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NEWS IN PHYSIOLOGICAL SCIENCES
The Estrogen Trinity: Membrane, Cytosolic, and
Nuclear Effects
Angel Nadal,1 Mario Díaz,2 and Miguel A. Valverde3
1
Institut de Bioenginyeria, Universitat Miguel Hernández, Campus de Sant Joan, 03550 Alacant; 2Laboratorio de Fisiologia Animal, Departamento de
Biologia Animal, Universidad de La Laguna, 38206 Tenerife; and 3Unitat de Senyalització Cellular, Departament de Ciències Experimentals i de la
Salut, Universitat Pompeu Fabra, C/Dr. Aiguader 80, 08003 Barcelona, Spain
Estrogens have a wide array of biological effects, targeting both genomic and nongenomic mechanisms.
Classically, the estrogen receptors activating the transcription machinery in the nucleus were thought
to be distinct from the extranuclear estrogen receptors. Recently, this conceptual wall has started
to be dismantled as the result of the identification of novel routes of estrogen action.
D
uring the past two decades, we have witnessed a scientific
revolution that has led to the identification of novel mechanisms of action for estrogens, typically termed “nongenomic
estrogenic effects,” a nomenclature that we consider inappropriate. Therefore, before continuing with the description of the
different mechanisms of action, we would like to clarify a few
concepts. We will refer to “nuclear effect” or “classic pathway”
when the action of estrogen occurs at the nucleus, involving
the direct participation of the estrogen receptors (ER) as transcription factors without other previous signaling steps
required for estrogen action. All other mechanisms of action of
estrogens will be named “alternative pathways.” These alternative pathways might be initiated at either membrane or cytosolic locations and result in either direct local effects (e.g., modulation of ion channel activity and cell excitability) or effects
such as the regulation of gene transcription secondary to the
activation of signaling cascades [e.g., cAMP or mitogen-activated protein kinase (MAPK) cascades].
The first indication of the existence of alternative steroid
actions was obtained by Hans Seyle in 1942, who reported the
rapid anesthetic effects of progesterone, an effect now identified as the result of progesterone interaction with γ-aminobutyric acid (GABA) and/or glutamate ionotropic receptors. To
date, the study of alternative actions of estrogens has been
extended to a wide range of different tissues, helping us to
understand the key roles of estrogens in the modulation of cell
growth, proliferation, and differentiation as well as the regulation of the activity of organs as diverse as the uterus, ovary,
testes, breast, bone, blood vessels, and brain. From these studies it is possible to identify typical characteristics of alternative
mechanisms of action of estrogens: 1) the rapid time course
(from seconds to minutes) of the primary effect is too fast to be
compatible with either RNA synthesis or protein translation; 2)
they might be dependent on the presence of classic ERs, a
mechanism typically identified by the inhibition of the effect
by specific ER antagonists such as ICI-182780, or might be ER
independent, i.e., insensitive to ICI-182780; and 3) the extracellular membrane-delimited primary effect might be achieved
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by estrogen conjugated to membrane-impermeant molecules
(usually large molecules like albumin or peroxidase), although
this assumption must be cautiously approached because it is
now recognized that these membrane-impermeant estrogen
conjugates may have free estrogen associated with them.
The huge number of reports referring to nongenomic effects
of estrogens has prompted the scientific community to find a
way to classify such novel mechanisms of action. A first
attempt at such a classification was launched at the First International Symposium on Rapid Responses of Steroid Hormones
(3). The present article will discuss the estrogen effects depending on the cellular location of the primary estrogen action:
membrane, cytosol, and nuclear effects. This review will also
focus on the main mechanisms of action of estrogens at the
three locations rather than an exhaustive listing of different
effects of estrogens. For a comprehensive account of estrogen’s
effects, we refer the readers to two excellent reviews (1, 3).
