- Top of page
Oestrogen is important for the development of neuroendocrine centres and other neural networks including limbic and motor systems. Later in adulthood, oestrogen regulates the functional performance of different neural systems and is presumably implicated in the modulation of cognitive efficiency. Although still a matter of controversial discussion, clinical and experimental studies point at a potential neuroprotective role of oestrogen. Concerning the concept of cellular oestrogen action, it is undisputed that it comprises the binding and activation of nuclear receptors. The last decades have, however, immensely broadened the spectrum of steroid signalling within a cell. Novel steroid-activated intracellular signalling mechanisms were described which are usually termed ‘non-classical’ or ‘non-genomic’. The brain appears to be a rich source of this new mode of oestrogen action. Studies from the past years have pinpointed non-classical oestrogen effects in many CNS regions. All available data support the view that non-classical oestrogen action requires interactions with putative membrane binding sites/receptors. In this article, we aim at compiling the most recent findings on the nature and identity of membrane oestrogen receptors with respect to the brain. We also attempt to turn readers attention to the coupling of these ‘novel’ receptors to distinct intracellular signalling pathways.
Oestrogen represents a pleiotropic factor to the CNS. It was demonstrated several decades ago that oestrogen is important for the regulation of neuroendocrine functions. Since then, additional target cells in the brain have been discovered and, to make a overdrawn statement, at present, it appears difficult to find a brain region which is not oestrogen-responsive at all (McEwen 2002). Because many of the oestrogen effects on neurones are stimulatory and ‘beneficial’, it is not surprising that several studies have also found a clear-established role for oestrogen as a neuroprotective factor (Garcia-Segura et al. 2001; Behl 2002). Besides fulfilling the task as a regulatory and protective factor in the adult brain, oestrogen is involved in the stimulation of ontogenetic processes in the CNS (Beyer 1999). Oestrogen promotes neuronal survival and co-ordinates cell differentiation (Naftolin et al. 1996; Sawada et al. 2000; Beyer et al. 2000). One particular attribute of oestrogen in the developing CNS is its potency to induce sexual differentiation of brain cells and neural circuits (Beyer 1999).
Despite the long history of oestrogen effects in the brain, the knowledge of oestrogen action at the cellular level is still scanty. It is beyond any question that steroids modulate gene transcription by interacting with nuclear receptors. Upon binding to its cognate receptor proteins, complex conformational changes occur at the receptor site and the composition of interacting proteins is altered, thus allowing the receptor to translocate to the nucleus and bind to specific sequences in the promoter region of target genes (Beato and Klug 2000). The timescale of this action varies from 1 h for aldosterone to several hours for other steroids. The ligand-dependent regulation of gene expression is generally sensitive to transcriptional and translational inhibitors as well as inhibitors of nuclear receptors. In the case of oestrogen, two types of nuclear receptors are known: oestrogen receptor-α (ER-α) and ER-β; ICI 182 780 represents the most frequently used antagonist for nuclear ERs (Kuiper et al. 1997). The two receptor subtypes share a similar structural organization into domains but reveal distinct differences in their binding affinities for different ligands and selective ER modulators (Gruber et al. 2002).
Besides genomic action, there is a growing body of evidence that oestrogen can achieve physiological effects through different signalling mechanisms which are usually subsumed as ‘non-genomic or non-classical’. Non-classical oestrogen effects are characterized by their rapid onset (s to min) and their insensitivity to transcriptional inhibitors. These effects are likely to be mediated by receptors integrated or associated with the plasma membrane and by an activation of distinct intracellular signalling cascades (Falkenstein et al. 2000; Küppers et al. 2001). Membrane action by and membrane receptors for oestrogen in the CNS were already postulated many years ago. The pioneering work of Kelly et al. (1976) described the rapid modulation of neuronal firing in the pre-optic region by oestrogen and posed the question of the existence of membrane receptors. At the same time, binding sites for oestrogen were described at the surface of endometrial cells (Pietras and Szego 1977). A few years later, high-affinity membrane-binding sites were found the in the uterus (Parikh et al. 1980) and in neurones (Towle and Sze 1983). Nowadays, membrane-associated oestrogen-binding sites are accepted, albeit controversially discussed. They reveal a widespread distribution in mammals in various organ systems including the uterus, the cardiovascular system, bone, smooth muscle and neural cells (Falkenstein et al. 2000; Levin 2001).
