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Estrogen Receptor-Mediated Rapid Signaling

Sections of Cardiovascular Medicine and Immunobiology, Vascular Biology and Transplantation Program, Investigative Medicine Program, and the Raymond and Beverly Sackler Foundation Cardiovascular Laboratory, Yale University School of Medicine, New Haven, Connecticut 06520

Address all correspondence and requests for reprints to: Jeffrey R. Bender, M.D., Divisions of Cardiovascular Medicine and Immunobiology, Yale University School of Medicine, 300 Cedar Street, New Haven, Connecticut 06520. E-mail: jeffrey.bender{at}yale.edu.

	Abstract

Top Abstract Introduction Evidence for Plasma Membrane... Molecular Features of Estrogen... Tissue-Specific, Rapid Responses... ER Splice Variants Controversies in Hormone... Conclusions References

 
In addition to nuclear-initiated (genomic) responses, estrogen receptors (ERs) have the ability to facilitate rapid, membrane-initiated, estrogen-triggered signaling cascades via a plasma membrane-associated form of the receptor. These rapid responses are dependent on assembly of membrane ER-centered multimolecular complexes, which can transduce ligand-activated signals to affect a variety of enzymatic pathways, often occurring in a cell-type-specific fashion with tissue-specific physiological outcomes. In some instances, cross-talk occurs between these membrane-initiated and nuclear responses, ultimately regulating transcriptional activation. The role of splice variants in membrane-initiated estrogen responses has been described, notably those within the vascular endothelium. In this review, we describe the evidence for membrane ERs, the molecular components of the aforementioned signaling complexes and pathways, the relevance of ER splice variants, and ER-mediated responses in specific tissues. Our growing understanding of ER-mediated actions at a molecular level will provide insight into the controversies surrounding hormone replacement therapy in postmenopausal women.

	Introduction

Top Abstract Introduction Evidence for Plasma Membrane... Molecular Features of Estrogen... Tissue-Specific, Rapid Responses... ER Splice Variants Controversies in Hormone... Conclusions References

 
OVARIAN STEROIDS ARE classical hormones with autocrine, paracrine, and distant effects on a wide variety of tissues. These effects modulate the phenotype and function of many cells, both in rapid, transient and slower, sustained fashion. Estrogen has distant and prominent effects on most organs, including those of the cardiovascular, nervous, reproductive, and musculoskeletal systems. In this regard, clinical data relating hormone treatment, often replacement therapy in postmenopausal women, and diseases of these various organ systems are rapidly emerging. In some cases, results of clinical trials have been at variance with the growing body of molecular and animal data. It has thus become increasingly important to dissect the molecular features of estrogen’s interactions with its receptors and define the wide array of cellular responses, many of which are tissue specific.

Historically, estrogen receptors (ERs) have been considered ligand-activated transcription factors, residing in the cytosol and translocating to the nucleus upon ligand binding and dimerization. As with many other steroid hormone receptors, when in the nucleus, ERs can either directly bind to their consensus target DNA sequence or interact with other nuclear proteins (coactivators or corepressors), thereby either enhancing or repressing gene activation. This has been referred to as a genomic or nuclear-initiated estrogen response. However, plasma membrane estrogen binding sites were described more than 25 yr ago. In the past 15 yr, data regarding estrogen-triggered rapid signaling responses, independent of nuclear localization and transcriptional effects, have been convincingly demonstrated. These have been referred to as nongenomic or membrane-initiated estrogen responses. The site of signal initiation is a useful nomenclature, because it has recently become clear that signals initiated at the plasma membrane can, in turn, trigger signaling cascades that result in nuclear events, without ER nuclear translocation. The recognition of this signal cross-talk has been important and will be discussed below.

