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Mutation of the palmitoylation site of estrogen receptor α in vivo reveals tissue-specific roles for membrane versus nuclear actions Marine Adlanmerini a , Romain Solinhac a,1 , Anne Abot a,1 , Aurélie Fabre a,1 , Isabelle Raymond-Letron b , Anne-Laure Guihot c , Frédéric Boudou a , Lucile Sautier a,b , Emilie Vessières c , Sung Hoon Kim d , Philippe Lière e , Coralie Fontaine a , Andrée Krust f , Pierre Chambon f,2 , John A. Katzenellenbogen d , Pierre Gourdy a , Philip W. Shaul g , Daniel Henrion c , Jean-François Arnal a,2 , and Françoise Lenfant a a Institut National de la Santé et de la Recherche Médicale U1048, Institut des Maladies Métaboliques et Cardiovasculaires, Université de Toulouse, 31 432 Toulouse Cedex 4, France; b Institut National Polytechnique, École Nationale Vétérinaire de Toulouse, Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Service 006, Université de Toulouse, F-31076 Toulouse, France; c Institut National de la Santé et de la Recherche Médicale U1083, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 6214, Université dAngers, 49000 Angers, France; d Department of Chemistry, University of Illinois at UrbanaChampaign, Urbana, IL 61820; e Institut National de la Santé et de la Recherche Médicale, Université Paris Sud, 94270 Kremlin Bicêtre, France; f Institut de Génétique et de Biologie Moléculaire et Cellulaire, Collège de France, Université de Strasbourg, FR-67404 Illkirch, France; and g Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX 75235 Contributed by Pierre Chambon, November 27, 2013 (sent for review October 7, 2013) Estrogen receptor alpha (ERα) activation functions AF-1 and AF-2 classically mediate gene transcription in response to estradiol (E2). A fraction of ERα is targeted to plasma membrane and elicits mem- brane-initiated steroid signaling (MISS), but the physiological roles of MISS in vivo are poorly understood. We therefore generated a mouse with a point mutation of the palmitoylation site of ERα (C451A-ERα) to obtain membrane-specific loss of function of ERα. The abrogation of membrane localization of ERα in vivo was con- firmed in primary hepatocytes, and it resulted in female infertility with abnormal ovaries lacking corpora lutea and increase in lutei- nizing hormone levels. In contrast, E2 action in the uterus was preserved in C451A-ERα mice and endometrial epithelial prolifer- ation was similar to wild type. However, E2 vascular actions such as rapid dilatation, acceleration of endothelial repair, and endo- thelial NO synthase phosphorylation were abrogated in C451A- ERα mice. A complementary mutant mouse lacking the transacti- vation function AF-2 of ERα (ERα-AF2 0 ) provided selective loss of function of nuclear ERα actions. In ERα-AF2 0 , the acceleration of endothelial repair in response to estrogendendrimer conjugate, which is a membrane-selective ER ligand, was unaltered, demon- strating integrity of MISS actions. In genome-wide analysis of uter- ine gene expression, the vast majority of E2-dependent gene regulation was abrogated in ERα-AF2 0 , whereas in C451A-ERα it was nearly fully preserved, indicating that membrane-to-nuclear receptor cross-talk in vivo is modest in the uterus. Thus, this work genetically segregated membrane versus nuclear actions of a ste- roid hormone receptor and demonstrated their in vivo tissue- specific roles. fertility | vascular effects | nongenomic effects | genomic actions A lthough estrogens classically serve as reproductive hor- mones, they induce cellular responses in almost all tissues in mammalian species. The biological effects of estrogens, and particularly of 17β-estradiol (E2), are initiated by their binding to intracellular estrogen receptors (ERs), ERα and ERβ, which classically serve as nuclear transcription factors (1, 2). The ERs regulate the transcription of hundreds of genes in a cell- and tissue-specific manner through their two activation functions (AFs), AF-1 and AF-2. The roles of the activation functions of ERα have been studied in vivo using mice deleted for ERαAF-1 or ERαAF-2 (35). These results, in particular the two models of ERαAF-2 inactivation (4, 6), suggested that many physiological functions strongly rely on nuclear ERα and gene transcription regulation. However, in addition to the nuclear, termed geno- mic actions of ER,the receptors stimulate rapid (from seconds to minutes), nonnuclear signal transduction, usually termed nongenomicor extranucleareffects. The rapid mobilization of intracellular calcium and the generation of cAMP by E2 were demonstrated several decades ago (7, 8). More recently, the modulation of potassium currents, phospholipase C activation, the increase in endothelial nitric oxide production, and the stimulation of protein kinase pathways (PI3K/Akt, Erk) have been described (913). These rapid effects have been attributed to cell membrane-initiated steroid signaling (MISS) by a sub- population of receptors associated with the plasma membrane. In the plasma membrane, ERα has been localized to caveolae/ lipid rafts by direct binding to caveolin-1 through Ser-522 or indirectly via the scaffold protein striatin, forming complexes with G proteins (Gα i and G βγ ) (1417). In addition, Cys-447 of human ERα is a site of palmitoylation that promotes plasma membrane Significance The in vivo roles of plasma membrane-associated estrogen receptor (ER)α, including cross-talk with nuclear ERα, are poorly understood. We created a mouse with a point mutation of the palmitoylation site of ERα (C451A-ERα) to obtain membrane- specific loss of function. A complementary mouse lacking the ERα activation function AF-2 (ERα-AF2 0 ) provided selective loss of function of nuclear ERα actions. Physiologic studies revealed critical requirements for membrane receptors in ovarian function and thereby in fertility, and in vascular physiology. In contrast, nuclear ERα actions mediate uterine responses to estrogen and genome-wide analysis indicates that membrane-to-nuclear receptor cross-talk in vivo is quite modest in uterus. These findings demonstrate for the first time critical tissue-specific roles for membrane versus nuclear actions of a steroid hor- mone receptor in vivo. Author contributions: J.-F.A. and F.L. designed research; M.A., R.S., A.A., A.F., I.R.-L., A.-L.G., F.B., L.S., E.V., P.L., and D.H. performed research; S.H.K., A.K., P.C., J.A.K., and P.W.S. contributed new reagents/analytic tools; M.A., R.S., A.A., A.F., I.R.-L., A.-L.G., F.B., L.S., E.V., P.L., C.F., D.H., J.-F.A., and F.L. analyzed data; and M.A., C.F., A.K., P.C., P.G., P.W.S., D.H., J.-F.A., and F.L. wrote the paper. The authors declare no conflict of interest. Data deposition: The data reported in this paper have been deposited in the Gene Ex- pression Omnibus (GEO) database, www.ncbi.nih.gov/geo (accession no. 53237). 1 R.S., A.A., and A.F. contributed equally to this work. 2 To whom correspondence may be addressed. E-mail: [email protected] or Jean-Francois. [email protected]. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1322057111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1322057111 PNAS | Published online December 26, 2013 | E283E290 PHYSIOLOGY PNAS PLUS

