Estradiol Induces Differential Neuronal Phenotypes by Activating Estrogen Receptor α or β 1

0013-7227/00/$03.00/0 Endocrinology Copyright © 2000 by The Endocrine Society

Vol. 141, No. 5 Printed in U.S.A.

Estradiol Induces Differential Neuronal Phenotypes by Activating Estrogen Receptor ␣ or ␤* CESARE PATRONE, GIUSEPPE POLLIO, ELISABETTA VEGETO, EVA ENMARK, IVAN DE CURTIS, J.-Å. GUSTAFSSON, AND ADRIANA MAGGI Center Milano Molecular Pharmacology Lab, Institute of Pharmacological Sciences, University of Milan (C.P., G.P., E.V., A.M.), 20133 Milan, Italy; Departments of Medical Nutrition and Biosciences, Karolinska Institute, Novum, Huddinge University Hospital (E.E., J.-A.G.), SM186 Huddinge, Sweden; and Cell Adhesion Unit, Department of Biological and Technological Research, San Raffaele Scientific Institute (I.d.C.), 20132, Milan, Italy ABSTRACT Estrogens are female sex steroids that have a plethora of effects on a wide range of tissues. These effects are mediated through two well characterized intracellular receptors: estrogen receptor ␣ and ␤ (ER␣ and ER␤, respectively). Because of their high structural homology, it has been argued whether these two receptors may elicit differential biochemical events in estrogen target cells. Here we examine the effect of 17␤-estradiol-dependent activation of ER␣ and ER␤ on neurite sprouting, a well known consequence of this sex hormone action in neural cells. In SK-N-BE neuroblastoma cells transfected with ER␣ or ER␤, 17␤-estradiol induces two distinct morphological phenotypes. ER␣ activation results in increased length and number of neurites,

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URING MAMMALIAN development, 17␤-estradiol (E2) exerts a critical influence over the architecture and survival of neural cells in both central and peripheral nervous systems. In the mature central nervous system, this hormone has a considerable activity on neural cell metabolism and plasticity, as documented by several studies carried out in vivo (1, 2) and in vitro (3, 4). Distinct effects of the hormone in developing and mature animals are also observed in other target organs, such as uterus, mammary gland, and bone (5– 8). To explain how a single molecule may elicit this multiplicity of functions, several hypotheses have been advanced: 1) formation of gradients of hormone concentration, 2) receptor heterogeneity, 3) variable receptor density, 4) ligand and unliganded activation of a single receptor (4), and 5) recruitment and availability of coactivators or corepressors. The recent description of a second intracellular estrogen receptor, ER␤ (9, 11), has spurred research on the biological relevance of ER heterogeneity. Localization studies have shown similarities, but also differences in ER␣ and ER␤ expression, supporting their differential function (12–16). Yet, evidence for a differential metabolic effect resulting from the E2 binding to ER␣ or ER␤ is still lacking. Received October 22, 1999. Address all correspondence and requests for reprints to: Dr. Adriana Maggi, Center MPL, Institute of Pharmacological Sciences, Via Balzaretti 9, I-20133 Milan, Italy. E-mail: [email protected]. * This work was supported by Telethon (E600), Italian Association of Cancer Research, BIOMED II (BMH4-CT-2286), Consiglio Nazionale Delle Ricerche (National Council for Research) Targeted Project Biotechnology, ISS Multiple Sclerosis Program, and the Italian Ministry of Education (CIP 9806267988).

whereas ER␤ activation modulates only neurite elongation. By the use of chimeric receptors we demonstrate that the presence of both transcription activation functions located in the NH2-terminus and COOH-terminus of the two ER proteins are necessary for maintaining the differential biological activity reported. ER␣-dependent, but not ER␤-dependent, morphological changes are observed only in the presence of the active form of the small G protein Rac1B. Our data provide the first clear evidence that, in a given target cell, ER␣ and ER␤ may play distinct biological roles and support the hypothesis that 17␤-estradiol activates selected intracellular signaling pathways depending on the receptor subtype bound. (Endocrinology 141: 1839 –1845, 2000)

