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Glucocorticoid receptor and sequential P53 activation by dexamethasone mediates apoptosis and cell cycle arrest of osteoblastic MC3T3-E1 cells.

Li H, Qian W, Weng X, Wu Z, Li H, Zhuang Q, Feng B, Bian Y - PLoS ONE (2012)

Bottom Line: Glucocorticoids play a pivotal role in the proliferation of osteoblasts, but the underlying mechanism has not been successfully elucidated.It was found that the inhibitory effects were largely attributed to apoptosis and G1 phase arrest.These data demonstrate that proliferation of MC3T3-E1 cell was significantly and directly inhibited by dexamethasone treatment via aberrant GR activation and subsequently P53 activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College, Beijing, China.

ABSTRACT
Glucocorticoids play a pivotal role in the proliferation of osteoblasts, but the underlying mechanism has not been successfully elucidated. In this report, we have investigated the molecular mechanism which elucidates the inhibitory effects of dexamethasone on murine osteoblastic MC3T3-E1 cells. It was found that the inhibitory effects were largely attributed to apoptosis and G1 phase arrest. Both the cell cycle arrest and apoptosis were dependent on glucocorticoid receptor (GR), as they were abolished by GR blocker RU486 pre-treatment and GR interference. G1 phase arrest and apoptosis were accompanied with a p53-dependent up-regulation of p21 and pro-apoptotic genes NOXA and PUMA. We also proved that dexamethasone can't induce apoptosis and cell cycle arrest when p53 was inhibited by p53 RNA interference. These data demonstrate that proliferation of MC3T3-E1 cell was significantly and directly inhibited by dexamethasone treatment via aberrant GR activation and subsequently P53 activation.

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DEX-induced MC3T3-E1 apoptosis and G0/G1 arrest are abolished by RU486 pre-treatment.(A) Assessment of apoptosis in MC3T3-E1 cells using flow cytometry with Annexin V-FITC/PI staining. The apoptotic cells to total cells ratio was significantly (P<0.05) higher in DEX group compared to the control group, RU486 group, DEX+RU group. DEX+RU group and RU486 group were implemented through 2-hour pretreatment of RU486 and then DEX/ethanol treatment. (B) The level of the cleaved-caspase 3 protein was significantly up-regulated in the DEX group, when compared to that in other groups. DEX+RU group and RU486 group (C) Cell cycle analysis using flow cytometry with PI staining, showing representive histograms of MC3T3-E1 cells in control group, DEX group, RU486 group, DEX+RU group. The distribution of the cell cycle phase was expressed as the percentage of cells in the G0–G1 phase, S phase and G2-M phase of the cell cycle. The proportion of cells in the S phase decreased markedly in the DEX group in comparison to the control group, RU486 group, DEX+RU group. The proportion of cells in the G0–G1 significantly increased in the DEX group.(P<0.05) Control group: cells treated with ethnol for 24hours. DEX group: cells treated with 1 µmol/L dexamethasone for 24 hours. RU486 group: cells pre-treated 2 hours by10µmol/L RU486 and then treated with ethnol for 24 hours. DEX+RU group: cells pre-treated 2 hours by10 µmol/L RU486 and then treated with 1 µmol/L dexamethasone for 24 hours.
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pone-0037030-g002: DEX-induced MC3T3-E1 apoptosis and G0/G1 arrest are abolished by RU486 pre-treatment.(A) Assessment of apoptosis in MC3T3-E1 cells using flow cytometry with Annexin V-FITC/PI staining. The apoptotic cells to total cells ratio was significantly (P<0.05) higher in DEX group compared to the control group, RU486 group, DEX+RU group. DEX+RU group and RU486 group were implemented through 2-hour pretreatment of RU486 and then DEX/ethanol treatment. (B) The level of the cleaved-caspase 3 protein was significantly up-regulated in the DEX group, when compared to that in other groups. DEX+RU group and RU486 group (C) Cell cycle analysis using flow cytometry with PI staining, showing representive histograms of MC3T3-E1 cells in control group, DEX group, RU486 group, DEX+RU group. The distribution of the cell cycle phase was expressed as the percentage of cells in the G0–G1 phase, S phase and G2-M phase of the cell cycle. The proportion of cells in the S phase decreased markedly in the DEX group in comparison to the control group, RU486 group, DEX+RU group. The proportion of cells in the G0–G1 significantly increased in the DEX group.(P<0.05) Control group: cells treated with ethnol for 24hours. DEX group: cells treated with 1 µmol/L dexamethasone for 24 hours. RU486 group: cells pre-treated 2 hours by10µmol/L RU486 and then treated with ethnol for 24 hours. DEX+RU group: cells pre-treated 2 hours by10 µmol/L RU486 and then treated with 1 µmol/L dexamethasone for 24 hours.

Mentions: To determine whether the inhibitory effect can be attributed to apoptosis and cell cycle arrest, and whether the effect results from GR activation, we treated MC3T3-E1 cells with PBS (control group), 1 µmol/L dexamethasone (DEX group), RU486 (RU486 group), and 1 µmol/L dexamethasone plus 10 µmol/L RU486 (DEX+RU group) respectively. The apoptotic cells to total cells ratio was significantly (P<0.05) higher in DEX group (12.1±0.04%) as compared to 3.01±0.04% in the control group, 3.54±0.05% in the RU486 group, and 5.36±0.03% in the DEX+RU group. (Figure 2 A) The level of the cleaved-caspase 3 protein was significantly up-regulated in the DEX group, when compared to that in other groups. (P<0.05) (figure 2B). By adding RU486 2 hours prior to dexamethasone treatment, we blocked the GR and the apoptosis effect associated with GR activation.