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Mutation of the palmitoylation site of estrogenreceptor α in vivo reveals tissue-specific rolesfor membrane versus nuclear actionsMarine Adlanmerinia, Romain Solinhaca,1, Anne Abota,1, Aurélie Fabrea,1, Isabelle Raymond-Letronb,Anne-Laure Guihotc, Frédéric Boudoua, Lucile Sautiera,b, Emilie Vessièresc, Sung Hoon Kimd, Philippe Lièree,Coralie Fontainea, Andrée Krustf, Pierre Chambonf,2, John A. Katzenellenbogend, Pierre Gourdya, Philip W. Shaulg,Daniel Henrionc, Jean-François Arnala,2, and Françoise Lenfanta

aInstitut National de la Santé et de la Recherche Médicale U1048, Institut des Maladies Métaboliques et Cardiovasculaires, Université de Toulouse, 31 432Toulouse Cedex 4, France; bInstitut National Polytechnique, École Nationale Vétérinaire de Toulouse, Institut National de la Santé et de la Recherche Médicale,Unité Mixte de Service 006, Université de Toulouse, F-31076 Toulouse, France; cInstitut National de la Santé et de la Recherche Médicale U1083, CentreNational de la Recherche Scientifique, Unité Mixte de Recherche 6214, Université d’Angers, 49000 Angers, France; dDepartment of Chemistry, University ofIllinois at Urbana–Champaign, Urbana, IL 61820; eInstitut National de la Santé et de la Recherche Médicale, Université Paris Sud, 94270 Kremlin Bicêtre,France; fInstitut de Génétique et de Biologie Moléculaire et Cellulaire, Collège de France, Université de Strasbourg, FR-67404 Illkirch, France; and gDepartmentof Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX 75235

Contributed by Pierre Chambon, November 27, 2013 (sent for review October 7, 2013)

Estrogen receptor alpha (ERα) activation functions AF-1 and AF-2classically mediate gene transcription in response to estradiol (E2).A fraction of ERα is targeted to plasma membrane and elicits mem-brane-initiated steroid signaling (MISS), but the physiological rolesof MISS in vivo are poorly understood. We therefore generateda mouse with a point mutation of the palmitoylation site of ERα(C451A-ERα) to obtain membrane-specific loss of function of ERα.The abrogation of membrane localization of ERα in vivo was con-firmed in primary hepatocytes, and it resulted in female infertilitywith abnormal ovaries lacking corpora lutea and increase in lutei-nizing hormone levels. In contrast, E2 action in the uterus waspreserved in C451A-ERα mice and endometrial epithelial prolifer-ation was similar to wild type. However, E2 vascular actions suchas rapid dilatation, acceleration of endothelial repair, and endo-thelial NO synthase phosphorylation were abrogated in C451A-ERα mice. A complementary mutant mouse lacking the transacti-vation function AF-2 of ERα (ERα-AF20) provided selective loss offunction of nuclear ERα actions. In ERα-AF20, the acceleration ofendothelial repair in response to estrogen–dendrimer conjugate,which is a membrane-selective ER ligand, was unaltered, demon-strating integrity of MISS actions. In genome-wide analysis of uter-ine gene expression, the vast majority of E2-dependent generegulation was abrogated in ERα-AF20, whereas in C451A-ERα itwas nearly fully preserved, indicating that membrane-to-nuclearreceptor cross-talk in vivo is modest in the uterus. Thus, this workgenetically segregated membrane versus nuclear actions of a ste-roid hormone receptor and demonstrated their in vivo tissue-specific roles.

fertility | vascular effects | nongenomic effects | genomic actions

Although estrogens classically serve as reproductive hor-mones, they induce cellular responses in almost all tissues in

mammalian species. The biological effects of estrogens, andparticularly of 17β-estradiol (E2), are initiated by their binding tointracellular estrogen receptors (ERs), ERα and ERβ, whichclassically serve as nuclear transcription factors (1, 2). The ERsregulate the transcription of hundreds of genes in a cell- andtissue-specific manner through their two activation functions(AFs), AF-1 and AF-2. The roles of the activation functions ofERα have been studied in vivo using mice deleted for ERαAF-1or ERαAF-2 (3–5). These results, in particular the two models ofERαAF-2 inactivation (4, 6), suggested that many physiologicalfunctions strongly rely on nuclear ERα and gene transcriptionregulation. However, in addition to the nuclear, termed “geno-mic actions of ER,” the receptors stimulate rapid (from seconds

to minutes), nonnuclear signal transduction, usually termed“nongenomic” or “extranuclear” effects. The rapid mobilizationof intracellular calcium and the generation of cAMP by E2 weredemonstrated several decades ago (7, 8). More recently, themodulation of potassium currents, phospholipase C activation,the increase in endothelial nitric oxide production, and thestimulation of protein kinase pathways (PI3K/Akt, Erk) havebeen described (9–13). These rapid effects have been attributedto cell membrane-initiated steroid signaling (MISS) by a sub-population of receptors associated with the plasma membrane.In the plasma membrane, ERα has been localized to caveolae/

lipid rafts by direct binding to caveolin-1 through Ser-522 orindirectly via the scaffold protein striatin, forming complexes withG proteins (Gαi and Gβγ) (14–17). In addition, Cys-447 of humanERα is a site of palmitoylation that promotes plasma membrane

Significance

The in vivo roles of plasma membrane-associated estrogenreceptor (ER)α, including cross-talk with nuclear ERα, are poorlyunderstood. We created a mouse with a point mutation of thepalmitoylation site of ERα (C451A-ERα) to obtain membrane-specific loss of function. A complementary mouse lacking theERα activation function AF-2 (ERα-AF20) provided selective lossof function of nuclear ERα actions. Physiologic studies revealedcritical requirements for membrane receptors in ovarian functionand thereby in fertility, and in vascular physiology. In contrast,nuclear ERα actions mediate uterine responses to estrogen andgenome-wide analysis indicates that membrane-to-nuclearreceptor cross-talk in vivo is quite modest in uterus. Thesefindings demonstrate for the first time critical tissue-specificroles for membrane versus nuclear actions of a steroid hor-mone receptor in vivo.

Author contributions: J.-F.A. and F.L. designed research; M.A., R.S., A.A., A.F., I.R.-L., A.-L.G.,F.B., L.S., E.V., P.L., and D.H. performed research; S.H.K., A.K., P.C., J.A.K., and P.W.S.contributed new reagents/analytic tools; M.A., R.S., A.A., A.F., I.R.-L., A.-L.G., F.B., L.S., E.V.,P.L., C.F., D.H., J.-F.A., and F.L. analyzed data; and M.A., C.F., A.K., P.C., P.G., P.W.S., D.H., J.-F.A.,and F.L. wrote the paper.

The authors declare no conflict of interest.

Data deposition: The data reported in this paper have been deposited in the Gene Ex-pression Omnibus (GEO) database, www.ncbi.nih.gov/geo (accession no. 53237).1R.S., A.A., and A.F. contributed equally to this work.2To whom correspondence may be addressed. E-mail: [email protected] or [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1322057111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1322057111 PNAS | Published online December 26, 2013 | E283–E290

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association of the receptor (18). A nonpalmitoylatable Cys447Alamutant form of ERα, or its C451A mutant mouse counterpart,expressed in cultured cells lacks interaction with caveolin-1 anddownstream activation of signaling pathways and cell pro-liferation (18, 19). Numerous cell culture experiments furthersuggest potentially important kinase-mediated cross-talk be-tween membrane and nuclear ERα that modifies genomic re-sponses to E2 (20, 21), including recent studies revealing thatMISS dependent on receptor palmitoylation influences receptornuclear actions (22). Our current understanding of these pro-cesses has relied primarily on experimentation in cell culture. Asa result, the in vivo roles of membrane-associated ERα in nu-merous physiologic processes are poorly understood.Using a pharmacological approach in cell-based assays,

