Role of Estrogen receptor/Sp1 Complexes in Estrogen-Induced Heat Shock Protein 27 Gene Expression

Mol Endocrinol. 1996 Nov;10(11):1371-8. doi: 10.1210/mend.10.11.8923463.

Abstract

Treatment of MCF-7 human breast cancer cells with 10 nM 17 beta-estradiol (E2) resulted in a 2-fold induction of heat shock protein (Hsp) 27 mRNA levels, and this response persisted for up to 24 h. The 5'-promoter region of the gene was further investigated to identify genomic sequences associated with E2 responsiveness. An Sp1 and half-palindromic estrogen response element (ERE) separated by 10 nucleotides, GGGCGGG(N)10GGTCA, were identified at -105 to -84, and formation of the Sp1/estrogen receptor (ER) complex was investigated by in vitro assays using synthetic Hsp 27-[32P]Sp1/ERE oligonucleotides in a gel mobility shift assay and transient transfection studies using short (-108/-84) and long (-108/+23) 5'-promoter sequences linked to a thymidine kinase promoter and the bacterial chloramphenicol acetyl transferase (CAT) reporter gene (Hsp-CATs and Hsp-CATl, respectively). Incubation of nuclear extracts from MCF-7 cells with an Hsp 27-[32P]Sp1/ERE oligonucleotide results in formation of an Sp1/ER complex. The formation of this complex was inhibited by coincubation with unlabeled Sp1/ERE, ERE, and Sp1 oligonucleotides and by preincubation with ER or Sp1 antibodies (immunodepletion). In addition, the complex was supershifted by coincubation with ER antibodies. Mutation of either Sp1 or ERE sites also decreases formation of the retarded band. E2 induced CAT activity in MCF-7 cells transiently transfected with either Hsp-CATs or Hsp-CATl plasmids. It was also demonstrated that E2 did not significantly induce CAT activity in MCF-7 cells transiently transfected with Hsp-CATl-containing mutations in both the Sp1 and ERE sites. The results of this study demonstrate that an Sp1/ER complex is involved in E2-induced Hsp 27 gene expression.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Antibodies
  • Base Sequence
  • Binding, Competitive
  • Breast Neoplasms / drug therapy
  • Breast Neoplasms / genetics
  • Breast Neoplasms / pathology
  • Chloramphenicol O-Acetyltransferase / genetics
  • Chloramphenicol O-Acetyltransferase / metabolism
  • Cloning, Molecular
  • DNA / metabolism
  • Electrophoresis / methods
  • Estradiol / pharmacology*
  • Female
  • Gene Expression Regulation, Neoplastic
  • Genes, Reporter
  • Heat-Shock Proteins / drug effects
  • Heat-Shock Proteins / genetics*
  • Heat-Shock Proteins / metabolism
  • Humans
  • Molecular Sequence Data
  • Promoter Regions, Genetic
  • RNA, Messenger / biosynthesis
  • RNA, Messenger / drug effects
  • Receptors, Estrogen / drug effects
  • Receptors, Estrogen / immunology
  • Receptors, Estrogen / metabolism*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sp1 Transcription Factor / genetics
  • Sp1 Transcription Factor / immunology
  • Sp1 Transcription Factor / metabolism*
  • TATA Box
  • Thymidine Kinase / genetics
  • Transfection

Substances

  • Antibodies
  • Heat-Shock Proteins
  • RNA, Messenger
  • Receptors, Estrogen
  • Recombinant Proteins
  • Sp1 Transcription Factor
  • Estradiol
  • DNA
  • Chloramphenicol O-Acetyltransferase
  • Thymidine Kinase