In this article, we will review the evidence for plasma membrane ERs, the nature of membrane ER-centered multimolecular complexes, and the signaling consequences of such receptor engagement. Because we believe that various ER splice forms are preferentially membrane targeted, we will discuss those splice variants. Finally, and in a clinically relevant context, we will discuss the cell/tissue specificity of estrogen-triggered signaling. Because our greatest experience has been with cells of the cardiovascular system, much of this review will describe existing and now well established complex formation and signaling responses in those cells. However, many of these vascular cell features apply to a wide range of other cells.

	Evidence for Plasma Membrane Receptors

Top Abstract Introduction Evidence for Plasma Membrane... Molecular Features of Estrogen... Tissue-Specific, Rapid Responses... ER Splice Variants Controversies in Hormone... Conclusions References

 
Evidence for membrane-associated steroid receptors first appeared in 1942, when a rapid anesthetic effect upon exposure to progesterone was described (1). After this, data supporting membrane-associated ERs, ultimately both ER and ERß isoforms, continued to accumulate, with rapid responses exhibited in many different tissues. Pietras and Szego (2) first described rapid effects of estrogen in 1975, noting immediate calcium fluxes in endometrial cells. Early estrogen responses in neuroendocrine tissue were also demonstrated, including a rapid rise in pituitary cell intracellular calcium, leading to action potential spikes and subsequent depolarization within 1 min (3). Rapid, estrogen-induced prolactin secretion from pituitary tumor cells has been described (4). Also, in these cells, there is evidence of surface estrogen receptors provided by the demonstration of a punctate immunofluorescent staining pattern, using anti-ER antibodies in nonpermeabilized cells. Rapid signaling has also been demonstrated in reproductive tissues such as breast (5, 6, 7, 8, 9), uterine (10), and ovarian (11, 12) cells as well as bone (13, 14, 15, 16) and neuronal tissue (17, 18, 19).

In addition to the aforementioned tissue types, membrane-associated ERs and rapid signaling are now well established within the vascular endothelium. We have used various membrane-impermeant forms of 17ß-estradiol (E2), including those covalently linked to either BSA or horseradish peroxidase. In human endothelial cells (EC), E2-BSA rapidly activates endothelial nitric oxide synthase (eNOS), leading to a 3-fold increase in intracellular cGMP within 5 min. A fluorophore-labeled E2-BSA produces a surface punctate membrane staining pattern in nonpermeabilized EC, similar to that described in pituitary tumor cells, above. These findings all strongly support ER localization at the EC plasma membrane (20).

Although the antibody reactivity data suggest that there is a receptor ectodomain, i.e. that ERs can exist as transmembrane molecules, it is the more accepted view that these receptors are membrane associated, anchored by scaffold proteins. Overexpression of striatin, a molecule that appears to facilitate the ER-centered, multimolecular complex formation, enhances ER plasma membrane localization (21). The Src-homology and collagen homology (Shc) adapter protein binds to docking sites on many growth factor receptors and associates with ERs through an interaction with IGF-I receptor (IGF-IR). Thus, in some cell types and settings, Shc appears to be an ER docking protein. Using small interfering RNA approaches, knockdown of either Shc or IGF-IR abrogates ER translocation to the plasma membrane induced by E2 (22). In addition to scaffold proteins, ER lipid modifications are involved in ER membrane targeting. Specifically, palmitoylation appears required, with cysteine 447 the apparent critical palmitoylation site (23). Furthermore, ERs are targeted to lipid rafts and, in ECs, the specialized membrane signaling organelles known as caveolae are the preferential sites of ER-centered complexes in EC (24, 25, 26). ER has been shown to interact with the caveolar structural protein, caveolin-1, through serine 522 of the ER E domain. This molecular interaction is critical for ER plasma membrane localization (27).