Like other members of the intracellular receptor superfamily, the ERs once bound to the natural ligand modulate the transcriptional activity of target genes by binding to DNA sequences in their promoter and cooperating with selected transcription proteins. In the modular organization of ER␣ and ER␤, the DNA-binding domain is localized centrally, and the two transcription activation functions are in the amino-terminal A/B domain (AF-1) and in the carboxylterminal E domain (AF-2) along with the hormone-binding site. The two ER subtypes share a high degree of homology; the DNA-binding site has 96% identity, and the hormonebinding site has 58% identity. This explains the strong similarities reported for the two receptors in pharmacological studies (17). The A/B domain, however, is poorly conserved (18%), suggesting the possibility of a differential AF-1 function and therefore a diversification of the transcriptional activities of the two receptors (9, 10). The potential for differential actions of ER␣ and ER␤ has been shown in the context of estrogen signaling through an AP-1 site in transiently transfected cells (15) and more recently in regulation of the osteopontin receptor through the SF-1 response element (16); however, no evidence has yet been provided that the two receptors can regulate diverse metabolic effects in a physiological context. The present study stems from previous work (18, 19) and from the studies in which we showed that SK-N-BE neuroblastoma cells transfected with ER␣ represent a suitable model for study of the well known effect of estrogen on neurite sprouting (19, 20) in cells of neural origin. By this model system we compare the consequences of agonistdependent activation of ER␣ and ER␤ on SK-N-BE cell mor-

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phology. Our results show that in the same cell line, E2 may induce clearly distinguishable morphologies by binding to ER␣ or ER␤. We also show that this effect is mediated by different intracellular signaling molecules, thus implying differential regulatory functions for the two ER subtypes in the same cell. Materials and Methods Reagents Unless otherwise specified, chemicals were purchased from Merck & Co., Inc. (Bracco, Milan, Italy), culture media and additives from Life Technologies, Inc. (Milan and Rome, Italy), and steroids from Sigma. Disposables and culture dishes were obtained from Corning, Inc.-Costar (Corning, NY). The ER␣, pCMVhER␣, and ER␤, PCMV5hER␤, expression plasmids were obtained from P. J. Kushner’s (21) and J.-Å. Gustafsson’s (9) laboratories. PMTmMOR, PMTmMOR-(182–599) and PMTmMOR-(1–339) were provided by M. Parker (22). All Rac expression plasmids were provided by I. De Curtis (23, 24).

Cell culture and transfection SK-N-BE cells were cultured in phenol red-free RPMI 1640 supplemented with 10% FCS (Oxoid, Milan, Italy), 50 U/ml penicillin G, 50 ␮g/ml streptomycin sulfate, 2 g/liter sodium carbonate, and 0.11 g/liter sodium piruvate at 37 C at 99% humidity and 5% CO2. Cells were split once a week and seeded in 100-mm diameter petri dishes at a density of 2.5 ⫻ 105. For transfection studies, 2 ⫻ 105 SK-N-BE cells were seeded in six-well plates and kept at 37 C in a humidified incubator for 24 h. After replacement of the culture maintenance medium with 1750 ␮l/ well phenol red-free DMEM with 10% dextran-coated charcoal-FCS, 1% mix of essential amino acids (aa), 50 U/ml penicillin G, 50 ␮g/ml streptomycin sulfate, 4 mm glucose, and 2.5 mm glutamine, cells were incubated for a minimum of 4 h. Transfection was performed using the calcium phosphate method as previously described (25). To identify the transfected cells, 1 ␮g/ml LacZ-containing plasmid (pCMV-␤gal, Promega Corp., Milan, Italy) or 3 ␮g/ml green fluorescence protein (GFP) expression plasmid (pEGFPN1, CLONTECH Laboratories, Inc., Palo Alto, CA) were transfected alone or with the specified concentration of the other plasmids. When necessary, carrier DNA was used to ensure a final concentration of transfected DNA of 6 ␮g/well. The coprecipitate was removed after 16 h, and cells were washed twice with PBS before addition of the medium phenol red-free RPMI with dextran-coated charcoal-10% FCS. Morphological differentiation was obtained by the addition of 10 nm E2 in the incubation medium for 7 days. For ␤-galactosidase staining, cells were fixed with 2% paraformaldehyde and 0.2% glutaraldehyde in PBS for 5 min at 4 C and incubated for 16 h at 37 C in the presence of a solution containing 5 mm K3Fe(CN)6, 5 mm K4Fe(CN) 6, 2 mm MgCl2x6H2O, and the cromophore substrate 5-bromo-4-chloro-3-indolyl-␤-galactopyranoside (X-GAL from Roche Molecular Biochemicals, Inc., Mannheim, Germany). Cells were then washed a few times with PBS and kept at 4 C in PBS with NaN3 (Carlo Erba, Milan, Italy) for future morphometric analysis. As previous results have shown that the concentration of E2 may influence the extent of morphological changes induced in SK-N-BE cells (19, 20), we carried out a series of experiments aimed at assessing the concentration of the hormone necessary for maximal stimulation of ER␣ and ER␤ with regard to SK-N-BE morphology. The concentration of the hormone that ensured the highest increase in neurite length was 10 nm. This concentration also ensured the maximal transcriptional activation in transient transfection of both receptors, as assessed by analysis of luciferase production from the pvERE-tkLUC reporter gene (25). This concentration of hormone was therefore used throughout the study. In addition, we tested whether any of the DNA plasmids used affected SK-N-BE viability. Thus, cells were transfected with the different mixtures of DNA used in the present study and then stained each day for up to 7 days, and the transfected cells were counted. No significant difference was observed in the experimental groups used here. The efficiency of transfection was determined by counting stained vs. unstained cells on the seventh day in culture. When possible (e.g. experiments presented in Figs. 5–7), the reproducibility of the transfection

