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Static mechanical stress induces apoptosis in rat endplate chondrocytes through MAPK and mitochondria-dependent caspase activation signaling pathways.

Kong D, Zheng T, Zhang M, Wang D, Du S, Li X, Fang J, Cao X - PLoS ONE (2013)

Bottom Line: Mechanical stress has detrimental effects on cartilaginous endplate chondrocytes due to apoptosis in vivo and in vitro.Treatment with inhibitors of JNK (SP600125), p38 MAPK (SB203580), and ERK (PD98059) prior to mechanical stimulation reversed both the static load-induced chondrocyte apoptosis and the activation of JNK, p38 MAPK, and ERK.Taken together, the data presented in this study demonstrate that mechanical stress induces apoptosis in rat cervical endplate chondrocytes through the MAPK-mediated mitochondrial apoptotic pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopedics, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.

ABSTRACT
Mechanical stress has detrimental effects on cartilaginous endplate chondrocytes due to apoptosis in vivo and in vitro. In this study, we investigated the possible apoptosis signaling pathways induced by mechanical stress in cultured rat cervical endplate chondrocytes. Static mechanical load significantly reduced cell viability in a time- and load-dependent manner, as demonstrated by the Cell Counting Kit-8 (CCK-8) assay. Chondrocyte apoptosis induced by mechanical stress was confirmed by annexin V/propidium iodide (PI) staining and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Western blot analysis revealed that static load-induced chondrocyte apoptosis was accompanied by increased phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK1/2), and p38 mitogen-activated protein kinase (MAPK). The loss of mitochondrial membrane potential (ΔΨm), increased Cytochrome c release, and activated Caspase-9 and Caspase-3, indicating that the mitochondrial pathway is involved in mechanical stress-induced chondrocyte apoptosis. Treatment with inhibitors of JNK (SP600125), p38 MAPK (SB203580), and ERK (PD98059) prior to mechanical stimulation reversed both the static load-induced chondrocyte apoptosis and the activation of JNK, p38 MAPK, and ERK. Taken together, the data presented in this study demonstrate that mechanical stress induces apoptosis in rat cervical endplate chondrocytes through the MAPK-mediated mitochondrial apoptotic pathway.

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Effect of mechanical stress on the expression of apoptosis-related proteins.Chondrocytes were loaded under 0.5 MPa for 24 h in the presence or absence of specific MAPK inhibitors, and the protein levels of Bax, Bcl-2, and Cytochrome c were assessed by western blot analysis. Expression of β-actin was used as an internal control. Data are presented as the mean ± SD of three independent experiments. *p<0.05; **p<0.01 versus unloaded cells, #p<0.05; ##p<0.01; ###p<0.001 versus loaded cells.
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pone-0069403-g005: Effect of mechanical stress on the expression of apoptosis-related proteins.Chondrocytes were loaded under 0.5 MPa for 24 h in the presence or absence of specific MAPK inhibitors, and the protein levels of Bax, Bcl-2, and Cytochrome c were assessed by western blot analysis. Expression of β-actin was used as an internal control. Data are presented as the mean ± SD of three independent experiments. *p<0.05; **p<0.01 versus unloaded cells, #p<0.05; ##p<0.01; ###p<0.001 versus loaded cells.

Mentions: To investigate the role of mitochondrial dysfunction in static compression-induced chondrocyte apoptosis, we used the lipophilic dye JC-1 to detect the loss of ΔΨm by fluorescence microscopy. As shown in Figure 4, static compression resulted in a decrease of red fluorescence and an increase of green fluorescence in the loaded chondrocytes compared with the control cells, indicative of a loss of ΔΨm. In addition, we evaluated the expression of Bcl-2 family proteins by western blot analysis. As illustrated in Figure 5A, mechanical load treatment resulted in an increased level of Bax protein compared with unloaded cells, whereas the level of Bcl-2 protein was significantly reduced. We next examined the release of Cytochrome c from the mitochondria into the cytosol. As shown in Figure 5B, the levels of cytosolic Cytochrome c had significantly increased after static compression, indicating that a mitochondrial pathway is involved in mechanical stress-induced chondrocyte apoptosis.


