It is a well-known fact that in ER-expressing cells, the non-activated ERs are associated in the cytoplasm with the Hsp (e.g., Hsp90, Hsp70, and Hsp56). Upon E2 binding, allosteric transitions occur in the ER, the Hsps dissociate and the receptors translocate to the nucleus, where ERE-based gene transcription occurs by recruiting cofactor proteins (i.e., co-activators and corepressors), and the basal transcription machinery (Kumar and Chambon, 1988; McKenna and O’Malley, 2001; Ascenzi et al., 2006; Lonard and O’Malley, 2007; O’Malley and Kumar, 2009). The co-activators interact with the ERs through their leucine- (L)-rich motifs (i.e., LXXLL, where X is any amino acid; e.g., SRC-1) and facilitate ER transcriptional activity by triggering chromatin remodeling (e.g., CBP/p300 PRMT1, SWI/SNF), RNA polymerase II activation, and linking the ERs to the basal transcription machinery (Ascenzi et al., 2006), whereas corepressors [e.g., histone deacetylases (HDAC)] prevent ERs from inducing gene transcription (Jepsen and Rosenfeld, 2002).

In the nucleus, ERα cycles on and off the ERE-containing gene promoters. Although this process occurs even in the absence of E2, E2 shifts these coordinated and transcriptionally unproductive ERα–DNA association/dissociation cycles toward a productive transcriptional response (Métivier et al., 2003; Reid et al., 2003). Interestingly, the discovery that E2-activated ERβ possesses a similar cyclic nature (Picard et al., 2008) indicates that this molecular circuitry is conserved. Therefore, the de-regulation of any step of the transcriptional cycle (e.g., alteration in ER: co-activators interaction) could lead to malignant transformation (Lonard and O’Malley, 2006).

In addition to direct interaction with ERE, ERs can regulate gene transcription without directly binding to DNA. In this indirect genomic mechanism, ERα associates with specific transcription factors, such as Sp1 and AP-1, which play an important role in the transcriptional activation of multiple growth regulatory genes in breast cancer cells. ERβ/Sp1 is primarily associated with decreased ligand-dependent gene expression (Safe, 2001; Pearce and Jordan, 2004). The interaction between ERs and nuclear factor-κB induces the E2-mediated inhibition of gene transcription (Ascenzi et al., 2006).

The nuclear actions of E2 occur after at least 2-h time lag post-E2 stimulation (Métivier et al., 2003; Reid et al., 2003) and explain some E2 functions in physiological and pathological situations (see Farach-Carson and Davis, 2003; Ascenzi et al., 2006). However, E2 elicits some cellular effects that are too rapid (i.e., in seconds to minutes) to be mediated by the activation of the nuclear ERs (Szego and Davis, 1967). These effects are refractory to transcription and translation inhibitors (Losel et al., 2003) and are induced by membrane-impermeable E2 conjugates (e.g., E2-BSA; Ascenzi et al., 2006). The E2-evoked rapid signals involve the activation of several signal transduction pathways (e.g., phospholipase C (PLC)/protein kinase C (PKC); Src/extracellular-activated kinase (ERK), phosphatidyl-inositol 3 kinase (PI3K)/AKT, p38/mitogen-activated protein kinase (MAPK); Janus kinase/signal transducers and activators of transcription (JAK/STAT); p21-activated kinase 1 (Pak1); casein kinase I-g2; sphingosine kinase; RhoA/ROCK-2/moesin; Losel et al., 2003; Ascenzi et al., 2006; Hammes and Levin, 2007; Sanchez et al., 2009) in E2-sensitive cells, and some of these signaling pathways could be cell type specific. Interestingly, both ERα and ERβ trigger the activation of the rapid E2-induced effects although there appears to be some degree of specificity in the isoform-mediated extra-nuclear signaling.