Nuclear effects
Many of the members of the steroid/thyroid receptor family
shuttle between the cytoplasm and the nucleus in the absence
of ligands. On binding to the agonist, they translocate to the
nucleus where, acting as a transcription factor, they initiate
transcription (1). The binding of estrogen to the ER occurs with
high affinity and promotes conformational changes in the
receptor-protein complex and dimerization, leading to its activation. This process enables the complex to bind to selective
sites on the DNA known as estrogen-responsive elements (ERE)
within the promoter region of target genes. Through its binding
to ERE, the activated estrogen-receptor complex triggers the
synthesis of specific mRNAs and the production of a number
of proteins that are responsible for the effects of the hormone
in the different cell types (Fig. 1). There are two receptors identified so far: ERα and ERβ (8). The ERs are multidomain proteins
with A-B domains containing a ligand-independent transcription activation function 1 (AF-1). The C region contains a
highly conserved DNA binding domain with two zinc fingers
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251
FIGURE 1. Scheme of the different mechanisms of action of estrogens. Estrogen targets appear at 3 different cellular levels. First, there are membrane targets,
including estrogen and receptor(s) related to the classic nuclear estrogen receptors (ER), nonclassic ERs, and ligand- and voltage-activated ion channels. The activation of some of these receptors triggers signaling events (e.g., intracellular messengers), resulting in the activation of different kinases. Second, there are cytosolic targets, mainly represented by the classic ERα and ERβ, which activate different signaling pathways. Activation of both membrane and cytosolic targets might
ultimately determine a change in gene expression. Finally, there are nuclear targets, defined as the direct modulation of gene expression by the interaction of the
ER-estrogen complex with the estrogen response element on the DNA. CREB, cAMP response element binding protein.
that are essential for DNA binding. Domain D is the hinge
region of the receptors, and domain E is responsible for specific ligand recognition and binding. The E domain also contains a ligand-dependent AF-2, and along with domain C it
contains a 90-kDa heat shock protein binding function. ERβ, of
which different isoforms have been identified, shares high
homology with ERα in certain domains, such as the DNA binding domain, but differs in the ligand-binding domain and the
NH2 terminal A-B domains. These differences generated considerable interest because of the underlying assumption that
ERα and ERβ would regulate different target genes. It was soon
observed that they can give rise to opposing regulatory signals
in response to 17β-estradiol. There are also differences in the
binding affinities for estrogenic substances. ERβ receptors have
a lower affinity for 17β-estradiol than ERα (8). ERs can form
homo- or heterodimers, raising the possibility that ERα and
ERβ act synergistically or as antagonists via homo- or heterodimerization on the activation step of a common responsive
element.
Alternatively, modulation of transcription might also occur
via protein-protein interactions of the activated ER complex
with coactivators and corepressors (1). A different way of regulating the transcription of genes that lack classic ERE involves
the modulation of the activity of other transcription factors
such as activator protein-1 or nuclear factor-κB (1). In a completely different fashion, estrogens may activate gene expression by acting outside the nucleus (the alternative pathway),
triggering signaling cascades that will ultimately induce tran252
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scription via the activation of cAMP response elements or the
phosphorylation and activation of transcription factors (6).
Membrane effects
In the past two decades, evidence has accumulated in favor
of membrane effects of estrogens after interaction with the cellular surface (3). These membrane actions could conceivably
be due to either a change in some membrane properties or to
specific binding to membrane targets. Nonspecific effects are
most commonly associated with a change in membrane fluidity. Nonetheless, concentrations at which nonspecific effects
are manifested are, in general, supramicromolar and therefore
will not be achieved in a physiological situation.
Specific effects after binding to membrane proteins have
been studied in practically all physiological systems, including
the endocrine, vascular, and nervous systems. Membrane proteins involved in rapid estrogen actions include specific ERs,
ion channels, membrane enzymes, and other ligand receptors.
Direct binding to neurotransmitter receptors and ion
channels
Steroids have been shown to act as allosteric coagonists or
antagonists of neurotransmitter receptors. The best-known
example is the allosteric modulation of the GABAA receptorchloride channel by neurosteroids, which mimics the effect of
barbiturates. Allosteric modulation of neurotransmitter recep-
tors by 17β-estradiol has also been studied, and its relevance
for the nervous system is highlighted by the modulation of
synaptic activity by estradiol. Here, 17β-estradiol behaves as
an allosteric antagonist of N-methyl-D-aspartate receptors and
5-hydroxytryptamine receptors (3). Recently, the existence of
an estrogen binding site within a voltage-gated ion channel has
been demonstrated (15). Binding of 17β-estradiol to an extracellular site of the β-subunit of the large-conductance calciumdependent potassium channel increases its activity, an effect
that might explain, at least in part, the estrogen-induced acute
vasodilation (14).
membrane are able to stimulate signaling pathways after interacting with G proteins, leading to the activation of phospholipase C and adenylyl cyclase, which increase inositol-1,4,5trisphosphate and cAMP levels, respectively (13). Therefore,
some of the membrane actions triggered by estrogens are likely
mediated by a membrane receptor, with a structure resembling
that of the classic ER. In respect to the localization of ER to the
plasma membranes of cells overexpressing ER, a cautionary
note should be taken into consideration. In our experience,
overexpression of various proteins result in their unusual location, e.g., the presence in the plasma membrane of proteins
normally found in intracellular locations.