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efficiency was measured by cotransfecting a plasmid for the constitutive expression of luciferase (pvERE-tkLUC). Within the same experiment the efficiency was very reproducible (the sd was never higher than 20% of the average). In the various experiments the percentage of transfected/total cells was between 8 –25%.

Quantitative analysis of luciferase activity Luciferase activity was measured by integrating the luminescence signal for 15 sec; experimental values are expressed as arbitrary luminescence units normalized for the protein concentration of the extract (25).

Immunocytochemistry SK-N-BE cells were grown and transfected in 24-well plates on 5% gelatin-coated coverslips. After fixation for 10 min in 4% paraformaldehyde in 0.1 m PBS (pH 7.5), cells were washed three times with PBS and incubated for 20 min at room temperature with blocking solution (5% horse serum and 0.1% Triton X-100 in PBS). After three washes in PBS, cells were incubated with 100 ␮l of a 1:500 PBS dilution of the antihuman ER␣ rat monoclonal antibody H222 (provided by G. Greene) overnight at 4 C. Cells were washed three times before incubation with the secondary Texas Red-conjugated antirat IgG antibody (Vector Laboratories, Inc., Burlingame, CA). ER␣ immunoreactivity and GFP fluorescence were examined using a Carl Zeiss Axiovert inverted microscope (New York, NY) fitted with a ⫻10 eyepiece and a ⫻32 objective.

Morphometric analysis Experiments were generally carried out in duplicate. Neurite length or number was evaluated in a blind fashion on ␤-galactosidase-stained cells using a Carl Zeiss Axiovert microscope with a ⫻32 objective and a ⫻10 eyepiece connected to a CCD videocamera module using the NIH Image program 1.52. For each experimental group, 20 –30 fields were chosen at random. All cells present in the field were used in the analysis. In each experiment a minimum of 20 cells/dish were evaluated. Each experiment was repeated 3 times.

Generation of the ER␣/␤ and ER␤/␣ chimera The pMT-hER␣/rER␤ was generated as follows. The N-terminal domain of human ER␣ (aa 1–186) was amplified with PCR, and an SpeI site was created at the 3⬘-end to enable ligation to rat ER␤ DNA and the ligand-binding domain. This fragment was ligated into an SpeI-site in the 3⬘-end of the N-terminal domain of rat ER␤ (aa 92 of the 485-aa form), simultaneously deleting aa 1–92 of rat ER␤ (485 aa). For the pMT-rER␤/ hER␣, a fragment containing most of the N-terminal domain of rat ER␤ (aa 1–92 of the 485-aa form) was ligated to a construct containing the DNA- and ligand-binding domains of human ER␣, starting at aa 176 (directly 5⬘ of the DNA-binding domain) by use of a synthetic SpeI/ BamHI linker.

Statistical analysis Data are expressed as the mean ⫾ sem of the number of experiments indicated and were analyzed using a computerized package [Systat 5.1 (Systat, Evanston, IL) for MacIntosh (Apple Computers, Inc., Cupertino, CA)]. Statistically significant differences were determined by two-way ANOVA.