Remarkably, accumulating evidence identifies at least two conserved pathways in membrane ERα-based E2 rapid signaling. The E2-dependent activation of the ERK component of the MAPK family and the AKT kinase in the PI3K axis occur in a variety of normal or transformed cell lines (e.g., vascular or epithelial cells; breast cancer cell; hepatoma cells). The mechanism by which the E2–ERα complex activates these two transduction pathways involves the E2-induced association of membrane ERα with several proteins involved in the ERK/MAPK pathway (i.e., Shc, Src, Ras) leading to Shc/Src/Ras/ERK activation (Song et al., 2004) and the PI3K/AKT pathway [i.e., p85, integrin-linked kinase (ILK), glycogen synthase kinase 3β (GSK3β)] leading to PI3K/ILK/AKT/GSK3β activation and nitric oxide (NO) release (Simoncini et al., 2000; Medunjanin et al., 2005; Acconcia et al., 2006). Interestingly, in cancer cells either signaling cascade transduces the E2 proliferative and anti-apoptotic effects by modulating E2-induced DNA synthesis, resulting in cell cycle progression and cell proliferation (Marino et al., 2002, 2003; Song et al., 2004) as well as cell migration (Acconcia et al., 2006).

The ability of the E2–ERβ complex to activate rapid extra-nuclear mechanisms has received less consideration. Our work identified the E2-dependent activation of the p38 component of the MAPK family as a conserved pathway in membrane ERβ-based E2 rapid signaling. Indeed, the E2-induced ERβ-mediated activation of the p38/MAPK occurs in ERβ-transfected HeLa cells and in ERβ containing rat myoblasts, colon adenocarcinoma cells, and neuroblastoma cells (Acconcia et al., 2005a; Galluzzo et al., 2007, 2009; De Marinis et al., 2010). In any case, the E2-dependent activation of the p38/MAPK pathway requires the physical association between membrane ERβ and p38 and results in triggering the E2 anti-proliferative and pro-apoptotic effects in colon cancer cell lines (Galluzzo et al., 2007).

The data reported here strongly demonstrate that the membrane-initiated effects of E2 can regulate different and opposite cellular processes, such as proliferation, survival, apoptosis, and are operative in transformed and malignant cells. Moreover, they represent conserved molecular circuitries by which E2 exerts its pleiotropic effects through the ERα and the ERβ. In turn, the de-regulation of ERα- and ERβ-mediated rapid signal transduction would lead to pathological consequences, such as cancer. In this respect, it is noteworthy that ERα-positive breast tumors are treated with drugs aimed at interfering with the availability of endogenous E2 (e.g., aromatase inhibitors) or ERα transcriptional activity (e.g., 4OH-tamoxifen; Keen and Davidson, 2003). The same drugs also act on the ERβ rapid signaling, but not because ERβ expression is inversely correlated with the development of several cancer (i.e., breast and colon cancer; Marino and Ascenzi, 2008; Barone et al., 2010; Warner and Gustafsson, 2010).

Therefore, one would expect this mechanism to be highly regarded as a target for cancer prevention or treatment. However, this is not the case most likely because of concerns about the way the membrane-initiated effects of E2 are elicited after E2–ER engagement.

Careful inspection of the literature reveals that the discovery that E2 elicits rapid effects in vivo was reported (Szego and Davis, 1967) soon after the formal definition that the sex steroid hormone E2 acts through a receptor protein (Jensen, 1965), but before the formulation of the concept that steroid hormones acted on DNA to regulate gene transcription (O’Malley and McGuire, 1968); the intravenous administration of physiological doses of E2 to ovariectomized rats induces within 15 s a dramatic increase of uterine cyclic AMP, which lasts for 5 min and is reduced to basal levels within 1 h. Interestingly, these effects are mimicked by another estrogenic molecule (i.e., diethylstilbestrol) and are unaffected by 17α-estradiol. The physiological relevance of this is apparent because there is early evidence of estrogenic stimulation, including the liberation of sequestered histamine and the accompanying expansion of the microcirculation and associated alterations in membrane functions (Szego and Davis, 1967).

