Marine Adlanmerini

Chanaëlle Febrissy

Rana Zahreddine

Emilie Vessières

Melissa Buscato

Romain Solinhac

Julie Favre

Typhaine Anquetil

Anne-Laure L Guihot

Frederic Boudou

; M Buscato

T Anquetil

I Raymond-Letron

P Chambon

P Gourdy

C Ohlsson

H Laurell

C Fontaine

R Metivier

M Le Romancer

D Henrion

J F Arnal

F Lenfant

Mutation of Arginine 264 on ERα (Estrogen Receptor Alpha) Selectively Abrogates the Rapid Signaling of Estradiol in the Endothelium Without Altering Fertility

published or not. The documents may come

Introduction

ERα, a member of the nuclear receptor family, is implicated in many physiological processes including reproduction, metabolism or cardio-vascular protection. ERα classically mediates its biological functions through regulation of gene expression via recruitment of cofactors to the AF1 and AF2 transactivation functions. These ERα-induced transcriptional changes occur in a cell-and tissue-specific manner, through either direct DNA interactions with Estrogen-responsive Elements (EREs), or indirectly, through protein-protein tethering with transcription factors such as c-Fos/c-Jun (AP-1) and SP1 [START_REF] Heldring | Estrogen receptors: How do they signal and what are their targets[END_REF] . However, besides this traditionally activity acting as a nuclear transcription factor, ERα can also mediate Membrane Initiated Steroid Signaling (MISS), usually termed "nongenomic" or "extranuclear" effects. These rapid effects of estrogen, such as the mobilization of intracellular calcium and generation of cAMP, were first described several decades ago [START_REF] Pietras | Specific binding sites for oestrogen at the outer surfaces of isolated endometrial cells[END_REF][START_REF] Szego | Adenosine 3',5'-monophosphate in rat uterus: Acute elevation by estrogen[END_REF] . In vitro studies allowed defining more precisely this extranuclear signaling of ERα. In particular, palmitoylation of human Cys447 was found to promote interaction with caveolin-1 and plasma membrane localization [START_REF] Acconcia | Palmitoylation-dependent estrogen receptor alpha membrane localization: Regulation by 17betaestradiol[END_REF][START_REF] Acconcia | S-palmitoylation modulates human estrogen receptor-alpha functions[END_REF][START_REF] Pedram | A conserved mechanism for steroid receptor translocation to the plasma membrane[END_REF] . In response to estrogens, this plasma membrane localization favored the activation of protein kinase pathways (PI3K/Akt, Erk), endothelial nitric oxide (NO) synthase and phospholipase C [START_REF] Hammes | Minireview: Recent advances in extranuclear steroid receptor actions[END_REF][START_REF] Kim | Membrane-initiated actions of estrogen on the endothelium[END_REF][START_REF] Wu | Non-nuclear estrogen receptor signaling in the endothelium[END_REF][START_REF] Simoncini | Non-genomic actions of sex steroid hormones[END_REF][START_REF] Simoncini | Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-oh kinase[END_REF] , implicated in cell survival and migration. These rapid effects can also lead to gene expression changes [START_REF] Rosa | Palmitoylation regulates 17beta-estradiolinduced estrogen receptor-alpha degradation and transcriptional activity[END_REF][START_REF] Madak-Erdogan | Nuclear and extranuclear pathway inputs in the regulation of global gene expression by estrogen receptors[END_REF] . The physiological role of these membraneinitiated ERα actions in vivo began to be elucidated recently with the generation of the mouse model mutated for the palmitoylation site of ERα (C451A-ERα), demonstrating the importance of membrane actions for the fertility but also for some rapid vascular effects of E2, such as acceleration of reendothelialization and endothelial NO synthase phosphorylation and activation [START_REF] Adlanmerini | Mutation of the palmitoylation site of estrogen receptor alpha in vivo reveals tissue-specific roles for membrane versus nuclear actions[END_REF] . Finally, two pharmacological tools, the estrogen-dendrimer conjugate (EDC) and "pathway preferential estrogens" (PaPEs), showed that activation of MISS is sufficient to elicit these rapid endothelial effects [START_REF] Chambliss | Non-nuclear estrogen receptor alpha signaling promotes cardiovascular protection but not uterine or breast cancer growth in mice[END_REF][START_REF] Madak-Erdogan | Design of pathway preferential estrogens that provide beneficial metabolic and vascular effects without stimulating reproductive tissues[END_REF][START_REF] Arnal | Membrane and nuclear estrogen receptor alpha actions: From tissue specificity to medical implications[END_REF] .

