Progesterone up-regulates p27 through an increased binding of the progesterone receptor-A-p53 protein complex onto the non-canonical p53 binding motif in HUVEC

研究成果: 雜誌貢獻文章

摘要

We previously demonstrated that progesterone (P4) up-regulated p53 expression, which in turn increased p21 and p27 expression, and finally resulted in proliferation inhibition in human umbilical vein endothelial cells (HUVEC). While a direct transcriptional activation of p21 by p53 protein has been clearly elucidated, the mechanism by which p53 induces p27 expression has not been documented. In this study, we identified three putative p53 protein binding domains at the p27 promoter. Luciferase assay showed that the activity of ectopically introduced p27 promoter constructs containing the potential p53 protein binding region was significantly increased by P4. Immunoblotting analysis indicated that P4 increased the level of p53 protein. Treatment with pifithrin-α-HBr (PFTα), a specific blocker of p53-responsive gene transactivation, reduced the P4-increased p27 promoter activity and p27 protein expression. Transfection with dominant-negative mutants of p53 (C135Y, R175H and R248 W) abolished the P4-increased p27 promoter activity. Moreover, deletion or TCCT nucleotide sequence fill-in at the core site of any of p53 protein binding domains led to the irresponsiveness of the p27 promoter to P4 treatment. Interestingly, immunoprecipitation and chromatin-immunoprecipitation analyses demonstrated that P4 increased the complex of p53-P4 receptor (PR) protein in the nucleus and the assembly of PR protein to the p53 protein binding region of the p27 promoter. Ectopic co-overexpression of p53 and PR-A constructs further augmented the P4-increased p27 promoter activity. Taken together, the results from the present study suggest that P4-increased p53 expression might directly up-regulate p27 transactivation, and PR-A protein might promote this effect by forming complex with p53 protein.
原文英語
期刊Journal of Steroid Biochemistry and Molecular Biology
DOIs
出版狀態接受/付印 - 一月 1 2018

指紋

Endothelial cells
Human Umbilical Vein Endothelial Cells
Progesterone
Up-Regulation
Protein Binding
Proteins
Transcriptional Activation
Chromatin Immunoprecipitation
p53 Genes
progesterone receptor A
Luciferases
Immunoprecipitation
Immunoblotting
Genetic Promoter Regions
Chromatin
Transfection
Assays
Nucleotides
Genes
Chemical activation

ASJC Scopus subject areas

  • Endocrinology, Diabetes and Metabolism
  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Endocrinology
  • Clinical Biochemistry
  • Cell Biology

引用此文

@article{14014d8dd780472296048c01feadac2f,
title = "Progesterone up-regulates p27 through an increased binding of the progesterone receptor-A-p53 protein complex onto the non-canonical p53 binding motif in HUVEC",
abstract = "We previously demonstrated that progesterone (P4) up-regulated p53 expression, which in turn increased p21 and p27 expression, and finally resulted in proliferation inhibition in human umbilical vein endothelial cells (HUVEC). While a direct transcriptional activation of p21 by p53 protein has been clearly elucidated, the mechanism by which p53 induces p27 expression has not been documented. In this study, we identified three putative p53 protein binding domains at the p27 promoter. Luciferase assay showed that the activity of ectopically introduced p27 promoter constructs containing the potential p53 protein binding region was significantly increased by P4. Immunoblotting analysis indicated that P4 increased the level of p53 protein. Treatment with pifithrin-α-HBr (PFTα), a specific blocker of p53-responsive gene transactivation, reduced the P4-increased p27 promoter activity and p27 protein expression. Transfection with dominant-negative mutants of p53 (C135Y, R175H and R248 W) abolished the P4-increased p27 promoter activity. Moreover, deletion or TCCT nucleotide sequence fill-in at the core site of any of p53 protein binding domains led to the irresponsiveness of the p27 promoter to P4 treatment. Interestingly, immunoprecipitation and chromatin-immunoprecipitation analyses demonstrated that P4 increased the complex of p53-P4 receptor (PR) protein in the nucleus and the assembly of PR protein to the p53 protein binding region of the p27 promoter. Ectopic co-overexpression of p53 and PR-A constructs further augmented the P4-increased p27 promoter activity. Taken together, the results from the present study suggest that P4-increased p53 expression might directly up-regulate p27 transactivation, and PR-A protein might promote this effect by forming complex with p53 protein.",
keywords = "p27, p53, Progesterone, Progesterone receptor, Promoter activity",
author = "Hsu, {Sung Po} and Lin, {Po Han} and Chou, {Chih Ming} and Lee, {Wen Sen}",
year = "2018",
month = "1",
day = "1",
doi = "10.1016/j.jsbmb.2018.08.011",
language = "English",
journal = "Journal of Steroid Biochemistry and Molecular Biology",
issn = "0960-0760",
publisher = "Elsevier Limited",

