s t e r o i d s 7 1 ( 2 0 0 6 ) 91–95

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Perspective

Nongenomic effects of estrogen: Why all the uncertainty?

a r t i c l e i n f o

Article history:

Received 4 August 2005

Available online 25 October 2005

Keywords:

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a b s t r a c t

It is clear that estradiol has profound, rapid effects on the conformation of the estrogen

receptors (ERs), ER� and ER�, which mediate the transcriptional effects of estradiol. Estro-

gen can elicit many other rapid changes in cells including changes in ion fluxes across

membranes and stimulation of kinases and phosphatases. The proteins which are the tar-

gets of these actions are the subject of intense investigation. One of the issues that have not

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strogen receptors

ongenomic effects

timulation

been satisfactorily resolved is whether ER� or ER� can reside in the plasma membrane and

participate in the rapid effects of estrogen. In the present commentary, we take a careful

look at some of the published data in an attempt to understand why it is so difficult to obtain

a definitive answer to this question.

© 2005 Elsevier Inc. All rights reserved.

ongenomic effects of estrogen refer to those events whichan be measured within seconds or minutes of administrationf estrogen, i.e. too rapidly to be mediated by transcriptionalctivation of genes. These effects are usually measured asncrease in phosphorylation of proteins, or increase in intra-ellular calcium. The controversies in this field are not abouthether or not estradiol-17� (E2) has nongenomic effects, but

ather whether or not all of these effects are of physiologicalelevance and whether or not they are mediated by the nuclearstrogen receptors (ERs). There is no argument that, in orderor E2 to initiate its genomic effects, it first has profound non-ranscriptional effects on the conformation, protein interac-ions, subcellular localization and DNA binding properties ofts receptors [1]. ERs are not the only proteins whose con-ormation is altered by E2. Another well-characterized pro-ein, alpha-fetoprotein, is an E2 transporter with a growthuppressive domain which is unmasked upon E2 binding [2].t would, therefore, not be surprising if there were kinases,hosphatases and/or ion channels with selective E2-bindingockets whose conformation, activation or inactivation wereodulated by E2.There has been much written on whether the nonge-

omic effects of E2 are mediated by membrane-bound ERsnd whether or not these receptors are identical to the nuclear

into cells, it is not necessary for estrogen receptors (what-ever their nature) to be membrane-bound in order for themto be activated by E2 and trigger changes in ion channels orkinases at the cell surface. However, the nature and locationof the receptor might have a profound effect on its affinityfor E2 and this might explain why many rapid effects of E2are observed at concentrations higher than 1 nM, which is theconcentration of E2 at which maximal activity of the nuclearreceptor is achieved. Of much greater concern than if the con-centrations of E2 needed to elicit rapid changes are differentfrom those needed for nuclear ER activation, is the questionwhether the concentrations of E2 used in experiments arephysiologically relevant. Maximal E2 level achieved in cyclingwomen is 2 nM and in rodents 0.1 nM. In the present discus-sion, studies where E2 concentrations used are higher thanthose that are physiologically relevant, are classified as phar-macological or toxicological.

One of the well-known rapid physiological responses ofthe uterus to E2 is water imbibition. This response has beendescribed as a pro-inflammatory action of E2 and is charac-terized by infiltration of eosinophils and tissue edema [3].In our own studies, the rapid water imbibition in the uterusin response to E2 was clearly evident in ER��−/− mice withintact ovaries [4]. The ER�−/− mouse which we used is not a

Rs. Since the plasma membrane is not a barrier for E2 entry complete knockout and fragments of ER are expressed in the

039-128X/$ – see front matter © 2005 Elsevier Inc. All rights reserved.

oi:10.1016/j.steroids.2005.09.001

92 s t e r o i d s 7 1 ( 2 0 0 6 ) 91–95

uterus [5,6]. As is evident from the phenotype of the ER�−/−mice, the truncated ER is insufficient for maintenance ofuterine growth and function. The results from this ER�−/−mouse might indicate that the full length ER is not requiredfor the rapid effect of E2. However, in the more recently pro-duced complete ER�−/− mouse [7], the uterus was completelynon-responsive to E2 and this indicates that some part ofER� is necessary for E2 to have rapid effects in the uterus.The mechanism behind E2-induced water imbibition is notclearly understood. It is an inflammatory response which canbe completely blocked by dexamethasone [8]. Inflammatoryresponses are mediated by pro-inflammatory cytokines, andinduction of COX-2 [9]. COX-2 is one of those proteins whoselevels are rapidly inducible through post-transcriptionalmechanisms such as mRNA stabilization and increase inmRNA translatability. Since MAPK and ERK are involved inCOX-2 stabilization [10], activation of these kinases by E2would be one pathway for rapid increase in COX-2. If thisis the case, the rapid effects of E2 in the uterus might bemediated by E2 activation of MAP kinase.