	Molecular Features of Estrogen-Mediated Rapid Signaling Responses

Top Abstract Introduction Evidence for Plasma Membrane... Molecular Features of Estrogen... Tissue-Specific, Rapid Responses... ER Splice Variants Controversies in Hormone... Conclusions References

 
Estrogen is able to evoke specific physiological responses in many tissues within seconds to minutes after ligand binding. Although this is not proof of membrane receptor localization, the rapidity of consequent events is certainly consistent with such a distribution. Many intracellular signaling cascades have been shown to be triggered by estrogen. Examples include activation of MAPK (5, 6, 13, 14, 20, 28, 29, 30) and phosphatidylinositol 3-kinase (PI3K)/Akt pathways (31, 32, 33, 34, 35), induction of ion channel fluxes (36, 37, 38), G-protein-coupled receptor-mediated second messenger generation (cAMP and calcium) (11, 16, 19, 39, 40, 41, 42, 43), as well as stimulation of growth factor receptors (44, 45). Activation of these pathways occurs in a cell-type-specific manner to alter downstream effectors and produce rapid physiological responses in target tissues.

We now consider ERs a central component of a membrane "signalsome" containing numerous molecules, the orchestration of which results in rapid signaling cascades. Because ERs do not have intrinsic kinase activity, these molecular interactions are critical to direct estrogen-stimulated rapid action and may occur in a cell-type-dependent fashion. Candidate molecules involved in formation of these signaling complexes include G-proteins (Gi), heat shock protein 90 (Hsp90), caveolin-1, matrix metalloproteinases (MMPs), Shc, modulator of nongenomic activity of the ER (MNAR), and c-Src. In addition to its potential as a docking protein for membrane-associated ER, E2 stimulates Shc phosphorylation, leading to Shc-Grb2-Sos complex formation, which in turn is critical to transduction of MAPK pathways (5). MNAR (also known as PELP1), is an important scaffold molecule in many tissues but may be preferentially expressed in rapidly proliferating cell types, as demonstrated in cancer-derived cell lines (46). MNAR acts as a scaffold protein and is anchored to c-Src, a nonreceptor tyrosine kinase, via its SH3 domain interaction with a PXXP motif on MNAR. This complex is further stabilized by the binding of c-Src’s SH2 domain to a phosphorylated tyrosine 537 of ER and binding of ER to LXXLL motifs on MNAR. Consequent to this series of interactions is activation of c-Src, leading to downstream signaling in the MAPK pathway through Ras and Raf (47).

	Tissue-Specific, Rapid Responses to Estrogen

Top Abstract Introduction Evidence for Plasma Membrane... Molecular Features of Estrogen... Tissue-Specific, Rapid Responses... ER Splice Variants Controversies in Hormone... Conclusions References

 
Within the cardiovascular system, rapid signaling pathways initiated by ER engagement have now been well defined. One method by which estrogen exerts rapid modulation of the vascular endothelium is via the enhanced production of nitric oxide (NO), a vasoprotective molecule important in maintaining vascular health. Critical to the transduction of this rapid signaling cascade is ER’s use of scaffold molecules and localization to caveolae (25, 26), as described above. The critical steps in E2-stimulated NO release include activation of eNOS via both PI3K/Akt and MAPK signaling pathways. E2 stimulation of EC leads to ER antagonist-inhibitable c-Src activation, via a tyrosine 530 dephosphorylation and tyrosine 416 autophosphorylation, followed by PI3K activation and recruitment of the serine/threonine kinase Akt to PI3K-generated membrane phospholipids. Akt phosphorylation then occurs on serine 473 and threonine 308. eNOS is a key Akt substrate, which then becomes phosphorylated on serine 1177, leading to stimulated enzyme activity and enhanced NO production in the presence of sufficient substrate and cofactor levels (31, 32) (Fig. 1). Signaling pathways leading to additional eNOS phosphorylation/activation responses can be achieved via MAPK (20), G- proteins (39), and alterations in intracellular calcium (40) and potassium (37). The relevant outcome is enhanced NO production, which promotes maintenance of vascular homeostasis through vasodilation, inhibition of platelet aggregation, leukocyte adhesion, and smooth muscle cell proliferation as well as playing a significant role in angiogenic responses (48). In vivo correlates have been described, with estrogen-induced femoral arterial vasodilatory responses to estrogen occurring within minutes. In some models, these responses are MAPK dependent (49).

