Mutation of the palmitoylation site of estrogen PNAS PLUS receptor α in vivo reveals tissue-specific roles for membrane versus nuclear actions

Marine 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, 31 432 Toulouse 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, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 6214, Université d’Angers, 49000 Angers, France; dDepartment of Chemistry, University of Illinois 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 gDepartment of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX 75235

Contributed by Pierre Chambon, November 27, 2013 (sent for review October 7, 2013) alpha (ERα) activation functions AF-1 and AF-2 to minutes), nonnuclear signal transduction, usually termed classically mediate gene transcription in response to estradiol (E2). “nongenomic” or “extranuclear” effects. The rapid mobilization A fraction of ERα is targeted to plasma membrane and elicits mem- of intracellular calcium and the generation of cAMP by E2 were brane-initiated steroid signaling (MISS), but the physiological roles demonstrated several decades ago (7, 8). More recently, the of MISS in vivo are poorly understood. We therefore generated modulation of potassium currents, phospholipase C activation, α a mouse with a point mutation of the palmitoylation site of ER the increase in endothelial nitric oxide production, and the (C451A-ERα) to obtain membrane-specific loss of function of ERα. PHYSIOLOGY α stimulation of protein kinase pathways (PI3K/Akt, Erk) have The abrogation of membrane localization of ER in vivo was con- been described (9–13). These rapid effects have been attributed firmed in primary hepatocytes, and it resulted in female infertility to cell membrane-initiated steroid signaling (MISS) by a sub- with abnormal ovaries lacking corpora lutea and increase in lutei- population of receptors associated with the plasma membrane. nizing hormone levels. In contrast, E2 action in the uterus was α preserved in C451A-ERα mice and endometrial epithelial prolifer- In the plasma membrane, ER has been localized to caveolae/ ation was similar to wild type. However, E2 vascular actions such lipid rafts by direct binding to caveolin-1 through Ser-522 or as rapid dilatation, acceleration of endothelial repair, and endo- indirectly via the scaffold protein striatin, forming complexes with α – thelial NO synthase phosphorylation were abrogated in C451A- G proteins (G i and Gβγ)(14 17). In addition, Cys-447 of human ERα mice. A complementary mutant mouse lacking the transacti- ERα is a site of palmitoylation that promotes plasma membrane vation function AF-2 of ERα (ERα-AF20) provided selective loss of function of nuclear ERα actions. In ERα-AF20, the acceleration of Significance endothelial repair in response to estrogen–dendrimer conjugate, which is a membrane-selective ER ligand, was unaltered, demon- The in vivo roles of plasma membrane-associated estrogen strating integrity of MISS actions. In genome-wide analysis of uter- receptor (ER)α, including cross-talk with nuclear ERα, are poorly ine , the vast majority of E2-dependent gene understood. We created a mouse with a point mutation of the α 0 α regulation was abrogated in ER -AF2 , whereas in C451A-ER it palmitoylation site of ERα (C451A-ERα) to obtain membrane- was nearly fully preserved, indicating that membrane-to-nuclear specific loss of function. A complementary mouse lacking the receptor cross-talk in vivo is modest in the uterus. Thus, this work ERα activation function AF-2 (ERα-AF20) provided selective loss genetically segregated membrane versus nuclear actions of a ste- of function of nuclear ERα actions. Physiologic studies revealed roid hormone receptor and demonstrated their in vivo tissue- critical requirements for membrane receptors in ovarian function specific roles. and thereby in fertility, and in vascular physiology. In contrast, nuclear ERα actions mediate uterine responses to estrogen and fertility | vascular effects | nongenomic effects | genomic actions genome-wide analysis indicates that membrane-to- cross-talk in vivo is quite modest in uterus. These lthough estrogens classically serve as reproductive hor- findings demonstrate for the first time critical tissue-specific Amones, they induce cellular responses in almost all tissues in roles for membrane versus nuclear actions of a steroid hor- mammalian species. The biological effects of estrogens, and mone receptor in vivo. particularly of 17β-estradiol (E2), are initiated by their binding to α β Author contributions: J.-F.A. and F.L. designed research; M.A., R.S., A.A., A.F., I.R.-L., A.-L.G., intracellular estrogen receptors (ERs), ER and ER , which 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. classically serve as nuclear transcription factors (1, 2). The ERs 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., regulate the transcription of hundreds of genes in a cell- and 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., tissue-specific manner through their two activation functions and F.L. wrote the paper. (AFs), AF-1 and AF-2. The roles of the activation functions of The authors declare no conflict of interest. ERα have been studied in vivo using mice deleted for ERαAF-1 Data deposition: The data reported in this paper have been deposited in the Gene Ex- α – pression Omnibus (GEO) database, www.ncbi.nih.gov/geo (accession no. 53237). or ER AF-2 (3 5). These results, in particular the two models of 1 α R.S., A.A., and A.F. contributed equally to this work. ER AF-2 inactivation (4, 6), suggested that many physiological 2 α To whom correspondence may be addressed. E-mail: [email protected] or Jean-Francois. functions strongly rely on nuclear ER and gene transcription [email protected]. “ regulation. However, in addition to the nuclear, termed geno- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. mic actions of ER,” the receptors stimulate rapid (from seconds 