (LiCl) or 10μM SB216763 (SB) treatment (“n” of experiments detailed within brackets for LiCl and SB216763) of the “fold change increase” in comparison of not stimulated samples (NS) of γH2AX (7 patients for LiCl and 5 for SB216763), GADD45β (6,2), p21 (7,3). IKKα (8,4) was also evaluated. 4C, 16 hours LiCl treatment significantly increased gene expression of IKKα (n = 3) and of its target gene MMP-10 (n = 3). 4D, Cumulative correlation analysis of the fold changes protein expression values, considering all the samples independently of time and stimulus, indicated the strong association between the hypertrophy marker GADD45β and both γH2AX (left graph: Spearman r value = 0.48, p = 0.0066, n = 25) and IKKα (right graph: Spearman r value = 0.37, p = 0.024, n = 30). *P < 0.05; **P < 0.01;***P < 0.001.
Mentions: As expected, LiCl or SB216763 were effective in increasing the extent of phosphorylated GSK3β. Noteworthy, the treatment also induced a slight increase of total GSK3β expression, that also appeared to change in non stimulated cells at 16 and 24 hours, reflecting cell cycle progression. We then performed western blot analysis to investigate whether the LiCl induced increased S phase could be dependent on an activated intra S checkpoint following DNA damage [26]. As shown in the representative case in Fig 4A, LiCl leads to a DNA damage response (DDR), with increased expression of γH2AX, GADD45β and p21. Fig 4B and S4 File show the cumulative densitometric analysis of the level of these proteins following either 5mM LiCl or 10μM SB216763 treatment. γH2AX level was increased at 8 hours by both LiCl and SB216763 and at 24 hours by LiCl. Moreover, γH2AX increase was an effective stimulus for the early induction of GADD45β, significantly increased by SB216763 at 8 hours and by LiCl at 24 hours. GADD45β dependence on γH2AX was further confirmed by the strong correlation between the two proteins (Fig 4D, left graph and S4 File). GADD45β, in turns, led to an increased expression of p21, a marker of senescence [27], significantly induced at 16 hours by both LiCl and SB216763. The effects of GSK3β inactivation on cell growth prompted us to investigate regulation of IKKα, involved in chondrocyte proliferation [28]. Fig 4B shows the cumulative results of 8 (LiCl) or 4 (SB216763) independent experiments and indicates that LiCl induces a modest, yet significant increase of IKKα protein expression at both 8 and 16 hours. In keeping with these findings, at 16 hours stimulation, 5mM LiCl but not 10 μM SB216763 significantly increases IKKα mRNA expression (Fig 4C and S4 File), and of its target gene MMP-10 (Fig 4C and S4 File). IKKα was also found to correlate with GADD45β expression (Fig 4D, right graph and S4 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