View larger version (36K): [in this window] [in a new window]   FIG. 1. ER-centered, eNOS-activating molecular complex in caveolae within vascular endothelium. Depicted at the bottom are molecular features of plasma membrane targeting, including palmitoylation, caveolin-1, and c-Src interactions. [Reproduced with permission from K. H. Kim and J. R. Bender: Science STKE 288: pe 28, 2005, (76 ).]

This regulation of cell growth highlights the cross-talk between estrogen’s nuclear-initiated (previously called genomic) and membrane-initiated, rapid signaling cascades. For example, PELP1/MNAR, the aforementioned scaffold protein, is involved in rapid, estrogen-stimulated Src/MAPK activation in breast cancer cells, enhancing growth factor-induced phosphorylation of signal transducer and activator of transcription 3 (STAT3). Phospho-STAT3 then may play a role in tumorigenesis through transcriptional enhancement of oncogenic, proproliferative genes, such as Cyclin D-1, c-Myc, and c-Fos (51). Transcription of Cyclin D1, important in cellular progression through the G1 phase of the cell cycle, can also be enhanced by the estrogen-mediated rapid activation of the Src/PI3K pathway (33) as well as activation of the cAMP/protein kinase A (PKA) pathway leading to cAMP response element (CRE)-mediated transcriptional activation (41).

Estrogen also exerts neuroprotective properties in the brain by means of rapid signaling, as well as by similar membrane-to-nuclear cross-talk events (52), through a membrane-associated ER observed in many neuronal cell types. In vitro studies demonstrate E2-mediated neuroprotection against glutamate excitotoxicity (28, 29, 53) and ß-amyloid peptide (31) via MAPK pathways. E2 can also confer activation of the cAMP/PKA pathway (42) in neuronal cells leading to CRE binding protein phosphorylation and activation of CRE-mediated transcription. This converges with MAPK-triggered transcription, affecting genes such as neurotensin/neuromedin (54). Neuroprotection can also be mediated by the estrogen-stimulated insulin receptor/PI3K/Akt pathway in the retina (34). Similarly, E2 can act via the Akt pathway to prevent injury-induced apoptosis in a model of ischemia using metabolically inhibited cortical explant cultures (35). Estrogen-induced rapid modulation of intracellular calcium has been observed in multiple neuronal cell types, such as astrocytes (43), granulosa cells (11), and striatal neurons (19).

Estrogen-triggered rapid signaling events in osteocytes confers protection against bone loss. Osteoblast MAPK phosphorylation is observed within 5 min of E2 stimulation (13). Antiapoptotic mechanisms may be induced by signaling through activation of Src/Shc/ERK (MAPK subfamily) pathways and subsequent nuclear ERK accumulation, which alters kinase-dependent transcription factors, thereby affecting transcription (14). The kinetics of ERK phosphorylation and length of time retained in the nucleus determine its pro- vs. antiapoptotic effects in osteoclasts and osteoblasts/osteocytes, respectively (55). ER may also interact with PKC and c-Src to affect osteoblast differentiation (15). Rapid PKC activation responses to E2-BSA have also been observed in chondrocytes through a mechanism dependent on G protein coupling to phospholipase C (16).

Thus, there now exists a large amount of data supporting rapid responses to estrogen in a wide variety of cells and tissues. Furthermore, the diversity of responses is amplified by consequential effects on transcription, initiated by signals generated at the plasma membrane (Table 1).