While palmitoylation of ERα was found to be critical for its membrane targeting, a sequence in the C-domain of human ERα (aa251-260) [START_REF] Krust | The chicken oestrogen receptor sequence: Homology with v-erba and the human oestrogen and glucocorticoid receptors[END_REF] was also reported to allow the segregation between non-nuclear and nuclear actions of E2 [START_REF] Wu | Point mutations in the eralpha galphai binding domain segregate nonnuclear from nuclear receptor function[END_REF] . Indeed, this motif was shown to interact with Gαi and be involved in rapid NO production in transfected COS cells. Interestingly, some point mutations within this domain, such as R256A, K257, D258A and R260A all abrogated the activation of eNOS and ERK phosphorylation in response to E2. By contrast, the E2-induced transcriptional response on ERE, SP1 and AP1-Luc gene reporter assays remained totally preserved, with the exception of R260A that abolished the response in an AP1 reporter gene assay. Furthermore, Le Romancer and coll. [START_REF] Romancer | Regulation of estrogen rapid signaling through arginine methylation by prmt1[END_REF] demonstrated that the same arginine 260 residue was transiently methylated by PRMT1 following E2 activation, and that this methylation was required to trigger ERα interaction with the p85 subunit of PI3K and Src. The complex ERα/PI3K/src was found to be overexpress in a subset of breast tumors and is an independent marker of poor prognosis [START_REF] Poulard | Activation of rapid oestrogen signalling in aggressive human breast cancers[END_REF] . Nevertheless, the specific roles of this arginine 260 of ERα remained to be determined in vivo.

To that end, a knock-in mouse model of ERα mutated for the arginine 264 (the mouse counterpart of human arginine 260) into alanine was generated by homologous recombination (designated R264A-ERα). In contrast to several other mouse models mutated for ERα, the R264A female mice are totally fertile, with normal hormonal serum levels and a normal uterine and ovarian morphology. However, the rapid vascular effects of estrogens recognized to be initiated at the plasma membrane, such as rapid endothelial repair, eNOS activation and dilatation of arteries were lost in R264-ERα mice.

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Material and Methods Mice. All procedures were performed in accordance with the principles established by the INSERM and were approved by the local Ethical Committee of Animal Care N° CEEA-122-2014-55 and CEEA PdL 2012.105 for dilation and arterial remodeling. The R264A-ERα knock-in mouse line was generated on a C57BL6/N background at the Mouse Clinical Institute (Illkirch, France) by homologous recombination, as described in Supplementary Fig1. R264A-ERα and their corresponding wild-type littermates were ovariectomized at 4 weeks of age. For acute E2 treatment, they were subcutaneously (s.c.) injected with vehicle (castor oil) or 17β-estradiol (E2, 8 µg/kg) 3 weeks after ovariectomy. For chronic E2 treatment, ovariectomized mice were aleatory randomized to be implanted with s.c. pellets releasing either vehicle or E2 (8 or 80 µg/kg/day as indicated, 60-days release; Innovative Research of America, Florida).

Evaluation of estrous cycle. Estrous cycle was determined by vaginal cytology method [START_REF] Caligioni | Assessing reproductive status/stages in mice[END_REF] in 3 month-old adult mice for 10-20 consecutive days. Briefly, every morning, vaginal enema was performed with 20μL of Phosphate-buffered saline and cytology was determined by microscopic observations: predominant nucleate epithelial cells characterize proestrus phase; predominant corneous anucleate epithelial cells characterize estrus phase; leucocytes, as well as mix of nucleate and corneous anucleate epithelial cells characterize metestrus and diestrus phase.

Histological evaluation of ovarian function.

Ovaries were dissected, fixed in 10% neutral buffered formalin for 24h, processed and embedded in paraffin. Slides were prepared by using transverse 4µm sections, deparaffinized and then stained by hematoxylin and eosin as previously described [START_REF] Abot | The af-1 activation function of estrogen receptor alpha is necessary and sufficient for uterine epithelial cell proliferation in vivo[END_REF] .

Immunohistochemistry:

Paraffin-embedded transverse sections (4 µm) from formalin-fixed uterine were stained as previously described [START_REF] Abot | The af-1 activation function of estrogen receptor alpha is necessary and sufficient for uterine epithelial cell proliferation in vivo[END_REF] with anti-Ki67 (RM-9106; Thermo-scientific, culture supernatant, dilution 1/200), or with anti-ERα (MC-20, 50 ng/ml, Santa Cruz). Sections were examined after digitization using a NanoZoomer Digital Pathology ® . To examine the proliferative effects of each treatment, the ratio of Ki67-positive epithelial cell/total cell number from two microscopic fields of measurement at x20 magnification for each uterine section was evaluated.

Hormone serums levels measurements. Serum levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were determined using the Multiplex Immunoassay Technology Xmap (MILLIPLEX, Millipore, St Quentin en Yvelines, France). Testosterone, Progesterone and 17β-estradiol were measured on serum of intact 10-12 week-old female mice using a gas chromatography-tandem mass spectrometry (GC-MS/MS) method as recently described [START_REF] Nilsson | Measurement of a comprehensive sex steroid profile in rodent serum by high-sensitive gas chromatography-tandem mass spectrometry[END_REF] .

