Stable overexpression of human HSF‐1 in murine cells suggests activation rather than expression of HSF‐1 to be the key regulatory step in the heat shock gene expression

Nahid F Mivechi, Xaio‐You ‐Y Shi, George M. Hahn

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Abstract

Transcription of the heat shock genes is regulated by the activation of the heat shock transcription factor (HSF‐1). After heat shock, HSF‐1 forms oligomers and binds to the heat shock element (HSE), which consists of several repeats of NGAAN located in the promoter region of the heat shock genes. HSF‐1 is then phosphorylated, leading to the enhanced transcription of the heat shock genes likely by transactivation. We have stably overexpressed the human heat shock transcription factor‐1 (HSF‐1) in murine cells to investigate whether the regulation of the expression of the heat shock genes may partly reside at the level of HSF‐1 expression. Human HSF‐1 cDNA was cloned into a retroviral vector (pvhhsf‐1) and was overexpressed in a murine fibroblast cell line. The overexpressed human HSF‐1 is found in both the cytoplasm and nucleus of control cells but is translocated into the nucleus upon heat shock. Electrophoretic mobility shift analysis suggests that the human HSF‐1 has constitutive DNA binding ability and its DNA binding ability is increased upon heat shock. Cross‐linking experiments indicate that the overexpressed human HSF‐1 is mainly a monomer under control conditions and forms oligomers upon heat shock. Immunoblotting shows that the human HSF‐1 is phosphorylated upon heat shock and its apparent molecular weight is shifted up by at least 10 kDa. In spite of both the DNA binding ability and phosphorylation, the overexpression of human HSF‐1 does not increase the transcription of murine HSP‐70 mRNA or increase the synthesis of other HSPs after heat shock beyond that observed in control untransfected cells. An exception is the enhanced synthesis of a 47–50 kDa protein after heat shock and an apparent lack of induction of one HSP‐70 kDa species when the protein pattern is analyzed by isoelectric focusing. Interestingly, cells overexpressing human HSF‐1 show a 4‐fold increase in the basal expression of luciferase when the plasmids containing the human HSP‐70 promoter ligated to the luciferase reporter gene are transiently expressed in these cells. Murine cells overexpressing human HSF‐1 are more resistant to the cytotoxic effects of heat when compared to the control untransfected cells, but the kinetics of thermotolerance development and decay is similar between HSF‐1 transfected and untransfected cells. In conclusion, human HSF‐1 protein in murine fibroblasts is modified in a similar fashion as the endogenous mouse HSF‐1 after heat shock. However, the overexpression of HSF‐1 does not result in overproduction of heat shock proteins after heat shock, perhaps because these cells contain abundant amounts of endogenous HSF‐1. © 1995 Wiley‐Liss, Inc.

Original languageEnglish (US)
Pages (from-to)266-280
Number of pages15
JournalJournal of Cellular Biochemistry
Volume59
Issue number2
DOIs
StatePublished - Jan 1 1995
Externally publishedYes

Fingerprint

Transcription
Gene expression
Shock
Hot Temperature
Chemical activation
Gene Expression
Genes
Heat-Shock Proteins
Fibroblasts
Luciferases
Oligomers
DNA

Keywords

  • HSF‐1
  • heat shock genes
  • monomer
  • murine cells
  • murine fibroblasts
  • retroviral vector