	ER Splice Variants

Top Abstract Introduction Evidence for Plasma Membrane... Molecular Features of Estrogen... Tissue-Specific, Rapid Responses... ER Splice Variants Controversies in Hormone... Conclusions References

The discussion of ER splice forms within vascular endothelium has mainly been restricted to the ER gene, located on chromosome 6. It contains eight exons that encode a 66-kDa full-length receptor. Like other steroid hormone receptors, ER can be divided into A-F motifs. The N-terminal AF-1 domain consists of the ligand-independent, transactivation A/B region. The C region is integral to DNA binding, whereas the D region functions in nuclear localization and dimerization. The AF-2 domain, or hormone-inducible transactivation domain, consists of the E/F region, responsible for dimerization, membrane targeting, and modulation of transcriptional activity. Multiple anti-ER antibody immunoreactive species have been described in numerous cell types. Subsequently, multiple transcripts of varying length have been correlated with the gene products of different sizes. In vivo gene targeting facilitated our understanding of ER variants. The first ER knockout (ERKO) mouse was generated by Lubahn et al. (63) in 1993 through gene targeting of exon 1. Subsequently known as the ERKO Chapel Hill, both male and female mice unexpectedly displayed relatively normal sexual development, although fertility was affected (63). When crossed with the ERß knockout (ßERKO) mouse, vascular responses to estrogen were largely maintained (64, 65). The retention of ER variants (55 and 46 kDa) was observed and provided the explanation for maintenance of estrogen responses. This was thought to be the consequence of a splice site and an internal translation initiation site, allowing for the production of ER short forms encoded by transcripts excluding exon 1 (66). The complete ERKO mouse was then generated by gene targeting at exon 2. This ERKO Strasbourg mouse, crossed with the ßERKO, loses a majority of its vascular responses to estrogen (67). This series of experiments, in multiple laboratories, demonstrated that ER variants are functional in vascular tissue.

ER RNA splicing mechanisms involve both alternative promoter usage and the more typical but variable intron-exon splicing. Within the vascular endothelium, generation of a full-length, mature RNA transcript involves splicing of upstream promoter exons B–F to an acceptor site 70 nucleotides upstream of exon 1, resulting in a 66-kDa receptor. However, a 46-kDa splice variant is abundant in EC (58, 61). Creation of this variant likely involves splicing of upstream promoter exons E–F to a translation initiation ATG within exon 2. This alternative splicing creates an N-terminal truncated receptor, devoid of the A/B regions but which appears fully functional to generate ligand-dependent, rapid signaling responses (58, 68).

Both ER66 and its truncated counterpart, ER46, can associate with the plasma membrane in osteoblasts (15, 69) and EC. They can form heterodimers, potentially competitively inhibiting the maximally efficient DNA binding achieved by ligand-bound ER66 homodimers. We have shown that ER46 is the predominant splice form expressed in the immortalized EC line EAhy.926. Although fully capable of mediating rapid signaling in these cells, ER46 is unable to support estrogen-dependent estrogen response element transactivation. Membrane-impermeant E2 stimulates rapid ER46 mobilization to caveolae microdomains, with consequent NO release. In ER-negative COS-7 cells transfected with ER46 or ER66 and eNOS, ER46 more efficiently transduces E2-stimulated eNOS phosphorylation responses than ER66. In fact, upon cotransfection, ER66 appears to reduce the noted ligand-triggered activation, suggesting a repressive or competitive effect of ER66 on rapid, ER46-mediated signaling. At the very least, these results confirm that there exists a signaling hierarchy and that selective receptor isoforms are likely to be more functional for specific responses in a given tissue (61).

	Controversies in Hormone Replacement Therapy

Top Abstract Introduction Evidence for Plasma Membrane... Molecular Features of Estrogen... Tissue-Specific, Rapid Responses... ER Splice Variants Controversies in Hormone... Conclusions References

 
Despite our growing understanding of the molecular controls on estrogen responses in numerous tissues, debates over the potentially beneficial and harmful effects of hormone replacement therapy (HRT) have intensified. This controversy is perhaps most evident with regard to estrogen use in cardiovascular disease prevention. Earlier retrospective studies supported a cardioprotective effect of estrogen in postmenopausal women. However, more recent prospective, randomized clinical trials have challenged this concept. The Heart and Estrogen/Progestin Replacement Study, which enrolled postmenopausal women with established coronary artery disease, failed to demonstrate a benefit of estrogen in secondary cardiovascular prevention. In fact, there was an increase in cardiac events in the hormone-treated group within the first study year (70, 71). The more recent, widely publicized Women’s Health Initiative (WHI), evaluating hormone effects on primary cardiovascular prevention, was terminated early because of a small but significant increase in cardiovascular events, with adverse outcomes, in the hormone-treated group (72).