Analysis of mRNA levels by RT-qPCR. Tissues were homogenized using a Precellys tissue homogenizer (Bertin Technol., Cedex, France), and total RNA from tissues was prepared using TRIzol (Invitrogen, Carlsbad, CA). RNA (0.5-1.0 µg) was reverse transcribed (RT) at 25°C for 10 min and then at 37°C for 2 h in 20 μL final volume using the High Capacity cDNA reverse transcriptase kit (Applied Biosystems). qPCR was performed on the equivalent of 7.5-20 ng of template using SsoFast EvaGreen Supermix (Bio-Rad) with primers validated for PCR efficiency and melt curve analysis (Supplementary Table 1). Results were normalized against the gene encoding the tumor protein, translationallycontrolled 1 (Tpt1) (aorta) or Gapdh/Hprt1 (uterus) using the geometric mean of relative quantities [START_REF] Vandesompele | Accurate normalization of real-time quantitative rt-pcr data by geometric averaging of multiple internal control genes[END_REF] . The data shown are geometrical means of relative quantities (2 -Cq +/linearized log2-based SEM) n=4/group) [START_REF] Taylor | The ultimate qpcr experiment: Producing publication quality, reproducible data the first time[END_REF] .

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Micro-array. Total RNA was extracted using TriPure reagent (Roche, Lyon, France). Microarray data were obtained from 200 ng of uterine total RNA labeled with Cy3 using Low Input Quickamp Labeling Kit (Agilent, Massy, France) and hybridized to Agilent SurePrint G3 Mouse GE (8X60K) following the manufacturer's instructions. All experimental details and data are available in the Gene Expression Omnibus (GEO) database under accession GSE152344 . Data were analyzed with the LIMMA package in R (R 2.13., www.Rproject.org). Hierarchical clustering was applied to the samples and the probes using 1-Pearson correlation coefficient as distance and the Ward's criterion for agglomeration. Functional analysis was carried out using DAVID Bioinformatics Resources 6.7 (http://david.abcc.ncifcrf.gov), and comparisons are realized with Venn Diagrams plug-in based upon the Venny tool developed by J.C Oliveros (GenoToul Bioinfo, Toulouse).

Integration of ChIP-seq data. We used the ER ChIP-seq data obtained from cultured cells of uteri (GSE36455) available at the GEO website [START_REF] Hewitt | Research resource: Whole-genome estrogen receptor alpha binding in mouse uterine tissue revealed by chip-seq[END_REF] . Reads were aligned onto the indexed chromosomes of the mm9 genome using bowtie 0.12.7 [START_REF] Li | The sequence alignment/map format and samtools[END_REF] with parameters allowing at most two mismatches, and selected for unique mapping onto the genome. Peak calling was next performed as previously described 29 using samtools 0.1.12a [START_REF] Langmead | Ultrafast and memory-efficient alignment of short DNA sequences to the human genome[END_REF] and MACS 1.4.1 [START_REF] Zhang | Model-based analysis of chip-seq (macs)[END_REF] with default parameters. Identification of ERBss located at the vicinity of genes sub-populations and on genes TSSs used home-written intersection scripts and algorithms from the cistrome web-platform (http://cistrome.org/ap/; 32 ). Motif analysis was performed using SeqPos tool on the cistrome web-platform. Motifs were declared enriched when p-values were <0.05 and Z-score>2.

Mouse carotid artery injury. Perivascular carotid artery electric injury was performed as previously described [START_REF] Adlanmerini | Mutation of the palmitoylation site of estrogen receptor alpha in vivo reveals tissue-specific roles for membrane versus nuclear actions[END_REF][START_REF] Billon-Gales | The transactivating function 1 of estrogen receptor alpha is dispensable for the vasculoprotective actions of 17beta-estradiol[END_REF] , [START_REF] Toutain | Estrogen receptor alpha expression in both endothelium and hematopoietic cells is required for the accelerative effect of estradiol on reendothelialization[END_REF] on ovariectomized mice treated or not for 2 weeks before surgery with E2 (80 µg/kg/day in 60 day-release pellets). Briefly, the left common carotid artery was exposed via an anterior cervicotomy. The electric injury was applied to the distal part (3 mm precisely) of the common carotid artery with a bipolar microregulator. The percentage of reendothelialization was calculated relative to the initial de-endothelialized area by assessing Evans blue dye uptake 3 days after injury. Images were acquired under a DMR 300 Leica microscope, using Leica Application Suite V3.8 and ImageJ softwares.

Vascular reactivity of isolated mesenteric arteries. As previously described [START_REF] Kauffenstein | Central role of p2y6 udp receptor in arteriolar myogenic tone[END_REF][START_REF] Begorre | Microvascular vasodilator properties of the angiotensin ii type 2 receptor in a mouse model of type 1 diabetes[END_REF] , five-mm long segments of second-order mesenteric arteries from both male and female mice were dissected and mounted between 2 glass cannulae and bathed in a physiological salt solution (pH 7.4, pO2 160 mm Hg, and pCO2 37 mm Hg). Pressure was controlled by a servoperfusion system, and diameter changes were measured continuously using a videomonitored system (Living Systems Instruments). Artery viability was assessed using KCl (80 mM) and endothelium integrity with acetylcholine (1 µM)-dependent dilation after precontraction with phenylephrine to approximately 50% of the maximal contractile response (Supplementary Figures 3A and3B). E2-dependent dilation (0.01-1 µM) was then assessed after precontraction with phenylephrine to approximately 50% of the maximal contractile response obtained with KCl (80 mM). E2-dependent dilatation was repeated after treatment of the arterial segments with the NO synthesis blocker N-omega-Nitro-L-arginine (L-NNA, 0.1 mM). In a separate series of experiments, segments of second-order mesenteric arteries were mounted in an arteriograph as described above and incubated with pertussis toxin (200 ng/ml) for 2 hours. E2-dependent dilation (0.01-1 µM) was then tested after precontraction with phenylephrine to 70%.