Actions through a membrane receptor related to classic
ERα
Actions through nonclassic ERs
Following the Mannheim classification of nongenomic
(alternative) steroid actions (3), two kinds of specific membrane steroid receptors might exist: 1) a membrane receptor
with a molecular structure related to the classic steroid nuclear
receptor and 2) a nonclassic steroid membrane receptor.
The existence of proteins in the plasma membrane that
cross-react with antibodies raised against classic estrogen
receptor ERα has been described (discussed in Ref. 12). The
membrane receptor involved appears to be very similar in
structure to ERα, since it is recognized by nine different antibodies raised against different epitopes of the ERα. Moreover,
expression of ERα and ERβ in Chinese hamster ovary cells
demonstrate that both ERα and ERβ located at the plasma
Classic ERs are not the only membrane receptors involved in
the generation of an estrogen response. Strong evidence for the
existence of different types of estrogen binding sites exists. 17βestradiol affects neuronal excitability in ERα knockout mice (4).
The possibility that ERβ is responsible for 17β-estradiol actions
is ruled out by the absence of effect of the specific antiestrogen
ICI-182780. It is now apparent that many of the membranedelimited estrogen responses are mediated by the activation of
phospholipase C/adenylyl cyclase pathways via the modulation of G protein-coupled receptors (7). However, the characterization and location of the membrane receptor involved in
these types of responses has not always been possible. An
example of successful identification of the membrane estrogen
FIGURE 2. Modulation of insulin secretion by estrogen. A: calcium imaging of a pancreatic islet of Langerhans exposed to 8 mM glucose and 1 nM 17β-estradiol. The color image of a fluo 3-loaded islet shows the fluorescence intensity (blue corresponds to low and red to high intensity) of the peripheral cells, with the
boundaries of each cell clearly visible. B: record of relative fluorescence intensity vs. time obtained from a cell within the islet shown in A. Note the increase in
the calcium spikes triggered by the addition of 1 nM 17β-estradiol. C: estrogen signaling in pancreatic β-cells. Binding of 17β-estradiol to the membrane γ-adrenergic receptor (γ) activates a guanylyl cyclase (GC) and subsequently the protein kinase G (PKG)-dependent phosphorylation of ATP-sensitive potassium channels.
The channel phosphorylation results in its closing, leading to membrane depolarization and opening of voltage-gated calcium channels. Calcium entry ultimately
triggers insulin secretion. The effect of 17β-estradiol, which requires the presence of glucose, potentiates the insulin secretion triggered by the latter.
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binding site has recently been reported for pancreatic β-cells
(Fig. 2). In this particular type of cell, 17β-estradiol modulation
of insulin secretion (12) is mediated by a membrane receptor
with a pharmacological profile of the so-called γ-adrenergic
receptor but unrelated to the classic ER, as manifested by
immunohistochemistry and binding experiments (11). The
effect of estrogen in pancreatic β-cells determines the closing
of ATP-dependent potassium channels and the subsequent
potentiation of calcium signals and insulin secretion via the
activation of a cGMP signaling cascade (discussed in Ref. 11).
“Estrogens...can also interact with targets
within the cytosol.”
Other examples of estrogen modulation of membrane properties involve the modulation of intracellular calcium homeostasis. Depending on the cell type, estrogens can either
increase (12) or decrease (14) intracellular calcium levels. The
actual mechanisms of action associated with calcium modulation by estrogens are not fully understood, but both modulation of intracellular calcium stores and regulation of plasma
membrane calcium channels have been proposed.
Cytoplasmic interactions
Estrogens, in addition to their actions at the membrane and
nuclear locations, can also interact with targets within the
cytosol. In this section, we will briefly discuss the nature of
these cytosolic targets. One of the best-studied examples of
cytosolic actions of estrogens is the activation of the
Src/Ras/extracellular signal-regulated kinase (ERK; also known
as MAPK) pathway (10), a mechanism of action that might be
related to the proliferative effect of estrogens.