Results

Initially, we conducted our experiments by transfecting SK-N-BE cells with ER␣ expression plasmid along with a DNA coding for GFP as a marker. In these experiments we calculated the percentage of cells expressing both proteins at different ratios of marker/ER. At a 4:1 marker/ER␣ DNA ratio, the highest ratio used, 74 –77% of the cells, depending on the experiment, expressed both proteins (Fig. 1, a and b). This allowed us to conclude that the majority of the cells

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FIG. 1. ER␣ or ER␤ activation induces a differential morphology in transfected SK-N-BE cells. Cells cotransfected with ER and GFP cDNAs at a 3:1 ratio coexpress the plasmid-encoded proteins, as shown by fluorescence analysis (a) and immunocytochemistry with the anti-ER antibody H222 (b). After E2 treatment, ER␤-transfected cells (c) show a fuse-shaped soma and extend one or two thin neurites, which are generally unbranched. ER␣-expressing cells (d) extend from the soma numerous neurites that are branched and terminate in large growth cones (Carl Zeiss Axiovert inverted microscope fitted with ⫻10 eyepieces and ⫻32 objective).

expressing the marker protein coexpressed the receptor of interest. Cells were then transfected with ER␣/GFP or ER␤/ GFP and treated with 10 nm E2. Crude morphological examination over the days in culture showed that in the two experimental groups the hormone induced clearly distinguishable morphological changes. ER␣-transfected cells responded to the hormonal treatment by assuming a multipolar neuron-like morphology with a polygonal soma and multiple, highly branched neurites (Fig. 1d), whereas ER␤transfected cells generally differentiated as bipolar neural cells with oval perykaryon and a single process emanating from the cell body (Fig. 1c). We then transfected SK-N-BE cells with the ␤-galactosidase gene (␤-gal) for identification of the transfected cells and increasing concentration of either ER␣ or ER␤ and performed morphometric analysis to assess the effect of the treatment on neurite growth and sprouting. The choice of ␤-gal was dictated by the higher sensitivity of this detection system, which allowed us to transfect minute amounts of the plasmid containing this marker complementary DNA (cDNA). Figure 2, upper panels, shows that in the absence of transfected ERs, E2 treatment did not cause changes in neurite length or number. In the ER␣-transfected cells, treatment with 10 nm E2 induced a significant increment in the length and number of neurites

per cell. The extent of both effects correlated with the amount of ER␣ plasmid transfected. At the highest concentration of plasmid, neurite length and number in E2-treated cells were 2.9- and 1.6-fold greater than those in ␤-gal transfected cells, respectively. Image analysis of cells transfected with identical concentrations of ER␤ inserted in the same plasmid of ER␣ demonstrated that E2 induced a similar increase in neurite length (maximal effect, 2.8-fold); however, no augmentation of neurites per cell was observed. In both cases, the plasmid titration experiment showed that the receptor dosage is important for the quantitative, but not the qualitative, effect of the hormone on SK-N-BE cells. This experiment showed that 0.15 ␮g of both ER␣ and ER␤ produced the half-maximal effect. As RT-PCR analysis shows that the two plasmids express the same concentration of receptor messenger RNA (mRNA; not shown) and conceivably protein, it was concluded that in the presence of 10 nm E2, the two receptors have the same potency with regard to SK-N-BE cell differentiation. In view of the low homology of the amino-terminus of the two receptors, we next tested whether the differential effects above reported could be attributed to this region by a domain-swapping experiment. Figures 3 and 4 show that in the chimera ER␣/␤, the presence of the ER␣ A/B domain ca-

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FIG. 2. Differential effects of ER␣ and ER␤ activation on SK-N-BE neuritogenesis. One microgram of the ␤-gal plasmid, was transfected alone (⫺) or was cotransfected with increasing concentrations (25, 50, 100, 250, 500, 1000, and 2000 ng) of the expression plasmids for ER␣ or ER␤ (pCMVhER␣ and pCMV5hER␤, respectively). To induce morphological differentiation, cells were grown for 7 days after transfection in the growth medium alone (empty bars) or with 10 nM E2 (filled bars). Fixed cells were stained with 5bromo-4-chloro-3-indolyl-␤-D-galactopyranoside. Error bars correspond to the SE of 3 independent experiments in each of which 20 –50 cells/experimental group were scored (Carl Zeiss Axiovert microscope fitted with a ⫻10 eyepiece and a ⫻32 objective). *, Significant difference between estrogen-treated and untreated cells (P ⱕ 0.001), by two-way ANOVA.