Static mechanical stress induces apoptosis in rat endplate chondrocytes through MAPK and mitochondria-dependent caspase activation signaling pathways.

Kong D, Zheng T, Zhang M, Wang D, Du S, Li X, Fang J, Cao X - PLoS ONE (2013)

Effect of mechanical stress on the expression of apoptosis-related proteins.Chondrocytes were loaded under 0.5 MPa for 24 h in the presence or absence of specific MAPK inhibitors, and the protein levels of Bax, Bcl-2, and Cytochrome c were assessed by western blot analysis. Expression of β-actin was used as an internal control. Data are presented as the mean ± SD of three independent experiments. *p<0.05; **p<0.01 versus unloaded cells, #p<0.05; ##p<0.01; ###p<0.001 versus loaded cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3716647&req=5

pone-0069403-g005: Effect of mechanical stress on the expression of apoptosis-related proteins.Chondrocytes were loaded under 0.5 MPa for 24 h in the presence or absence of specific MAPK inhibitors, and the protein levels of Bax, Bcl-2, and Cytochrome c were assessed by western blot analysis. Expression of β-actin was used as an internal control. Data are presented as the mean ± SD of three independent experiments. *p<0.05; **p<0.01 versus unloaded cells, #p<0.05; ##p<0.01; ###p<0.001 versus loaded cells.
Mentions: To investigate the role of mitochondrial dysfunction in static compression-induced chondrocyte apoptosis, we used the lipophilic dye JC-1 to detect the loss of ΔΨm by fluorescence microscopy. As shown in Figure 4, static compression resulted in a decrease of red fluorescence and an increase of green fluorescence in the loaded chondrocytes compared with the control cells, indicative of a loss of ΔΨm. In addition, we evaluated the expression of Bcl-2 family proteins by western blot analysis. As illustrated in Figure 5A, mechanical load treatment resulted in an increased level of Bax protein compared with unloaded cells, whereas the level of Bcl-2 protein was significantly reduced. We next examined the release of Cytochrome c from the mitochondria into the cytosol. As shown in Figure 5B, the levels of cytosolic Cytochrome c had significantly increased after static compression, indicating that a mitochondrial pathway is involved in mechanical stress-induced chondrocyte apoptosis.

Bottom Line: Mechanical stress has detrimental effects on cartilaginous endplate chondrocytes due to apoptosis in vivo and in vitro.Treatment with inhibitors of JNK (SP600125), p38 MAPK (SB203580), and ERK (PD98059) prior to mechanical stimulation reversed both the static load-induced chondrocyte apoptosis and the activation of JNK, p38 MAPK, and ERK.Taken together, the data presented in this study demonstrate that mechanical stress induces apoptosis in rat cervical endplate chondrocytes through the MAPK-mediated mitochondrial apoptotic pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopedics, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.

ABSTRACT
Mechanical stress has detrimental effects on cartilaginous endplate chondrocytes due to apoptosis in vivo and in vitro. In this study, we investigated the possible apoptosis signaling pathways induced by mechanical stress in cultured rat cervical endplate chondrocytes. Static mechanical load significantly reduced cell viability in a time- and load-dependent manner, as demonstrated by the Cell Counting Kit-8 (CCK-8) assay. Chondrocyte apoptosis induced by mechanical stress was confirmed by annexin V/propidium iodide (PI) staining and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Western blot analysis revealed that static load-induced chondrocyte apoptosis was accompanied by increased phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK1/2), and p38 mitogen-activated protein kinase (MAPK). The loss of mitochondrial membrane potential (ΔΨm), increased Cytochrome c release, and activated Caspase-9 and Caspase-3, indicating that the mitochondrial pathway is involved in mechanical stress-induced chondrocyte apoptosis. Treatment with inhibitors of JNK (SP600125), p38 MAPK (SB203580), and ERK (PD98059) prior to mechanical stimulation reversed both the static load-induced chondrocyte apoptosis and the activation of JNK, p38 MAPK, and ERK. Taken together, the data presented in this study demonstrate that mechanical stress induces apoptosis in rat cervical endplate chondrocytes through the MAPK-mediated mitochondrial apoptotic pathway.

Show MeSH
Related in: MedlinePlus