These data raise the question about the identity and functionality of membrane ER. One might immediately claim the presence of novel ERs as it was done by several groups (reviewed by Wong et al. 1996; Falkenstein et al. 2000). Pancreatic β-cells appear to express an ER at their surface which resembles in its pharmacological profile the γ-adrenergic receptor (Nadal et al. 2000). In the CNS, interactions between oestrogen and catecholamine receptors were described in the 1980s (Paden et al. 1982). It appears that the competitive binding of oestrogen for these receptors is based on the catechol ring structure (Watts and Sutherland 1987). This is in line with observations showing dopamine-mediated regulation of motor behaviour to be sensitive to rapid oestrogen modulation through dopamine receptors (Becker and Beer 1986). Using a different experimental approach, Toran-Allerand et al. (2002) postulated the existence of a novel ER-X in the plasma membrane of cortical cells. ER-X appears to be enriched in caveolar-like microdomains of neuronal membranes. Pharmacological data reveal a high-affinity and saturable oestradiol binding with a Kd of approximately 1.6 nm. On the functional side, this type of cortical membrane ER is likely to be involved in the rapid oestrogen-dependent activation of mitogen-activating protein (MAP) kinases (Singh et al. 2000). The authors claimed in their study that this type of membrane ER is different from the known classical nuclear ERs, as it is also detectable in ER-α knockout mice. Such a conclusive statement, however, needs to be handled with care. The knockout model used in this study is known to have residual ER-α activity documented best by the presence of ER-α transcripts in different tissues, although showing rather reduced levels compared to the wild-type (Couse et al. 1997). Therefore, low expression levels of ER-α in these animals may account for membrane ERs. This is also suggested by the fact that (i) the membrane ER-X was immunprecipitated with an antiserum raised against the full-length ER-α and (ii) ER-X can be visualized by in situ hybridization using an oligonucleotide probe delineating a part of the ligand binding domain of ER-α (Toran-Allerand et al. 2002.)
Notwithstanding the importance of these data and the possibility of more than one potential membrane-associated protein that can interact with oestrogen and cause fast physiological changes, we will focus here on the alternative that classical nuclear ERs conform to the demands of a membrane receptor and may account for many of the observed non-classical oestrogen effects. Evidence to support this hypothesis came first from in vitro studies using conventional immunocytochemistry (ICC). Using antisera against nuclear ERs, Pappas et al. (1995) observed a labelling of the plasma membrane in rat pituitary tumour cells. The membrane localization of ERs is in concordance with the known rapid oestrogen-induced prolactin release in this cell line. Several studies then demonstrated classical nuclear receptors to be located at the plasma membrane of a variety of cell types, including endothelial cells (Caulin-Glaser et al. 1997) and spermatozoa (Luconi et al. 1999). This supports the view that membrane oestrogen action represents a universally rather than exotic phenomenon. Membrane ERs can be visualized by different experimental approaches. In the CNS, ICC and confocal microscopy show a predominant localization of nuclear ERs in close proximity to the plasma membrane throughout the neurone, including the soma and neurite (Clarke et al. 2000) and in dendritic spines and axon terminals (McEwen et al. 2001). Our studies using either fluorescence-labelled oestrogenic (E-BSA) or antibodies against different epitopes of nuclear ERs confirm these observations. We were able to visualize specific binding sites for E-BSA at the neuronal plasma membrane (Fig. 1a). Similar data were obtained when we applied antisera against ERs (Fig. 1b). These findings imply that classical ERs may act within a cell in a dynamic way and suggest they ERs can be found in various subcellular structures. This is supported by the demonstration that oestrogen is capable to bind and interact with proteins in the mitochondrial membrane (Ramirez et al. 2001) and that ERs are associated with pre-synaptic structures thereby controlling synaptic transmission (Adams et al. 2002).
Figure 1. Confocal analysis of ligand-labelling and immunocytochemical staining of cultured mouse midbrain neurones. (a) Living cells were exposed to 17-β-oestradiol coupled to hemisuccinate-BSA-FITC at a steroid concentration of approximately 1 n m for 5 min followed by a brief washing step and subsequent fixation. Note the presence of labelled clusters at the surface of the cell soma (arrowheads). Pre-incubation with unlabelled oestrogen completely prevented this staining. (b) Immunocytochemistry with an polyclonal antiserum specific for the nuclear oestrogen receptor-α. The arrows point at clusters of ER-α associated with the neuronal surface (magnification × 550).