These two studies (and others) have directed the avoidance of HRT in postmenopausal women, at least in the context of cardiovascular disease prevention. However, these conclusions have created a conundrum. As described above, there are numerous cellular and molecular studies supporting that estrogen promotes a favorable vascular profile, and many animal studies correlate with these in vitro results. Most notable is a macaque atherosclerosis study, in which ovariectomized monkeys were placed on a high-cholesterol diet in the absence or presence of HRT. Those started on HRT at the time of surgical menopause had a 70% reduction in the burden of coronary atherosclerosis. In contrast, when HRT was delayed 2 yr, the beneficial effect was lost (73). This underscores a critical timing issue, which can be related to the aforementioned recent clinical trials. The average age at enrollment in the WHI was 62.5 yr, or an average of 12 yr post menopause. If the beneficial effects of estrogen are in prevention of early vascular pathology, as many of us believe, initiating HRT when atherosclerosis is likely already established would not be expected to provide the protection seen in the earlier observational studies. Certainly, the Heart and Estrogen/Progestin Replacement Study trial enrolled women with preestablished cardiovascular disease. Furthermore, subgroup WHI analysis does, in fact, display a beneficial cardiovascular effect of hormone therapy in women 50–59 yr of age (74), although the data are underpowered to achieve statistical significance, based on the subgroup size (75). It is clear that clinical trials for women within the menopausal transition are required to clarify whether HRT can provide cardiovascular protection.

	Conclusions

Top Abstract Introduction Evidence for Plasma Membrane... Molecular Features of Estrogen... Tissue-Specific, Rapid Responses... ER Splice Variants Controversies in Hormone... Conclusions References

 
Our understanding of the membrane-initiated responses to estrogen has grown dramatically in the last 25 yr. These responses are primarily driven by ER-centered, multimolecular complexes, often localized to specialized signaling organelles within the plasma membrane. The distinction between genomic and nongenomic responses has blurred, because there are now many examples of membrane-initiated signals that, through signaling cascades, result in transcriptional events. The prevalent clinical controversies in HRT make further molecular dissection imperative. Key ER molecular details that will direct therapeutic targeting include those related to tissue specificity, dominance of receptor splice form expression, and predominant molecular interaction partners within a given cell. Understanding these tissue-specific intricacies will greatly enhance our ability to favorably manipulate vascular responses, regulate cellular proliferation and apoptosis in hormone-responsive cancers, achieve neuroprotection, and positively impact bone remodeling.

	Footnotes

 
This work was supported by National Institutes of Health Grants HL61782 and T3207950 and by the Raymond and Beverly Sackler Foundation.

Disclosure summary: All authors have nothing to declare.

First Published Online August 31, 2006

Abbreviations: CRE, cAMP response element; E2, 17ß-estradiol; EC, endothelial cells; ER, estrogen receptor; ERKO, ER knockout; GPR30, G protein-coupled receptor 30; HRT, hormone replacement therapy; IGF-IR, IGF-I receptor; MNAR, modulator of nongenomic activity of the ER; PI3K, phosphatidylinositol 3-kinase; PKA, protein kinase A; Shc, Src-homology and collagen homology; STAT3, signal transducer and activator of transcription 3; WHI, Women’s Health Initiative.

Received May 31, 2006.

Accepted for publication July 6, 2006.

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Top Abstract Introduction Evidence for Plasma Membrane... Molecular Features of Estrogen... Tissue-Specific, Rapid Responses... ER Splice Variants Controversies in Hormone... Conclusions References

 

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