Regulation of cell physiology through the modulation of estrogen receptors activities by natural and synthetic compounds

17-?-estradiol (E2) is the most active estrogen in humans and exerts profound effects on the growth, differentiation, and functioning of many reproductive and non-reproductive tissues. A number of synthetic substances known as xenoestrogens show estrogenic effects; among them, bisphenol A (BPA) is one of the best characterized. Also naturally plant-produced molecules [e.g., naringenin (Nar)] are known to display a mild estrogenic activity. Because of their ability to interfere with many aspects of natural hormones-dependent control of body homeostasis they are also known as endocrine disruptors (EDs). Two estrogens receptors (ER) are known in humans to interact with endogenous E2 and EDs: these two isoforms, the ER? and the ER?, modify the expression of specific genes acting as ligandactivated transcription factors. In addition, rapid effects of E2 depend on the presence of the ERs to the plasma membrane, which is determined by Palmitoylation of the ERs and lead to the activation of ERK/MAPK, PI3K/AKT and p38/MAPK pathways. Also EDs can bind and modulate genomic and rapid ERs activities acting as agonist or antagonist depending on the isoform they bind. Different layers of regulation modulate the complexity of this signaling network, through the numerous ER post-translational modifications. Indeed, beside palmitoylation, in response to E2 binding ERs are also phosphorylated on serine (S) residues. Another layer of complexity is introduced by the control of ERs intracellular levels; the binding of E2 produces ER? ubiquitination, an event that leads to the 26S-proteasome-mediated receptor degradation, drastically lowering the protein half life. This mechanism is, however, not fully understood and many steps of the process are still to be cleared. Because all the effects of E2 occurs through the above-mentioned ligand-dependent modulation of ERs intracellular content, the goal of the present project was to understand the mechanisms underlying the ligand-dependent modulation of ER intracellular levels to better clarify their modulation abilities in ER?- and ER?-driven physiological processes. Results obtained with a wide spectrum of approaches demonstrate that Nar and BPA affect ER? and ER? protein intracellular content in this way influencing the resulting ERs-dependent effects. In particular, while BPA mimics E2 effects in inducing the 26S proteasome-dependent ER? degradation, Nar induces the receptor accumulation; importantly, this modulation seems to be connected with degradation, as both E2 as well as EDs induce the ER? mRNA levels down-regulation. Studying ER? palmitoylation, we found that this modification is the upstream structural determinant that guarantees the physiological balance of the ER? protein levels. Our finding demonstrates that E2 maintains both a constant level of S118 phosphorylation, whereas it triggers a significant reduction in total ER? content and a parallel increase in ER? gene transcription and that the activation of the PI3K/AKT pathway but not of the ERK/MAPK pathway regulates ER? phosphorylation. Reduction in S118 phosphorylation also correlates with a faster E2-induced receptor elimination and is paralleled with ER? transcription impairment. Indeed both palmitoylation and phosphorylation control ER? activity and stability and are linked each other in a consequent process Our experiments also show that the EDs produce as E2 an increase in ERE-mediated transcription and that the ER? partial antagonist Nar determines ER? phosphorylation as well as the E2 mimetic BPA, that partially stabilizes the receptor. Analysis of the modality by which Nar and BPA affect ER? protein intracellular content reveals that BPA mimics the E2 effects in inducing the 26S proteasome-dependent ER? degradation while Nar induces the receptor accumulation by blocking ER? proteolytic degradation. More importantly, we also found that the Nar-dependent accumulation of ER? results in an increased receptor transcriptional activity and that, upon Nar stimulation, E2 looses its capacity to regulate ER? turnover and to physiologically control ER? gene transcription and that both EDs raise ER? cellular content alone and in co-administration with E2. These discoveries indicate that in a cellular context exposed to Nar the absolute physiological receptor response or the one in response to E2 is changed because of dysregulated receptor expression. Thus, Nar modulation of ER? cellular content could further affect the E2-dependent regulation of specific cellular processes leading to scenarios that strongly diverge from the physiological ones and that the fine hormone-dependent modulation of ERs intracellular levels is intrinsically connected with all the aspects of the molecular mechanisms (i.e., genomic and extra-nuclear) that ERs uses to transduce the physiological E2 intracellular message.