1073/pnas.1322057111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1322057111 PNAS Early Edition | 1of8 Downloaded by guest on September 27, 2021 association of the receptor (18). A nonpalmitoylatable Cys447Ala As an initial characterization of this mouse model, we evalu- mutant form of ERα, or its C451A mutant mouse counterpart, ated the expression of ERα protein in various organs from lit- expressed in cultured cells lacks interaction with caveolin-1 and termate wild-type (WT-C451) and mutant C451A-ERα mice downstream activation of signaling pathways and cell pro- (Fig. 1 A–C). ERα protein abundance in the uterus, aorta, and liferation (18, 19). Numerous cell culture experiments further hepatocytes was similar in the two groups. Plasma membranes suggest potentially important kinase-mediated cross-talk be- were then prepared from primary cultures of hepatocytes that tween membrane and nuclear ERα that modifies genomic re- can be obtained in sufficient quantities to allow sucrose gradient sponses to E2 (20, 21), including recent studies revealing that isolation of plasma membranes. Whereas ERα protein was MISS dependent on receptor palmitoylation influences receptor abundant in plasma membrane fractions (2–4) prepared from nuclear actions (22). Our current understanding of these pro- WT-C451 hepatocytes, a 60% decrease was observed in fraction cesses has relied primarily on experimentation in cell culture. As α α 4 of the mutant C451A-ER hepatocytes (Fig. 1D). Functionally, a result, the in vivo roles of membrane-associated ER in nu- we also found that phosphorylation of adenosine monophosphate- merous physiologic processes are poorly understood. activated protein kinase (AMPK) was increased twofold in re- Using a pharmacological approach in cell-based assays, sponse to E2 in WT-C451 mice but unchanged in C451A-ERα Harrington et al. (23) synthetized estrogen–macromolecule (Fig. 1E). These data reveal that the C451A mutation effectively conjugates (EDCs) to provide a gain-of-function strategy. EDC α consists of estrogen attached to a large, positively charged non- alters the plasma membrane localization of ER in vivo, which degradable poly(amido)amine dendrimer via hydrolytically sta- further abrogates the signaling pathway. ble linkages. Studies in breast cells clearly showed that Membrane-Associated ERα Is Required for Ovarian Function. When EDC was highly effective in stimulating nonnuclear signaling but eight C451A-ERα female mice were mated with WT-C451 males, inefficient in stimulating nuclear ER target gene expression be- no litters were observed despite continuous mating over a 5-mo cause EDC does not enter the nucleus (23). Importantly, in vivo administration of EDC fully stimulated carotid artery reendo- period. To investigate the basis of this infertility, histomorphologic thelialization but not uterine proliferation, suggesting for the analysis was performed on ovaries from 10- to 12-wk-old female first time that activating nonnuclear ERα signaling was sufficient mice. There was an excess of large, hemorrhagic, and/or cystic α to promote beneficial vascular effects of estrogen (24). Mem- follicles originating from antral follicles in C451A-ER ovaries, brane only ERα mice expressing the ERα E-domain under the along with an almost total absence of typical mature corpora lutea − − CytoMegaloVirus promoter in an ERα / background were also (Fig. 2 A and B). Counting of the number of follicles at different generated, but the vascular phenotype, such as NO production or stages revealed that ovaries from C451A-ERα mice displayed re- reendothelialization, was not assessed in this model (25). duced frequency of primordial follicles but normal percentages of To investigate the physiologic importance of membrane-initi- primary, secondary, and tertiary follicles (Fig. S2A). Wall com- ated ERα actions in vivo, we created a knock-in mouse model position of the tertiary follicle was also altered, with a decrease in with a selective loss of function of membrane ERα action by the percentage of the follicle area composed of granulosa cells, mutating the palmitoylation site at mouse ERα Cys451 to Ala whereas the percentage of the thecal cell area was conserved (Fig. (designated C451A-ERα). A complementary mutant mouse S2B). Additionally, there was a marked reduction in the density of model lacking the activation function AF-2 of ERα (designated glandular interstitial cells when WT-C451 were compared with ERα-AF20) (4, 6) provided a selective loss of function of nuclear C451A-ERα ovaries, which appeared loosely arranged and asso- ERα actions. Physiological studies in these two models revealed ciated with areas of hemorrhage. critical requirements for membrane receptors in ovarian function and thereby in fertility, and in vascular physiology. In contrast, genome-wide analysis of uterine gene expression demonstrated that nuclear ERα actions mediate uterine responses to estrogen, A Uterus Aorta Hepatocytes and also that membrane-to-nuclear receptor cross-talk in vivo is WT C451A -/- WT C451A ERα ERα ERα WT C451A modest in this tissue. These findings demonstrate critical tissue- 66kDa 66kDa specific roles for membrane versus genomic actions of a steroid Acn Gapdh hormone receptor in vivo. BCWT WT C451A ERα -66kDa Results E2 -+ -+ Annexin α p_AMPKα The C451A Mutation of ER in Vivo Alters the Membrane Localization C451A of ERα. Compared with the three other cysteines present in the ERα -66kDa AMPKα ligand-binding domain of ERα, C447/451 is the least reactive to Annexin Gapdh iodoacetic acid, suggesting a nonexposed position of this amino F1 F2 F3 F4 300 acid (26), and consequently the least likely candidate for lipid WT ** C451A 3 modifications. Nevertheless, two independent studies have clearly 200 shown that the palmitoylation of C447/451 is required for mem- 2 α brane localization of ER (18, 19). It is likely that palmitoylation 100 (AU) 1 of ERα C447/451 occurs before ligand binding, when the ligand- 0 0 pAMPK/AMPK pAMPK/AMPK