Analyses of Atheromatous Lesions. Since sex differences have been frequently reported in mouse atherosclerosis studies [START_REF] Robinet | Consideration of sex differences in design and reporting of experimental arterial pathology studies-statement from atvb council[END_REF] with a most widely reported sex effect on atherosclerosis in female mice, the E2 protective effect on atherosclerosis was only studied here in female mice, using the guidelines for experimental atherosclerosis studies described in the AHA A c c e p t e d m a n u s c r i p t Statement [START_REF] Daugherty | Recommendation on design, execution, and reporting of animal A c c e p t e d m a n u s c r i p t atherosclerosis studies: A scientific statement from the american heart association[END_REF] . Bilateral ovariectomy was performed, as previously described [START_REF] Adlanmerini | Mutation of the palmitoylation site of estrogen receptor alpha in vivo reveals tissue-specific roles for membrane versus nuclear actions[END_REF] . Two weeks after surgery, mice were switched to a hypercholesterolemic-atherogenic diet (1.25% cholesterol, 6% fat, no cholate; TD96335; Harlan Teklad, Madison, WI) and concomitantly received long-term estrogenic treatments. Mice were implanted with subcutaneous pellets releasing either placebo or 17β-estradiol (60-day release, 0.1 mg [ie, 80 µg/kg per day]) at weeks 6 and 12. At the end of the protocol, overnight fasted mice were anesthetized with a combination of ketamine hydrochloride (100 mg/kg; Panpharma) and xylazine (5 mg/kg; Sigma-Aldrich) via intraperitoneal injection, and blood was collected from retro-orbital venous plexus. After euthanasia, the heart, the thoracic aorta, the liver, and the uterus were carefully dissected. Lipid deposition size was estimated at the aortic sinus, as previously described [START_REF] Billon-Gales | The transactivating function 1 of estrogen receptor alpha is dispensable for the vasculoprotective actions of 17beta-estradiol[END_REF] . Briefly, each heart was frozen on a cryostat mount with optimal cutting temperature compound. One hundred 10-µm-thick sections were prepared from the top of the left ventricle, where the aortic valves were first visible, up to a position in the aorta where the valve cusps were just disappearing from the field. After drying for 2 hours, the sections were stained with oil red O and counterstained with Mayer's hematoxylin. One 10 µm-thick cut-section was collected from each 100 μm-thick zone was used for specific morphometric evaluation of intimal lesions using a computerized Biocom morphometry system. The first and most proximal section to the heart was taken 90 µm distal to the point where the aorta first becomes rounded. The mean lesion size (expressed in µm2) in these 10 sections was used to evaluate the lesion size of each animal.

Determination of Plasma Lipids. Overnight fasted mice were anesthetized, and blood samples were collected from the retro-orbital venous plexus. Total plasma cholesterol was assayed using the CHOD-PAD kit (Horiba ABX, Montpellier, France). The high-density lipoprotein fraction was isolated from 10 µl of serum and assayed using the "C-HDL+Third generation" kit (Roche, Lyon, France).

Blood Flow-Mediated Arterial Remodeling. Increase in blood flow, applied to the mesenteric artery, was performed, as previously described, on 4-to 5-month-old female mice 39 . Briefly, 3 consecutive first-order mesenteric arteries were used, and surgery consisted of ligatures of second-order branches. The artery located between the 2 ligated arteries was designated as the high-flow artery. Arteries located at a distance of the ligated arteries were used as controls (normal flow). After 14 days, mice were euthanized and mesenteric arteries were collected. Arteries were cannulated in an arteriograph as described above (vascular reactivity) and bathed in a Ca2+-free physiological salt solution containing ethylene-bis-(oxyethylenenitrolo) tetra-acetic acid (EGTA, 2 mmol/L) and sodium nitroprusside (SNP, 10 µmol/L). Pressure steps (10 to 125 mmHg) were then performed in order to determine passive arterial diameter. In each protocol, animals were anesthetized with isoflurane (2.5%). They were treated with buprenorphine (Temgesic; 0.1 mg/kg, SC) before and after surgery..

Western-blotting.

Mouse endothelial cells were isolated from aorta of wild-type and R264A-ERα mice, as previously described [START_REF] Vion | Autophagy is required for endothelial cell alignment and atheroprotection under physiological blood flow[END_REF] . These mouse endothelial cells obtained from primary cultures and fresh tissues from uteri or testis were homogenized on Cell Lysis Buffer (TRis 80mM, pH=8; 5% SDS, 10% Glycerol) using a Precellys 24 (Bertin, France) with beads for the tissues. Total proteins (50 µg for testis and 10µg for uteri, all lysate for murine endothelial cells) were separated on Criterion TGX Stain-Free precast polyacrylamide 4-20% gels (Biorad) and transferred to nitrocellulose membranes using Trans Blot Turbo RTA Transfer Kit Midi 0.2µm Nitrocellulose. The following primary antibodies were used: anti-ER (60C, 04-820, Millipore, culture supernatant, dilution 1/2000) and anti-β-actin (#BA3R, at 0.5 µg/ml, Arigo Biolaboratories) (Supplementary Table 2). Bands were revealed using HRP-conjugated secondary antibodies and visualized through ECL Prime detection, according to the manufacturer's instructions (GE Healthcare), using a ChemiDoc Imaging System (Bio-Rad). Bands were quantified using densitometry in the ImageJ software.