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

@article{b5a78658a70e418cad61e48eeaa4224d,
title = "Stable overexpression of human HSF‐1 in murine cells suggests activation rather than expression of HSF‐1 to be the key regulatory step in the heat shock gene expression",
abstract = "Transcription of the heat shock genes is regulated by the activation of the heat shock transcription factor (HSF‐1). After heat shock, HSF‐1 forms oligomers and binds to the heat shock element (HSE), which consists of several repeats of NGAAN located in the promoter region of the heat shock genes. HSF‐1 is then phosphorylated, leading to the enhanced transcription of the heat shock genes likely by transactivation. We have stably overexpressed the human heat shock transcription factor‐1 (HSF‐1) in murine cells to investigate whether the regulation of the expression of the heat shock genes may partly reside at the level of HSF‐1 expression. Human HSF‐1 cDNA was cloned into a retroviral vector (pvhhsf‐1) and was overexpressed in a murine fibroblast cell line. The overexpressed human HSF‐1 is found in both the cytoplasm and nucleus of control cells but is translocated into the nucleus upon heat shock. Electrophoretic mobility shift analysis suggests that the human HSF‐1 has constitutive DNA binding ability and its DNA binding ability is increased upon heat shock. Cross‐linking experiments indicate that the overexpressed human HSF‐1 is mainly a monomer under control conditions and forms oligomers upon heat shock. Immunoblotting shows that the human HSF‐1 is phosphorylated upon heat shock and its apparent molecular weight is shifted up by at least 10 kDa. In spite of both the DNA binding ability and phosphorylation, the overexpression of human HSF‐1 does not increase the transcription of murine HSP‐70 mRNA or increase the synthesis of other HSPs after heat shock beyond that observed in control untransfected cells. An exception is the enhanced synthesis of a 47–50 kDa protein after heat shock and an apparent lack of induction of one HSP‐70 kDa species when the protein pattern is analyzed by isoelectric focusing. Interestingly, cells overexpressing human HSF‐1 show a 4‐fold increase in the basal expression of luciferase when the plasmids containing the human HSP‐70 promoter ligated to the luciferase reporter gene are transiently expressed in these cells. Murine cells overexpressing human HSF‐1 are more resistant to the cytotoxic effects of heat when compared to the control untransfected cells, but the kinetics of thermotolerance development and decay is similar between HSF‐1 transfected and untransfected cells. In conclusion, human HSF‐1 protein in murine fibroblasts is modified in a similar fashion as the endogenous mouse HSF‐1 after heat shock. However, the overexpression of HSF‐1 does not result in overproduction of heat shock proteins after heat shock, perhaps because these cells contain abundant amounts of endogenous HSF‐1. {\circledC} 1995 Wiley‐Liss, Inc.",
keywords = "HSF‐1, heat shock genes, monomer, murine cells, murine fibroblasts, retroviral vector",
author = "Mivechi, {Nahid F} and Shi, {Xaio‐You ‐Y} and Hahn, {George M.}",
year = "1995",
month = "1",
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doi = "10.1002/jcb.240590215",
language = "English (US)",
volume = "59",
pages = "266--280",
journal = "Journal of Cellular Biochemistry",
issn = "0730-2312",
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TY - JOUR

T1 - Stable overexpression of human HSF‐1 in murine cells suggests activation rather than expression of HSF‐1 to be the key regulatory step in the heat shock gene expression

AU - Mivechi, Nahid F

AU - Shi, Xaio‐You ‐Y

AU - Hahn, George M.