When the multifaceted and intertwined nature of ERs signaling network was already known, the group of Dr Jameson Jakacka et al., 2002 created a mouse model in which a point mutation was created in the first zinc finger of the DBD of the endogenous ERα. This mutation (E207A/G208A in mouse ERα) abolishes receptor to ERE binding and the activation of ERE-containing reporter genes, but preserves all the other ERα-based signals (Jakacka et al., 2002; Pedram et al., 2009). The analysis of the phenotype for the ER knock-in mouse model revealed that the knock-in female mice are infertile; indeed, their ovaries did not contain any corpora lutea and the stromal cells contained abundant lipid droplets. In addition, the uteri were enlarged with a clear evidence of cystic endometrial hyperplasia, whereas the mammary glands were hypo-plastic (Jakacka et al., 2002). Because the ERα knock-in mice phenotypes were significantly different from the ERα knock-out mice phenotypes, the observed defects demonstrate the complexity of the E2 signaling in whole-animal physiology and highlight how the extra-nuclear ERα signaling modulates different E2-dependent effects in different tissues. Interestingly, the alteration in the steroidogenesis (i.e., fat accumulation in ovary stromal cells) in the ERα knock-in mouse model is phenocopied by the accumulation of abdominal visceral and other depots of fat (i.e., overall increased body weight), which is observed in the transgenic mice expressing only a functional E domain of ERα at the plasma membrane (Pedram et al., 2009). Thus, the fact that E2 determines such a dramatic imbalance in the female mice physiology by acting on ERα, which is incapable to trigger ERE-mediated E2-induced gene transcription, and on the E domain of the receptor, which is critical for extra-nuclear E2 signaling activation, further demonstrates the activity of the membrane-initiated ERα pathway in vivo (Jakacka et al., 2002; Pedram et al., 2009). Nonetheless, although both ERα knock-in mice and transgenic ERα E domain mice retain the E2-induced activation of ERK/MAPK and AKT as the wild type counterparts, the analysis of their phenotype demonstrates that the nuclear ERα is critical for normal development in mice (Jakacka et al., 2002; Pedram et al., 2009), thus confirming that there is both nuclear and membrane-initiated ERα signaling cross-talk in vivo. Unfortunately, none of these studies analyzed whether these animals were more or less prone to develop tumors. Considering that ERα membrane-initiated signals are critical for DNA synthesis and cell cycle progression, one might expect an increased incidence in tumors in at least some E2-sensitive tissues.

Chambliss et al. (2010) recently used a more physiological approach to demonstrate that activation of rapid, membrane-initiated ERα signals is important in vivo. The use of an E2-dendrimer conjugate, that activates membrane-associated ERs, but excludes them from the nucleus (Harrington et al., 2006), revealed that, in vivo, the vascular protective effects of E2 are mediated exclusively through membrane-initiated ERα signal pathways (Chambliss et al., 2010). These data are, of course, extremely important because they represent the first demonstration that extra-nuclear E2-mediated effects are operative in mice. In turn, this evidence gives the opportunity to pharmacologically target extra-nuclear E2 signaling in vivo through the development of extra-nuclear specific drugs. Nonetheless, the E2-dendrimer conjugates are synthetic and the presence of the dendrimer may render the interpretation of the data incomplete. For this reason, understanding how a non-membrane localized receptor (e.g., the palmitoylation-defective mutant) works in vivo under the stimulation of the physiological hormone remains a critical issue.

Finally, it is important to note here that an in vivo model in which the ERβ is manipulated to study its membrane-initiated signals is not available at the present. Moreover, caution should be used in the interpretation of the abovementioned data because they were all studied in an ERβ-wild type background.

As the emerging data concerning membrane ERs signaling facilitates our understanding of E2 actions, the integration of this information with the clinic-pathological data of patients becomes imperative. For the moment, only nuclear ERs localization tests are utilized in daily hospital practices to obtain predictive diagnosis and to choose pharmacological therapies.