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Statistical analyses. Results are expressed as the mean ± SEM. To test the effect of treatments or genotypes, Mann -Whitney test or unpaired t-test with Welch's correction were performed, as specified. To test the interaction between treatments and genotypes, a 2-way ANOVA was carried out. When an interaction was observed between 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 as statistically significant (*: P<0.05; **: P<0.01; ***: P<0.001).

Results

R264A mice are fertile and present a normal reproductive profile.

To study the potential role of arginine 264 of ERα in the rapid signaling in vivo, a knock-in strategy was employed in which the arginine 264, the murine counterpart of the human arginine 260, was mutated into alanine using a targeting construct containing two base-pair changes in exon 5 (Figure 1A and Supplementary Figures S1A andS1B). The R264A mutation was confirmed by PCR using tail DNA as template (Supplementary Figure S1C). Since the fertility is usually altered in mice mutated for ERα, including the mice mutated specifically for the palmitoylation site [START_REF] Adlanmerini | Mutation of the palmitoylation site of estrogen receptor alpha in vivo reveals tissue-specific roles for membrane versus nuclear actions[END_REF] , the reproductive capacity of R264A mice was first evaluated. The R264A-ERα female mice were found totally fertile when continuously mated with wild type males (0.8 litters/month/female; 6.5 pups/litter) as compared to WT mice (0.6 litters/month/female; 6.5 pups/litter). To further evaluate the ovarian function, estrous cyclicity was then evaluated by daily vaginal cytology (Figure 1B). As illustrated in Figure 1B, R264A-ERα females alternated proestrus, estrus, met/diestrus phases in a similar way than WT mice with regular estrous cycles. Histomorphological analysis of the ovaries showed normal structure, with in particular many corpus luteum and absence of any cystic or hemorrhagic follicles (Figure 1C). Uteri were also normal on their gross appearance and the uterine weights of WT and R264A-ERα mice remained similar (Figures 2A and2B). As an initial characterization of this mouse model, we also evaluated the expression of ERα protein in uterus from littermate wildtype (WT-ERα) and mutant R264A-ERα mice. ERα protein abundance detected by western blot in the uterus was similar in the two groups (Figures 2C-D). Moreover, immunodetection of ERα in uterus clearly detect ERα on uterine epithelial and stromal cells without any difference between nuclear and extra-nuclear staining between the 2 genotypes (Figure 2E). Serum levels of gonadotropin and ovarian sexual hormones were finally measured in intact 10-12 week old female mice (Table 1). All these hormones were present in similar levels in the serum of R264A-ERα mice as compared to their WT littermates.

Altogether, these data demonstrate that the R264A-ERα mice are fertile with no detectable perturbation of the hypothalamic-pituitary-ovary axis and steroidogenesis.

Nuclear actions of R264A-ERα are widely conserved in the uterus

Previous studies performed in vitro in transfected HeLa cells have shown that this specific human counterpart mutant ERα-R260A displays normal capacity to activate gene transcription via direct ERE-mediated binding to DNA as well as indirect, tethered, DNA binding via the transcription factors Sp1. In contrast, AP-1-mediated transcriptional transactivation was not activated by E2 binding to this ERα-R260A [START_REF] Wu | Point mutations in the eralpha galphai binding domain segregate nonnuclear from nuclear receptor function[END_REF] . Furthermore, O'Brien et al. ( 2006) [START_REF] O'brien | Estrogen-induced proliferation of uterine epithelial cells is independent of estrogen receptor alpha binding to classical estrogen response elements[END_REF] suggested that this non-ERE transcriptional response impacted the estrogeninduced proliferation of uterine epithelial cells. We therefore studied the uterotrophic response to E2 by comparing uterine wet weights in ovariectomized female mice 24 hours after a single subcutaneous injection of vehicle or 8µg/kg of E2 . Acute E2 treatment caused a slightly but significantly greater increase in uterine weight in R264A-ERα as compared to WT-ERα mice. Ki-67 labeling was also performed 24h following E2 (Figure 2F). A slightly but significant increased epithelial proliferation was also observed in R264-ERα mice as compared to WT mice (92.9±1.7 for R264A-ERα as compared to 76.9± 7.6 for WT, Inter. P=0.0375; Gen P=0.039; E2 P<0.0001) (Figures 2G-H).

