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

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 estro-gen 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 arid 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 (∼25-fold) CAT activity stimulated by E₂ alone was increased 2- to 3-fold (to ∼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+IBMX 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. In addition, when a vitellogenin promoter linked to the CAT reporter gene was transfected, TPA inhibited ER-mediated transcription in MCF-7 cells and had no effect on ER-mediated transcription in CHO cells, while CT+IBMX consistently enhanced this transcription in both cell types. These results demonstrate that TPA shows cell- and promoter-specific effects on ER-mediated transcription, while CT+IBMX consistently synergized with E₂ in ER-mediated transcription from both promoters in the two cell types. Synergistic stimulation of ER-mediated transcription by E₂ and protein kinase activators does not appear to result from changes in cellular ER content or in the binding affinity of ER for ligand or the ERE, but, rather, may be a consequence of a stabilization or facilitation of interaction with target components of the transcriptional machinery, possibly through changes in phosphorylation of ER or other proteins. Cross-talk between the ER and these signal transduction pathways may be important in modulating receptor-dependent transcription.