A fraction of the nuclear estrogen receptor ␣ (ER␣) is localized to the plasma membrane region of 17␤-estradiol (E2) target cells. We previously reported that ER␣ is a palmitoylated protein. To gain insight into the molecular mechanism of ER␣ residence at the plasma membrane, we tested both the role of palmitoylation and the impact of E2 stimulation on ER␣ membrane localization. The cancer cell lines expressing transfected or endogenous human ER␣ (HeLa and HepG2, respectively) or the ER␣ nonpalmitoylable Cys447Ala mutant transfected in HeLa cells were used as experimental models. We found that palmitoylation of ER␣ enacts ER␣ association with the plasma membrane, interaction with the membrane protein caveolin-1, and nongenomic activities, including activation of signaling pathways and cell proliferation (i.e., ERK and AKT activation, cyclin D 1 promoter activity, DNA synthesis). Moreover, E2 reduces both ER␣ palmitoylation and its interaction with caveolin-1, in a time-and dose-dependent manner. These data point to the physiological role of ER␣ palmitoylation in the receptor localization to the cell membrane and in the regulation of the E2-induced cell proliferation. INTRODUCTIONThe sex steroid 17␤-estradiol (E2) acts by binding to its nuclear receptors (i.e., ER␣ and ER␤) that then transactivate target genes. In addition, E2 induces rapid, nongenomic actions involving plasma membrane-associated signaling that require a membrane ER (Coleman and Smith, 2001;Kelly and Levin, 2001;Jakacka et al., 2002;Marino et al., 2002). Although different structural and functional properties have been reported for the membrane-associated ER by comparison with nuclear ER␣ and ER␤ (Ropero et al., 2002;Toran-Allerand et al., 2002;Deecher et al., 2003), immunocytochemical studies revealed the presence of a significant fraction of nuclear ER also on the plasma membrane (Pappas et al., 1995;Norfleet et al., 1999;Dan et al., 2003;Razandi et al., 2003;Arvanitis et al., 2004;Song et al., 2004). In addition, a single mRNA originates a similarly sized nuclear and membrane ER in ER␣-transfected Chinese hamster ovary and HeLa cells (Razandi et al., 1999;Marino et al., 2002Marino et al., , 2003. Thus, ER␣ localizes to both the nucleus and the plasma membrane. Moreover, the membrane ER␣ is emerging as the primary endogenous mediator of E2 rapid responses important in cell proliferation (Marino et al., 1998(Marino et al., , 2002Castoria et al., 1999Castoria et al., , 2001Razandi et al., 1999Razandi et al., , 2000Lobenhofer et al., 2000;Acconcia et al., 2004a;Fernando and Wimalasena, 2004).Debate is open regarding the structural bases and the mechanisms for ER␣ maintenance at and translocation to the plasma membrane. ER␣ does not display any intrinsic transmembrane domain (Song et al., 2004); thus, ER␣ interaction with specific membrane proteins have been proposed to explain its membrane localization Migliaccio et al., 2002;Razandi et al., 2002Razandi et al., , 2003Toran-Allerand et al., 2002;Arvanitis et al., 2004). In particular, the Ser522 re...

The capability of 17beta-estradiol (E2) to induce the non-genomic activities of its receptors (ER alpha and ER beta) and to evoke different signaling pathways committed to the regulation of cell proliferation has been analyzed in different cell cancer lines containing transfected (HeLa) or endogenous (HepG2, DLD1) ER alpha or ER beta. In these cell lines, E2 induced different effects on cell growth/apoptosis in dependence of ER isoforms present. The E2-ER alpha complex rapidly activated multiple signal transduction pathways (i.e., ERK/MAPK, PI3K/AKT) committed to both cell cycle progression and apoptotic cascade prevention. On the other hand, the E2-ER beta complex induced the rapid and persistent phosphorylation of p38/MAPK which, in turn, was involved in caspase-3 activation and cleavage of poly(ADP-ribose)polymerase, driving cells into the apoptotic cycle. In addition, the E2-ER beta complex did not activate any of the E2-ER alpha-activated signal molecules involved in cell growth. Taken together, these results demonstrate the ability of ER beta isoform to activate specific signal transduction pathways starting from plasma membrane that may justify the effect of E2 in inducing cell proliferation or apoptosis in cancer cells. In particular this hormone promotes cell survival through ER alpha non-genomic signaling and cell death through ER beta non-genomic signaling.