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Despite similar uterine weights observed in intact 3-months old female mice, the differences in uterine weights and epithelial proliferation observed in acute experiments (Figures 2F and2H) led us to extensively evaluate the capacity of R264A-ERα to activate gene transcription in vivo in uteri 6 hours after acute s.c. E2 treatment (8μg/Kg). The E2regulated genes in R264A-ERα and WT mice as compared to placebo is presented in the volcano plot in Figure 3A (P<0.05). Cluster analysis of all of the tested conditions identified two major patterns of gene expression corresponding to genes down-or up-regulated genes by E2 (Figure 3B). The patterns of genes regulated by E2 by the R264A mutant are very similar to control WT littermates treated by E2. Importantly, 15,409 probes are regulated by E2 treatment in WT mice, which is almost identical to the 15,247 E2-regulated genes in R264A mice (absolute Fold change >1.5; P value <0.05). In terms of fully Ensembl annotated genes, the Venn-diagram in Figure 3C indicates that 7,283 genes are commonly regulated genes from totals of 8,102 and 8,039 genes regulated by E2 in WT or R264A mice, respectively. By consequence, only a small number of genes were regulated in an ERα-R264A dependent manner: 756 genes (around 8.5% of total regulated genes) as compared to 819 genes regulated in an ERα-WT dependent manner (9.2% of total regulated genes). However, only 3 probes were significantly differentially regulated by E2 between the 2 genotypes, corresponding to Gpsm2, Il11 and Fam178b (Figure 3B). At a lower stringency, one other gene, Kalrn was also declared as being differentially expressed. Since the number of differentially dysregulated genes by E2 was so low between WT and R264A, we reanalyzed these data by performing the BH correction on P-values after the selection of probes on their expression (Pval2 in the Table provided within Figure 3D). This procedure similarly identified Fam178b and Il-11 as well as Kalrn with a lower stringency. From this total of 6 genes, statistical differences between WT and R264A-ERα gene expression for 4 of these genes, i.e Gpsm2, Nmur2, Kalrn and IL11 were validated by RT-qPCR (Figure 3E). The Gpsm2 (G-protein signaling modulator 2) and Nmur2 (encoding a protein for the Neuromedin U receptor, which is part of a G-protein coupled receptor 1 family) were less expressed in the uteri from E2-treated R264A-ERα mice as compared to WT (fold change -1.5 and 2, respectively). By contrast, the Il11 (interleukin 11) is highly induced in response to E2, and 2-fold more induced in R264A-ERα mice as compared to WT mice, while the Kalrn (encoding a Kalirin RhoGEF Kinase) is more highly induced by E2 in R264A-ERα as compared to the WT.

While AP1-mediated transcriptional activation by E2 has been reported to be deficient 19 in HeLa cells transfected with the human counterpart of R264A (i.e. R260A) , we thus aimed at determining whether this observation could translate into the fact that, despite a very low number of differentially regulated genes between the 2 genotypes. 819 and 756 genes were identified as specifically E2-regulated genes in either WT or R264A mice respectively. We took advantage of the published ERα cistrome (entirety of ER binding events on chromatin) in uterine WT cells [START_REF] Hewitt | Research resource: Whole-genome estrogen receptor alpha binding in mouse uterine tissue revealed by chip-seq[END_REF] to assess whether these WT or R264A-specific E2 regulated genes were at the vicinity or not of these ERBSs (Estrogen Receptor Binding Sites). Similar number of ERBs in a 10 kb window centered at their Transcription start site (TSS) were found in genes regulated in WT or R26AA conditions (Supplementary Figures S2A-C and Table S2) while analysis of the transcription factor motifs included within the ERBSs found at the proximity of these genes indicate that AP1 (ATF2/Jun heterodimers TFs) motifs are absent from the ERBSs located at the vicinity of genes specifically regulated by E2 in R264A-ERα mice only.

Taken together, considering that only 4 genes were differentially regulated by E2 between R264A-ERα and its wild type littermates as compared to the 8,000 genes on average regulated by E2 in both genotypes, we conclude that R264A has minor impact on gene expression regulation in the uterus in response to E2.

Membrane-initiated vascular actions of ERα, such as acute E2-induced dilation are abrogated in R264A-ERα mice.

