Evaluation of potential implication of membrane estrogen binding sites on ERE-dependent transcriptional activity and intracellular estrogen receptor-alpha regulation in MCF-7 breast cancer cells.
The potential involvement of membrane estrogen binding sites in the induction of ERE-dependent transcriptional activity as well as in the regulation of intracellular estrogen receptor alpha (ER-alpha) level under estradiol (E2) stimulation was investigated. Our approach relied upon the use of two DCC-treated E2-BSA (bovine serum albumin) solutions (E2-6-BSA and E2-17-BSA). The absence of detectable free E2 in these solutions was established. Both E2-BSA conjugates led to a transient dose-dependent stimulation of the expression of ERE-luciferase (LUC) reporter gene in MVLN cells (MCF-7 cells stably transfected with a pVit-tk-LUC reporter plasmid), a property not recorded with free E2, which maintained enhanced transcriptional activity during the whole experiment. A very low concentration of E2 (10 pM) synergistically acted with E2-BSA conjugates. Hence, ERE-dependent transcriptional activity induced by these conjugates appeared to result from their known interactions with membrane estrogen binding sites. Anti-estrogens (AEs: 4-OH-TAM and RU 58,668), which antagonize genomic ER responses, abrogated the luciferase activity induced by E2-BSA conjugates, confirming a potential relationship between membrane-related signals and intracellular ER. Moreover, induction of luciferase was recorded when the cells were exposed to IBMX (3-isobutyl-1-methylxanthine) and cyclic nucleotides (cAMP/cGMP), suggesting the implication of the latter in the signal transduction pathway leading to the expression of the reporter gene. Growth factors (IGF-I, EGF and TGF-alpha) also slightly stimulated luciferase and synergistically acted with 10 pM E2, or 1 microM E2-BSA conjugates, in agreement with the concept of a cross-talk between steroids and peptides acting on the cell membrane. Remarkably, E2-BSA conjugates, IBMX and all investigated growth factors failed to down-regulate intracellular ER in MCF-7 cells, indicating the need for a direct intracellular interaction of the ligand with the receptor to regulate its level. ER elimination was, however, found in the presence of conditioned media (CMs) prepared from cells pre-exposed to E2-BSA conjugates, suggesting that they may produce (a) modulator(s) that may enhance receptor down-regulation when released within the medium.
Seo
Hye Sook
HS
Laboratoire J.-C. Heuson de Cancérologie Mammaire, Service de Médecine Interne, Institut Jules Bordet, Université Libre de Bruxelles, Brussels, Belgium.
Leclercq
Guy
G
D015056:1-Methyl-3-isobutylxanthine / Q000378:metabolism; D001943:Breast Neoplasms; D004791:Enzyme Inhibitors / Q000378:metabolism; D004958:Estradiol / Q000378:metabolism*; D020847:Estrogen Receptor Modulators / Q000378:metabolism; D047628:Estrogen Receptor alpha; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006133:Growth Substances / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D011960:Receptors, Estrogen / Q000378:metabolism*; D020218:Response Elements / Q000235:genetics*; D012710:Serum Albumin, Bovine / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D004791:Enzyme Inhibitors; D020847:Estrogen Receptor Modulators; D047628:Estrogen Receptor alpha; D006133:Growth Substances; D008024:Ligands; D011960:Receptors, Estrogen; D012710:Serum Albumin, Bovine; D004958:Estradiol; D008156:Luciferases; D015056:1-Methyl-3-isobutylxanthine