1. Does E2 activate kinases and is ERlocated in the plasma membrane?

Definitive identification of a membrane bound ER in normalcells is an extremely difficult task and it has not been achieved

other questions. The nuclear ER, in the absence of E2, is in aninactive state bound to HSP90 and other chaperones. Physi-ologically, a receptor homodimer is formed when the cell isexposed to E2 [16]. When nuclear receptors are expressed inthe absence of HSP90, their affinity for their ligands is veryreduced [17]. The data of Razandi et al. suggest that there areconditions when a homodimer of ER� gets to the plasma mem-brane in the absence of E2 and still maintains a very highaffinity for E2. Until the methodological issues are clarified,the data in these studies cannot be used as conclusive prooffor the presence of ER in the plasma membrane.

Another laboratory modified ER� so that it is targeted tothe plasma membrane and studied its function in the ER�-negative MDA-MB-231 breast cancer cell line [18]. In the mod-ified ER (mER), the nuclear localization signal was removedfrom ER� and fatty acid acylation sites were introduced. Themodifications did not alter the affinity of the receptor forE2 and the mER� was found on the cell membrane. Unlikethe normal ER�, mER� did not increase transcription of EREreporter genes but surprisingly, upon addition of E2 to thecells expressing mER, there was no increase in phosphoryla-tion of MAPK and there was a decrease in Akt phosphorylation.The reason for the decrease in phosphorylation of Akt by E2in the presence of both ER� and mER� is not clear. Akt isa survival factor [19] which, in the presence of PI3, is acti-vated by a phosphorylation. It is inactivated by the tumorsuppressor phosphatase, PTEN. It has been reported that E2

yet. One laboratory has been most dedicated in its attemptsto demonstrate functional membrane bound ER� [11–15]. Theconclusion from this laboratory is that a small fraction ofnuclear ER� is located at the plasma membrane in caveolaewhere it regulates phosphorylation of proteins in MAPK andERK signaling pathways. Although the studies are publishedin good journals, there are some methodological questionswhich have to be clarified before the data can be used to sup-port the claim that ER is located in the plasma membrane. Asan example, Razandi et al. [14] have presented evidence thatnuclear ER� is located in the plasma membrane as a homod-imer and this plasma membrane receptor has a kD for E2 of0.2 nM. The evidence for monomers and dimers in the plasmamembranes was obtained by resolving plasma membraneson a non-denaturing gel. Plasma membrane proteins, in theabsence of any detergent, would not easily enter an 8% poly-acrylamide gel and if they did, their migration would certainlynot depend on their size. The electrophoretic migration of pro-teins in the absence of SDS is driven by the native charge of theproteins themselves. Proteins with an isoelectric point of 8.8,whether they are large or small, will not migrate very far at apH 8.8. In another approach to show the presence of ER� in themembrane [11], ER� was cross-linked with its ligand, [125I]17�-E2 and the complex analysed by SDS-PAGE. It is not clearhow incubation of plasma membranes in 4% formaldehydefor 24 h would cross-link ER to estradiol. Such cross-linkingwould require amino groups which are not present in estra-diol. In addition, under these conditions, membrane proteinswould become cross-linked to each other and it would not bepossible to say with certainty that any 125I-labeled band on theSDS gel was ER�.

If the technical difficulties are put aside, the presence ofmonomers and dimers of ER in the plasma membranes raises

increases phosphorylation of Akt [20]. In the study referred to,it is not clear whether E2 induced a phosphatase, decreased akinase, or decreased PI3. The lack of phosphorylation of MAPKcould be interpreted to mean that location at the cell sur-face is insufficient criterion for E2 to signal through MAPK orthat MDA-MB-231 cells are inappropriate vehicles for study ofmembrane ER signaling because the MAPK pathway is alreadyfully activated. Thus, although these experiments are techni-cally excellent, they do not necessarily help us to advance ourunderstanding of the rapid effects of E2.