Download figure to PowerPoint
Despite the physiological and structural evidence of membrane ERs in the CNS, several questions remain to be answered: (i) are both subtypes of classical ERs (α/β) equally located at the plasma membrane and (ii) how are membrane ERs integrated/attached to the membrane and finally linked to signal transduction systems? Relating to the first point, Razandi et al. (1999) used a Chinese hamster ovary (CHO) cell transfection assay and found that both ERs are located at the plasma membrane. It is noteworthy that, although only a small percentage of transfected ERs are transported into the membrane, these receptors are in the position to rapidly trigger IP3 and cAMP formation. Other studies using conventional staining methods detected solely ER-α in the plasma membrane of neurones (Clarke et al. 2000; Watson et al. 2002). In order to pursue this line, we have applied confocal and fluorescence-activated cell sorting (analysis) (FACS) scan analysis of neuronal cell cultures from various brain regions. Using a set of different antisera against ER-α and ER-β for ICC, we have detected only ER-α allocated at the plasma membrane (Fig. 1b). ER-β labelling at the surface was never observed. This is in agreement with FACS data from our lab. Using fluorescein isothiocyanate (FITC)-conjugated oestrogen-BSA, a subpopulation of neurones (approximately 30%) was positively labelled (Fig. 2). Specific labelling at the neuronal plasma membrane was also obtained with ER-α antibodies, whereas no such effect was seen with ER-β antisera (unpublished data). These findings confirm a recently published FACS study with human endothelial cells (Russell et al. 2000). Taken together, the available information suggests that the classical ER-α is associated predominantly with the neuronal plasma membrane and is responsible for rapid non-classical oestrogen action. Nevertheless, independent investigations point at the possibility that there may exist additional membrane receptors for oestrogen besides ER-α such as aminergic receptors and novel, yet unidentified, binding proteins. This point warrants further investigation, as this diversity of membrane ERs may critically contribute to the well-known differences in rapid physiological responses of cells upon oestrogen stimulation.
Figure 2. FACS analysis of primary midbrain neuronal cell cultures. (a) Background gating of living neural cells showing autofluorescence. (b) Treatment of cells with a FITC-conjugated BSA construct revealed no specific labelling of cells. (c) Exposure of neurones to a FITC-conjugated 17β-oestradiol-BSA (FITC-E-BSA) construct resulted in a specific labelling and shifting of a subpopulation of cells (white arrow) which represents approximately 20–30% of all analysed cells. (d) Propidium iodide staining was used to detect dying or dead cells (black arrowhead) which are different from FITC-E-BSA-positive cells (white arrow).
Download figure to PowerPoint
Referring to the second question, an important issue is how ER-α is affixed to the plasma membrane and coupled to intracellular signalling processes. At present, no convincing data are available which could explain the membrane location of classical Ers, such as myristolation/palmitolation sites. This calls for the presence of interacting proteins which confirm that classical ERs do not possess lipophilic domains nor do they contain transport and fix classical ER-α to the surface. To further prove this hypothesis, we have started immunoprecipitation experiments with ER-α-specific antisera using neural membrane preparations. This approach yielded several not yet fully characterized proteins to be co-precipitated with ER-α. The composition of these associated proteins, which range from 20 to 160 kDa, appears to be tissue- as well as cell type-specific. This could be a hint to a better understanding of the known tissue specificity of rapid oestrogen effects on intracellular signalling cascades. Recently, Wong et al. (2002) discovered and characterized one of these ER-interacting proteins which appears to be the missing link connecting ERs to the MAP-kinase pathways. This protein with a molecular weight of approximately 120 kDa which was detected in a human tumour cell line and termed MNAR (modulator of non-genomic activity of oestrogen receptor) represents a member of the Src family of tyrosine kinases which is capable of phosphorylating members of the MAP kianse family such as Erk1/2. We have detected a corresponding mouse homologue, which is selectively expressed in neural cells (unpublished data) and implicated in the rapid activation of MAP kinases by oestrogen (Ivanova et al. 2001a). MNAR contains a protein motif which can interact with a hydrophobic groove in the ligand binding domain of nuclear hormone receptors. This allows MNAR to act as a scaffold protein that can incorporate ERs. Futhermore, MNAR is also found outside the nucleus localized close to the plasma membrane. It is still speculation, but we assume that MNAR belongs to a distinct new class of nuclear receptor-interacting proteins with several yet unidentified members. This makes them promising cellular targets being responsible for the coupling of membrane ERs to the different intracellular signalling cascades.
