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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.

Show MeSH
GRα Gene Silencing inhibited MC3T3-E1 G0–G1 arrest and apoptosis induced by DEX.(A) Real time PCR examination of MC3T3-E1 cells in which the GRαgene function was silenced by siRNA (siGR-1, siGR-2) targeting GRαmRNA; the mRNA expression level of GRαin the siGR-1and siGR-2 groups decreased significantly (P<0.05) compared to that in the FBS group and the siC group. (B) Examination of the protein expression level of the GRαgene by Western blotting following treatment with the indicated siRNA molecules siGR-1 and siGR-2. The protein expression level of GRαsignificantly decreased in the siGR -1, and siGR -2 groups compared to that in the siC groups and FBS groups. SiGR-1 and siGR -2 group: Cells treated with siRNA molecules siGR -1 and siGR -2, respectively. siC group: Cells treated with a siRNA which had a randomized nucleotide sequence that had no significant homology to any part of the human genome. FBS group: Cells without any treatment. (C) The level of the cleaved-caspase 3 protein was significantly up-regulated in the DEX group, when compared to that in the control group, the siGR-1 group,DEX+ siGR-1 group. (D) Visualization of apoptotic cells by the TUNEL (DAB/haemotox) assay in MC3T3-E1 cells. The cell nuclei was stained with DAPI (blue) (4′,6-diamidino-2-phenylindole).And the apoptotic cells were visualized by TUNEL staining(brown). The apoptotic cells in DEX group is significantly increased compared to the control group, the siGR-1 group,DEX+siGR-1 group. (E) Assessment of apoptosis in MC3T3-E1 cell using flow cytometry with Annexin V-FITC/PI staining. Annexin V-FITC(+)PI(−) cells were considered as early apoptotic cells. The apoptotic cells to total cells ratio was significantly (P<0.05) higher in DEX group compared to the control group, the siGR-1 group,DEX+siGR-1 group. (F) Cell cycle analysis using flow cytometry with PI staining, showing representive histograms of MC3T3-E1 cells in control group, DEX group, the siGR-1 group,DEX+ siGR-1 group. The proportion of cells in the S phase decreased markedly in the DEX group in comparison to the control group, the siGR-1 group,DEX+ siGR-1 group. The proportion of cells in the G0–G1 significantly increased in the DEX group. (P<0.05) Control group: cells treated with PBS. DEX group: cells cells treated with 1 µmol/L dexamethasone. siGR-1 group: cells treated with siRNA molecules siGR -1. DEX+ siGR-1 group: cells cells treated with 1µmol/L dexamethasone plus siRNA molecules siGR -1.
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pone-0037030-g003: GRα Gene Silencing inhibited MC3T3-E1 G0–G1 arrest and apoptosis induced by DEX.(A) Real time PCR examination of MC3T3-E1 cells in which the GRαgene function was silenced by siRNA (siGR-1, siGR-2) targeting GRαmRNA; the mRNA expression level of GRαin the siGR-1and siGR-2 groups decreased significantly (P<0.05) compared to that in the FBS group and the siC group. (B) Examination of the protein expression level of the GRαgene by Western blotting following treatment with the indicated siRNA molecules siGR-1 and siGR-2. The protein expression level of GRαsignificantly decreased in the siGR -1, and siGR -2 groups compared to that in the siC groups and FBS groups. SiGR-1 and siGR -2 group: Cells treated with siRNA molecules siGR -1 and siGR -2, respectively. siC group: Cells treated with a siRNA which had a randomized nucleotide sequence that had no significant homology to any part of the human genome. FBS group: Cells without any treatment. (C) The level of the cleaved-caspase 3 protein was significantly up-regulated in the DEX group, when compared to that in the control group, the siGR-1 group,DEX+ siGR-1 group. (D) Visualization of apoptotic cells by the TUNEL (DAB/haemotox) assay in MC3T3-E1 cells. The cell nuclei was stained with DAPI (blue) (4′,6-diamidino-2-phenylindole).And the apoptotic cells were visualized by TUNEL staining(brown). The apoptotic cells in DEX group is significantly increased compared to the control group, the siGR-1 group,DEX+siGR-1 group. (E) Assessment of apoptosis in MC3T3-E1 cell using flow cytometry with Annexin V-FITC/PI staining. Annexin V-FITC(+)PI(−) cells were considered as early apoptotic cells. The apoptotic cells to total cells ratio was significantly (P<0.05) higher in DEX group compared to the control group, the siGR-1 group,DEX+siGR-1 group. (F) Cell cycle analysis using flow cytometry with PI staining, showing representive histograms of MC3T3-E1 cells in control group, DEX group, the siGR-1 group,DEX+ siGR-1 group. The proportion of cells in the S phase decreased markedly in the DEX group in comparison to the control group, the siGR-1 group,DEX+ siGR-1 group. The proportion of cells in the G0–G1 significantly increased in the DEX group. (P<0.05) Control group: cells treated with PBS. DEX group: cells cells treated with 1 µmol/L dexamethasone. siGR-1 group: cells treated with siRNA molecules siGR -1. DEX+ siGR-1 group: cells cells treated with 1µmol/L dexamethasone plus siRNA molecules siGR -1.

Mentions: To investigate GRαgene function, we tried to silence this gene in MC3T3-E1 cells using siRNA targeting GRαmRNA. The elimination of the GRαmRNA and protein in MC3T3-E1 cell cultures was achieved as determined by real-time PCR (Fig. 3A) and Western blotting (Fig. 3B), respectively. When compared to those in untreated and siRNA control (siC) group, the mRNA and protein expression level of GRα decreased significantly (P<0.05) in the siGR-1 and siGR-2 group. On the other hand, the β-actin did not vary significantly between the groups.