Bisphenol A (BPA) exposure has been associated with serious endocrine-disrupting effects in humans and wildlife. Toxicological and epidemiological studies evidenced that BPA increases body mass index and disrupts normal cardiovascular physiology by interfering with endogenous hormones in rodents, nonhuman primates, and cell culture test systems. The BPA concentration derived from these experiments were used by government regulatory agencies to determine the safe exposure levels of BPA in humans. However, accumulating literature in vivo and in vitro indicate that at concentrations lower than that reported in toxicological studies, BPA could elicit a different endocrine-disrupting capacity. To further complicate this picture, BPA effects rely on several and diverse mechanisms that converge upon endocrine and reproductive systems. If all or just few of these mechanisms concur to the endocrine-disrupting potential of low doses of BPA is at present still unclear. Thus, taking into account that the incidence and/or prevalence of health problems associated with endocrine disruption have increased worldwide, the goal of the present review is to give an overview of the many mechanisms of BPA action in order to decipher whether different mechanisms are at the root of the effect of low dose of BPA on endocrine system.

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Estrogens induce cell proliferation in target tissues by stimulating progression through the G1 phase of the cell cycle. Activation of cyclin D(1) gene expression is a critical feature of this hormonal action. The existence of rapid/nongenomic estradiol-regulated protein kinase C (PKC-alpha) and extracellular signal-regulated kinase (ERK) signal transduction pathways, their cross talk, and role played in DNA synthesis and cyclin D(1) gene transcription have been studied herein in human hepatoma HepG2 cells. 17Beta-estradiol was found to rapidly activate PKC-alpha translocation and ERK-2/mitogen-activated protein kinase phosphorylation in this cell line. These actions were independent of each other, preceding the increase of thymidine incorporation into DNA and cyclin D(1) expression, and did not involve DNA binding by estrogen receptor. The results obtained with specific inhibitors indicated that PKC-alpha pathway is necessary to mediate the estradiol-induced G1-S progression of HepG2 cells, but it does not exert any effect(s) on cyclin D(1) gene expression. On the contrary, ERK-2 cascade was strongly involved in both G1-S progression and cyclin D(1) gene transcription. Deletion of its activating protein-1 responsive element motif resulted in attenuation of cyclin D(1) promoter responsiveness to estrogen. These results indicate that estrogen-induced cyclin D(1) transcription can occur in HepG2 cells independently of the transcriptional activity of estrogen receptor, sustaining the pivotal role played by nongenomic pathways of estrogen action in hormone-induced proliferation.

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.

Recently, we reported that human neuroglobin (NGB) is a new player in the signal transduction pathways that lead to 17β-estradiol (E2)-induced neuron survival. Indeed, E2 induces in neuron mitochondria the enhancement of NGB level, which in turn impairs the activation of a pro-apoptotic cascade. Nowadays, the existence of a similar pathway activated by E2 in non-neuronal cells is completely unknown. Here, the role of E2-induced NGB upregulation in tumor cells is reported. E2 induced the upregulation of NGB in a dose- and time-dependent manner in MCF-7, HepG2, SK-N-BE, and HeLa cells transfected with estrogen receptor α (ERα), whereas E2 was unable to modulate the NGB expression in the ERα-devoid HeLa cells. Both transcriptional and extranuclear ERα signals were required for the E2-dependent upregulation of NGB in MCF-7 and HepG2 cell lines. E2 stimulation modified NGB intracellular localization, inducing a significant reduction of NGB in the nucleus with a parallel increase of NGB in the mitochondria in both HepG2 and MCF-7 cells. Remarkably, E2 pretreatment did not counteract the H2O2-induced caspase-3 and poly (ADP-ribose) polymerase 1 (PARP-1) cleavage, as well as Bcl-2 overexpression in MCF-7 and HepG2 cells in which NGB was stably silenced by using shRNA lentiviral particles, highlighting the pivotal role of NGB in E2-induced antiapoptotic pathways in cancer cells. Present results indicate that the E2-induced NGB upregulation in cancer cells could represent a defense mechanism of E2-related cancers rendering them insensitive to oxidative stress. As a whole, these data open new avenues to develop therapeutic strategies against E2-related cancers.