binding domain is less tightly folded (27). Fig. S1A displays ER α of membrane WT C451A a model of palmitoylation of C447/451 showing that despite the expression Relative buried nature of this residue a considerable portion of the at- Fig. 1. Validation of the C451A-ERα mouse model. (A)ERα protein levels in tached palmitoyl chain extends beyond the surface of the ERα uterus, aorta, and hepatocytes homogenates from ovariectomized WT-C451 and C451A-ERα mice. (B) Representative Western blot of plasma membrane- ligand-binding domain, and therefore could function as a mem- α α brane tether. To study the role of MISS actions of ERα, a knock- associated ER isolated from WT-C451 or C451A-ER hepatocytes by sucrose gradient. Quantification of plasma membrane-associated ERα in WT-C451 or in strategy was used in which the palmitoylation site Cys451 was C451A-ERα hepatocytes, n = 3. Results are normalized to annexin II expres- mutated to alanine using a targeting construct containing two base sion. (C) Phospho-MAPK/MAPK abundance in hepatocytes treated or not pair changes in exon 7 (Fig. S1B). The C451A mutation was with E2 (10−7 M, 15 min), from WT-C451 and C451A-ERα mice, n = 3. Rep- confirmed by PCR using tail DNA (Fig. S1C). resentative Western blot is shown. AU, arbitrary units.

2of8 | www.pnas.org/cgi/doi/10.1073/pnas.1322057111 Adlanmerini et al. Downloaded by guest on September 27, 2021 injection. A robust proliferative response was observed, similar PNAS PLUS ABWT-C451 C451A-ERα Corpus Hemorrhagic Luteum or Cystic in both WT-C451 and C451A-ERα mice (Fig. 3 C and D), and CL (CL) Follicles E2 treatment caused the same increase of luminal epithelial α 8 ** 8 (HCF) height in the two groups (Fig. 3E). The global ER protein HCF abundance was equivalent in the uterus homogenates from CL 6 6 HCF ovariectomized untreated WT-ERα and C451A-ERα and simi- 4 4 HCF larly decreased on both genotypes after E2 treatment (Fig. S3A). CL 2 2 Changes in expression levels of ERβ and/or GPR30 after acute or CL % of follicles of % 0 0 chronic E2 treatment, as assessed by RT-quantitative PCR, were WT C451A WT C451A not significantly different in the two genotypes, and thereby do C not support the idea of a potential compensatory roles of these WT-C451 C451A-ERα p-value receptors (Fig. S3 B and C). Thus, the C451A mutation of ERα does not alter epithelial endometrial cell proliferation or the E2 (pg/ml) 13.9 ± 7.4 11.8 ± 6.3 0.72 uterotrophic response to E2, demonstrating that membrane- PG (ng/ml) 0.1 ± 0.02 0.03 ± 0.01 * 0.01 initiated effects of ERα are not essential for uterine actions of FSH (pg/ml) 494.9 ± 119.3 812.2 ± 94.5 0.067 the hormone. LH (pg/ml) 319.5 ± 45.0 1046 ± 216 *** 0.0005 Gene Expression Profiling Analysis Reveals Differential Roles of Fig. 2. Adult ovarian phenotype is disturbed in the C451A-ERα mice. (A) Membrane Versus Nuclear ERα in Gene Regulation. In contrast to Representative images of WT-C451 and C451A-ERα ovaries (×25). CL, corpus the C451A mutation, previous studies in mice lacking the ERα luteum; HCF, hemorrhagic cystic follicles. (B) Percentages of corpora lutea activation function AF-2 (ERα-AF20) revealed that the utero- and hemorrhagic/cystic follicles were determined in ovaries from 3-mo-old trophic response to E2 is entirely dependent on AF-2 (4, 6). We WT-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. therefore compared the gene expression profiles in the uteri of C451A-ERα and ERα-AF20 mice (4), including littermate con- trols, 6 h after administration of the vehicle versus E2 (8 μg/kg) We also measured steroid sex hormone levels in intact 10- to using pangenomic microarrays.

12-wk-old female mice (Fig. 2C). C451A-ERα mice exhibited The interaction between genotype and treatment effect is PHYSIOLOGY serum E2 levels similar to those of WT-C451 mice, whereas shown in the heat map in Fig. 4A (P < 0.05). Cluster analysis of progesterone levels were markedly decreased in mutant mice. all of the conditions tested identified two major patterns of gene The level of FSH tended to be higher (P = 0.067) and luteinizing expression. The first cluster contained genes regulated by E2 in hormone (LH) was markedly increased (P = 0.0005) in C451A- C451A-ERα mutant mice, and these genes were almost equiva- ERα mice in comparison with WT-C451 mice. Collectively, these lent to those regulated by E2 in the control wild-type littermates 0 findings indicate that membrane ERα is required for normal of either the C451A-ERα or the ERα-AF2 mice. The patterns ovarian morphology and function. of genes expressed in all of the vehicle-treated animals were very similar, and they closely resembled the gene pattern observed in Membrane Actions of ERα Are Dispensable for Uterine Responses E2-treated ERα-AF20 mice, demonstrating an absence of tran- to Estrogen. The uteri of intact WT-C451 and C451A-ERα mice scriptional response to E2 in ERα-AF20 mice, at least in this were found to be normal in gross appearance (Fig. 3A). The tissue. Accordingly, E2 affected a very high number of genes in uterotrophic response to E2 was also similar in the two geno- the two sets of wild-type mice: 17,248 genes in WT-AF2 and types when the uterine wet weights were compared in ovariec- 18,112 genes in WT-C451A (Fig. S4A). The slight differences – – tomized female mice treated or not for 28 d by E2 (8 μg·kg 1·d 1 may be due to the fact that the wild-type mice are, respectively, s.c.) (Fig. 3B). To evaluate the impact of E2 on uterine epithelial on C57BL/6N and C57BL/6J background. More importantly, proliferation, Ki-67 labeling was performed 24 h following E2 16,659 genes were altered by E2 treatment in C451A-ERα