A c c e p t e d m a n u s c r i p t

Non-genomic actions of estrogens has been found to be particularly implicated in some protective vascular effects of E2, i.e. rapid dilatation and acceleration of re-endothelialization 14-17 . So, we first evaluated whether expression of ERα protein level was conserved in primary culture of endothelial cells of WT and R264-ERα mice. As shown in Figures 4A-B, R264A-ERα protein abundance was similar to WT-ERα. We then evaluated the acute dilation following addition of stepwise dosesE2 (0.01-1 µM) in WT and R264A mesenteric arteries from female mice, after precontraction with phenylephrine. Typical tracings of the results are shown in Figure 4C. We first checked that the KCl-mediated contraction of the arteries and the precontraction level of the arteries before E2-mediated dilation were similar between the two genotypes (Supplementary Figures 3A-B). In female WT mice, E2 induced a concentration-dependent dilation, which was significant at an E2 concentrations of 10 -7 M (20.3± 4.3%) and 10 -6 M (40.7± 3.7%) compared to vehicle-treated WT vessels (8.6± 3.6% and 10.3± 3.6%, respectively) (Figure 4D). A significant effect of E2 was found in the WT-ERα at 10 -7 and 10 -6 (* (P<0.05) and *** (P<0.001) respectively), while E2-induced dilatation was completely absent in R264A-ERα arteries treated with 10 -6 M E2 (13.0 ± 2.2% compared to vehicle: 10.3 ± 2.2%). L-NNA, an inhibitor of NO synthesis, blocked E2-induced dilation of WT arteries (-0.5 ± 3.6%) (Figure 4E), but had any additional inhibitory effect on the unresponsive R264-ERα arteries to E2 (8.1 ± 6.0%). The L-NNA inhibitory effect on the E2mediated dilation of mesenteric arteries suggested that these rapid membrane effects of estrogens are essentially mediated through the rapid stimulation of eNOS-dependent production of NO as previously shown in the human coronary artery [START_REF] Guetta | The role of nitric oxide in coronary vascular effects of estrogen in postmenopausal women[END_REF] .The L-NNA inhibitor effect of e-NOS was also tested on acetylcholine-induced dilation of WT and R264A-ERα mice (Figure 4F) to confirm that the NO signaling is operating properly in the R264A-ERα while it is only altered by the arginine mutation in response to E2. Sex differences in the E2induced acute dilation of mesenteric arteries were then tested, using mesenteric arteries from male mice. Typical tracings and results (Supplementary Figure 4) totally matched results obtained in female mice, with a significant E2-induced acute dilation in male WT arteries (40.3± 4.8 % with E2 10 -6 M) and an absence of dilation in R264A-ERα mice in response to E2 10 -6 M (12.9± 2.6 %) as compared to WT-EtOH and R264-ERα-EtOH (18.3± 9 % and 18.6± 4.1, respectively). Fertility of these male mice was also totally normal, with no impact of the R264A-ERα mutation on the reproductive tissues (Supplementary Figure 5). Previous studies performed in both an immortalized endothelial cell line and in transfected COS cells have shown that the C-domain of human ERα (aa251-260) was required to interact with Gαi and be involved in rapid NO production [START_REF] Wyckoff | Plasma membrane estrogen receptors are coupled to endothelial nitric-oxide synthase through galpha(i)[END_REF] . To further investigate the importance of R264A-ERα in this interaction and in this E2-acute induced dilation, mesenteric arteries from female WT-ERα mice were pretreated with pertussis toxin (PTX) for 120 min and further stimulated with E2. Figure 4G indicates that this pre-treatment with PTX totally abrogated the rapid E2-induced dilation, revealing the important role of R264A-ERα in the interaction with G proteins.

Membrane-initiated vascular actions of ERα, such as acceleration of reendothelialization after electric carotid injury are abrogated in R264A-ERα mice.

The role of R264 was then investigated in vivo by evaluating re-endothelialization of the carotid artery after electric injury in response to E2, an effect previously demonstrated to be exclusive membrane ERα effects [START_REF] Adlanmerini | Mutation of the palmitoylation site of estrogen receptor alpha in vivo reveals tissue-specific roles for membrane versus nuclear actions[END_REF][START_REF] Chambliss | Non-nuclear estrogen receptor alpha signaling promotes cardiovascular protection but not uterine or breast cancer growth in mice[END_REF][START_REF] Arnal | Membrane and nuclear estrogen receptor alpha actions: From tissue specificity to medical implications[END_REF] (Figure 5A). As observed on the photographs of the Evans Blue staining and on the corresponding quantifications of re-endothelialization, E2 treatment caused 2-fold increase in endothelial repair in WT mice (37.6 ± 3.3 % and 19.4 ± 2.7 % in WT mice treated or not with E2, respectively), whereas it had no effect in R264A-ERα mice (21.1 ± 1.9 % as compared to 17.7 ± 2.4 %). We previously demonstrated that this E2 action required ERα in both the endothelial and hematopoietic cells [START_REF] Toutain | Estrogen receptor alpha expression in both endothelium and hematopoietic cells is required for the accelerative effect of estradiol on reendothelialization[END_REF] . To further explore the role of hematopoietic cells ERα in this process, WT mice were irradiated and grafted with either WT or R264A-ERα bone marrow (Figure 5B). The E2 effect was still present in WT mice grafted whatever the genotype, indicating that the non-genomic actions of ERα in the bone marrow are not required for the accelerative effect of E2, suggesting a role for the non-A c c e p t e d m a n u s c r i p t genomic effects of ERα in the vasculature rather than in the hematopoietic cells for the acceleration of re-endothelialization.

Altogether these results demonstrate that arginine 264 is crucial for the membrane initiated endothelial effects of ERα, namely vascular dilatation and acceleration of reendothelialization in response to E2.

Effect of E2 on atheroma and flow-mediated remodeling in R264A-ERα mice.