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)

GRα Gene Silencing inhibited MC3T3-E1 G0–G1 arrest and apoptosis induced by DEX.(A) Real time PCR examination of MC3T3-E1 cells in which the GRαgene function was silenced by siRNA (siGR-1, siGR-2) targeting GRαmRNA; the mRNA expression level of GRαin the siGR-1and siGR-2 groups decreased significantly (P<0.05) compared to that in the FBS group and the siC group. (B) Examination of the protein expression level of the GRαgene by Western blotting following treatment with the indicated siRNA molecules siGR-1 and siGR-2. The protein expression level of GRαsignificantly decreased in the siGR -1, and siGR -2 groups compared to that in the siC groups and FBS groups. SiGR-1 and siGR -2 group: Cells treated with siRNA molecules siGR -1 and siGR -2, respectively. siC group: Cells treated with a siRNA which had a randomized nucleotide sequence that had no significant homology to any part of the human genome. FBS group: Cells without any treatment. (C) The level of the cleaved-caspase 3 protein was significantly up-regulated in the DEX group, when compared to that in the control group, the siGR-1 group,DEX+ siGR-1 group. (D) Visualization of apoptotic cells by the TUNEL (DAB/haemotox) assay in MC3T3-E1 cells. The cell nuclei was stained with DAPI (blue) (4′,6-diamidino-2-phenylindole).And the apoptotic cells were visualized by TUNEL staining(brown). The apoptotic cells in DEX group is significantly increased compared to the control group, the siGR-1 group,DEX+siGR-1 group. (E) Assessment of apoptosis in MC3T3-E1 cell using flow cytometry with Annexin V-FITC/PI staining. Annexin V-FITC(+)PI(−) cells were considered as early apoptotic cells. The apoptotic cells to total cells ratio was significantly (P<0.05) higher in DEX group compared to the control group, the siGR-1 group,DEX+siGR-1 group. (F) Cell cycle analysis using flow cytometry with PI staining, showing representive histograms of MC3T3-E1 cells in control group, DEX group, the siGR-1 group,DEX+ siGR-1 group. The proportion of cells in the S phase decreased markedly in the DEX group in comparison to the control group, the siGR-1 group,DEX+ siGR-1 group. The proportion of cells in the G0–G1 significantly increased in the DEX group. (P<0.05) Control group: cells treated with PBS. DEX group: cells cells treated with 1 µmol/L dexamethasone. siGR-1 group: cells treated with siRNA molecules siGR -1. DEX+ siGR-1 group: cells cells treated with 1µmol/L dexamethasone plus siRNA molecules siGR -1.
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Related In: Results  -  Collection

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pone-0037030-g003: GRα Gene Silencing inhibited MC3T3-E1 G0–G1 arrest and apoptosis induced by DEX.(A) Real time PCR examination of MC3T3-E1 cells in which the GRαgene function was silenced by siRNA (siGR-1, siGR-2) targeting GRαmRNA; the mRNA expression level of GRαin the siGR-1and siGR-2 groups decreased significantly (P<0.05) compared to that in the FBS group and the siC group. (B) Examination of the protein expression level of the GRαgene by Western blotting following treatment with the indicated siRNA molecules siGR-1 and siGR-2. The protein expression level of GRαsignificantly decreased in the siGR -1, and siGR -2 groups compared to that in the siC groups and FBS groups. SiGR-1 and siGR -2 group: Cells treated with siRNA molecules siGR -1 and siGR -2, respectively. siC group: Cells treated with a siRNA which had a randomized nucleotide sequence that had no significant homology to any part of the human genome. FBS group: Cells without any treatment. (C) The level of the cleaved-caspase 3 protein was significantly up-regulated in the DEX group, when compared to that in the control group, the siGR-1 group,DEX+ siGR-1 group. (D) Visualization of apoptotic cells by the TUNEL (DAB/haemotox) assay in MC3T3-E1 cells. The cell nuclei was stained with DAPI (blue) (4′,6-diamidino-2-phenylindole).And the apoptotic cells were visualized by TUNEL staining(brown). The apoptotic cells in DEX group is significantly increased compared to the control group, the siGR-1 group,DEX+siGR-1 group. (E) Assessment of apoptosis in MC3T3-E1 cell using flow cytometry with Annexin V-FITC/PI staining. Annexin V-FITC(+)PI(−) cells were considered as early apoptotic cells. The apoptotic cells to total cells ratio was significantly (P<0.05) higher in DEX group compared to the control group, the siGR-1 group,DEX+siGR-1 group. (F) Cell cycle analysis using flow cytometry with PI staining, showing representive histograms of MC3T3-E1 cells in control group, DEX group, the siGR-1 group,DEX+ siGR-1 group. The proportion of cells in the S phase decreased markedly in the DEX group in comparison to the control group, the siGR-1 group,DEX+ siGR-1 group. The proportion of cells in the G0–G1 significantly increased in the DEX group. (P<0.05) Control group: cells treated with PBS. DEX group: cells cells treated with 1 µmol/L dexamethasone. siGR-1 group: cells treated with siRNA molecules siGR -1. DEX+ siGR-1 group: cells cells treated with 1µmol/L dexamethasone plus siRNA molecules siGR -1.
Mentions: To investigate GRαgene function, we tried to silence this gene in MC3T3-E1 cells using siRNA targeting GRαmRNA. The elimination of the GRαmRNA and protein in MC3T3-E1 cell cultures was achieved as determined by real-time PCR (Fig. 3A) and Western blotting (Fig. 3B), respectively. When compared to those in untreated and siRNA control (siC) group, the mRNA and protein expression level of GRα decreased significantly (P<0.05) in the siGR-1 and siGR-2 group. On the other hand, the β-actin did not vary significantly between the groups.

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