However, at least for breast cancer, mounting evidence points to the expression of the membrane ERα as a tumor exclusivity. In fact, the adjacent non-cancerous tissues are negative for membrane ERα (Kampa et al., 2008). Interestingly, the analysis of 219 primary breast cancers by immuno-histochemical staining reveals that membrane ERα is detected in 34% of the cases in which the tumor is classified as ERα-negative on the basis of the ERα nuclear expression. Moreover, breast tumor samples, expressing membrane ERα, also display a positive correlation with phosphorylated AKT and HER-2 over-expression (Kim et al., 2006). Remarkably, another study reports that, in ERα-positive breast specimens derived from patients after surgical resection, the plasma membrane localized ERα is phosphorylated on the serine residue 118 (i.e., activated), whereas it is absent from normal breast tissue. The phosphorylated, membrane-bound ER appears to define the “invasive carcinoma state” in human fresh tissue specimens because the analysis of the downstream signaling pathways in the cytosol of the same samples shows an hyper-activated status of the signaling kinases ERK/MAPK and AKT, which is correlated with an increase in anti-apoptotic signals (Mintz et al., 2008). Furthermore, other phosphorylated forms of the ERα have been found in tumors but their correlation with membrane-initiated signaling was not studied (Murphy et al., 2011). Data concerning the expression of membrane ERs in tumor specimens are limited to ERα, while no studies for membrane ERβ in tumor samples have been performed.

Nonetheless, at present, the mechanism by which an increased fraction of the activated ERα is localized at the cell plasma membrane in the tumor cells is still not known. De-regulation of the enzyme that palmitoylates receptors may, however, account for it. In addition, activated ERα can be forced to localize outside the nucleus and/or in close proximity of the plasma membrane by virtue of the over-expression of some specific ERα interactors. The spliced form of the metastasis tumor antigen (MTA1s), the modulator of extra-nuclear activity of estrogen receptor (MNAR, also known as PELP1), and activated growth factor receptors are the prototype of such a protein (Acconcia and Kumar, 2006). Indeed, MTA1s sequesters the ERα in the cytoplasm, is highly expressed in ERα-negative but not in ERα-positive breast tumors, and an increased level of MTA1s in ERα-nuclear negative tumors is associated with the presence of cytoplasmic ERα in tumor cells (Acconcia and Kumar, 2006). Similarly, PELP1 functions as a scaffold that brings together the ERα and the signaling proteins required for the activation of the extra-nuclear E2 effects in close proximity of the plasma membrane. As in the case of MTA1s, PELP1 is over-expressed in aggressive and metastatic breast cancers (Acconcia and Kumar, 2006). Finally, the microtubule-binding protein hematopoietic PBX-interaction protein (HPIP) also contributes to E2 extra-nuclear signaling by recruiting the ERα and its signaling partners in the cytoplasm of the breast cancer cells (Manavathi et al., 2006). In addition to these extra-nuclear ERα interactors, the de-regulation of the ERα membrane signaling might occur as a consequence of the over-expression of specific ERα splice variants (e.g., ERα36; ERα46), which also localize into the cytoplasm or at the plasma membrane and hyper-activate the proliferative and anti-apoptotic E2 signals (Kim and Bender, 2009; Ohshiro et al., 2010).

It is, therefore, evident that the molecular circuitries, which are conserved in cell lines, are also operative in a tumor environment, and the ERα membrane-initiated signals are active both in nuclear ERα-positive and in nuclear ERα-negative breast tumors. Consequently, although the presence of the ERβ at the plasma membrane of tumor specimens has not yet been reported, given the high degree of conservation in both structure and mechanism of action of either ER isoforms, it is conceivable that ERβ expressing tumors also display a fraction of the receptor located at the plasma membrane. In this respect, on the basis that membrane ERα in tumors heavily contributes to uncontrolled proliferation through the activation of the ERK/MAPK and AKT pathways (Kim et al., 2006; Kampa et al., 2008; Mintz et al., 2008), ERβ in tumor cell membrane may be associated with increased p38/MAPK activation and an increased E2-dependent apoptosis (Galluzzo et al., 2007). In support of this hypothesis, ERβ expression is lost during the progression of colon carcinoma and breast tumors expressing ERβ have usually a better prognosis (Galluzzo et al., 2007; Barone et al., 2010).