Harrington et al. (23) synthetized estrogen–macromoleculeconjugates (EDCs) to provide a gain-of-function strategy. EDCconsists of estrogen attached to a large, positively charged non-degradable poly(amido)amine dendrimer via hydrolytically sta-ble linkages. Studies in breast cancer cells clearly showed thatEDC was highly effective in stimulating nonnuclear signaling butinefficient in stimulating nuclear ER target gene expression be-cause EDC does not enter the nucleus (23). Importantly, in vivoadministration of EDC fully stimulated carotid artery reendo-thelialization but not uterine proliferation, suggesting for thefirst time that activating nonnuclear ERα signaling was sufficientto promote beneficial vascular effects of estrogen (24). Mem-brane only ERα mice expressing the ERα E-domain under theCytoMegaloVirus promoter in an ERα−/− background were alsogenerated, but the vascular phenotype, such as NO production orreendothelialization, was not assessed in this model (25).To investigate the physiologic importance of membrane-initi-

ated ERα actions in vivo, we created a knock-in mouse modelwith a selective loss of function of membrane ERα action bymutating the palmitoylation site at mouse ERα Cys451 to Ala(designated C451A-ERα). A complementary mutant mousemodel lacking the activation function AF-2 of ERα (designatedERα-AF20) (4, 6) provided a selective loss of function of nuclearERα actions. Physiological studies in these two models revealedcritical requirements for membrane receptors in ovarian functionand thereby in fertility, and in vascular physiology. In contrast,genome-wide analysis of uterine gene expression demonstratedthat nuclear ERα actions mediate uterine responses to estrogen,and also that membrane-to-nuclear receptor cross-talk in vivo ismodest in this tissue. These findings demonstrate critical tissue-specific roles for membrane versus genomic actions of a steroidhormone receptor in vivo.

ResultsThe C451A Mutation of ERα in Vivo Alters the Membrane Localizationof ERα. Compared with the three other cysteines present in theligand-binding domain of ERα, C447/451 is the least reactive toiodoacetic acid, suggesting a nonexposed position of this aminoacid (26), and consequently the least likely candidate for lipidmodifications. Nevertheless, two independent studies have clearlyshown that the palmitoylation of C447/451 is required for mem-brane localization of ERα (18, 19). It is likely that palmitoylationof ERα C447/451 occurs before ligand binding, when the ligand-binding domain is less tightly folded (27). Fig. S1A displaysa model of palmitoylation of C447/451 showing that despite theburied nature of this residue a considerable portion of the at-tached palmitoyl chain extends beyond the surface of the ERαligand-binding domain, and therefore could function as a mem-brane tether. To study the role of MISS actions of ERα, a knock-in strategy was used in which the palmitoylation site Cys451 wasmutated to alanine using a targeting construct containing two basepair changes in exon 7 (Fig. S1B). The C451A mutation wasconfirmed by PCR using tail DNA (Fig. S1C).

As an initial characterization of this mouse model, we evalu-ated the expression of ERα protein in various organs from lit-termate wild-type (WT-C451) and mutant C451A-ERα mice(Fig. 1 A–C). ERα protein abundance in the uterus, aorta, andhepatocytes was similar in the two groups. Plasma membraneswere then prepared from primary cultures of hepatocytes thatcan be obtained in sufficient quantities to allow sucrose gradientisolation of plasma membranes. Whereas ERα protein wasabundant in plasma membrane fractions (2–4) prepared fromWT-C451 hepatocytes, a 60% decrease was observed in fraction4 of the mutant C451A-ERα hepatocytes (Fig. 1D). Functionally,we also found that phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) was increased twofold in re-sponse to E2 in WT-C451 mice but unchanged in C451A-ERα(Fig. 1E). These data reveal that the C451A mutation effectivelyalters the plasma membrane localization of ERα in vivo, whichfurther abrogates the signaling pathway.

Membrane-Associated ERα Is Required for Ovarian Function. Wheneight C451A-ERα female mice were mated with WT-C451 males,no litters were observed despite continuous mating over a 5-moperiod. To investigate the basis of this infertility, histomorphologicanalysis was performed on ovaries from 10- to 12-wk-old femalemice. There was an excess of large, hemorrhagic, and/or cysticfollicles originating from antral follicles in C451A-ERα ovaries,along with an almost total absence of typical mature corpora lutea(Fig. 2 A and B). Counting of the number of follicles at differentstages revealed that ovaries from C451A-ERα mice displayed re-duced frequency of primordial follicles but normal percentages ofprimary, secondary, and tertiary follicles (Fig. S2A). Wall com-position of the tertiary follicle was also altered, with a decrease inthe percentage of the follicle area composed of granulosa cells,whereas the percentage of the thecal cell area was conserved (Fig.S2B). Additionally, there was a marked reduction in the density ofglandular interstitial cells when WT-C451 were compared withC451A-ERα ovaries, which appeared loosely arranged and asso-ciated with areas of hemorrhage.

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Fig. 1. Validation of the C451A-ERα mouse model. (A) ERα protein levels inuterus, aorta, and hepatocytes homogenates from ovariectomized WT-C451and C451A-ERα mice. (B) Representative Western blot of plasma membrane-associated ERα isolated from WT-C451 or C451A-ERα hepatocytes by sucrosegradient. Quantification of plasma membrane-associated ERα in WT-C451 orC451A-ERα hepatocytes, n = 3. Results are normalized to annexin II expres-sion. (C) Phospho-MAPK/MAPK abundance in hepatocytes treated or notwith E2 (10−7 M, 15 min), from WT-C451 and C451A-ERα mice, n = 3. Rep-resentative Western blot is shown. AU, arbitrary units.

E284 | www.pnas.org/cgi/doi/10.1073/pnas.1322057111 Adlanmerini et al.

We also measured steroid sex hormone levels in intact 10- to12-wk-old female mice (Fig. 2C). C451A-ERα mice exhibitedserum E2 levels similar to those of WT-C451 mice, whereasprogesterone levels were markedly decreased in mutant mice.The level of FSH tended to be higher (P = 0.067) and luteinizinghormone (LH) was markedly increased (P = 0.0005) in C451A-ERαmice in comparison with WT-C451 mice. Collectively, thesefindings indicate that membrane ERα is required for normalovarian morphology and function.

Membrane Actions of ERα Are Dispensable for Uterine Responsesto Estrogen. The uteri of intact WT-C451 and C451A-ERα micewere found to be normal in gross appearance (Fig. 3A). Theuterotrophic response to E2 was also similar in the two geno-types when the uterine wet weights were compared in ovariec-tomized female mice treated or not for 28 d by E2 (8 μg·kg–1·d–1s.c.) (Fig. 3B). To evaluate the impact of E2 on uterine epithelialproliferation, Ki-67 labeling was performed 24 h following E2

injection. A robust proliferative response was observed, similarin both WT-C451 and C451A-ERα mice (Fig. 3 C and D), andE2 treatment caused the same increase of luminal epithelialheight in the two groups (Fig. 3E). The global ERα proteinabundance was equivalent in the uterus homogenates fromovariectomized untreated WT-ERα and C451A-ERα and simi-larly decreased on both genotypes after E2 treatment (Fig. S3A).Changes in expression levels of ERβ and/orGPR30 after acute orchronic E2 treatment, as assessed by RT-quantitative PCR, werenot significantly different in the two genotypes, and thereby donot support the idea of a potential compensatory roles of thesereceptors (Fig. S3 B and C). Thus, the C451A mutation of ERαdoes not alter epithelial endometrial cell proliferation or theuterotrophic response to E2, demonstrating that membrane-initiated effects of ERα are not essential for uterine actions ofthe hormone.

