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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
p53 gene silienc by siRNA can reverse DEX induced apoptosis and cell cycle arrest of MC3T3-E1 cells.(A) Real time PCR examination of MC3T3-E1 cells in which the p53 gene function was silenced by siRNA (sip53-1, sip53-2) targeting p53mRNA; the mRNA expression level of p53 in the sip53-1and sip53-2 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 p53αgene by Western blotting following treatment with the indicated siRNA molecules sip53-1 and sip53-2. The protein expression level of P53 significantly decreased in the sip53 -1, and sip53 -2 groups compared to that in the siC groups and FBS groups. Sip53-1 and sip53 -2 group: Cells treated with siRNA molecules sip53 -1 and sip53 -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 sip53 -1 group DEX+ sip53 -1 group. (D) Apoptosis as demonstarted by 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 percentage of apoptotic cells in the DEX group was higher than in the control group, the sip53 -1 group DEX+ sip53 -1 group. (E) Assessment of apoptosis in MC3T3-E1 cell 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, the sip53 -1 group,DEX+ sip53 -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 sip53 -1 group,DEX+ sip53 -1 group. The proportion of cells in the S phase decreased markedly in the DEX group in comparison to the control group, the sip53 -1 group,DEX+ sip53 -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. sip53 -1 group: cells treated with siRNA molecules sip53 -1. DEX+ sip53 -1 group: cells cells treated with 1 µmol/L dexamethasone plus siRNA molecules sip53 -1.
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pone-0037030-g005: p53 gene silienc by siRNA can reverse DEX induced apoptosis and cell cycle arrest of MC3T3-E1 cells.(A) Real time PCR examination of MC3T3-E1 cells in which the p53 gene function was silenced by siRNA (sip53-1, sip53-2) targeting p53mRNA; the mRNA expression level of p53 in the sip53-1and sip53-2 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 p53αgene by Western blotting following treatment with the indicated siRNA molecules sip53-1 and sip53-2. The protein expression level of P53 significantly decreased in the sip53 -1, and sip53 -2 groups compared to that in the siC groups and FBS groups. Sip53-1 and sip53 -2 group: Cells treated with siRNA molecules sip53 -1 and sip53 -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 sip53 -1 group DEX+ sip53 -1 group. (D) Apoptosis as demonstarted by 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 percentage of apoptotic cells in the DEX group was higher than in the control group, the sip53 -1 group DEX+ sip53 -1 group. (E) Assessment of apoptosis in MC3T3-E1 cell 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, the sip53 -1 group,DEX+ sip53 -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 sip53 -1 group,DEX+ sip53 -1 group. The proportion of cells in the S phase decreased markedly in the DEX group in comparison to the control group, the sip53 -1 group,DEX+ sip53 -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. sip53 -1 group: cells treated with siRNA molecules sip53 -1. DEX+ sip53 -1 group: cells cells treated with 1 µmol/L dexamethasone plus siRNA molecules sip53 -1.

Mentions: To investigate the p53 gene function, we tried to silence this gene in MC3T3-E1 cells with siRNA targeting p53 mRNA. The knockdown of the p53 mRNA in MC3T3-E1 cell cultures was achieved as verified by RT-PCR and western blot (Fig. 5A, B). The protein expression level of p53 decreased significantly (P<0.05) in the sip53-1 and sip53-2 groups compared to that in the siRNA negative control groups and untreated groups. For comparison, the β-actin protein 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)

p53 gene silienc by siRNA can reverse DEX induced apoptosis and cell cycle arrest of MC3T3-E1 cells.(A) Real time PCR examination of MC3T3-E1 cells in which the p53 gene function was silenced by siRNA (sip53-1, sip53-2) targeting p53mRNA; the mRNA expression level of p53 in the sip53-1and sip53-2 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 p53αgene by Western blotting following treatment with the indicated siRNA molecules sip53-1 and sip53-2. The protein expression level of P53 significantly decreased in the sip53 -1, and sip53 -2 groups compared to that in the siC groups and FBS groups. Sip53-1 and sip53 -2 group: Cells treated with siRNA molecules sip53 -1 and sip53 -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 sip53 -1 group DEX+ sip53 -1 group. (D) Apoptosis as demonstarted by 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 percentage of apoptotic cells in the DEX group was higher than in the control group, the sip53 -1 group DEX+ sip53 -1 group. (E) Assessment of apoptosis in MC3T3-E1 cell 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, the sip53 -1 group,DEX+ sip53 -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 sip53 -1 group,DEX+ sip53 -1 group. The proportion of cells in the S phase decreased markedly in the DEX group in comparison to the control group, the sip53 -1 group,DEX+ sip53 -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. sip53 -1 group: cells treated with siRNA molecules sip53 -1. DEX+ sip53 -1 group: cells cells treated with 1 µmol/L dexamethasone plus siRNA molecules sip53 -1.
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Related In: Results  -  Collection

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pone-0037030-g005: p53 gene silienc by siRNA can reverse DEX induced apoptosis and cell cycle arrest of MC3T3-E1 cells.(A) Real time PCR examination of MC3T3-E1 cells in which the p53 gene function was silenced by siRNA (sip53-1, sip53-2) targeting p53mRNA; the mRNA expression level of p53 in the sip53-1and sip53-2 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 p53αgene by Western blotting following treatment with the indicated siRNA molecules sip53-1 and sip53-2. The protein expression level of P53 significantly decreased in the sip53 -1, and sip53 -2 groups compared to that in the siC groups and FBS groups. Sip53-1 and sip53 -2 group: Cells treated with siRNA molecules sip53 -1 and sip53 -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 sip53 -1 group DEX+ sip53 -1 group. (D) Apoptosis as demonstarted by 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 percentage of apoptotic cells in the DEX group was higher than in the control group, the sip53 -1 group DEX+ sip53 -1 group. (E) Assessment of apoptosis in MC3T3-E1 cell 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, the sip53 -1 group,DEX+ sip53 -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 sip53 -1 group,DEX+ sip53 -1 group. The proportion of cells in the S phase decreased markedly in the DEX group in comparison to the control group, the sip53 -1 group,DEX+ sip53 -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. sip53 -1 group: cells treated with siRNA molecules sip53 -1. DEX+ sip53 -1 group: cells cells treated with 1 µmol/L dexamethasone plus siRNA molecules sip53 -1.
Mentions: To investigate the p53 gene function, we tried to silence this gene in MC3T3-E1 cells with siRNA targeting p53 mRNA. The knockdown of the p53 mRNA in MC3T3-E1 cell cultures was achieved as verified by RT-PCR and western blot (Fig. 5A, B). The protein expression level of p53 decreased significantly (P<0.05) in the sip53-1 and sip53-2 groups compared to that in the siRNA negative control groups and untreated groups. For comparison, the β-actin protein 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