Interazioni tra le attività del recettore per gli estrogeni alfa e la rete di segnali basati sull'ubiquitina

17β-estradiol (E2) controls a plethora of physiological processes but plays also a critical role in breast cancer progression, thus the fine comprehension of the mechanisms that control E2-induced cell proliferation would help to identify new putative druggable targets for the treatment of breast cancer. The ubiquitin (Ub)- system is gaining much attention for cancer therapies because it allows to build a complex interactions network that is critical for signal transduction to many cellular processes. The Ub-based network depends on non-covalent binding between ubiquitinated proteins and proteins that possess an ubiquitin binding domain (UBD). Interestingly, recent papers reported that the Ub-system deeply impacts the E2 signalling by modulating the estrogen receptor (ER) α sub-type, which is the principal mediator of the E2 mitogenic effects. In particular, while ERα polyubiquitination controls the receptor turnover and transcriptional activity, ERα monoubiquitination is required for the E2-dependent activation of rapid signalling to cell proliferation. Even if it has not been clarified how the Ub modification on ERα modulates the receptor activities, one attractive possibility is that ERα could recognize and transduce the Ub modification on itself or on interacting proteins through an UBD. Thus, the main goal of the present PhD project was to understand the noncovalent Ub-binding abilities of ERα and their regulatory role(s) in E2-dependent cellular processes. To this purpose, initial experiments performed in vitro showed that ERα has two different Ub-binding surfaces (UBSs): in A/B and E domains. By focusing on the E domain Ub-binding ability we identified the structural determinants required for ERα to non-covalently associate to Ub (i.e., L429, A430) in vitro and in cell lines. Next, we analyzed the regulatory role of the ERα-UBS in E2-dependent cellular processes. In particular, we found that even if the L429A,A430G (LAAG) mutation did not alter ERα ability to bind E2, cells expressing the mutant ERα did not proliferate after E2 treatment. Because the E2-dependent cell proliferation depends on the activation of extra-nuclear signalling kinases, we evaluated the activating phosphorylation of some signalling pathways activated by E2. Our results indicate that the ERα-UBS mutation impairs the E2-induced activation of the PI3K/AKT as well as the E2-induced PI3K/AKT-dependent ERα Ser118 phosphorylation. The finding that this residue is not phosphorylated in LAAG ERα cells after E2 treatment further suggested an impairment of the ERα-UBS mutant ability to mediate E2 target genes expression. DNA Micro Arrays experiments definitively demonstrated that the LAAG mutant ERα was less transcriptionally active than the wt receptor. Interestingly, the Ingenuity Pathway Analysis helped us identifying CREB1 as another transcription factor activated by E2 through wt but not throught LAAG ERα. We found that E2 triggered CREB1 transcriptional activation through the PI3K/AKT activation and the mutations of the ERα-UBS impair this pathway. Thus, it is possible that an ERα-UBSdependent membrane complex, responsible for the E2-triggered PI3K/AKT signalling activation, controls cell proliferation through the regulation of ERα and CREB1 activation required for gene transcription. Overall, the data reported in this PhD project indicate that the ERα possesses an UBS on its E domain that plays a critical role for E2-induced nuclear and extranuclear signalling to cell proliferation. In conclusion, our findings open new avenues in the field of E2-activated molecular mechanisms to physiological effects that now have to include also the noncovalent Ub-binding abilities of ERα. Given the key role played by ERα in breast cancer progression, the comprehension of the regulatory role of the ERα-UBS on E2 mitogenic effects reveals new putative druggable target. In this respect, the interference of the UBD:Ub interaction by using specific small molecules has been already proposed as a future pharmacological target against cancer

Identification of an estrogen receptor alpha non-covalent ubiquitin binding surface: role in 17beta-estradiol-induced transcriptional activity

""Ubiquitin (Ub)-binding domains (UBDs) located in Ub receptors decode the ubiquitination signal by non-covalently engaging the Ub modification on their binding partners and transduce the Ub signalling through Ub-based molecular interactions. In this way, inducible protein ubiquitination regulates diverse biological processes. The estrogen receptor alpha (ERα) is a ligand-activated transcription factor that mediates the pleiotropic effects of the sex hormone 17β-estradiol (E2). Fine regulation of E2 pleiotropic actions depends on E2-dependent ERα association with a plethora of binding partners and\\\/or on the E2 modulation of receptor ubiquitination. Indeed, E2-induced ERα polyubiquitination triggers receptor degradation and transcriptional activity, and E2-dependent reduction in ERα monoubiquitination is crucial for E2 signalling. Monoubiquitinated proteins often contain UBDs, but whether non-covalent Ub-ERα binding could occur and play a role in E2-ERα signalling is unknown. Here, we report an Ub-binding surface within the ERα ligand binding domain that directs in vitro the receptor interaction with both ubiquitinated proteins and recombinant Ub chains. Mutational analysis reveals that ERα residues leucine 429 and alanine 430 are involved in Ub binding. Moreover, impairment of ERα association to ubiquitinated species strongly affects E2-induced ERα transcriptional activity. Considering the importance of UBDs in the Ub-based signalling network and the central role of different ERα binding partners in the modulation of E2-dependent effects, our discoveries provide novel insights into ERα activity that could also be relevant for ERα-dependent diseases."