PY - 1995/1/1

Y1 - 1995/1/1

N2 - Transcription of the heat shock genes is regulated by the activation of the heat shock transcription factor (HSF‐1). After heat shock, HSF‐1 forms oligomers and binds to the heat shock element (HSE), which consists of several repeats of NGAAN located in the promoter region of the heat shock genes. HSF‐1 is then phosphorylated, leading to the enhanced transcription of the heat shock genes likely by transactivation. We have stably overexpressed the human heat shock transcription factor‐1 (HSF‐1) in murine cells to investigate whether the regulation of the expression of the heat shock genes may partly reside at the level of HSF‐1 expression. Human HSF‐1 cDNA was cloned into a retroviral vector (pvhhsf‐1) and was overexpressed in a murine fibroblast cell line. The overexpressed human HSF‐1 is found in both the cytoplasm and nucleus of control cells but is translocated into the nucleus upon heat shock. Electrophoretic mobility shift analysis suggests that the human HSF‐1 has constitutive DNA binding ability and its DNA binding ability is increased upon heat shock. Cross‐linking experiments indicate that the overexpressed human HSF‐1 is mainly a monomer under control conditions and forms oligomers upon heat shock. Immunoblotting shows that the human HSF‐1 is phosphorylated upon heat shock and its apparent molecular weight is shifted up by at least 10 kDa. In spite of both the DNA binding ability and phosphorylation, the overexpression of human HSF‐1 does not increase the transcription of murine HSP‐70 mRNA or increase the synthesis of other HSPs after heat shock beyond that observed in control untransfected cells. An exception is the enhanced synthesis of a 47–50 kDa protein after heat shock and an apparent lack of induction of one HSP‐70 kDa species when the protein pattern is analyzed by isoelectric focusing. Interestingly, cells overexpressing human HSF‐1 show a 4‐fold increase in the basal expression of luciferase when the plasmids containing the human HSP‐70 promoter ligated to the luciferase reporter gene are transiently expressed in these cells. Murine cells overexpressing human HSF‐1 are more resistant to the cytotoxic effects of heat when compared to the control untransfected cells, but the kinetics of thermotolerance development and decay is similar between HSF‐1 transfected and untransfected cells. In conclusion, human HSF‐1 protein in murine fibroblasts is modified in a similar fashion as the endogenous mouse HSF‐1 after heat shock. However, the overexpression of HSF‐1 does not result in overproduction of heat shock proteins after heat shock, perhaps because these cells contain abundant amounts of endogenous HSF‐1. © 1995 Wiley‐Liss, Inc.

AB - Transcription of the heat shock genes is regulated by the activation of the heat shock transcription factor (HSF‐1). After heat shock, HSF‐1 forms oligomers and binds to the heat shock element (HSE), which consists of several repeats of NGAAN located in the promoter region of the heat shock genes. HSF‐1 is then phosphorylated, leading to the enhanced transcription of the heat shock genes likely by transactivation. We have stably overexpressed the human heat shock transcription factor‐1 (HSF‐1) in murine cells to investigate whether the regulation of the expression of the heat shock genes may partly reside at the level of HSF‐1 expression. Human HSF‐1 cDNA was cloned into a retroviral vector (pvhhsf‐1) and was overexpressed in a murine fibroblast cell line. The overexpressed human HSF‐1 is found in both the cytoplasm and nucleus of control cells but is translocated into the nucleus upon heat shock. Electrophoretic mobility shift analysis suggests that the human HSF‐1 has constitutive DNA binding ability and its DNA binding ability is increased upon heat shock. Cross‐linking experiments indicate that the overexpressed human HSF‐1 is mainly a monomer under control conditions and forms oligomers upon heat shock. Immunoblotting shows that the human HSF‐1 is phosphorylated upon heat shock and its apparent molecular weight is shifted up by at least 10 kDa. In spite of both the DNA binding ability and phosphorylation, the overexpression of human HSF‐1 does not increase the transcription of murine HSP‐70 mRNA or increase the synthesis of other HSPs after heat shock beyond that observed in control untransfected cells. An exception is the enhanced synthesis of a 47–50 kDa protein after heat shock and an apparent lack of induction of one HSP‐70 kDa species when the protein pattern is analyzed by isoelectric focusing. Interestingly, cells overexpressing human HSF‐1 show a 4‐fold increase in the basal expression of luciferase when the plasmids containing the human HSP‐70 promoter ligated to the luciferase reporter gene are transiently expressed in these cells. Murine cells overexpressing human HSF‐1 are more resistant to the cytotoxic effects of heat when compared to the control untransfected cells, but the kinetics of thermotolerance development and decay is similar between HSF‐1 transfected and untransfected cells. In conclusion, human HSF‐1 protein in murine fibroblasts is modified in a similar fashion as the endogenous mouse HSF‐1 after heat shock. However, the overexpression of HSF‐1 does not result in overproduction of heat shock proteins after heat shock, perhaps because these cells contain abundant amounts of endogenous HSF‐1. © 1995 Wiley‐Liss, Inc.

KW - HSF‐1

KW - heat shock genes

KW - monomer

KW - murine cells

KW - murine fibroblasts

KW - retroviral vector

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DO - 10.1002/jcb.240590215

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