FIG. 3. Immunocytochemistry of transfected SK-N-BE cells. SK-N-BE cells were cotransfected with 1 ␮g of the marker (␤-gal, pCMV␤-gal) and 4 ␮g of the empty vector or the plasmids containing ER␣, ER␤, or the chimeras ER␣/␤ and ER␤/␣. The experiment was performed using the same ER constructs made in the same vector (ER␣, pMTmMOR; ER␤, ERpMThER␤530, the longer form of ER␤; ER␣/␤, pMTERh␣/r␤; ER␤/␣, pMTERr␤/h␣). In ER␣/␤, the A/B amino-terminal domain was replaced with the same domain from ER␣; in ER␤/␣, the opposite substitution was made as described in Materials and Methods. After transfection, cells were grown and fixed for ␤-galactosidase staining. Images of ␤-gal-stained cells were collected on a Carl Zeiss Axiovert inverted microscope fitted with a ⫻10 eyepiece and a ⫻32 objective.

pacitates ER␤ to modulate the number of neurites per neuron. However, the chimeric receptor in repeated experiments displayed, on the average, a significant 25% reduced maximal activity with respect to ER␣, indicating that other sequences located in the more carboxyl-terminus may be necessary for the full activity on differentiation. This hypothesis was further supported by the observation that the ER␤/␣ chimera, in which the A/B domain was substituted with the ER␤ corresponding domain, is still determining the increase in both length and number of neurites per cell. Remarkably, the E2-induced morphology of the cells transfected with the ER␣/␤ chimera was hybrid; the cell body was fusiform, but the neurites had a certain degree of complexity and growth cones typical of ER␣-transfected cells. On the other hand, the ER␤/␣ chimera had a morphology very similar to that of the ER␣-expressing cells, with stellate somata and numerous neurites terminating in large, palmate growth cones (Fig. 3). Interestingly, when cells were transfected with ER␣ deleted in the amino-terminal or carboxyl-terminal, we failed to see any E2-dependent effect on neurite outgrowth (Fig. 5). This

observation indicates that sequences located in both A and E regions of the two ERs are necessary to induce the described morphological effects. Several recent studies have addressed the molecular mechanisms underlying cytoarchitectonic modifications that generally occur via local control of actin polymerization. These mechanisms, although still largely unknown, seem to involve small guanosine triphosphatases of the Rho family (26). We therefore tested whether the differential effects of E2-activated ER␣ or ER␤ on SK-N-BE cells could be mediated by some of these proteins. We chose to examine the role of Rac1b in the E2-induced neuritogenesis of SK-N-BE cells because this monomeric GTP-binding protein was recently shown to specifically increase the number of neurites per cell and dramatically expand neurite branching in primary neurons cultured in vitro (23). In addition, recent studies have shown that the highest mRNA expression of the human homolog of this gene (Rac3) is in the brain (27). To investigate whether ER␣ induced SK-N-BE morphological changes by stimulating Rac1B, we studied the effects of ER␣ activation

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FIG. 4. Quantitative analysis of the morphogenetic effects of transfected ER chimeras. Cells were transfected and treated as specified in Fig. 3. In each experiment, the morphometric analysis was made on a minimum of 20 ␤-galexpressing cells/experimental group. Results represent the mean ⫾ SEM of 3 independent experiments. Filled bars, Estrogen-differentiated cells; empty bars, controls. Statistical analysis was performed by two-way ANOVA with Tukey’s multiple range test at 95% and 99% for post-hoc comparison. *, Significant difference between estrogentreated and untreated cells (P ⱕ 0.001), by two-way ANOVA. ⌬, Significant difference among the groups indicated (P ⱕ 0.05)