Clathrin Heavy Chain Interacts With Estrogen Receptor α and Modulates 17β-Estradiol Signaling

17β-estradiol (E2)-induced signaling and control of estrogen receptor (ER)α degradation both play a major role in breast cancer cell proliferation. We recently reported the involvement of lysosomal function in both E2-dependent ERα breakdown and E2-induced cell proliferation and thus hypothesized a role for endocytic proteins in ERα signaling. An small interfering RNA screen identified proteins that regulate intracellular endocytic traffic and whose silencing alters E2-induced ERα degradation. One such protein was the clathrin heavy chain (CHC), whose role in E2:ERα signaling to cell proliferation is unknown. Here, we show that CHC physically interacts with ERα in the cytoplasm of breast cancer cells and regulates E2-induced cell proliferation. Surprisingly, the CHC:ERα interaction is required to sustain E2 signaling but is dispensable for ERα degradation. Our data also demonstrate that many membrane trafficking proteins contribute to the regulation of ERα degradation, thus unraveling the contribution of endocytic proteins in E2:ERα signaling

Palmitoylation regulates 17β-estradiol-induced estrogen receptor-α degradation and transcriptional activity

The estrogen receptor-α (ERα) is a transcription factor that regulates gene expression through the binding to its cognate hormone 17β-estradiol (E2). ERα transcriptional activity is regulated by E2-evoked 26S proteasome-mediated ERα degradation and ERα serine (S) residue 118 phosphorylation. Furthermore, ERα mediates fast cell responses to E2 through the activation of signaling cascades such as the MAPK/ERK and phosphoinositide-3-kinase/v-akt murine thymoma viral oncogene homolog 1 pathways. These E2 rapid effects require a population of the ERα located at the cell plasma membrane through palmitoylation, a dynamic enzymatic modification mediated by palmitoyl-acyl-transferases. However, whether membrane-initiated and transcriptional ERα activities integrate in a unique picture or represent parallel pathways still remains to be firmly clarified. Hence, we evaluated here the impact of ERα palmitoylation on E2-induced ERα degradation and S118 phosphorylation. The lack of palmitoylation renders ERα more susceptible to E2-dependent degradation, blocks ERα S118 phosphorylation and prevents E2-induced ERα estrogen-responsive element-containing promoter occupancy. Consequently, ERα transcriptional activity is prevented and the receptor addressed to the nuclear matrix subnuclear compartment. These data uncover a circuitry in which receptor palmitoylation links E2-dependent ERα degradation, S118 phosphorylation, and transcriptional activity in a unique molecular mechanism. We propose that rapid E2-dependent signaling could be considered as a prerequisite for ERα transcriptional activity and suggest an integrated model of ERα intracellular signaling where E2-dependent early extranuclear effects control late receptor-dependent nuclear actions. © 2012 by The Endocrine Society.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Palmitoylation regulates 17β-estradiol-induced estrogen receptor-α degradation and transcriptional activity.

""The estrogen receptor-α (ERα) is a transcription factor that regulates gene expression through the binding to its cognate hormone 17β-estradiol (E2). ERα transcriptional activity is regulated by E2-evoked 26S proteasome-mediated ERα degradation and ERα serine (S) residue 118 phosphorylation. Furthermore, ERα mediates fast cell responses to E2 through the activation of signaling cascades such as the MAPK\\\/ERK and phosphoinositide-3-kinase\\\/v-akt murine thymoma viral oncogene homolog 1 pathways. These E2 rapid effects require a population of the ERα located at the cell plasma membrane through palmitoylation, a dynamic enzymatic modification mediated by palmitoyl-acyl-transferases. However, whether membrane-initiated and transcriptional ERα activities integrate in a unique picture or represent parallel pathways still remains to be firmly clarified. Hence, we evaluated here the impact of ERα palmitoylation on E2-induced ERα degradation and S118 phosphorylation. The lack of palmitoylation renders ERα more susceptible to E2-dependent degradation, blocks ERα S118 phosphorylation and prevents E2-induced ERα estrogen-responsive element-containing promoter occupancy. Consequently, ERα transcriptional activity is prevented and the receptor addressed to the nuclear matrix subnuclear compartment. These data uncover a circuitry in which receptor palmitoylation links E2-dependent ERα degradation, S118 phosphorylation, and transcriptional activity in a unique molecular mechanism. We propose that rapid E2-dependent signaling could be considered as a prerequisite for ERα transcriptional activity and suggest an integrated model of ERα intracellular signaling where E2-dependent early extranuclear effects control late receptor-dependent nuclear actions."

