Synergistic activation of estrogen receptor-mediated transcription by estradiol and protein kinase activators

Research output: Contribution to journalArticle

Abstract

Since we have observed effects of growth factors and cAMP as well as estradiol (E2) on regulation of expression of some genes stimulated by the estrogen receptor (ER), we have undertaken studies to examine directly whether activators of protein kinases can modulate transcriptional activity of the ER. We find that activators of protein kinase-A [cholera toxin plus 3-isobutyl-1-methylxanthine (CT+IBMX)] and protein kinase-C [12-O-tetradecanoylphorbol-13-acetate (TPA)] markedly synergize with E2 in ER-mediated transcriptional activation. When a reporter plasmid [with a minimal promoter containing a TATA region and estrogen-responsive elements (ERE) linked to a chloramphenicol acetyltransferase (CAT) reporter gene] was transfected into MCF-7 human breast cancer cells, which contain endogenous ER, E2 evoked a dose-dependent increase in CAT activity. While treatment with protein kinase-A or -C activator alone evoked only very low CAT activity, the maximal (approximately 25-fold) CAT activity stimulated by E2 alone was increased 2- to 3-fold (to approximately 60 times the control value) upon cotreatment with either of the protein kinase activators. Interestingly, antiestrogen abolished all of the CAT activity induced by E2 and protein kinase activators. Immunoblots showed that TPA reduced ER levels to 30% of control values after 24 h, while CT+IBMX increased levels about 1.5-fold. Scatchard binding analysis revealed no change in the binding affinity of E2 to ER by these agents. Gel mobility shift competition assays with extracts prepared from cells that had been treated with E2 and protein kinase activators did not reveal any quantitative or qualitative changes in the binding of ER to the ERE in vitro. In ER-deficient Chinese hamster ovary (CHO) cells transfected with the reporter gene and varying amounts of an ER expression vector, the level of CAT activity obtained by cotreatment with E2 and CT+IMBX was 3-fold higher than that observed with E2 alone over the range of different ER amounts tested. This ER-mediated synergism was still retained in an amino-terminal A/B-domain-deleted ER mutant lacking the hormone-independent transcriptional activation function (TAF-1), but was greatly reduced in two hormone-binding domain (region E) mutants that exhibit significantly diminished ligand-dependent transcriptional activation. TPA did not show any synergistic activation with E2 in CHO cells, indicating differences in responses between cell types.

Original languageEnglish (US)
Pages (from-to)441-452
Number of pages12
JournalMolecular Endocrinology
Volume7
Issue number3
DOIs
StatePublished - Mar 1993

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Estrogen Receptors
Protein Kinases
Estradiol
Chloramphenicol O-Acetyltransferase
1-Methyl-3-isobutylxanthine
Tetradecanoylphorbol Acetate
Transcriptional Activation
Cyclic AMP-Dependent Protein Kinases
Cricetulus
Reporter Genes
Ovary
Estrogens
Hormones
Estrogen Receptor Modulators
Cholera Toxin
Gene Expression Regulation
Electrophoretic Mobility Shift Assay
Cell Extracts
Protein Kinase C
Intercellular Signaling Peptides and Proteins

ASJC Scopus subject areas

  • Molecular Biology
  • Endocrinology

Cite this

Synergistic activation of estrogen receptor-mediated transcription by estradiol and protein kinase activators. / Cho, Hyeseong; Katzenellenbogen, Benita S.

In: Molecular Endocrinology, Vol. 7, No. 3, 03.1993, p. 441-452.

Research output: Contribution to journalArticle

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