in SK-N-BE cells transfected with ER␣ cDNA together with a dominant negative form of cRac1b, Rac1bN17, which ensured the blockade of this signaling pathway (24). Quantitative analysis of neurite lengths and number of neurites per cell (Fig. 6, upper and middle panels) showed that increasing concentrations of the dominant negative Rac1b progressively reduced the morphogenetic activity of E2. At the highest concentration of vector that could be transfected, the dominant negative Rac1b reduced about 80% the effect of E2 on neurite growth and sprouting. The effect of Rac1b appears to be specific, as the cotransfection of the dominant negative form of another Rac protein, Rac1aN17, did not interfere with the effects of ER␣. When the same experiment was carried out by cotransfecting the ER␤ containing plasmid, neither of the two dominant negative forms, Rac1bN17 or Rac1aN17, interfered with the E2 activity (Fig. 6, lower panel), suggesting that both small G proteins are not involved in the intracellular signaling of ER␤. At the highest concentration tested, the dominant negative and the constitutively active form, Rac1bV12, of the small G protein alone did not have any significant effect on SK-N-BE morphology. As expected, in these transfections, E2 treatment did not affect the number of neurites per cell (not shown). Northern analysis studies performed in SK-N-BE cells stably transfected with ER␣ did not show any effect of E2 treatment on Rac1b mRNA content (not shown). To verify whether the lack of Rac1bN17 on ER␤ activity on SK-N-BE differentiation was not due to a decreased transfection efficiency, the pvERE-tkLUC reporter gene was included in the different transfection DNA mixtures. As shown in Fig. 7, the transcription of the reporter gene was significantly increased by E2-activated ER␣ and ER␤ (⫹4.2- and 3.9-fold, respectively). The expression of Rac and its mutant forms did not alter the extent of activation of the reporter by neither of the two receptors. Discussion

Ligand binding studies have shown more similarities than differences between ER␣ and ER␤. In particular, the natural

ligand E2 binds both receptors with the same high affinity (0.05– 0.1 nm) (28). In addition, both E2-activated receptors recognize the same estrogen response element and display similar transcriptional activity. In contrast with these observations, the generation of knockout mice might suggest that the two receptor subtypes have distinct physiological functions. For instance, the ablation of ER␣ (29, 30), but not of ER␤, results in loss of E2-induced uterine hyperplasia (31, 32). In addition, ER␣ KO mice have a severe reproductive and behavioral phenotype, resulting in complete infertility. ER␤ KO mice, on the contrary, have normal sexual behavior and are fertile. Breast development is impaired in ERKO, whereas in ER␤ KO, breasts develop normally, and females can lactate (32). These models together with the observation that ER␣ and ER␤ have distinct and only partially overlapping patterns of expression (12, 13, 33) suggest that the two receptors might have distinct physiological functions. Supporting this view are studies carried out by Paech and Vanacker (15, 16) that show that these receptors signal in an opposite way on the activating protein-1 site and have a different behavior on the SFRE site. These latter studies, however, are limited to the observation of the transcriptional effects of the two proteins on synthetic reporter genes. The present study shows that when expressed in the same cell type, the two receptors can trigger differential intracellular signals, leading to distinct physiological and metabolic responses. Thus, E2 may induce the assembly of diverse transcription activation factors working on selected promoters. This is strongly suggested by affinity selection studies carried out with collections of peptides that were shown to discriminate between the two E2liganded receptors (34). The findings reported here may be of significance for understanding of the effects of E2 in the development of neural cells. The shape of E2-induced neurites in ER␤-transfected cells is typically tapered with few filopodia, in contrast with ER␣-transfected SK-N-BE in which the neurites often terminate in large, complex growth cones. These morphologies were described by Smith and Skene (35) in embryonic dorsal root ganglia neurons grown in vitro, who propose that the

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FIG. 6. ER␣, but not ER␤, morphogenetic effects are blocked by the dominant negative mutant of Rac1B. Upper and middle panels, SKN-BE cells were cotransfected with 1 ␮g ER␣ plasmid (ER␣) with increasing concentrations (100, 1000, and 3000 ng) of the dominant negative mutant of Rac1b, cRac1bN17 (1bN17), or a fixed concentration of the dominant negative form of Rac 1A (3000 ng; 1aN17). As a control, cells were also transfected with the expression plasmids for ␤-gal alone (1 ␮g), ER␣, and the dominant negative or the constitutively active (1bV12) mutants of Rac1b alone. Morphometric analysis was performed after 7 days of growth in medium deprived of estrogens (empty bars) or supplemented with 10 nM E2 (filled bars). The experiment was repeated three times. The variability of the transfection efficiency in the various experiments ranged from 9 –20%, with no significant effect on the final result. Lower panels, ER␤ activity on SK-N-BE cells was examined as described above. *, P ⱕ 0.001 ⫾ SEM, E2 vs. untreated cells, by two-way ANOVA.