Under nonstimulatory conditions, the catalytic domains of
the Src kinase are in an inactive state as a result of intramolecular interactions. Binding of estradiol to either cytosolic ERα or
ERβ receptors determines the interaction of ER with Src, changing the conformation of the kinase to an active state and the
activation of the Src/Ras/ERK signaling cascade. Similar results
have also been reported for the androgen and progesterone
receptors (10), although the latter requires the participation of
the ER as well. These studies provide a good example of crosstalk between different steroid receptors as well as between different signaling pathways, i.e., estrogens activate the signaling
pathways (Src/Ras/ERK) normally associated with growth factors (e.g., epidermal growth factor or insulin). Moreover, there
is a real two-way cross-talk, because many growth factors (6)
have been involved in ER activation in the absence of estradiol,
normally via the phosphorylation of ER. However, the ability
to induce ER-mediated transcription in response to growth factors depends on the cell context and on the target genes.
Another example of a cytosolic effect of estrogen is the modulation of nitric oxide (NO) bioavailability in vascular cells.
Estrogen’s effects on the vasculature are related to a reduced
risk of coronary heart disease in women (discussed in Ref. 9).
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These vascular effects involve both the systemic modulation of
lipid metabolism and local effects leading to vasodilation (9).
The latter has been shown to include rapid increase in the production of the vasodilator NO (9) and/or the modulation of
smooth muscle ion channels (14). It is known that estrogen
activates endothelial NO synthase (eNOS, the enzyme responsible for NO production) without altering its expression in vascular endothelium, an effect that appears to require ERα with
an intact hormone-binding domain. This effect cannot be
reproduced by binding of estrogen to ERβ (5), although both
forms (ERα and ERβ) are expressed in vascular cells (9). The
activation of NOS by estrogen has been classically associated
with the interaction of estrogen with a plasma membrane
receptor, a hypothesis supported by the fact that nonpermeable
estrogen conjugated to albumin also reproduces the effect.
Recent reports (Ref. 5 and references therein) have demonstrated that the activation of NOS in endothelial cells by ERα
involves phosphatidylinositol 3-kinase (PI3K), and Chambliss
and co-workers (2) have localized functional ERα-eNOS signaling complex to endothelial caveolae. These structures form
signal-transducing membrane microdomains that, in endothelial cells, in addition to ERα and eNOS, also contain PI3K. This
new, and very interesting, model for signal transduction moves
the site of action of estrogen from the external to the internal
side of the membrane. Binding of estrogen to the ERα via a
direct physical interaction with the regulatory p85 subunit of
PI3K activates serine/threonine protein kinase B (also termed
Akt), which in turn will phosphorylate and activate NOS (5).
However, there are still observations that require further study
to fully understand the role of ERα in the activation of NOS. It
seems that activation of NOS by estrogen, like the activation of
the Src/Ras/ERK pathway described above, requires calcium
(5). Typically, a calcium peak precedes the increase in NO production. How can we integrate these two observations? Does
estrogen, alone or bound to ERα, mobilize calcium before
interacting with PI3K? These are questions that, undoubtedly,
will be resolved in the near future.
Summary
The existence of one of the three mechanisms in a particular cell is not exclusive. In this way, estrogens can elicit diverse
cellular effects that may depend on their concentration
because most of the estrogen binding sites present different
affinities. In general, binding to ion channels and allosteric
effects through other ligand receptors are triggered at supraphysiological doses, which could be achieved locally or after
accumulation in the lipid bilayer. Other properties of estrogens, e.g., antioxidant properties, also occur at supraphysiological concentrations. Actions via binding to putative membrane ER are produced at concentrations within the physiological range (10−12−10−9 M). Nonetheless, high estrogen concentrations might be reached locally in certain areas of the brain,
and the lipophilicity of steroids would contribute to the enrichment of estrogens at the membrane-receptor interface.
In the future, binding sites are likely to be identified in different channels or membrane receptors, and the molecular
mechanisms of estrogen action at the three cellular compart-
ments will be unveiled. This knowledge will help us to understand the endocrine regulation of different body systems.
We gratefully acknowledge the support of CICYT/FEDER (A. Nadal and M.