Gene Expression Profiling Analysis Reveals Differential Roles ofMembrane Versus Nuclear ERα in Gene Regulation. In contrast tothe C451A mutation, previous studies in mice lacking the ERαactivation function AF-2 (ERα-AF20) revealed that the utero-trophic response to E2 is entirely dependent on AF-2 (4, 6). Wetherefore compared the gene expression profiles in the uteri ofC451A-ERα and ERα-AF20 mice (4), including littermate con-trols, 6 h after administration of the vehicle versus E2 (8 μg/kg)using pangenomic microarrays.The interaction between genotype and treatment effect is

shown in the heat map in Fig. 4A (P < 0.05). Cluster analysis ofall of the conditions tested identified two major patterns of geneexpression. The first cluster contained genes regulated by E2 inC451A-ERα mutant mice, and these genes were almost equiva-lent to those regulated by E2 in the control wild-type littermatesof either the C451A-ERα or the ERα-AF20 mice. The patternsof genes expressed in all of the vehicle-treated animals were verysimilar, and they closely resembled the gene pattern observed inE2-treated ERα-AF20 mice, demonstrating an absence of tran-scriptional response to E2 in ERα-AF20 mice, at least in thistissue. Accordingly, E2 affected a very high number of genes inthe two sets of wild-type mice: 17,248 genes in WT-AF2 and18,112 genes in WT-C451A (Fig. S4A). The slight differencesmay be due to the fact that the wild-type mice are, respectively,on C57BL/6N and C57BL/6J background. More importantly,16,659 genes were altered by E2 treatment in C451A-ERα

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Fig. 2. Adult ovarian phenotype is disturbed in the C451A-ERα mice. (A)Representative images of WT-C451 and C451A-ERα ovaries (×25). CL, corpusluteum; HCF, hemorrhagic cystic follicles. (B) Percentages of corpora luteaand hemorrhagic/cystic follicles were determined in ovaries from 3-mo-oldWT-C451 and C451A-ERα mice, n = 4. (C) Serum E2, progesterone (PG), FSH,and LH concentrations in 3-mo-old WT-C451 and C451A-ERα mice, n = 9.

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Fig. 3. Uterine proliferative response is maintained in C451A-ERα mice. (A) Representative images of intact uterus. (B) Uterine wet weight in ovariectomizedWT-C451 and C451A-ERα mice treated or not with chronic (28 d) E2 treatment (8 μg/kg per day), n = 4–6. (C) Representative Ki-67 immunodetection in uterussections from WT-C451A and C451A-ERα mice treated or not with E2 for 24 h. (Scale bar, 100 μm.) (D) Percentages of Ki-67–positive cells in epithelium and (E)luminal epithelial height (LEH) were measured, n = 4–8. For B, D, and E, statistical results of two-way ANOVA are shown, explaining the effect of treatment(Treat.), genotype (Gen.), and the interaction (Inter.) between these two variables.

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mice, with 14,907 commonly regulated in WT-C451, which repre-sents 82%of the genes targeted byE2 inWT-C451mice. In contrast,only 800 genes were regulated by E2 in ERα-AF20, with 640 genesin common to those altered by E2 in WT-AF2 uterus (3.7%).A Venn diagram was generated to assess the overlap of up-

and down-regulated genes found in the genotype and treatmentgroups. The vast majority of genes (7,551 and 7,201 genes ingroups C and D) were respectively up-regulated or down-regu-lated in a specific ERα-AF2-dependent manner (Fig. 4C). Incontrast, a small number of genes were regulated specifically inan ERα-C451 palmitoylation site-dependent manner (35 up-regulated and 67 down-regulated, groups A and B). Finally,a very limited number of genes was either repressed or activatedby E2 via both AF2- or C451-dependent processes in the same oropposite manner (groups E–H). Therefore, there is modest, ifany, cross-talk between the two receptor populations in uterus.Gene ontology analysis (Fig. S4B and Dataset S1) indicatedthat the limited number of palmitoylation-dependent genes(groups A and B) are involved in membrane-, transmembrane-,or extracellularly related processes or lipid transport pathways.The large number of AF2-dependent genes (groups C and D)are primarily involved in nuclear, phosphoprotein-related, orsplicing events.

Altogether, the genome-wide analysis of the uterus responseto E2 demonstrates that most of gene regulation by ERα is re-lated to AF2-dependent, transcriptional nuclear processes, whereasmembrane ERα signaling has a minor impact, affecting only avery limited number of genes in this tissue.

Membrane-Initiated Vascular Actions of ERα Are Abrogated in C451A-ERα Mice. The membrane-initiated actions of E2 have been par-ticularly explored in the vasculature, where estrogen can acutelyinduce vasodilation in humans (28), at least in part through therapid stimulation of endothelial nitric oxide synthase (eNOS)activity in an ERα-dependent manner (29, 30). Studies usingEDC, which selectively activates nonnuclear ER, have also pre-viously suggested that nonnuclear ERα signaling is important forthe endothelial effects of E2 in vivo (24). We then evaluated thevasodilatory effects of both E2 and EDC in WT-ERα andC451A-ERα mice by measuring dilatory responses of isolatedphenylephrine-preconstricted mesenteric arteries (Fig. S5A).Within minutes, E2 (1 μM) increased the diameter of arteriesfrom wild-type mice by 32%, whereas there was no significantvasodilatory response in arteries from ERα−/− or C451A-ERαmice (Fig. 5A, Left). EDC also induced rapid dilation of mes-enteric arteries from WT-C451 with a magnitude similar to thatobserved with E2, and no dilation of arteries from ERα−/− orC451A-ERα mice (Fig. 5A, Right). Because rapid dilatation inresponse to E2 is the consequence of stimulation of eNOSthrough Akt-mediated phosphorylation of eNOS S1177 (31),phosphorylation of eNOS in response to E2 was evaluated inisolated carotid arteries and found to be increased in WT-C451arteries, but not in arteries from ERα−/− or C451A-ERα mice(Fig. 5B).To directly evaluate how ERα C451 palmitoylation and the

resulting plasma membrane targeting of the receptor does affecta functional response of endothelial cells to E2, endothelial cellmigration was tested in scratch assays using primary cells isolatedfrom the aorta of WT-C451 and C451A-ERα mice. Indeed,activation of endothelial cell migration by E2 is mediated byplasma membrane-associated ERα coupled to eNOS via GαI(24). E2 stimulated migration of WT-C451 endothelial cells,but not of C451A-ERα endothelial cells (Fig. 5C). The role ofmembrane-associated ERα in the vascular actions of E2 wasthen investigated in vivo by evaluating reendothelialization ofthe carotid artery (Fig. 5D). E2 treatment caused an increase inendothelial repair in control WT-C451mice, whereas it had noeffect in C451A-ERα mice (Fig. 5D).Previous cell culture studies indicated that disrupting the

palmitoylation of ERα resulted in a receptor that is more sen-sitive to E2-dependent degradation (22). We therefore evaluatedpossible alterations in ER abundance, in presence of E2 treat-ment, in the arteries of C451A-ERα mice. First, ERα and ERβmRNA expression was assessed in aorta from ovariectomizedC451A-ERα and WT-C451 mice treated with vehicle versus E2(8 μg/kg) for 6 h or 4 wk (Fig. S5 B and C). Aortas from bothvehicle and E2-treated C451A-ERα mice displayed ERα andERβ mRNA levels similar to those of WT mice. ERα proteinabundance was also equivalent in aorta from untreated ovari-ectomized C451A-ERα and WT-C451 mice, and a similar de-crease in the amount of the receptor was observed after E2treatment (80 μg·kg–1·d–1 for 2 wk) in the two genotypes (Fig.S5D). Thus, although previous in vitro experiments suggestedthat ERα palmitoylation influences the abundance of the re-ceptor protein (22), this was not detected in vivo in arteriesand uterus.Altogether, these findings indicate that preventing membrane-

initiated ERα signaling by targeting the palmitoylation site ofERα abrogates major vascular actions of E2, including rapidartery dilation and the promotion of endothelial repair, despitethe presence of a global similar ERα arterial abundance.