2. Nitric oxide synthase (NOS) as a targetfor E2

Stirone et al. [21] demonstrated that within 30 min of itsaddition to intact mouse cerebral vessels ex vivo, 10 nM E2caused a 1.5-fold increase in NO production. This was accom-panied by a 1.5-fold increase in cerebrovascular levels ofphosphorylated-eNOS, but not in total eNOS protein. Interest-ingly, stimulation of NO release by E2 could only be detectedin vessels from ovariectomized animals, not from intact or E2-treated ovariectomized mice. In E2-replete mice, basal levelsof phosphorylated-eNOS and phosphorylated Akt in cerebralblood vessels were 1.5-fold higher than in E2-deficient mice.Thus, in this set of experiments, at a concentration of E2higher than that needed to saturate the nuclear ER, there is arapid E2-induced phosphorylation not involving transcription.This elevation of NO by E2 is, however, difficult to reconcilewith the known ER�-dependent sex difference in endothelialnitric oxide synthesis. The basal release of nitric oxide in aor-tic rings is significantly higher in males than in females. InER�−/− male mice, NO synthase is reduced and the sex differ-

s t e r o i d s 7 1 ( 2 0 0 6 ) 91–95 93

ence is lost. The concentration of E2 used in the Stirone study(10 nM), though quite low, is 100-fold higher than the plasmalevels in a mouse at estrus and the effects observed may bepharmacological, not physiological.

3. CREB as a target for E2

In another ex vivo model, Zhao et al. [22] investigated the rapideffects of E2 on neurite outgrowth in primary hippocampalneurons. They found that E2 (30 nM) activates Ca2+ influx inprimary hippocampal neurons within 50 s of its administra-tion and the Ca2+ remained elevated throughout the durationof the observation (300 s). This leads to activation of the tran-scription factor, cyclic AMP response element-binding protein(CREB) and neurite growth. Both the calcium increase and thephosphorylation of CREB were transient and, upon continuedexposure to E2, returned to baseline by 60 min. The concentra-tion of E2 used in the Zhao study is more than 100-fold higherthan would be needed to activate the mouse nuclear ER andno claims are made that ER is involved. Yet, Abraham et al. [23]have provided strong evidence that CREB phosphorylation inresponse to E2 requires the presence of ERs: in vivo admin-istration of E2 (1 �g per mouse) does not elicit an increase inCREB phosphorylation in any brain region in the absence ofER, i.e. in ER��−/− mice.

Since E2 signaling is not identical to cAMP signaling, howcZbgegtaps

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sient. It is not clear how long the levels were sustained orwhether a second dose of estrogen elicited further response.As in the case of MDA-MB-231 cells, COS7 cells might not bethe most appropriate for study of the membrane effects of E2because they do not normally express ER� and may not havethe components of the pathway needed to complete the sig-naling.

5. A completely new estrogen receptor

Toran-Allerand et al. recently reported on the existence of anovel protein which they have named “ER-X”. It represents ahigh-affinity, saturable, E2 binding site (Kd ∼ 1.6 nM) found incaveolar domains in the plasma membrane [27]. It is clearlydifferent from nuclear ERs since it binds both 17�- and 17�-E2with similar affinity. Both steroids elicit rapid and sustainedphosphorylation and activation of MAP kinase isoforms aswell as ERK1 and ERK2. “ER-X” is also recognized by someER� antibodies. The hypothesis is that this pathway mediatesinfluences of estrogen on neuronal differentiation, survivaland plasticity in the developing brain.

If E2, independent of ER, has important functions inthe brain, there should be significant differences in braindevelopment between aromatase knockout mice and ER�−/−mice. The role of E2 in the developing brain is under intenseinvestigation in many laboratories. But it appears that in

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an a cell use CREB for both E2 and cAMP mediated pathways?hang et al. [24] have shown that CREB occupies a large num-er of promoters in many different human tissues, but in anyiven cell type, only a small selected number of these promot-rs is activated by cAMP. Clearly, activation of cAMP regulatedenes is determined by the cellular profile of other transcrip-ion factors besides CREB. The phosphorylation of CREB asn intermediate in rapid E2 signaling introduces many novelossibilities to explain some of the cell specificity in rapid E2ignaling.