Diaz), Fundación Navarro-Tripodi (A. Nadal), and the Human Frontiers Science Program, Spanish Ministry of Science and Technology and Distinció de
la Generalitat de Catalunya per a la Promoció de la Recerca Universitaria to
M. A. Valverde.
References
1. Beato M and Klug J. Steroid hormone receptors: an update. Hum Reprod
Update 6: 225−236, 2000.
2. Chambliss KL, Yuhanna IS, Mineo C, Liu P, German Z, Sherman TS,
Mendelsohn ME, Anderson RG, and Shaul PW. Estrogen receptor alpha
and endothelial nitric oxide synthase are organized into a functional signaling module in caveolae. Circ Res 87: E44−E52, 2000.
3. Falkenstein E, Tillmann HC, Christ M, Feuring M, and Wehling M. Multiple actions of steroid hormones−a focus on rapid, nongenomic effects.
Pharmacol Rev 52: 513−556, 2000.
4. Gu Q, Korach KS, and Moss RL. Rapid action of 17beta-estradiol on
kainate-induced currents in hippocampal neurons lacking intracellular
estrogen receptors. Endocrinology 140: 660−666, 1999.
5. Hisamoto K, Ohmichi M, Kurachi H, Hayakawa J, Kanda Y, Nishio Y,
Adachi K, Tasaka K, Miyoshi E, Fujiwara N, Taniguchi N, and Murata Y.
Estrogen induces the Akt-dependent activation of endothelial nitric oxide
synthase in vascular endothelial cells. J Biol Chem 276: 3459−3467, 2001.
6. Kato S, Masuhiro Y, Watanabe M, Kobayashi Y, Takeyama KI, Endoh H, and
Yanagisawa J. Molecular mechanism of a cross-talk between oestrogen
and growth factor signalling pathways. Genes Cells 5: 593−601, 2000.
7. Kelly MJ and Wagner EJ. Estrogen modulation of G-protein-coupled receptors. Trends Endocrinol Metab 10: 369−374, 1999.
8. Kuiper GG, Carlsson B, Grandien K, Enmark E, Haggblad J, Nilsson S, and
Gustafsson JA. Comparison of the ligand binding specificity and transcript
tissue distribution of estrogen receptors alpha and beta. Endocrinology
138: 863−870, 1997.
9. Mendelsohn ME and Karas RH. The protective effects of estrogen on the
cardiovascular system. N Engl J Med 340: 1801−1811, 1999.
10. Migliaccio A, Castoria G, Di Domenico M, de Falco A, Bilancio A, Lombardi M, Barone MV, Ametrano D, Zannini MS, Abbondanza C, and Auricchio F. Steroid-induced androgen receptor-oestradiol receptor beta-Src
complex triggers prostate cancer cell proliferation. EMBO J 19: 5406−
5417, 2000.
11. Nadal A, Ropero AB, Laribi O, Maillet M, Fuentes E, and Soria B. Nongenomic actions of estrogens and xenoestrogens by binding at a plasma
membrane receptor unrelated to estrogen receptor alpha and estrogen
receptor beta. Proc Natl Acad Sci USA 97: 11603−11608, 2000.
12. Nadal A, Rovira JM, Laribi O, Leon-quinto T, Andreu E, Ripoll C, and Soria
B. Rapid insulinotropic effect of 17beta-estradiol via a plasma membrane
receptor. FASEB J 12: 1341−1348, 1998.
13. Razandi M, Pedram A, Greene GL, and Levin ER. Cell membrane and
nuclear estrogen receptors (ERs) originate from a single transcript: studies
of ERalpha and ERbeta expressed in Chinese hamster ovary cells. Mol
Endocrinol 13: 307−319, 1999.
14. Ruehlmann DO, Steinert JR, Valverde MA, Jacob R, and Mann GE. Environmental estrogenic pollutants induce acute vascular relaxation by
inhibiting L-type Ca2+ channels in smooth muscle cells. FASEB J 12: 613−
619, 1998.
15. Valverde MA, Rojas P, Amigo J, Cosmelli D, Orio P, Bahamonde MI, Mann
GE, Vergara C, and Latorre R. Acute activation of Maxi-K channels (hSlo)
by estradiol binding to the beta subunit. Science 285: 1929−1931, 1999.
News Physiol. Sci. • Volume 16 • December 2001
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