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Fig. 4. Large-scale gene analysis on response to E2 on C451A-ERα and ERα-AF20 mice. (A) Gene expression profiles were obtained by microarray analysis(Agilent SurePrint G3 Mouse GE, 8 × 60K) of uterine samples from C451A-ERα, ERα-AF20 mice or their littermate WT controls treated with E2 (8 μg/kg,6 h) or vehicle (Veh), n = 3–4. The heat map represents the data obtained onall samples for the differentially expressed probes exhibiting interactionbetween genotype and treatment (BH adjusted P < 0.001). (B) The Venndiagram illustrates the overlaps between the genes significantly (P < 0.05)up- or down-regulated in the uterus following E2 treatment in ERα-AF20

and C451A-ERα mice. (C) The Venn diagram illustrates the overlaps betweenthe genes significantly (BH adjusted P < 0.05) up- or down-regulated inthe uterus following interaction between E2 treatment and genotypes, re-spectively, ERα-AF20 and C451A-ERα mice.

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Membrane-Initiated Vascular Actions of ERα Are Conserved in ERα-AF20 Mice. The marked contrast between ERα function in theuterus of C451-ERα and ERα-AF20 mice suggests that parallelstudies in the two models may reveal potential tissue-specificroles for nuclear versus nonnuclear receptors. We have pre-viously shown that the activation function AF2 is dispensable forthe accelerating effect of E2 on endothelial healing (4). Here, wefirst confirmed a similar E2-induced acceleration of reendothe-lialization after carotid injury in WT-AF2 and ERα-AF20 mice(Fig. 6). We further directly tested whether membrane-initiatedERα function is retained in ERα-AF20 mice and found that in-deed EDC caused similar promotion of endothelial repair inWT-AF2 and ERα-AF20 mice (Fig. 6). Therefore, whereas nu-clear ERα actions mediated by AF-2 are abrogated, nonnuclearERα function is intact in the ERα-AF20 mice, indicating that theconformation of the membrane ERα-AF20 allows its activationby E2 as well as by EDC.

DiscussionIn the present study we investigated the physiological roles ofa pool of ERα localized at the plasma membrane by mutating inmice the ERα C451 recognized as a key palmitoylation site invitro. This genetic loss-of-function model revealed that ERαC451 is absolutely required for plasma membrane location andsignals that are critical for arterial effects of E2, including thestimulation of vasodilation and the promotion of endothelial re-pair, but also female fertility. In contrast, the uterine proliferationin response to E2 occurs independently of membrane ERα.Complementary studies in ERα-AF20 mice further demonstrateda preservation of membrane ERα action and vascular function inthis model. Moreover, genome-wide analysis of uterine gene ex-pression revealed that membrane ERα signaling has only a minorimpact on gene expression in this sex target tissue. These obser-vations reveal tissue-specific roles for membrane versus nuclearERα in vivo. In that broader context, this work genetically segre-gated nonnuclear and nuclear actions of a steroid hormone re-ceptor and demonstrated their tissue specificity.We first determined whether mutation of the palmitoylation

site of mouse ERα alters the expression level of the protein invivo, because in vitro studies (22) had shown that such a mutatedhuman ERα (C447A) was more sensitive to E2-dependent deg-radation. The mutated C451A-ERα protein was normal inabundance in the different murine tissues analyzed. With respectto the abrogation of vascular E2 effects in the C451A-ERαmouse, it was also important to demonstrate that the abundanceof the C451A-ERα protein in the vasculature in vivo is normalunder basal and E2-stimulated conditions. Furthermore, theanticipated decrease in plasma membrane association of themutant protein in vivo mirrors in vitro observations (18, 19), andit validates the C451A-ERα mouse as a selective loss-of-functionmodel attenuating membrane receptor localization.Our study on the reproduction of the C451A-ERα mice

demonstrates the crucial role of membrane ERα in female fer-tility. This is at least in part due to alterations in ovarian func-tion, with the ovaries of adult C451A-ERα mice being almostcompletely devoid of corpora lutea and displaying a high number

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Fig. 5. Membrane-initiated endothelial effects of E2 are abrogated in C451A-ERα mice. (A) Percentage of relaxation of mesenteric arteries from WT-C451,ERα −/−, and C451A-ERα mice following addition of E2 (1 μM) or EDC (ethinyl-estradiol dendrimer conjugate, 1 μM), n = 8–9. (B) Phospho-eNOS/eNOSabundance in carotid arteries from WT-C451, ERα −/−, and C451A-ERα mice treated or not with E2 (10−8 M, 30 min), n = 6. Representative Western blot isshown. AU, arbitrary units. (C) Percentage of migration of primary endothelial cells treated or not with E2 (10−9 M, 16 h), n = 3–4. (D) Carotid arteryreendothelialization in ovariectomized WT-C451 and C451A-ERα mice treated or not with E2 (80 μg·kg–1·d–1, 2 wk), n = 8–10. For C and D, statistical results oftwo-way ANOVA are shown, reporting the statistical interaction between treatment and genotype.

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Fig. 6. Membrane effects of ERα are preserved in ERα-AF20 mutant mice.Carotid artery reendothelialization in ovariectomized WT-AF2 and AF2-ERαmice treated with E2 (80 μg·kg–1·d–1, 2 wk) or vehicle (Veh) and EDC(240 μg·kg–1·d–1, 2 wk) or control dendrimer, n = 8–10. Statistical results oftwo-way ANOVA are shown, explaining the effect of treatment, genotype,and interaction between these two variables.

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of hemorrhagic and/or cystic follicles, indicating that the matu-ration of follicles into corpora lutea (luteinization, which allowsthe transition to the next estrous cycle) is abnormal. The capacityfor ovulation per se has not yet been directly evaluated inC451A-ERα mice. The absence of corpora lutea, which produceprogesterone, likely underlies the low serum progesterone foundin C451-ERα mice. Furthermore, the level of LH in intact micewas increased, suggesting that the control of LH production bythe hypothalamic–pituitary axis is adversely affected in C451-ERα mice. Female mice deficient in either ERαAF-1 or -2(named ERα-AF10 and ERα-AF20, respectively) are also sterile,primarily owing to absence of uterine hypertrophy (3, 4, 6). Thus,both plasma membrane and nuclear actions of ERα are abso-lutely required for female fertility.We then explored to what extent membrane ERα is important

for estrogen vascular effects. We found that both E2 and EDCelicit a rapid vasorelaxant effect in isolated mesenteric arteriesfrom wild-type mice, whereas these effects are lost in ERα−/−and C451A-ERα mice. eNOS1177 phosphorylation in response toE2 was also abolished in arteries from C451A-ERα mice, as wasthe accelerative action of E2 on endothelial healing both in vivoand in vitro. In the past, the selective membrane ER activatorEDC was shown to be sufficient to stimulate endothelial cellmigration and the acceleration of reendothelialization (24).Furthermore, the acceleration of endothelial repair by E2 orEDC in ERα-AF20 mice was preserved, demonstrating that theMISS effects are fully activable in this model. Altogether, thecurrent findings indicate that the activation of membrane ERα isboth necessary and sufficient to promote endothelial repair andcontribute to the integrity of the endothelial monolayer.In contrast, C451A-ERα mice also revealed that receptor

palmitoylation and membrane targeting are dispensable foruterine growth responses to E2. Previous work showed that theselective activation of membrane ERα by EDC does not induceuterine hypertrophy (24), suggesting that MISS activation aloneis not sufficient to promote a proliferative response in uterineepithelium. Using a loss-of-function strategy, we now show thatMISS effects involving ERα palmitoylation are also not neces-sary. In contrast, previous studies have demonstrated the im-portance of genomic (i.e., transcriptional) activity of ERα in thisprocess, because E2 does not induce a uterine proliferative re-sponse in ERα-AF10 or in ERα-AF20 mice (5, 6).We further explored the gene expression profiles of ERα-

