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Mutation of the Palmitoylation Site of Estrogen Receptor Α in Vivo Reveals

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Mutation of the Palmitoylation Site of Estrogen Receptor Α in Vivo Reveals 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) Estrogen receptor 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 gene expression, 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-nuclear receptor 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 cancer 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.
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