WT-C451 C451A-ERα Veh E2 24h 100 A C D WT 80 C451A 60

-positive 40

67 20 -

-C451 0

epithelial cells Veh E2 24h % Ki WT Treat.=***, Gen.=ns, Inter.=ns B Chronic treatment 150 E 20 (mg) WT ns WT C451A 15 C451A

100 (µm) α 10

weight 50

LEH 5 0 0 Veh E2 4 w. Veh E2 24h C451A-ER Treat.=***, Gen.= ns, Inter.=ns

Uterine Treat.=***, Gen.=ns, Inter.=ns

Fig. 3. Uterine proliferative response is maintained in C451A-ERα mice. (A) Representative images of intact uterus. (B) Uterine wet weight in ovariectomized WT-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 uterus sections 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.

Adlanmerini et al. PNAS Early Edition | 3of8 Downloaded by guest on September 27, 2021 Altogether, the genome-wide analysis of the uterus response WT-AF2 – E2 A Group 1 to E2 demonstrates that most of gene regulation by ERα is re-

C451A-ERα – E2 lated to AF2-dependent, transcriptional nuclear processes, whereas membrane ERα signaling has a minor impact, affecting only a WT-C451 – E2 very limited number of genes in this tissue.

WT-AF2 – Veh Membrane-Initiated Vascular Actions of ERα Are Abrogated in C451A- ERα Mice. The membrane-initiated actions of E2 have been par- WT-C451 – Veh Group 2 Group ticularly explored in the vasculature, where estrogen can acutely induce vasodilation in humans (28), at least in part through the C451A-ERα – Veh rapid stimulation of endothelial nitric oxide synthase (eNOS) activity in an ERα-dependent manner (29, 30). Studies using ERα-AF20 – E2 EDC, which selectively activates nonnuclear ER, have also pre- ERα-AF20 – Veh viously suggested that nonnuclear ERα signaling is important for the endothelial effects of E2 in vivo (24). We then evaluated the C vasodilatory effects of both E2 and EDC in WT-ERα and B E2 treatment in C451 mice AF2 dependent C451A dependent C451A-ERα mice by measuring dilatory responses of isolated WT-C451 1752 DOWN UP phenylephrine-preconstricted mesenteric arteries (Fig. S5A). (18112) μ D Within minutes, E2 (1 M) increased the diameter of arteries 14907 from wild-type mice by 32%, whereas there was no significant 7201 A − − G 35 vasodilatory response in arteries from ERα / or C451A-ERα 3205 C451A ER C (16659) 28 B mice (Fig. 5A, Left). EDC also induced rapid dilation of mes- 7551 67 enteric arteries from WT-C451 with a magnitude similar to that E2 treatment in AF2 mice α−/− E F observed with E2, and no dilation of arteries from ER or α WT-AF2 37 102 C451A-ER mice (Fig. 5A, Right). Because rapid dilatation in (17248) 160 H response to E2 is the consequence of stimulation of eNOS 640 29 through Akt-mediated phosphorylation of eNOS S1177 (31), 16608 AF2 dependent C451A dependent phosphorylation of eNOS in response to E2 was evaluated in ER AF20 isolated carotid arteries and found to be increased in WT-C451 UP DOWN − − (800) arteries, but not in arteries from ERα / or C451A-ERα mice Fig. 4. Large-scale gene analysis on response to E2 on C451A-ERα and ERα- (Fig. 5B). AF20 mice. (A) Gene expression profiles were obtained by microarray analysis To directly evaluate how ERα C451 palmitoylation and the (Agilent SurePrint G3 Mouse GE, 8 × 60K) of uterine samples from C451A- resulting plasma membrane targeting of the receptor does affect ERα,ERα-AF20 mice or their littermate WT controls treated with E2 (8 μg/kg, a functional response of endothelial cells to E2, endothelial cell 6 h) or vehicle (Veh), n =3–4. The heat map represents the data obtained on migration was tested in scratch assays using primary cells isolated all samples for the differentially expressed probes exhibiting interaction α < from the aorta of WT-C451 and C451A-ER mice. Indeed, between genotype and treatment (BH adjusted P 0.001). (B) The Venn activation of endothelial cell migration by E2 is mediated by diagram illustrates the overlaps between the genes significantly (P < 0.05) α up- or down-regulated in the uterus following E2 treatment in ERα-AF20 plasma membrane-associated ER coupledtoeNOSviaGαI and C451A-ERα mice. (C) The Venn diagram illustrates the overlaps between (24). E2 stimulated migration of WT-C451 endothelial cells, the genes significantly (BH adjusted P < 0.05) up- or down-regulated in but not of C451A-ERα endothelial cells (Fig. 5C). The role of the uterus following interaction between E2 treatment and genotypes, re- membrane-associated ERα in the vascular actions of E2 was spectively, ERα-AF20 and C451A-ERα mice. then investigated in vivo by evaluating reendothelialization of the carotid artery (Fig. 5D). E2 treatment caused an increase in endothelial repair in control WT-C451mice, whereas it had no mice, with 14,907 commonly regulated in WT-C451, which repre- effect in C451A-ERα mice (Fig. 5D). sents 82% of the genes targeted by E2 in WT-C451 mice. In contrast, Previous cell culture studies indicated that disrupting the only 800 genes were regulated by E2 in ERα-AF20, with 640 genes palmitoylation of ERα resulted in a receptor that is more sen- in common to those altered by E2 in WT-AF2 uterus (3.7%). sitive to E2-dependent degradation (22). We therefore evaluated A Venn diagram was generated to assess the overlap of up- possible alterations in ER abundance, in presence of E2 treat- and down-regulated genes found in the genotype and treatment ment, in the arteries of C451A-ERα mice. First, ERα and ERβ groups. The vast majority of genes (7,551 and 7,201 genes in mRNA expression was assessed in aorta from ovariectomized groups C and D) were respectively up-regulated or down-regu- C451A-ERα and WT-C451 mice treated with vehicle versus E2 lated in a specific ERα-AF2-dependent manner (Fig. 4C). In (8 μg/kg) for 6 h or 4 wk (Fig. S5 B and C). Aortas from both contrast, a small number of genes were regulated specifically in vehicle and E2-treated C451A-ERα mice displayed ERα and β α an ERα-C451 palmitoylation site-dependent manner (35 up- ER mRNA levels similar to those of WT mice. ER protein abundance was also equivalent in aorta from untreated ovari- regulated and 67 down-regulated, groups A and B). Finally, α a very limited number of genes was either repressed or activated ectomized C451A-ER and WT-C451 mice, and a similar de- crease in the amount of the receptor was observed after E2 by E2 via both AF2- or C451-dependent processes in the same or – – treatment (80 μg·kg 1·d 1 for 2 wk) in the two genotypes (Fig. opposite manner (groups E–H). Therefore, there is modest, if S5D). Thus, although previous in vitro experiments suggested any, cross-talk between the two receptor populations in uterus. that ERα palmitoylation influences the abundance of the re- Gene ontology analysis (Fig. S4B and Dataset S1) indicated ceptor protein (22), this was not detected in vivo in arteries that the limited number of palmitoylation-dependent genes and uterus. (groups A and B) are involved in membrane-, transmembrane-, Altogether, these findings indicate that preventing membrane- or extracellularly related processes or lipid transport pathways. initiated ERα signaling by targeting the palmitoylation site of The large number of AF2-dependent genes (groups C and D) ERα abrogates major vascular actions of E2, including rapid are primarily involved in nuclear, phosphoprotein-related, or artery dilation and the promotion of endothelial repair, despite splicing events. the presence of a global similar ERα arterial abundance.