AF20 and C451A-ERα mice in the uterus, a tissue that is highlyresponsive to E2. Genome-wide gene array studies revealed that,in contrast to nuclear ERα-mediated processes, membrane ERα-mediated signaling has only a minor impact on gene expression.A small set of genes was found to be regulated in the uterus byboth membrane and nuclear ERα. Altogether, we can proposethat C451A-ERα and ERα-AF20 mice can be considered asmouse models of membrane ERα and nuclear ERα loss offunction, respectively, but with preservation of nuclear ERα andmembrane ERα, respectively.Cell culture studies (18, 19, 22, 32) previously suggested that

rapid E2-dependent signaling may modify ERα transcriptionalactivity and thereby modulate final receptor-dependent nuclearactions, representing cross-talk between nonnuclear and nuclearreceptor subpopulations. The function of many transcriptionfactors is regulated through protein kinase-mediated phosphor-ylation, and these transcription factors may be targets for ex-tranuclear actions of estrogen (20, 33). An important role forERα MISS was reported in cancer cell proliferative responses toE2 (34–36), and ERK 2, a downstream effector of the MAPKpathway, cooperates in regulating gene transcription (21). Sim-ilarly, a cross-talk between membrane-initiated signaling of ste-roid receptors, such as progesterone or androgen receptor, andgene regulation and cell proliferation was also reported in variousculture models of cancer cells (37). However, in contrast to the

conclusions raised from these models of cultured cancer cells, ourlarge-scale gene analysis reveals that although MISS via ERα caninfluence the regulation of a small subset of genes in the uterus, theimpact of nonnuclear ERα on gene regulation is quite modest inthis organ, paradigmatic of the proliferative action of E2.This context-specific role for membrane-initiated signals fits

along the complexity of the palmitoylation process (38), em-phasizing the regulation and the spatiotemporal dynamics ofprotein palmitoylation. These levels of complexity should beaddressed in future studies, even if such in vivo studies seemparticularly difficult.The discoveries that were rendered possible via the generation

of the C451A-ERα mouse model indicate that membrane andnuclear actions of ERα in vivo are highly tissue-specific in somekey targets and functions. The C451A-ERα mouse now providesa valuable loss-of-function model for the study of the roleof plasma membrane-associated ERα in numerous additionalphysiological and pathophysiological processes affected by thereceptor, as well as a means to conceive new selective ERαmodulators with an optimized medical profile.

Materials and MethodsMice. All procedures involving experimental animals were performed in ac-cordancewith the principles established by the Institut National de la Santé etde la Recherche Médicale and were approved by the local Ethical Committeeof Animal Care. The C451A-ERα knock-in mouse line was generated ona C57BL6/N background through the strategy outlined in Fig. S1B at theMouse Clinical Institute (Illkirch, France). ERα −/−, ERα-AF20 mice have beenpreviously described (4, 39). C451A-ERα and their corresponding wild-typelittermates (WT-C451) were ovariectomized at 4 wk of age. For acute E2treatment, they were injected s.c. with vehicle (castor oil) or 17β-estradiol(E2, 8 μg/kg) 3 wk after ovariectomy. For chronic E2 treatment, ovariecto-mized mice were implanted with s.c. pellets releasing either vehicle or E2(8 or 80 μg·kg–1·d–1 as indicated, 60-d release; Innovative Research of America).

Isolation of Plasma Membranes. Primary hepatocyte cultures were isolatedfrom the livers of 8- to10-wk-old mice by a modification of the collagenasemethod (40). Briefly, livers from WT-C451 and C451A-ERα mice were per-fused using the portal vein with HBSS before collagenase perfusion (C5138;Sigma). Hepatocytes were then put in cultures in DMEM. Plasma membraneswere isolated by a discontinuous sucrose step gradient, from which 12fractions were recovered (41). The first fractions (1–4) contained the plasmamembrane proteins, which were identified after protein precipitation byWestern blot using polyclonal antibodies against either ERα or annexin II.

Mouse Carotid Artery Injury. Perivascular carotid artery electric injury wasperformed as previously described (3) in ovariectomized mice treated or notfor 2 wk before surgery with E2 (80 μg·kg–1·d–1 in 60-d-release pellets) orEDC (ethinyl-estradiol dendrimer conjugated, 240 μg·kg–1·d–1). Briefly, theleft common carotid artery was exposed via an anterior cervicotomy. Theelectric injury was applied to the distal part (3 mm precisely) of the commoncarotid artery with a bipolar microregulator. The percentage of reendo-thelialization was calculated relative to the initial deendothelialized area byassessing Evans blue dye uptake 3 d after injury. Images were acquired un-der a DMR 300 Leica microscope, using Leica Application Suite V3.8 andImageJ softwares.

Vascular Reactivity of Isolated Mesenteric Arteries. As previously described(42), 5-mm-long segments of second-order mesenteric arteries were dis-sected and mounted between two glass cannulae and bathed in a physio-logical salt solution (pH 7.4, PO2 160 mm Hg, and PCO2 37 mm Hg). Pressurewas controlled by a servo-perfusion system, and diameter changes weremeasured continuously using a video-monitored system (Living SystemsInstruments). Artery viability was assessed using KCl (80 mM) and endothe-lium integrity with acetylcholine (1 μM). EDC- (1 μM) and E2- (0.01–1 μM)dependent dilation was then assessed after precontraction with phenyl-ephrine to 70% of the maximal contractile response obtained with KCl(80 mM).

Mouse Endothelial Cell Migration Assay. Endothelial cells were isolated fromthe aortae of wild-type and C451A-ERαmice, and cell migration was assessedusing methods modified from those previously described (43). At first pas-

E288 | www.pnas.org/cgi/doi/10.1073/pnas.1322057111 Adlanmerini et al.

sage, the cells were transferred into six-well plates, and following growth toconfluence in EGM-2 medium (Lonza) containing 10% FBS, a defined linearregion of cells was removed using a pipette tip. The initial area devoid ofcells was marked and quantified on images obtained at baseline, andtreatments with vehicle or E2 (10−9 M) were initiated in phenol red-freeDMEM with 5% charcoal-stripped FBS. Sixteen hours later the cells werefixed and stained with Coomassie blue, and repeat images were obtained toquantify the remaining area devoid of cells. Cells were imaged using aninverted phase-contrast microscope (Nikon ECLIPSE TS100; Nikon Corpora-tion) and a digital camera (Infinity 1). The area devoid of cells was quantifiedusing Adobe ImageReady CS2 software. The degree of migration was cal-culated as the percentage of the initial area devoid of cells that was occu-pied by cells following 16-h treatment.

