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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 vivo detection of inactive GSK3β in osteoarthritic articular chondrocytes.1A, Upper row (4x, original magnification): pGSK3β immunohistochemical staining in normal cartilage and in cartilage from representative examples of a non-obese and an obese patient. Lower images (10x and 40x details): pGSK3β staining in superficial, mid-deep and calcified cartilage zones, in representative samples derived from a non-obese (left) or from an obese patient (right). 1B, High magnification images of pGSK3β staining obtained with confocal microscopy of chondrocytes in the superficial (upper row) or mid-deep layers (lower row) of cartilage derived from a non-obese (left column) or from an obese patient (right column). Bar = 10 μm. Graph: percentage of phospho-GSK3β positive cells in superficial or mid-deep layers in non obese (NO, white column) or obese patients (O, black columns) as assessed by confocal microscopy. 1C, Left graph: percentage of phospho-GSK3β positive chondrocytes in mid-deep layers of knee cartilage, represented as a function of the Body Mass Index of the patients. Right graph: percentage of phospho-GSK3β positive chondrocytes in mid-deep layers of knee cartilage, represented as a function of the age of the patients.
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pone.0143865.g001: In vivo detection of inactive GSK3β in osteoarthritic articular chondrocytes.1A, Upper row (4x, original magnification): pGSK3β immunohistochemical staining in normal cartilage and in cartilage from representative examples of a non-obese and an obese patient. Lower images (10x and 40x details): pGSK3β staining in superficial, mid-deep and calcified cartilage zones, in representative samples derived from a non-obese (left) or from an obese patient (right). 1B, High magnification images of pGSK3β staining obtained with confocal microscopy of chondrocytes in the superficial (upper row) or mid-deep layers (lower row) of cartilage derived from a non-obese (left column) or from an obese patient (right column). Bar = 10 μm. Graph: percentage of phospho-GSK3β positive cells in superficial or mid-deep layers in non obese (NO, white column) or obese patients (O, black columns) as assessed by confocal microscopy. 1C, Left graph: percentage of phospho-GSK3β positive chondrocytes in mid-deep layers of knee cartilage, represented as a function of the Body Mass Index of the patients. Right graph: percentage of phospho-GSK3β positive chondrocytes in mid-deep layers of knee cartilage, represented as a function of the age of the patients.

Mentions: The extent of phospho-GSK3β was investigated by confocal microscopy and immunohistochemistry onto a set of 15 different knee OA cartilage samples (from 7 patients; in case of replicate samples for the same patients the mean values were considered) and on samples from 4 normal subjects. Normal cartilage was negative to phospho-GSK3β staining among all layers, while OA cartilage had most phospho-GSK3β positive cells localized in mid-deep layers with an high (>30%) prevalence of positive cells in obese patients (BMI over 30) (Fig 1A and 1B and S1 File). The analysis indicated that the percentage of phospho-GSK3β positive chondrocytes showed a trend towards a positive correlation with BMI (7 patients: Spearman r = 0.631, p = 0.069, Fig 1C, left graph) and negative with the age of the patients (Spearman r = - 0.739, p = 0.066 Fig 1C, right graph). The data were also analyzed exploiting a contingency table that distinguished “obese” (BMI >30) and “non obese” patients and “phospho-GSK3β positive” (>30%) and “phospho-GSK3β negative” patients. The Fisher’s exact test was statistically significant (two tailed p value = 0.0476). phospho-GSK3β staining in chondrocytes had only an extranuclear pattern (Fig 1B). An high level of staining was also found in calcified cartilage areas, where terminally differentiated chondrocytes survive, suggesting that a similar phenotype had been improperly recapitulated in OA articular cartilage (bottom 10x and 40x detail of the IHC results shown in the lower panels of Fig 1A).


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 vivo detection of inactive GSK3β in osteoarthritic articular chondrocytes.1A, Upper row (4x, original magnification): pGSK3β immunohistochemical staining in normal cartilage and in cartilage from representative examples of a non-obese and an obese patient. Lower images (10x and 40x details): pGSK3β staining in superficial, mid-deep and calcified cartilage zones, in representative samples derived from a non-obese (left) or from an obese patient (right). 1B, High magnification images of pGSK3β staining obtained with confocal microscopy of chondrocytes in the superficial (upper row) or mid-deep layers (lower row) of cartilage derived from a non-obese (left column) or from an obese patient (right column). Bar = 10 μm. Graph: percentage of phospho-GSK3β positive cells in superficial or mid-deep layers in non obese (NO, white column) or obese patients (O, black columns) as assessed by confocal microscopy. 1C, Left graph: percentage of phospho-GSK3β positive chondrocytes in mid-deep layers of knee cartilage, represented as a function of the Body Mass Index of the patients. Right graph: percentage of phospho-GSK3β positive chondrocytes in mid-deep layers of knee cartilage, represented as a function of the age of the patients.
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pone.0143865.g001: In vivo detection of inactive GSK3β in osteoarthritic articular chondrocytes.1A, Upper row (4x, original magnification): pGSK3β immunohistochemical staining in normal cartilage and in cartilage from representative examples of a non-obese and an obese patient. Lower images (10x and 40x details): pGSK3β staining in superficial, mid-deep and calcified cartilage zones, in representative samples derived from a non-obese (left) or from an obese patient (right). 1B, High magnification images of pGSK3β staining obtained with confocal microscopy of chondrocytes in the superficial (upper row) or mid-deep layers (lower row) of cartilage derived from a non-obese (left column) or from an obese patient (right column). Bar = 10 μm. Graph: percentage of phospho-GSK3β positive cells in superficial or mid-deep layers in non obese (NO, white column) or obese patients (O, black columns) as assessed by confocal microscopy. 1C, Left graph: percentage of phospho-GSK3β positive chondrocytes in mid-deep layers of knee cartilage, represented as a function of the Body Mass Index of the patients. Right graph: percentage of phospho-GSK3β positive chondrocytes in mid-deep layers of knee cartilage, represented as a function of the age of the patients.
Mentions: The extent of phospho-GSK3β was investigated by confocal microscopy and immunohistochemistry onto a set of 15 different knee OA cartilage samples (from 7 patients; in case of replicate samples for the same patients the mean values were considered) and on samples from 4 normal subjects. Normal cartilage was negative to phospho-GSK3β staining among all layers, while OA cartilage had most phospho-GSK3β positive cells localized in mid-deep layers with an high (>30%) prevalence of positive cells in obese patients (BMI over 30) (Fig 1A and 1B and S1 File). The analysis indicated that the percentage of phospho-GSK3β positive chondrocytes showed a trend towards a positive correlation with BMI (7 patients: Spearman r = 0.631, p = 0.069, Fig 1C, left graph) and negative with the age of the patients (Spearman r = - 0.739, p = 0.066 Fig 1C, right graph). The data were also analyzed exploiting a contingency table that distinguished “obese” (BMI >30) and “non obese” patients and “phospho-GSK3β positive” (>30%) and “phospho-GSK3β negative” patients. The Fisher’s exact test was statistically significant (two tailed p value = 0.0476). phospho-GSK3β staining in chondrocytes had only an extranuclear pattern (Fig 1B). An high level of staining was also found in calcified cartilage areas, where terminally differentiated chondrocytes survive, suggesting that a similar phenotype had been improperly recapitulated in OA articular cartilage (bottom 10x and 40x detail of the IHC results shown in the lower panels of Fig 1A).

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