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or ß1
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
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: adriana.maggi{at}unimi.it
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß
(ER and ERß, 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 17ß-estradiol-dependent activation of ER and
ERß 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 ERß, 17ß-estradiol induces two distinct
morphological phenotypes. ER activation results in increased length
and number of neurites, whereas ERß 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 ERß-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 ERß may play distinct biological roles and support the
hypothesis that 17ß-estradiol activates selected intracellular
signaling pathways depending on the receptor subtype bound.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ERß expression,
supporting their differential function (12, 13, 14, 15, 16). Yet, evidence for a
differential metabolic effect resulting from the
E2 binding to ER or ERß is still
lacking.
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 ERß, 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 carboxyl-terminal 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 hormone-binding 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 ERß 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
agonist-dependent activation of ER and ERß on SK-N-BE cell
morphology. Our results show that in the same cell line,
E2 may induce clearly distinguishable
morphologies by binding to ER or ERß. 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, pCMVhER, and ERß, PCMV5hERß,
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 µg/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 x
105. For transfection studies, 2 x
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 µl/well
phenol red-free DMEM with 10% dextran-coated charcoal-FCS, 1% mix of
essential amino acids (aa), 50 U/ml penicillin G, 50 µg/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 µg/ml
LacZ-containing plasmid (pCMV-ßgal, Promega Corp.,
Milan, Italy) or 3 µg/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 µg/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 ß-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-ß-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 ERß 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 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%.
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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 µl 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 x10 eyepiece and a x32 objective.
Morphometric analysis
Experiments were generally carried out in duplicate. Neurite
length or number was evaluated in a blind fashion on
ß-galactosidase-stained cells using a Carl Zeiss
Axiovert microscope with a x32 objective and a x10 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/ß and ERß/ chimera
The pMT-hER/rERß 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 ERß 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 ERß (aa 92 of the 485-aa form), simultaneously deleting aa 1–92
of rat ERß (485 aa). For the pMT-rERß/hER, a fragment containing
most of the N-terminal domain of rat ERß (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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 expressing the marker protein
coexpressed the receptor of interest. Cells were then transfected with
ER/GFP or ERß/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 ERß-transfected cells
generally differentiated as bipolar neural cells with oval perykaryon
and a single process emanating from the cell body (Fig. 1c).
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or ERß and performed morphometric
analysis to assess the effect of the treatment on neurite growth and
sprouting. The choice of ß-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 ß-gal transfected cells, respectively. Image analysis
of cells transfected with identical concentrations of ERß 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 µg of both ER and ERß 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.
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and 4
show that in the chimera ER/ß, the presence of the ER A/B
domain capacitates ERß 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 ERß/ chimera, in which the A/B domain was
substituted with the ERß 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/ß 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
ERß/ 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.
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or ERß 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 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 ERß 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 ERß. 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 ERß 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 ERß (+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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ERß. 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 ERß, 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. ERß KO mice, on the contrary, have normal sexual
behavior and are fertile. Breast development is impaired in ERKO,
whereas in ERß KO, breasts develop normally, and females can lactate
(32). These models together with the observation that ER and ERß
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 E2-liganded
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
ERß-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 elongating mode
of growth (similar to that observed in the presence of ERß) 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 ERß 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 ERß in central nervous system
ontogeny might be in agreement with the observation of higher perinatal
ERß expression that coincides with a higher estrogen synthesis rate
and aromatase expression (38). Because of the 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 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 ERß on the signaling that regulates cytoskeletal
organization described here may therefore also exist in tumor
cells.
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Acknowledgments |
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Footnotes |
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Received October 22, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in vivo and in vitro. Biochem Biophys Res
Commun 243:122–126[CrossRef][Medline]
in the rat ovary.
Endocrinology 140:963–971 and ß during mouse embryogenesis. Mech Dev 8:163–167
receptor chimeras. Endocrinology 139:4513–4522 and ERß at AP1 sites. Science 277:1508–1510, but not by ERß. EMBO
J 18:4270–4279[CrossRef][Medline]
and ERß. J Steroid Biochem Mol
Biol 69:165–175[CrossRef][Medline]
in SK-N-BE neuroblastoma and COS-1 renal carcinoma cells. Mol
Endocrinol 12:835–841 and beta. Proc
Natl Acad Sci USA 96:7077–7082-knockout
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0.001 ±
SEM, E2 vs. untreated cells, by
two-way ANOVA.
, Significant difference among the groups indicated
(P