Hormone Assays. Serum levels of LH and FSH were determined using theMultiplex Immunoassay Technology Xmap (MILLIPLEX; Millipore). Pro-gesterone and 17β-estradiol were measured by gas chromatography–massspectrometry (44), with minor modifications. These assays were performedon intact 10- to12-wk-old female mice.

Immunohistochemistry. Paraffin-embedded transverse sections (4 μm) fromformalin-fixed uterine or ovary specimens were stained as previously de-scribed (5) with anti–Ki-67 antigen (RM-9106; Thermo-scientific). Sectionswere examined under a Nikon Eclipse E400 microscope using image analysissoftware Morpho Expert. For uterus, the luminal epithelial height andstromal height of endometrium is the mean of 10 measurements, whereasthe percentage of Ki-67–positive epithelial cells is the mean of three mea-surements in each transverse uterus section. Ovarian function was evaluatedin 3-mo-old adult mice by counting the number of follicles in each class (45).The number of follicles in each category was expressed as the percentage oftotal number of follicles. Analysis of the area of the granulosa and theca wasperformed on the largest tertiary follicle of each section, as previously de-scribed (46), and expressed as a percentage of the total area of follicle.

Microarray. Total RNA was extracted using TriPure reagent (Roche) and re-verse-transcribed (High-Capacity cDNA Reverse Transcription Kit; AppliedBiosystems). Microarray data were obtained from 200 ng of uterine total RNAlabeled with Cy3 using Low Input Quickamp Labeling Kit (Agilent) and hy-bridized to Agilent SurePrint G3 Mouse GE (8 × 60K) microarrays followingthe manufacturer’s instructions. All experimental details are available in theGene Expression Omnibus (GEO) database under accession no. 53237. Datawere analyzed under R (R 2.13; www.R-project.org). Hierarchical clusteringwas applied to the samples and the probes using 1-Pearson correlation co-efficient as distance and Ward’s criterion for agglomeration. Functionalanalysis was carried out using DAVID Bioinformatics Resources 6.7 (http://

david.abcc.ncifcrf.gov), and comparisons are realized with the Venn Dia-grams plug-in based upon the VENNY tool developed by J. C. Oliveros.

Western Blotting. Total proteins were separated on a 10% SDS/PAGE gel andtransferred to a nitrocellulose membrane. The primary antibodies used are asfollows: MC20 (sc-542; Santa Cruz Biotechnology), Gapdh (sc-32233; SantaCruz Biotechnology), pSer1177-eNOS (612392; BD Bioscience), eNOS (610297;BD Bioscience), pThr172-AMPK (2531; Cell Signaling), AMPK (2532; Cell Sig-naling), actin (A2066; Sigma), and annexin II (sc-9061; Santa Cruz Bio-technology). Revelation was performed using an HRP-conjugated secondaryantibody and visualized by ECL detection according to the manufacturer’sinstructions (Amersham Biosciences/GE Healthcare), using ChemiDoc Imag-ing System (Bio-Rad). Bands were quantified using ImageJ densitometry.

Statistical Analyses. Results are expressed as the mean ± SEM. To test theeffect of treatments or genotypes, t test or Mann–Whitney test was per-formed. To test the interaction between treatments and genotypes, a two-way ANOVA was carried out. When an interaction was observed betweentwo variables, the effect of treatment was studied in each genotype usingthe Bonferroni post hoc test. A value of P < 0.05 was considered statisticallysignificant (*P < 0.05; **P < 0.01; ***P < 0.001).

ACKNOWLEDGMENTS. The C451A-ERα mouse was generated at the MouseClinical Institute by M. C. Birling and coworkers. We thank Dr. ChristopherMayne for creating the model of ERα-C451A palmitate (Fig. S1A) and RosaSirianni, who performed the endothelial cell migration experiments. Wethank Dr. Benita Katzenellenbogen and Dr. Henrik Laurell for fruitful dis-cussions and suggestions for the writing of the manuscript. We also thankthe staff of the animal facility (J.-C. Albouys, M. J. Fouque, and G. Carcasses);C. Bleuart for anatomopathology at the École Nationale Vétérinaire de Tou-louse, the staff of the Anexplo platform (A. Desquennes and S. Legonidec);and M. Buscato [Institut National de la Santé et de la Recherche Médicale(INSERM) 1048] for skillful technical assistance. We also thank Y. Lippi andP. Martin for the excellent contribution to microarray analysis carried out atGeT-TRIX Genopole Toulouse facility. The work at I2MC-INSERM U1048 issupported by Institut National de la Santé et de la Recherche Médicale,Université et CHU de Toulouse, Faculté de Médecine Toulouse-Rangueil,Fondation pour la Recherche Médicale, Fondation de France, Fondation del’Avenir, Conseil Régional Midi-Pyrénées, and AVIESAN-Astra-Zeneca. Thework at INSERM U1083-Centre National de la Recherche Scientifique (CNRS)Unité Mixte de Recherche 6214 is supported by INSERM, CNRS, Centre Hos-pitalier Universitaire and Université d’Angers, Fondation de France, Fonda-tion de l’Avenir and Conseil Régional Pays de la Loire, Conseil RégionalMidi-Pyrénées, and Fondation pour la Recherche Médicale. M.A. and A.A.were supported by grants from the Ministère de la Recherche and Groupe deRéflexion sur la Recherche Cardiovasculaire, respectively. This work was sup-ported by National Institutes of Health Grants DK015556 and HL087564(to J.A.K. and P.W.S., respectively).

1. Hewitt SC, Harrell JC, Korach KS (2005) Lessons in estrogen biology from knockout

and transgenic animals. Annu Rev Physiol 67:285–308.2. Nilsson S, Koehler KF, Gustafsson JA (2011) Development of subtype-selective oes-

trogen receptor-based therapeutics. Nat Rev Drug Discov 10(10):778–792.3. Billon-Galés A, et al. (2009) The transactivating function 1 of estrogen receptor alpha

is dispensable for the vasculoprotective actions of 17beta-estradiol. Proc Natl Acad Sci

USA 106(6):2053–2058.4. Billon-Galés A, et al. (2011) Activation function 2 (AF2) of estrogen receptor-alpha is

required for the atheroprotective action of estradiol but not to accelerate endothelial

healing. Proc Natl Acad Sci USA 108(32):13311–13316.5. Abot A, et al. (2013) The AF-1 activation function of estrogen receptor α is necessary

and sufficient for uterine epithelial cell proliferation in vivo. Endocrinology 154(6):

2222–2233.6. Arao Y, et al. (2011) Estrogen receptor α AF-2 mutation results in antagonist reversal

and reveals tissue selective function of estrogen receptor modulators. Proc Natl Acad

Sci USA 108(36):14986–14991.7. Pietras RJ, Szego CM (1977) Specific binding sites for oestrogen at the outer surfaces

of isolated endometrial cells. Nature 265(5589):69–72.8. Szego CM, Davis JS (1967) Adenosine 3′,5′-monophosphate in rat uterus: acute ele-

vation by estrogen. Proc Natl Acad Sci USA 58(4):1711–1718.9. Hammes SR, Levin ER (2011) Minireview: Recent advances in extranuclear steroid

receptor actions. Endocrinology 152(12):4489–4495.10. Simoncini T (2009) Mechanisms of action of estrogen receptors in vascular cells: rel-

evance for menopause and aging. Climacteric 12(Suppl 1):6–11.11. Kim KH, Bender JR (2009) Membrane-initiated actions of estrogen on the endothe-

lium. Mol Cell Endocrinol 308(1-2):3–8.12. Wu Q, Chambliss K, Umetani M, Mineo C, Shaul PW (2011) Non-nuclear estrogen

receptor signaling in the endothelium. J Biol Chem 286(17):14737–14743.13. Ueda K, Karas RH (2013) Emerging evidence of the importance of rapid, non-nuclear

estrogen receptor signaling in the cardiovascular system. Steroids 78(6):589–596.