Glucocorticoid receptor and sequential P53 activation by dexamethasone mediates apoptosis and cell cycle arrest of osteoblastic MC3T3-E1 cells.

Li H, Qian W, Weng X, Wu Z, Li H, Zhuang Q, Feng B, Bian Y - PLoS ONE (2012)

DEX-induced MC3T3-E1 apoptosis and G0/G1 arrest are abolished by RU486 pre-treatment.(A) Assessment of apoptosis in MC3T3-E1 cells using flow cytometry with Annexin V-FITC/PI staining. The apoptotic cells to total cells ratio was significantly (P<0.05) higher in DEX group compared to the control group, RU486 group, DEX+RU group. DEX+RU group and RU486 group were implemented through 2-hour pretreatment of RU486 and then DEX/ethanol treatment. (B) The level of the cleaved-caspase 3 protein was significantly up-regulated in the DEX group, when compared to that in other groups. DEX+RU group and RU486 group (C) Cell cycle analysis using flow cytometry with PI staining, showing representive histograms of MC3T3-E1 cells in control group, DEX group, RU486 group, DEX+RU group. The distribution of the cell cycle phase was expressed as the percentage of cells in the G0–G1 phase, S phase and G2-M phase of the cell cycle. The proportion of cells in the S phase decreased markedly in the DEX group in comparison to the control group, RU486 group, DEX+RU group. The proportion of cells in the G0–G1 significantly increased in the DEX group.(P<0.05) Control group: cells treated with ethnol for 24hours. DEX group: cells treated with 1 µmol/L dexamethasone for 24 hours. RU486 group: cells pre-treated 2 hours by10µmol/L RU486 and then treated with ethnol for 24 hours. DEX+RU group: cells pre-treated 2 hours by10 µmol/L RU486 and then treated with 1 µmol/L dexamethasone for 24 hours.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3375272&req=5

pone-0037030-g002: DEX-induced MC3T3-E1 apoptosis and G0/G1 arrest are abolished by RU486 pre-treatment.(A) Assessment of apoptosis in MC3T3-E1 cells using flow cytometry with Annexin V-FITC/PI staining. The apoptotic cells to total cells ratio was significantly (P<0.05) higher in DEX group compared to the control group, RU486 group, DEX+RU group. DEX+RU group and RU486 group were implemented through 2-hour pretreatment of RU486 and then DEX/ethanol treatment. (B) The level of the cleaved-caspase 3 protein was significantly up-regulated in the DEX group, when compared to that in other groups. DEX+RU group and RU486 group (C) Cell cycle analysis using flow cytometry with PI staining, showing representive histograms of MC3T3-E1 cells in control group, DEX group, RU486 group, DEX+RU group. The distribution of the cell cycle phase was expressed as the percentage of cells in the G0–G1 phase, S phase and G2-M phase of the cell cycle. The proportion of cells in the S phase decreased markedly in the DEX group in comparison to the control group, RU486 group, DEX+RU group. The proportion of cells in the G0–G1 significantly increased in the DEX group.(P<0.05) Control group: cells treated with ethnol for 24hours. DEX group: cells treated with 1 µmol/L dexamethasone for 24 hours. RU486 group: cells pre-treated 2 hours by10µmol/L RU486 and then treated with ethnol for 24 hours. DEX+RU group: cells pre-treated 2 hours by10 µmol/L RU486 and then treated with 1 µmol/L dexamethasone for 24 hours.
Mentions: To determine whether the inhibitory effect can be attributed to apoptosis and cell cycle arrest, and whether the effect results from GR activation, we treated MC3T3-E1 cells with PBS (control group), 1 µmol/L dexamethasone (DEX group), RU486 (RU486 group), and 1 µmol/L dexamethasone plus 10 µmol/L RU486 (DEX+RU group) respectively. The apoptotic cells to total cells ratio was significantly (P<0.05) higher in DEX group (12.1±0.04%) as compared to 3.01±0.04% in the control group, 3.54±0.05% in the RU486 group, and 5.36±0.03% in the DEX+RU group. (Figure 2 A) The level of the cleaved-caspase 3 protein was significantly up-regulated in the DEX group, when compared to that in other groups. (P<0.05) (figure 2B). By adding RU486 2 hours prior to dexamethasone treatment, we blocked the GR and the apoptosis effect associated with GR activation.

Bottom Line: Glucocorticoids play a pivotal role in the proliferation of osteoblasts, but the underlying mechanism has not been successfully elucidated.It was found that the inhibitory effects were largely attributed to apoptosis and G1 phase arrest.These data demonstrate that proliferation of MC3T3-E1 cell was significantly and directly inhibited by dexamethasone treatment via aberrant GR activation and subsequently P53 activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College, Beijing, China.

ABSTRACT
Glucocorticoids play a pivotal role in the proliferation of osteoblasts, but the underlying mechanism has not been successfully elucidated. In this report, we have investigated the molecular mechanism which elucidates the inhibitory effects of dexamethasone on murine osteoblastic MC3T3-E1 cells. It was found that the inhibitory effects were largely attributed to apoptosis and G1 phase arrest. Both the cell cycle arrest and apoptosis were dependent on glucocorticoid receptor (GR), as they were abolished by GR blocker RU486 pre-treatment and GR interference. G1 phase arrest and apoptosis were accompanied with a p53-dependent up-regulation of p21 and pro-apoptotic genes NOXA and PUMA. We also proved that dexamethasone can't induce apoptosis and cell cycle arrest when p53 was inhibited by p53 RNA interference. These data demonstrate that proliferation of MC3T3-E1 cell was significantly and directly inhibited by dexamethasone treatment via aberrant GR activation and subsequently P53 activation.

Show MeSH