4of8 | www.pnas.org/cgi/doi/10.1073/pnas.1322057111 Adlanmerini et al. Downloaded by guest on September 27, 2021 A C PNAS PLUS 100 50 # 2E 50 # CDE Veh E2 80 * 40 40 ns 30 30 60 20 20 40 Migration of

% Dilatation % 10 10 20 % Dilatation % primary ECs (%) 0 0 WT -/- -/- 0 ERα C451A WT ERα C451A WT-C451 C451A-ERα B WT ERα-/- C451A D Interaction: * VehE2 Veh E2 Veh E2 50 p-eNOS Veh E2 WT-C451 * Veh eNOS 40 ns β-acn E2 30 WT ERα -/- C451A-ERα C451A-ERα (AU) 10 10 10 20 * Veh 8 8 ns 8 ns 6 6 6 4 4 4 10 E2 2 2 2 Re-endothelialized area (%) WT-C451 C451A-ERα 0 0 0 Veh E2 Veh E2 Veh E2 Interaction: * peNOS / eNOS

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/eNOS − − − abundance 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 is − shown. 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 artery – – reendothelialization 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 of two-way ANOVA are shown, reporting the statistical interaction between treatment and genotype. PHYSIOLOGY

Membrane-Initiated Vascular Actions of ERα Are Conserved in ERα- Discussion 0 AF2 Mice. The marked contrast between ERα function in the In the present study we investigated the physiological roles of 0 uterus of C451-ERα and ERα-AF2 mice suggests that parallel a pool of ERα localized at the plasma membrane by mutating in studies in the two models may reveal potential tissue-specific mice the ERα C451 recognized as a key palmitoylation site in roles for nuclear versus nonnuclear receptors. We have pre- vitro. This genetic loss-of-function model revealed that ERα viously shown that the activation function AF2 is dispensable for C451 is absolutely required for plasma membrane location and the accelerating effect of E2 on endothelial healing (4). Here, we signals that are critical for arterial effects of E2, including the first confirmed a similar E2-induced acceleration of reendothe- stimulation of vasodilation and the promotion of endothelial re- lialization after carotid injury in WT-AF2 and ERα-AF20 mice pair, but also female fertility. In contrast, the uterine proliferation (Fig. 6). We further directly tested whether membrane-initiated in response to E2 occurs independently of membrane ERα. ERα function is retained in ERα-AF20 mice and found that in- Complementary studies in ERα-AF20 mice further demonstrated deed EDC caused similar promotion of endothelial repair in a preservation of membrane ERα action and vascular function in WT-AF2 and ERα-AF20 mice (Fig. 6). Therefore, whereas nu- this model. Moreover, genome-wide analysis of uterine gene ex- clear ERα actions mediated by AF-2 are abrogated, nonnuclear pression revealed that membrane ERα signaling has only a minor ERα function is intact in the ERα-AF20 mice, indicating that the impact on gene expression in this sex target tissue. These obser- conformation of the membrane ERα-AF20 allows its activation vations reveal tissue-specific roles for membrane versus nuclear by E2 as well as by EDC. ERα 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 OVX+Veh OVX+E2 α Veh site of mouse ER alters the expression level of the protein in OVX+Dendrimer OVX+EDC 50 vivo, because in vitro studies (22) had shown that such a mutated ns ns E2 human ERα (C447A) was more sensitive to E2-dependent deg- *** radation. The mutated C451A-ERα protein was normal in 40 ** WT-AF2 *** ** EDC abundance in the different murine tissues analyzed. With respect α 30 to the abrogation of vascular E2 effects in the C451A-ER 0 Veh mouse, it was also important to demonstrate that the abundance of the C451A-ERα protein in the vasculature in vivo is normal 20 -AF2 E2 under basal and E2-stimulated conditions. Furthermore, the ER α 10 anticipated decrease in plasma membrane association of the 0 EDC Re-endothelialized (%) area WT-AF2 ERα-AF2 mutant protein in vivo mirrors in vitro observations (18, 19), and Treatment=***, Genotype=ns, Interaction=ns it validates the C451A-ERα mouse as a selective loss-of-function model attenuating membrane receptor localization. α α 0 Fig. 6. Membrane effects of ER are preserved in ER -AF2 mutant mice. Our study on the reproduction of the C451A-ERα mice Carotid artery reendothelialization in ovariectomized WT-AF2 and AF2-ERα – – α mice treated with E2 (80 μg·kg 1·d 1, 2 wk) or vehicle (Veh) and EDC demonstrates the crucial role of membrane ER in female fer- – – (240 μg·kg 1·d 1, 2 wk) or control dendrimer, n =8–10. Statistical results of tility. This is at least in part due to alterations in ovarian func- two-way ANOVA are shown, explaining the effect of treatment, genotype, tion, with the ovaries of adult C451A-ERα mice being almost and interaction between these two variables. completely devoid of corpora lutea and displaying a high number