FIG. 5. ER␣ deleted in the amino- and carboxyl-termini fails to induce neurite outgrowth. Cells were transfected with the ␤-gal plasmid alone (␤-GAL) or with ER␣; ER mutants [PMTmMOR-(182–599) carrying a deletion in the NH2 terminal, and PMTmMOR-(1–339) with a deletion in the COOH-terminal]. Empty bars, Controls; filled bars, cells treated for 7 days with 10 nM E2. Data represent measurements performed on a total of 50 cells/group in three separate experiments.

elongating mode of growth (similar to that observed in the presence of ER␤) may correspond to axonal regeneration, whereas the arborizing mode (as shown with ER␣) is more representative of sprouting. In view of the recent localization of both ERs in the peripheral nervous system (36, 37) and the fact that SK-N-BE cells are neural crest derivatives, our data might suggest that ER␤ receptors might cover a more relevant role for neural cell differentiation, whereas activation of ER␣ receptors would be more implicated in synaptic plasticity both during development and in the mature nervous system. Extending our results to potential effects of estrogens in the central nervous system, the hypothesis of a more pronounced role of ER␤ in central nervous system ontogeny might be in agreement with the observation of higher perinatal ER␤ expression that coincides with a higher estrogen synthesis rate and aromatase expression (38). Because of the

FIG. 7. ER␣ and ER␤ activities on the pvERE-tkLUC reporter are not affected by the presence of Rac1b or its mutated forms. SK-N-BE cells were transfected with 1 ␮g of either of the two receptor plasmids, 1 ␮g pvERE-tkLUC, and 1 ␮g ␤-gal. Where indicated, 3 ␮g Rac1b and its mutants plasmids were cotransfected. Extracts for luciferase activity were prepared at 48 h after transfection. Luciferase assay was carried out as described in Materials and Methods, and the enzyme activity was normalized on the proteins present in the extract.

well described involvement of Rac proteins in the formation of dendritic spines (39), the interaction between ER␣ and Rac1B protein revealed by the present study might support the hypothesis of ER␣ activity in mature neural cells and be of relevance to explain the mechanism of the described activity of E2 on dendritic spine synthesis (40, 41). We here propose that Rac1b is an important element for estrogen-differentiating potential in cells of neural origin. How ER␣ and Rac1b interact is only matter of speculation. Rac1b, similarly to the other components of the Rho family of proteins, is at least partially cytosolic and translocates to

ESTRADIOL INDUCTION OF DIFFERENTIAL NEURONAL PHENOTYPES

the cell membrane upon activation (26). Immunocytochemical studies (not shown) indicate that the ER␣ synthesized in transfected SK-N-BE cells is a nuclear protein. As our preliminary results fail to show any increase in Rac1b mRNA after E2 treatment, we hypothesize that the E2-ER␣ complex triggers Rac1b activity by augmenting the synthesis of the guanine nucleotide exchange factors or of other proteins capable of positive interaction with Rac1b. The implications of our study might extend beyond endocrine neurobiology alone. Recent studies in mammary tumor cells (42) have shown that in breast cancer cells (MCF-7) E2, after the binding of its intracellular receptor, triggers the phosphorylation of p190, a GAP protein that stabilizes GTP binding to p21ras, Rho, and Rac. In such cells this activity would have relevant functional consequences in coupling mitogenic signaling to the intracellular pathways regulating cytoskeletal organization and cell adhesion. The differential effect of ER␣ and ER␤ on the signaling that regulates cytoskeletal organization described here may therefore also exist in tumor cells. Acknowledgments We thank Thomas Barkhem at KaroBio for the preparation of the plasmids with chimeric ER and ER␤ 530, M. Parker for MOR plasmids and helpful discussion, Prof. Paolo Castano for advice on microphotography, Ms. Monica Rebecchi and Clara Meda for their excellent technical support, G. Rovati for statistical analysis, and P. Ciana, for his criticisms of the experimental work.

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