14. Lu Q, et al. (2004) Striatin assembles a membrane signaling complex necessary for

rapid, nongenomic activation of endothelial NO synthase by estrogen receptor alpha.

Proc Natl Acad Sci USA 101(49):17126–17131.15. Levin ER (2005) Integration of the extranuclear and nuclear actions of estrogen. Mol

Endocrinol 19(8):1951–1959.16. Simoncini T, et al. (2000) Interaction of oestrogen receptor with the regulatory sub-

unit of phosphatidylinositol-3-OH kinase. Nature 407(6803):538–541.17. Chambliss KL, et al. (2000) Estrogen receptor alpha and endothelial nitric oxide syn-

thase are organized into a functional signaling module in caveolae. Circ Res 87(11):

E44–E52.18. Acconcia F, et al. (2005) Palmitoylation-dependent estrogen receptor alpha mem-

brane localization: Regulation by 17beta-estradiol. Mol Biol Cell 16(1):231–237.19. Pedram A, et al. (2007) A conserved mechanism for steroid receptor translocation to

the plasma membrane. J Biol Chem 282(31):22278–22288.20. Lonard DM, O’Malley BW (2012) Nuclear receptor coregulators: Modulators of pa-

thology and therapeutic targets. Nat Rev Endocrinol 8(10):598–604.21. Madak-Erdogan Z, Lupien M, Stossi F, BrownM, Katzenellenbogen BS (2011) Genomic

collaboration of estrogen receptor alpha and extracellular signal-regulated kinase 2

in regulating gene and proliferation programs. Mol Cell Biol 31(1):226–236.22. La Rosa P, Pesiri V, Leclercq G, Marino M, Acconcia F (2012) Palmitoylation regulates

17β-estradiol-induced estrogen receptor-α degradation and transcriptional activity.

Mol Endocrinol 26(5):762–774.23. Harrington WR, et al. (2006) Estrogen dendrimer conjugates that preferentially ac-

tivate extranuclear, nongenomic versus genomic pathways of estrogen action. Mol

Endocrinol 20(3):491–502.24. Chambliss KL, et al. (2010) Non-nuclear estrogen receptor alpha signaling promotes

cardiovascular protection but not uterine or breast cancer growth in mice. J Clin In-

vest 120(7):2319–2330.25. Pedram A, et al. (2009) Developmental phenotype of a membrane only estrogen

receptor alpha (MOER) mouse. J Biol Chem 284(6):3488–3495.

Adlanmerini et al. PNAS | Published online December 26, 2013 | E289

PHYS

IOLO

GY

PNASPL

US

26. Hegy GB, et al. (1996) Carboxymethylation of the human estrogen receptor ligand-binding domain-estradiol complex: HPLC/ESMS peptide mapping shows that cysteine447 does not react with iodoacetic acid. Steroids 61(6):367–373.

27. Gee AC, Katzenellenbogen JA (2001) Probing conformational changes in the estrogenreceptor: Evidence for a partially unfolded intermediate facilitating ligand bindingand release. Mol Endocrinol 15(3):421–428.

28. Gilligan DM, Quyyumi AA, Cannon RO, 3rd (1994) Effects of physiological levels ofestrogen on coronary vasomotor function in postmenopausal women. Circulation89(6):2545–2551.

29. Lantin-Hermoso RL, et al. (1997) Estrogen acutely stimulates nitric oxide synthaseactivity in fetal pulmonary artery endothelium. Am J Physiol 273(1 Pt 1):L119–L126.

30. Caulin-Glaser T, García-Cardeña G, Sarrel P, Sessa WC, Bender JR (1997) 17 beta-estradiol regulation of human endothelial cell basal nitric oxide release, independentof cytosolic Ca2+ mobilization. Circ Res 81(5):885–892.

31. Guo X, Razandi M, Pedram A, Kassab G, Levin ER (2005) Estrogen induces vascularwall dilation: Mediation through kinase signaling to nitric oxide and estrogen re-ceptors alpha and beta. J Biol Chem 280(20):19704–19710.

32. Madak-Erdogan Z, et al. (2008) Nuclear and extranuclear pathway inputs in the regu-lation of global gene expression by estrogen receptors.Mol Endocrinol 22(9):2116–2127.

33. Björnström L, Sjöberg M (2002) Signal transducers and activators of transcriptionas downstream targets of nongenomic estrogen receptor actions. Mol Endocrinol16(10):2202–2214.

34. Acconcia F, Kumar R (2006) Signaling regulation of genomic and nongenomic func-tions of estrogen receptors. Cancer Lett 238(1):1–14.

35. Poulard C, et al. (2012) Activation of rapid oestrogen signalling in aggressive humanbreast cancers. EMBO Mol Med 4(11):1200–1213.

36. Razandi M, Pedram A, Levin ER (2000) Plasma membrane estrogen receptors signal toantiapoptosis in breast cancer. Mol Endocrinol 14(9):1434–1447.

37. Lange CA, Gioeli D, Hammes SR, Marker PC (2007) Integration of rapid signaling

events with steroid hormone receptor action in breast and prostate cancer. Annu Rev

Physiol 69(1):171–199.38. Salaun C, Greaves J, Chamberlain LH (2010) The intracellular dynamic of protein

palmitoylation. J Cell Biol 191(7):1229–1238.39. Dupont S, et al. (2000) Effect of single and compound knockouts of estrogen re-

ceptors alpha (ERalpha) and beta (ERbeta) on mouse reproductive phenotypes. De-

velopment 127(19):4277–4291.40. Berry MN, Friend DS (1969) High-yield preparation of isolated rat liver parenchymal

cells: A biochemical and fine structural study. J Cell Biol 43(3):506–520.41. Terrillon S, et al. (2003) Oxytocin and vasopressin V1a and V2 receptors form con-

stitutive homo- and heterodimers during biosynthesis. Mol Endocrinol 17(4):677–691.42. Tarhouni K, et al. (2013) Key role of estrogens and endothelial estrogen receptor α in

blood flow-mediated remodeling of resistance arteries. Arterioscler Thromb Vasc Biol

33(3):605–611.43. Sundgren NC, et al. (2011) Coupling of Fcγ receptor I to Fcγ receptor IIb by SRC kinase

mediates C-reactive protein impairment of endothelial function. Circ Res 109(10):

1132–1140.44. Liere P, et al. (2000) Validation of an analytical procedure to measure trace amounts

of neurosteroids in brain tissue by gas chromatography-mass spectrometry.

J Chromatogr B Biomed Sci Appl 739(2):301–312.45. Myers M, Britt KL, Wreford NG, Ebling FJ, Kerr JB (2004) Methods for quantifying

follicular numbers within the mouse ovary. Reproduction 127(5):569–580.46. Moore AM, Prescott M, Campbell RE (2013) Estradiol negative and positive feedback

in a prenatal androgen-induced mouse model of polycystic ovarian syndrome.

Endocrinology 154(2):796–806.

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