. GPR30 as a target for E2

n contrast to the need for ER in mediating the action of E2n CREB, the G protein coupled receptor, GPR30, appears toe activated by E2 in the absence of ER [25]. Revankar et al.

26] used COS7 cells, which do not naturally express GPR30 orR� and do not respond to E2 to study the role of GRP30 in2 signaling. When COS7 cells were forced to express GPR30,2 elicited a sustained mobilization of intracellular calcium,hich involved EGF receptor signaling. When ER� was intro-uced into COS7 cells, 1 nM E2 elicited a rapid increase in

ntracellular calcium but in contrast to the GPR response, theR response was mediated by PLC, not via EGFR signaling. Themplication here is that E2 can increase intracellular calciumhrough more than one pathway and it does not always requireR.

The magnitude of the calcium mobilization achieved with2 was similar to that obtained when the purinergic recep-or was stimulated, but unlike the effects of the purinergiceceptor agonist, which elicited a very transient increase inntracellular calcium, the effect of E2 on GPR30 was not tran-

the complete absence of E2 (aromatase knockout miceoverall development of the brain is normal although neuronasurvival in the adult brain is compromised [28]. It is quitechallenge to understand how E2 could play a role in the developing brain since the fetus is very well-protected against thactions of E2 [29] and the female brain would be masculinizeif it were exposed to E2 at the inappropriate time.

6. Physiological significance of rapid E2signaling

Perhaps questions should be redirected away from membranlocalization and towards the physiological significance of thrapid estrogen effects. When receptor ligands, such as neurotransmitters, induce rapid changes at the cell surface, causinchanges in intracellular levels of ions such as potassium ancalcium, the changes are not only rapid, they are also transient. There are multiple mechanisms at a synapse to terminate neurotransmitter action and rectify intracellular iohomeostasis. In the case of E2, plasma levels do not changrapidly and transiently and no extremely rapid metabolipathways to terminate estrogen action at the cell surface havbeen described. If estrogen induces rapid increase in intracellular calcium, how is its action terminated? How is thchange rectified? Is it possible that the presence of estrogesimply puts the cell under prolonged stress, causing the cell tcontinuously expend energy to return calcium levels to baseline? Or does the system desensitize rapidly after its initiaresponse? If so, what is the nature of this desensitizationDoes a cell rapidly respond to estrogen in an estrogen repletanimal or is this response an artifact resulting from estrogedepletion?

94 s t e r o i d s 7 1 ( 2 0 0 6 ) 91–95

7. Conclusions

What we can conclude from the data reviewed is that as theexperiments become more and more sophisticated, they alsobecome more and more artificial and the artificial systemsused to investigate the problem are introducing problems oftheir own. There are physiological systems that offer the pos-sibility to examine the rapid effects of E2. The uterus is one.Purves-Tyson and Keast [30] have shown that the peripheralnervous system is another. They have demonstrated that,in the peripheral nervous system, both dorsal root ganglion(DRG) and autonomic pelvic ganglion (PG) neurons expressERs. E2, apparently via ER, increases phosphorylation of CREBonly in DRG. Perhaps comparison of the DGR and PG wouldprovide some more information about the components of therapid signaling pathway. Another cell that is of potential usein this regard is the GnRH neuron. It is well-known that E2inhibits the release of GnRH except during the midcycle surgeof E2. At the peak of E2 concentrations in the plasma, there isa paradoxical release of GnRH [31]. One possible mechanismfor this could be an E2-sensitive pathway, which is activatedonly when E2 levels are very high. Such a system would alsoprovide an answer to the question as to how E2 can have rapideffects in an E2 replete system.

Overall, the data which have been analysed in this reviewindicate that the rapid effects of E2 are simply early steps

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Margaret WarnerJan-Ake Gustafsson∗

Department of Bioscience and Medical Nutrition, KarolinskaInstitute, NOVUM, Huddinge S-141 86, Sweden

∗Corresponding author. Tel.: +46 8 585 837 46;fax: +46 8779 87 95.

E-mail address: Jan-Ake.Gustafsson@mednut.ki.se(J.-A. Gustafsson)