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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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Association of oxidative damage, markers of DDR and senescence in mid-deep layers of cartilage of obese patients.8-oxo-dG, GADD45β, p21 and SA-β-Gal assessed with immunohistochemistry and colorimetric detection and bright field images at 4x original magnification: cases representative of non obese (upper panel) or obese (lower panel) patients. High magnification inset shows the prevalent cytoplasmic localization of GADD45β signal (red staining) with sybr green as a nuclear counterstaining. For each marker, experiments were carried out in the same experimental session to rule out biases in comparing the signal intensity. Below the pictures for each marker a cumulative assessment (with mean and standard error of mean) of the percentage of positive cells in superficial, intermediate and deep layers in non obese (NO, white column) or obese patients (O, black columns) is shown. *P < 0.05; **P < 0.01;***P < 0.001.
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pone.0143865.g005: Association of oxidative damage, markers of DDR and senescence in mid-deep layers of cartilage of obese patients.8-oxo-dG, GADD45β, p21 and SA-β-Gal assessed with immunohistochemistry and colorimetric detection and bright field images at 4x original magnification: cases representative of non obese (upper panel) or obese (lower panel) patients. High magnification inset shows the prevalent cytoplasmic localization of GADD45β signal (red staining) with sybr green as a nuclear counterstaining. For each marker, experiments were carried out in the same experimental session to rule out biases in comparing the signal intensity. Below the pictures for each marker a cumulative assessment (with mean and standard error of mean) of the percentage of positive cells in superficial, intermediate and deep layers in non obese (NO, white column) or obese patients (O, black columns) is shown. *P < 0.05; **P < 0.01;***P < 0.001.

Mentions: Oxidative DNA damage was investigated by mean of 8-oxo-dG staining [23]. Overall, in cartilage of obese patients we found association of higher staining of phospho-GSK3β (as detailed in Fig 1), 8-oxo-dG, GADD45β (with an exclusive cytoplasmic distribution, see inset in Fig 5) and of its target gene p21, and of immunologically detectable SA-β Gal (See Fig 5, lower right panel), suggesting that also in the tissue there is evidence of a mechanistic link between GSK3β inactivation, DNA damage response and chondrocyte senescence. Fig 5 shows correlated immunohistochemistry experiments on cartilage sections derived from two representative OA cartilage samples resulted negative (upper panel) or positive (lower panel,) for phospho-GSK3β. Bottom graphs show the image analysis of the percentage of positive cells for the different markers in the different superficial, intermediate and deep cartilage zones in samples derived from non obese and obese patients (S5 File). Obese patients presented significantly higher level of 8-oxo-dG, GADD45β and p21 in deep cartilage layers.


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)

Association of oxidative damage, markers of DDR and senescence in mid-deep layers of cartilage of obese patients.8-oxo-dG, GADD45β, p21 and SA-β-Gal assessed with immunohistochemistry and colorimetric detection and bright field images at 4x original magnification: cases representative of non obese (upper panel) or obese (lower panel) patients. High magnification inset shows the prevalent cytoplasmic localization of GADD45β signal (red staining) with sybr green as a nuclear counterstaining. For each marker, experiments were carried out in the same experimental session to rule out biases in comparing the signal intensity. Below the pictures for each marker a cumulative assessment (with mean and standard error of mean) of the percentage of positive cells in superficial, intermediate and deep layers in non obese (NO, white column) or obese patients (O, black columns) is shown. *P < 0.05; **P < 0.01;***P < 0.001.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4664288&req=5

pone.0143865.g005: Association of oxidative damage, markers of DDR and senescence in mid-deep layers of cartilage of obese patients.8-oxo-dG, GADD45β, p21 and SA-β-Gal assessed with immunohistochemistry and colorimetric detection and bright field images at 4x original magnification: cases representative of non obese (upper panel) or obese (lower panel) patients. High magnification inset shows the prevalent cytoplasmic localization of GADD45β signal (red staining) with sybr green as a nuclear counterstaining. For each marker, experiments were carried out in the same experimental session to rule out biases in comparing the signal intensity. Below the pictures for each marker a cumulative assessment (with mean and standard error of mean) of the percentage of positive cells in superficial, intermediate and deep layers in non obese (NO, white column) or obese patients (O, black columns) is shown. *P < 0.05; **P < 0.01;***P < 0.001.
Mentions: Oxidative DNA damage was investigated by mean of 8-oxo-dG staining [23]. Overall, in cartilage of obese patients we found association of higher staining of phospho-GSK3β (as detailed in Fig 1), 8-oxo-dG, GADD45β (with an exclusive cytoplasmic distribution, see inset in Fig 5) and of its target gene p21, and of immunologically detectable SA-β Gal (See Fig 5, lower right panel), suggesting that also in the tissue there is evidence of a mechanistic link between GSK3β inactivation, DNA damage response and chondrocyte senescence. Fig 5 shows correlated immunohistochemistry experiments on cartilage sections derived from two representative OA cartilage samples resulted negative (upper panel) or positive (lower panel,) for phospho-GSK3β. Bottom graphs show the image analysis of the percentage of positive cells for the different markers in the different superficial, intermediate and deep cartilage zones in samples derived from non obese and obese patients (S5 File). Obese patients presented significantly higher level of 8-oxo-dG, GADD45β and p21 in deep cartilage layers.

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