Despite the unresolved mechanism of anchoring of ERs to the membrane, it is unchallenged that these membrane receptors are in the position to activate distinct intracellular signalling cascades. We and other groups have proved that interactions between oestrogen and membrane ERs are apparently often linked to calcium-dependent signalling processes (Wong et al. 1996; Falkenstein et al. 2000; Beyer et al. 2002). In the CNS, this has been demonstrated for dopamine neurones (Beyer and Raab 1998) and in the hippocampus (Sawai et al. 2002). Oestrogen effects on calcium homeostasis can easily be tested by intracellular calcium measurements using microspectrofluorimetry and FURA-2 as a calcium indicator. Figures 3(a and b) summarize some of our previous experiments. Oestrogen produces a rapid, dose-dependent and stereo-specific rise in intracellular calcium levels (Beyer and Raab 1998). Pharmacological studies further reveal that this effect is mediated via IP3 formation. Thus, calcium released from intracellular stores upon stimulation of the PLC/IP3 cascade by oestrogen probably represents the first sustainable physiological cellular response (Beyer et al. 2002). Because oestrogen in equal measure can activate different intracellular signal transduction pathways such a cAMP, protein kinase A, protein kinase C, PI3-kinase/Akt in a calcium-sensitive and cell type-specific fashion (Beyer and Karolczak 2000; Falkenstein et al. 2000; Ivanova et al. 2001a, 2002), our present working hypothesis is that calcium transients are an important controlling entity which define further downstream signalling events. This concept is illustrated in Fig. 3(c) for our experimental model, i.e. the developing nigrostriatal system. In some cases, we were able to relate non-classical oestrogen action and its coupling to intracellular signalling cascades to distinct functional biological responses of these cells. One of the most intriguing observation for developmental biologists is that oestrogen can stimulate the expression of growth factors such as brain-derived neurotrophic factor (BDNF) and growth-derived neurotrophic factor (GDNF) in the embryonic brain through calcium-dependent signalling processes (Ivanova et al. 2001b, 2002).
Figure 3. Rapid non-classical stimulation of intracellular signalling processes in neural cells by oestrogen interactions with putative membrane receptors. (a and b) Intracellular free calcium recordings in cultured midbrain neurones by fast fluorescence photometry with the calcium-sensor FURA-2. (a) Dose-dependency of rapid and transient calcium signals after oestrogen application (E) at different steroid concentrations. (b) Stereo-specificity of oestrogen effects. Calcium spikes were only observed after an exposure to 17β-oestradiol (Eβ) but not to 17α-oestradiol. Potassium stimulation (K) characterize responsive cells as neurones. (c) Schematic illustration of the cell type-specificity of oestrogen-dependent activation of intracellular signal cascades and the appropriate biological effects in the midbrain. For further details see text. Data summarize previous observations ( Beyer and Raab 1998 ; Beyer and Karolczak 2000 ; Ivanova et al. 2001a, 2002, 2003 ).
Download figure to PowerPoint
In summary, it appears safe to conclude that oestrogen effects in the brain include complex cellular mechanisms ranging from classical nuclear to non-classical membrane-mediated action. Both ways of cell signalling may be activated separately, although there is good evidence that they are intertwined at several cellular instances and can affect each other reciprocally. An important, but yet not fully clarified, issue is the location of ERs. We propose a highly dynamic intracellular mobility of classical ERs being located at nuclear sites but also extranuclear in different cell compartments including the plasma membrane. Although elusive, a key to understand this motility in future will be the identification the cellular interaction partners associated with ERs. They might be responsible on the one hand for the intracellular translocation and positioning of ERs; alternatively, they may be necessary for the functional coupling of ERs to intracellular signalling cascades.