}

TY - JOUR

T1 - Progesterone up-regulates p27 through an increased binding of the progesterone receptor-A-p53 protein complex onto the non-canonical p53 binding motif in HUVEC

AU - Hsu, Sung Po

AU - Lin, Po Han

AU - Chou, Chih Ming

AU - Lee, Wen Sen

PY - 2018/1/1

Y1 - 2018/1/1

N2 - We previously demonstrated that progesterone (P4) up-regulated p53 expression, which in turn increased p21 and p27 expression, and finally resulted in proliferation inhibition in human umbilical vein endothelial cells (HUVEC). While a direct transcriptional activation of p21 by p53 protein has been clearly elucidated, the mechanism by which p53 induces p27 expression has not been documented. In this study, we identified three putative p53 protein binding domains at the p27 promoter. Luciferase assay showed that the activity of ectopically introduced p27 promoter constructs containing the potential p53 protein binding region was significantly increased by P4. Immunoblotting analysis indicated that P4 increased the level of p53 protein. Treatment with pifithrin-α-HBr (PFTα), a specific blocker of p53-responsive gene transactivation, reduced the P4-increased p27 promoter activity and p27 protein expression. Transfection with dominant-negative mutants of p53 (C135Y, R175H and R248 W) abolished the P4-increased p27 promoter activity. Moreover, deletion or TCCT nucleotide sequence fill-in at the core site of any of p53 protein binding domains led to the irresponsiveness of the p27 promoter to P4 treatment. Interestingly, immunoprecipitation and chromatin-immunoprecipitation analyses demonstrated that P4 increased the complex of p53-P4 receptor (PR) protein in the nucleus and the assembly of PR protein to the p53 protein binding region of the p27 promoter. Ectopic co-overexpression of p53 and PR-A constructs further augmented the P4-increased p27 promoter activity. Taken together, the results from the present study suggest that P4-increased p53 expression might directly up-regulate p27 transactivation, and PR-A protein might promote this effect by forming complex with p53 protein.

AB - We previously demonstrated that progesterone (P4) up-regulated p53 expression, which in turn increased p21 and p27 expression, and finally resulted in proliferation inhibition in human umbilical vein endothelial cells (HUVEC). While a direct transcriptional activation of p21 by p53 protein has been clearly elucidated, the mechanism by which p53 induces p27 expression has not been documented. In this study, we identified three putative p53 protein binding domains at the p27 promoter. Luciferase assay showed that the activity of ectopically introduced p27 promoter constructs containing the potential p53 protein binding region was significantly increased by P4. Immunoblotting analysis indicated that P4 increased the level of p53 protein. Treatment with pifithrin-α-HBr (PFTα), a specific blocker of p53-responsive gene transactivation, reduced the P4-increased p27 promoter activity and p27 protein expression. Transfection with dominant-negative mutants of p53 (C135Y, R175H and R248 W) abolished the P4-increased p27 promoter activity. Moreover, deletion or TCCT nucleotide sequence fill-in at the core site of any of p53 protein binding domains led to the irresponsiveness of the p27 promoter to P4 treatment. Interestingly, immunoprecipitation and chromatin-immunoprecipitation analyses demonstrated that P4 increased the complex of p53-P4 receptor (PR) protein in the nucleus and the assembly of PR protein to the p53 protein binding region of the p27 promoter. Ectopic co-overexpression of p53 and PR-A constructs further augmented the P4-increased p27 promoter activity. Taken together, the results from the present study suggest that P4-increased p53 expression might directly up-regulate p27 transactivation, and PR-A protein might promote this effect by forming complex with p53 protein.

KW - p27

KW - p53

KW - Progesterone

KW - Progesterone receptor

KW - Promoter activity

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U2 - 10.1016/j.jsbmb.2018.08.011

DO - 10.1016/j.jsbmb.2018.08.011

M3 - Article

AN - SCOPUS:85054138434

JO - Journal of Steroid Biochemistry and Molecular Biology

JF - Journal of Steroid Biochemistry and Molecular Biology

SN - 0960-0760

ER -