Nuclear and Extranuclear Pathway Inputs in the Regulation of Global Gene Expression by Estrogen Receptors

Abstract

Whereas estrogens exert their effects by binding to nuclear estrogen receptors (ERs) and directly altering target gene transcription, they can also initiate extranuclear signaling through activation of kinase cascades. We have investigated the impact of estrogen-mediated extranuclear-initiated pathways on global gene expression by using estrogen-dendrimer conjugates (EDCs), which because of their charge and size remain outside the nucleus and can only initiate extranuclear signaling. Genome-wide cDNA microarray analysis of MCF-7 breast cancer cells identified a subset of 17beta-estradiol (E2)-regulated genes ( approximately 25%) as EDC responsive. The EDC and E2-elicited increases in gene expression were due to increases in gene transcription, as observed in nuclear run-on assays and RNA polymerase II recruitment and phosphorylation. Treatment with antiestrogen or ERalpha knockdown using small interfering RNA abolished EDC-mediated gene stimulation, whereas GPR30 knockdown or treatment with a GPR30-selective ligand was without effect, indicating ER as the mediator of these gene regulations. Inhibitors of MAPK kinase and c-Src suppressed both E2 and EDC stimulated gene expression. Of note, in chromatin immunoprecipitation assays, EDC was unable to recruit ERalpha to estrogen-responsive regions of regulated genes, whereas ERalpha recruitment by E2 was very effective. These findings suggest that other transcription factors or kinases that are downstream effectors of EDC-initiated extranuclear signaling cascades are recruited to regulatory regions of EDC-responsive genes in order to elicit gene stimulation. This study thus highlights the importance of inputs from both nuclear and extranuclear ER signaling pathways in regulating patterns of gene expression in breast cancer cells.

Nuclear receptors: Genomic and non-genomic effects converge

“…Moreover, the generation of mice and cell lines lacking NR genes that did not show rapid responses, or expressing mutant NRs (for example, DNA binding-deficient) that were unable to mediate both the genomic and the non-genomic effects of their ligands, strongly argue against the existence of other receptors (reviewed in refs. [15][16][17][18]. Recent data, however, support a role of membrane-associated G protein-coupled ER (GPR30) in intracellular Ca 2+ oscillations induced by estrogen in primate LHRH neurones and in the proliferation and migration of ER-negative breast cancer cells.…”

“…8,9,12,18,30 In principle, extranuclear-initiated NR actions may regulate gene expression: (a) by the modulation of the transcriptional activity of NRs themselves or, (b) independently of NR transcription factor activity.…”

“…This is assessed by the increase in its phosphorylation and that of MSK substrates: the transcription factors cyclic AMP response element-binding protein In the case of ER the effects on gene expression by using nuclear or extranuclear pathways are different, that is, treating MCF-7 breast cancer cells with the usual 17β-estradiol (E2) or with E2-dendrimer conjugates (that remain outside the nucleus) distinct genes become regulated. 18 Most…”

Effets membranaires du récepteur alpha des œstrogènes

“…L'EDC, composé synthétique d'une part, et l'estétrol (E4), un oestrogène naturel produit uniquement pendant la grossesse d'autre part, semblent à cet égard tout à fait intéressants. L'EDC qui, comme nous l'avons déjà évoqué, a permis l'étude in vivo des effets membranaires du récepteur, a également permis d'évaluer in vitro, l'impact de l'activation du ER membranaire, sur la régulation de l'expression génique des cellules du cancer du sein MCF-7 [45]. L'analyse à large échelle du génome, par biopuces (microarray), des cellules stimulées, a conduit à l'identification, parmi les gènes induits par E2, d'un sous-ensemble de gènes (25 %) également sensibles à l'EDC.…”

“…l'activation de kinases ou autres signaux, l'EDC initie des modifications au niveau des facteurs de transcription qui sont recrutés sur les régions régulatrices des gènes régulés [45]. L'estétrol (E4) est un oestrogène produit uniquement par le foie foetal durant la grossesse chez l'homme [46].…”