Adlanmerini et al. PNAS Early Edition | 5of8 Downloaded by guest on September 27, 2021 of hemorrhagic and/or cystic follicles, indicating that the matu- conclusions raised from these models of cultured cancer cells, our ration of follicles into corpora lutea (luteinization, which allows large-scale gene analysis reveals that although MISS via ERα can the transition to the next estrous cycle) is abnormal. The capacity influence the regulation of a small subset of genes in the uterus, the for ovulation per se has not yet been directly evaluated in impact of nonnuclear ERα on gene regulation is quite modest in C451A-ERα mice. The absence of corpora lutea, which produce this organ, paradigmatic of the proliferative action of E2. progesterone, likely underlies the low serum progesterone found This context-specific role for membrane-initiated signals fits in C451-ERα mice. Furthermore, the level of LH in intact mice along the complexity of the palmitoylation process (38), em- was increased, suggesting that the control of LH production by phasizing the regulation and the spatiotemporal dynamics of the hypothalamic–pituitary axis is adversely affected in C451- protein palmitoylation. These levels of complexity should be ERα mice. Female mice deficient in either ERαAF-1 or -2 addressed in future studies, even if such in vivo studies seem (named ERα-AF10 and ERα-AF20, respectively) are also sterile, particularly difficult. primarily owing to absence of uterine hypertrophy (3, 4, 6). Thus, The discoveries that were rendered possible via the generation both plasma membrane and nuclear actions of ERα are abso- of the C451A-ERα mouse model indicate that membrane and lutely required for female fertility. nuclear actions of ERα in vivo are highly tissue-specific in some We then explored to what extent membrane ERα is important key targets and functions. The C451A-ERα mouse now provides for estrogen vascular effects. We found that both E2 and EDC a valuable loss-of-function model for the study of the role elicit a rapid vasorelaxant effect in isolated mesenteric arteries of plasma membrane-associated ERα in numerous additional −/− from wild-type mice, whereas these effects are lost in ERα physiological and pathophysiological processes affected by the 1177 and C451A-ERα mice. eNOS phosphorylation in response to receptor, as well as a means to conceive new selective ERα E2 was also abolished in arteries from C451A-ERα mice, as was modulators with an optimized medical profile. the accelerative action of E2 on endothelial healing both in vivo and in vitro. In the past, the selective membrane ER activator Materials and Methods EDC was shown to be sufficient to stimulate endothelial cell Mice. All procedures involving experimental animals were performed in ac- migration and the acceleration of reendothelialization (24). cordance with the principles established by the Institut National de la Santé et Furthermore, the acceleration of endothelial repair by E2 or de la Recherche Médicale and were approved by the local Ethical Committee EDC in ERα-AF20 mice was preserved, demonstrating that the of Animal Care. The C451A-ERα knock-in mouse line was generated on a C57BL6/N background through the strategy outlined in Fig. S1B at the MISS effects are fully activable in this model. Altogether, the − − α Mouse Clinical Institute (Illkirch, France). ERα / ,ERα-AF20 mice have been current findings indicate that the activation of membrane ER is α both necessary and sufficient to promote endothelial repair and previously described (4, 39). C451A-ER and their corresponding wild-type littermates (WT-C451) were ovariectomized at 4 wk of age. For acute E2 contribute to the integrity of the endothelial monolayer. β α treatment, they were injected s.c. with vehicle (castor oil) or 17 -estradiol In contrast, C451A-ER mice also revealed that receptor (E2, 8 μg/kg) 3 wk after ovariectomy. For chronic E2 treatment, ovariecto- palmitoylation and membrane targeting are dispensable for mized mice were implanted with s.c. pellets releasing either vehicle or E2 uterine growth responses to E2. Previous work showed that the (8 or 80 μg·kg–1·d–1 as indicated, 60-d release; Innovative Research of America). selective activation of membrane ERα by EDC does not induce uterine hypertrophy (24), suggesting that MISS activation alone Isolation of Plasma Membranes. Primary hepatocyte cultures were isolated is not sufficient to promote a proliferative response in uterine from the livers of 8- to10-wk-old mice by a modification of the collagenase epithelium. Using a loss-of-function strategy, we now show that method (40). Briefly, livers from WT-C451 and C451A-ERα mice were per- MISS effects involving ERα palmitoylation are also not neces- fused using the portal vein with HBSS before collagenase perfusion (C5138; sary. In contrast, previous studies have demonstrated the im- Sigma). Hepatocytes were then put in cultures in DMEM. Plasma membranes portance of genomic (i.e., transcriptional) activity of ERα in this were isolated by a discontinuous sucrose step gradient, from which 12 fractions were recovered (41). The first fractions (1–4) contained the plasma process, because E2 does not induce a uterine proliferative re- α 0 α 0 membrane proteins, which were identified after protein precipitation by sponse in ER -AF1 or in ER -AF2 mice (5, 6). Western blot using polyclonal antibodies against either ERα or annexin II. We further explored the gene expression profiles of ERα- 0 AF2 and C451A-ERα mice in the uterus, a tissue that is highly Mouse Carotid Artery Injury. Perivascular carotid artery electric injury was responsive to E2. Genome-wide gene array studies revealed that, performed as previously described (3) in ovariectomized mice treated or not – – in contrast to nuclear ERα-mediated processes, membrane ERα- for 2 wk before surgery with E2 (80 μg·kg 1·d 1 in 60-d-release pellets) or – – mediated signaling has only a minor impact on gene expression. EDC (ethinyl-estradiol dendrimer conjugated, 240 μg·kg 1·d 1). Briefly, the A small set of genes was found to be regulated in the uterus by left common carotid artery was exposed via an anterior cervicotomy. The both membrane and nuclear ERα. Altogether, we can propose electric injury was applied to the distal part (3 mm precisely) of the common that C451A-ERα and ERα-AF20 mice can be considered as carotid artery with a bipolar microregulator. The percentage of reendo- mouse models of membrane ERα and nuclear ERα loss of thelialization was calculated relative to the initial deendothelialized area by assessing Evans blue dye uptake 3 d after injury. Images were acquired un- function, respectively, but with preservation of nuclear ERα and α der a DMR 300 Leica microscope, using Leica Application Suite V3.8 and membrane ER , respectively. ImageJ softwares. Cell culture studies (18, 19, 22, 32) previously suggested that rapid E2-dependent signaling may modify ERα transcriptional Vascular Reactivity of Isolated Mesenteric Arteries. As previously described activity and thereby modulate final receptor-dependent nuclear (42), 5-mm-long segments of second-order mesenteric arteries were dis- actions, representing cross-talk between nonnuclear and nuclear sected and mounted between two glass cannulae and bathed in a physio- receptor subpopulations. The function of many transcription logical salt solution (pH 7.4, PO2 160 mm Hg, and PCO2 37 mm Hg). Pressure factors is regulated through protein kinase-mediated phosphor- was controlled by a servo-perfusion system, and diameter changes were ylation, and these transcription factors may be targets for ex- measured continuously using a video-monitored system (Living Systems Instruments). Artery viability was assessed using KCl (80 mM) and endothe- tranuclear actions of estrogen (20, 33). An important role for μ μ – μ ERα MISS was reported in cancer cell proliferative responses to lium integrity with acetylcholine (1 M). EDC- (1 M) and E2- (0.01 1 M) – dependent dilation was then assessed after precontraction with phenyl- E2 (34 36), and ERK 2, a downstream effector of the MAPK ephrine to 70% of the maximal contractile response obtained with KCl pathway, cooperates in regulating gene transcription (21). Sim- (80 mM). ilarly, a cross-talk between membrane-initiated signaling of ste- roid receptors, such as progesterone or androgen receptor, and Mouse Endothelial Cell Migration Assay. Endothelial cells were isolated from gene regulation and cell proliferation was also reported in various the aortae of wild-type and C451A-ERα mice, and cell migration was assessed culture models of cancer cells (37). However, in contrast to the using methods modified from those previously described (43). At first pas-

6of8 | www.pnas.org/cgi/doi/10.1073/pnas.1322057111 Adlanmerini et al. Downloaded by guest on September 27, 2021 sage, the cells were transferred into six-well plates, and following growth to david.abcc.ncifcrf.gov), and comparisons are realized with the Venn Dia- PNAS PLUS confluence in EGM-2 medium (Lonza) containing 10% FBS, a defined linear grams plug-in based upon the VENNY tool developed by J. C. Oliveros. region of cells was removed using a pipette tip. The initial area devoid of cells was marked and quantified on images obtained at baseline, and Western Blotting. Total proteins were separated on a 10% SDS/PAGE gel and − treatments with vehicle or E2 (10 9 M) were initiated in phenol red-free transferred to a nitrocellulose membrane. The primary antibodies used are as DMEM with 5% charcoal-stripped FBS. Sixteen hours later the cells were follows: MC20 (sc-542; Santa Cruz Biotechnology), Gapdh (sc-32233; Santa fixed and stained with Coomassie blue, and repeat images were obtained to Cruz Biotechnology), pSer1177-eNOS (612392; BD Bioscience), eNOS (610297; quantify the remaining area devoid of cells. Cells were imaged using an BD Bioscience), pThr172-AMPK (2531; ), AMPK (2532; Cell Sig- inverted phase-contrast microscope (Nikon ECLIPSE TS100; Nikon Corpora- naling), actin (A2066; Sigma), and annexin II (sc-9061; Santa Cruz Bio- tion) and a digital camera (Infinity 1). The area devoid of cells was quantified technology). Revelation was performed using an HRP-conjugated secondary using Adobe ImageReady CS2 software. The degree of migration was cal- antibody and visualized by ECL detection according to the manufacturer’s culated as the percentage of the initial area devoid of cells that was occu- instructions (Amersham Biosciences/GE Healthcare), using ChemiDoc Imag- pied by cells following 16-h treatment. ing System (Bio-Rad). Bands were quantified using ImageJ densitometry.