Diverse post-translational modifications of estrogen receptor α cross-talk in the coordination of 17 β-estradiol-dependent cell proliferation

""The sex hormone 17-estradiol (E2) exerts its pleiotropic effects through the binding to the ligand-activated transcription factor estrogen receptor alpha (ERThe E2:ER complex regulates several physiological processes including cell survival and proliferation through transcriptional [i.e., estrogen responsive element (ERE)-based gene transcription] and non-transcriptional membrane-initiated effects (i.e., activation of signalling cascades).. Many post-translational modifications occur on ER and are regulated by E2. Indeed, E2 induces ER phosphorylation that facilitates ER-dependent gene transcription while the hormone reduces ER palmitoylation, thus modulating the amount of the receptor located at the plasma membrane and the E2 signalling to cell proliferation. The ER is also an ubiquitinated protein: ER polyubiquitination (polyUbq) increases upon E2 binding and E2-dependent ER degradation occurs in parallel to the appearance of the E2-evoked physiological effects.. However, the role of ER post-translational modifications in the regulation of the E2-dependent cell proliferation is poorly appreciated. Therefore, we analyzed here how ER phosphorylation, palmitoylation and ubiquitination influence E2-induced cell proliferation in an integrated manner.. Our results demonstrate that the polyUbq-based ER degradation cross-talks with receptor phosphorylation and palmitoylation and is required for the E2-dependent control of cell proliferation. Furthermore, the lack of ER palmitoylation fastens E2-induced polyUbq-dependent ER degradation and prevents both receptor phosphorylation and E2-dependent cell proliferation. Therefore, these data demonstrate that a code of diverse post-translational modifications occurs on ER and uncover a new model of E2:ER cellular signalling in which the E2-dependent control of ER post-translational modifications finely coordinates the E2 ability to regulate cell proliferation.. "

The involvement of lysosomes in E2-induced ERα degradation in MCF-7 cells.

<p>(A) XTT assay in growing MCF-7 cells treated for 24 hrs with different doses of chloroquine (Clo). (B) Western blot and relative densitometric analyses (C’) of ERα cellular levels in MCF-7 cells treated for 2 hrs with E2 (10 nM) in the presence of different concentrations of chloroquine (Clo). (C) Western blot and relative densitometric analyses (C’) of EGF-R cellular levels in HeLa cells treated for 2 hrs with EGF (1 µg/ml) in the presence of different concentrations of chloroquine (Clo). (D) Western blot and relative densitometric analyses of ERα cellular levels in MCF-7 cells treated with chloroquine (Clo 10 µM) in the presence of different doses of E2. (E–F) Western blot and relative densitometric analyses of ERα cellular levels in MCF-7 cells treated for with E2 (10 nM) at different time points in the presence of chloroquine (Clo 10 µM). Inhibitor alone was administrated for 2 hours and 30 min. Loading control was done by evaluating vinculin expression in the same filter. *indicates significant differences with respect to the control sample (0 or -); °indicates significant differences with respect to the corresponding E2-treated or EGF-treated samples (<i>p</i><0.05). Figure shows representative blots of at least three independent experiments.</p

The involvement of lysosomes in E2-induced ERα activities.

<p>(A) RT-qPCR analysis of pS2/TIFF (pS2), cathepsin D (CatD) and progesterone receptor (PR) mRNA expression normalized on the GAPDH mRNA expression in MCF-7 cells treated with E2 (10 nM) for 24 hrs both in the presence and in the absence of chloroquine (Clo–10 µM) treatment. Western blot (B) and relative densitometric (B’) ERK1/2 and AKT phosphorylation in MCF-7 cells treated with E2 (10 nM) at different time points. Where indicated, cells were treated chloroquine (Clo–10 µM) 30 min before E2 administration. Loading control was done by evaluating vinculin expression in the same filter. *indicates significant differences with respect to the relative control (0) sample; °indicates significant differences with respect to the corresponding E2 sample (<i>p</i><0.05). Figure shows representative blots of three independent experiments.</p

ERα localization to lysosomes.

<p>(A) Growing MCF-7 cells were co-stained with anti-ERα Sp-1 and D-12 antibodies. (B) Growing pc DNA flag ERα -transfected HeLa cells were co-stained with anti-ERα HC-20 and flag antibodies. (C) Western blot analysis of ERα cellular levels in growing MCF-7 cells treated with cycloheximide (CHX - 1 mg/ml) for 24 hrs with both anti-ERα Sp-1 and D-12 antibodies. Loading control was done by evaluating vinculin expression in the same filter. (D) Anti-ERα Sp-1 staining of MCF-7 cells treated for with cycloheximide (CHX - 1 mg/ml) for 24 hrs. MCF-7 cells were co-stained with anti-ERα Sp-1 antibody together with either LAMP-2 antibody (E) or lysotracker (F) both in the presence and in the absence of E2 (10 nM–2 hrs). pc DNA flag ERα (Nessi)-transfected HeLa cells were co-stained with anti-ERα HC-20 antibody together with either LAMP-2 antibody (G) or lysotracker (H) both in the presence and in the absence of E2 (10 nM–2 hrs). Figures show one unique confocal plane. All co-staining procedures were described in details in the Material and Methods section.</p