Hormone Assays. Serum levels of LH and FSH were determined using the Statistical Analyses. Results are expressed as the mean ± SEM. To test the Multiplex Immunoassay Technology Xmap (MILLIPLEX; Millipore). Pro- effect of treatments or genotypes, t test or Mann–Whitney test was per- gesterone and 17β-estradiol were measured by gas chromatography–mass formed. To test the interaction between treatments and genotypes, a two- spectrometry (44), with minor modifications. These assays were performed way ANOVA was carried out. When an interaction was observed between on intact 10- to12-wk-old female mice. two variables, the effect of treatment was studied in each genotype using the Bonferroni post hoc test. A value of P < 0.05 was considered statistically Immunohistochemistry. Paraffin-embedded transverse sections (4 μm) from significant (*P < 0.05; **P < 0.01; ***P < 0.001). formalin-fixed uterine or ovary specimens were stained as previously de- scribed (5) with anti–Ki-67 antigen (RM-9106; Thermo-scientific). Sections ACKNOWLEDGMENTS. The C451A-ERα mouse was generated at the Mouse were examined under a Nikon Eclipse E400 microscope using image analysis Clinical Institute by M. C. Birling and coworkers. We thank Dr. Christopher α software Morpho Expert. For uterus, the luminal epithelial height and Mayne for creating the model of ER -C451A palmitate (Fig. S1A) and Rosa stromal height of endometrium is the mean of 10 measurements, whereas Sirianni, who performed the endothelial cell migration experiments. We thank Dr. Benita Katzenellenbogen and Dr. Henrik Laurell for fruitful dis- the percentage of Ki-67–positive epithelial cells is the mean of three mea- cussions and suggestions for the writing of the manuscript. We also thank surements in each transverse uterus section. Ovarian function was evaluated the staff of the animal facility (J.-C. Albouys, M. J. Fouque, and G. Carcasses); in 3-mo-old adult mice by counting the number of follicles in each class (45). C. Bleuart for anatomopathology at the École Nationale Vétérinaire de Tou- The number of follicles in each category was expressed as the percentage of louse, the staff of the Anexplo platform (A. Desquennes and S. Legonidec); PHYSIOLOGY total number of follicles. Analysis of the area of the granulosa and theca was and M. Buscato [Institut National de la Santé et de la Recherche Médicale performed on the largest tertiary follicle of each section, as previously de- (INSERM) 1048] for skillful technical assistance. We also thank Y. Lippi and scribed (46), and expressed as a percentage of the total area of follicle. P. Martin for the excellent contribution to microarray analysis carried out at GeT-TRIX Genopole Toulouse facility. The work at I2MC-INSERM U1048 is supported by Institut National de la Santé et de la Recherche Médicale, Microarray. Total RNA was extracted using TriPure reagent (Roche) and re- Université et CHU de Toulouse, Faculté de Médecine Toulouse-Rangueil, verse-transcribed (High-Capacity cDNA Reverse Transcription Kit; Applied Fondation pour la Recherche Médicale, Fondation de France, Fondation de Biosystems). Microarray data were obtained from 200 ng of uterine total RNA l’Avenir, Conseil Régional Midi-Pyrénées, and AVIESAN-Astra-Zeneca. The labeled with Cy3 using Low Input Quickamp Labeling Kit (Agilent) and hy- work at INSERM U1083-Centre National de la Recherche Scientifique (CNRS) bridized to Agilent SurePrint G3 Mouse GE (8 × 60K) microarrays following Unité Mixte de Recherche 6214 is supported by INSERM, CNRS, Centre Hos- ’ the manufacturer’s instructions. All experimental details are available in the pitalier Universitaire and Université d Angers, Fondation de France, Fonda- tion de l’Avenir and Conseil Régional Pays de la Loire, Conseil Régional Gene Expression Omnibus (GEO) database under accession no. 53237. Data Midi-Pyrénées, and Fondation pour la Recherche Médicale. M.A. and A.A. were analyzed under R (R 2.13; www.R-project.org). Hierarchical clustering were supported by grants from the Ministère de la Recherche and Groupe de was applied to the samples and the probes using 1-Pearson correlation co- Réflexion sur la Recherche Cardiovasculaire, respectively. This work was sup- efficient as distance and Ward’s criterion for agglomeration. Functional ported by National Institutes of Health Grants DK015556 and HL087564 analysis was carried out using DAVID Bioinformatics Resources 6.7 (http:// (to J.A.K. and P.W.S., respectively).

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8of8 | www.pnas.org/cgi/doi/10.1073/pnas.1322057111 Adlanmerini et al. Downloaded by guest on September 27, 2021