Estrogens have numerous well-known protective vascular effects, besides their effects on re-endothelialization and arterial remodeling. E2 is, in particular, involved in the prevention of atheroma and in flow-mediated remodeling that are known to involved nuclear actions of estrogens [START_REF] Guivarc'h E | Predominant role of nuclear versus membrane estrogen receptor alpha in arterial protection: Implications for estrogen receptor alpha modulation in cardiovascular prevention/safety[END_REF] . We first analyzed the nuclear response of E2 in aorta, by comparing gene expression profile in the aorta from R264A-ERα with their littermate WT controls, 6 hours after acute E2 administration. As illustrated in Figure 6A, all genes tested were similarly regulated in both genotypes as revealed by absence of interaction between genotype and treatment after 2-way ANOVA analysis for all the genes analyzed, neither for up-regulated genes (Grem2, Gatnl2, Myc, Cam2kb, Tiparp) nor the down-regulated ones (Shisha2). We then investigated the impact of the R264A-ERα mutation on arteries submitted to an atherogenic stress. Our previous works demonstrated that atheroprotection is entirely dependent on nuclear ERα 45 . To this aim, R264A-ERα mice were bred with LDLr -/-mice and fed an hypercholesterolemic diet for 12 weeks. E2 treatment similarly decreased fatty streak deposits at the aortic sinus of both LDLr -/-and R264A-ERα/LDLr -/-mice (Figures 6B and6C). We also assessed the role of R264A-ERα in the lipid profile (Table 2). As previously described [START_REF] Billon-Gales | The transactivating function 1 of estrogen receptor alpha is dispensable for the vasculoprotective actions of 17beta-estradiol[END_REF] , both plasma cholesterol and HDL cholesterol fractions were decreased by E2 and these effects were similarly observed in R264A-ERα mice. As expected, ovariectomy led to uterine atrophy. Exogenous E2 replacement induced uterine hypertrophy in both R264A-ERα/LDLr -/-and their littermate controls, with body weights being similar whatever the genotype (Table 2).

Finally, we investigated flow-mediated remodeling of resistance arteries from R264A-ERα mice and their wild type littermates. Two weeks after adjacent arterial ligation in vivo, arterial diameter was determined in vitro in response to stepwise increases in intraluminal pressure (Figures 6D and6E). The increase in passive arterial diameter in response to increases in pressure was significant and similar in high-flow arteries from WT mice, and from R264-ERα mice.

Altogether, these results demonstrated that R264 is not necessary to mediate these two vascular effects of E2, recognized as being dependent on nuclear, but not MISS, ERα actions [START_REF] Guivarc'h E | Predominant role of nuclear versus membrane estrogen receptor alpha in arterial protection: Implications for estrogen receptor alpha modulation in cardiovascular prevention/safety[END_REF] , segregating the nuclear/membrane vascular effects of E2.

Discussion

In the present study, we used the R264A-ERα mice to determine whether the point mutation of arginine 264 into alanine into murine ERα affects the physiological functions of this steroid receptor in vivo. Our findings reveal that the amino acid R264 is not crucial neither for female or male fertilities nor for ERα-regulated gene expression regulation in aorta and uterus. In contrast, R264 is selectively required for the vascular membrane initiated actions of ERα. Indeed, R264A mice presented an absence of E2-induced acceleration of reendothelialization and rapid NO-induced vasodilatation while it is dispensable for prevention of atherosclerosis and arterial remodeling. Thus, R264A mouse is the first model without any endocrine and reproductive alteration that genetically segregates nuclear and membrane effects of ERα in the vasculature.

We have first determined the reproductive phenotype of female R264A mice, looking at the estrous cycle, the ovarian function and quantifying the sexual hormone levels. R264A-ERα mice are totally fertile with regular estrous cycle profiles. The ovarian function is entirely preserved, as attested by the presence of corpus luteum which confirmed that ovulation has occurred. LH serum levels, which are typically affected in all previous ERα mutants, are A c c e p t e d m a n u s c r i p t normal in R264A-ERα mice. Moreover, circulating ovarian hormones levels such as estradiol, progesterone and testosterone are completely normal as compared to WT mice, suggesting the absence of any deregulation of ERα functions in steroidogenesis and hypothalamicpituitary-axis (HPO). This result is at first glance surprising, because all ERα mutant mice generated so far are infertile, including the previous mouse models C451A and NOER, mutated on the palmitoylation site C451 of ERα that abrogated the membrane effects of estrogens [START_REF] Hamilton | Estrogen hormone physiology: Reproductive findings from estrogen receptor mutant mice[END_REF] . Indeed, the C451A mice presented polycystic and hemorrhagic ovaries and an increase in LH serum levels, clearly demonstrating the role of membrane ERα in fertility [START_REF] Adlanmerini | Mutation of the palmitoylation site of estrogen receptor alpha in vivo reveals tissue-specific roles for membrane versus nuclear actions[END_REF] . Nevertheless, only another mouse model reported as impaired for membrane ERα signaling remained fertile: the DPM mouse model, in which membrane ERα signaling is impeded by overexpression of a peptide that prevents ERα interaction with striatin [START_REF] Moens | Rapid estrogen receptor signaling is essential for the protective effects of estrogen against vascular injury[END_REF] . Striatin directly binds to aa176-253 of ERα and serves as a scaffold protein to form and stabilize ERα caveolar complex with eNOS [START_REF] Lu | Striatin assembles a membrane signaling complex necessary for rapid, nongenomic activation of endothelial no synthase by estrogen receptor alpha[END_REF] . Overexpression of disrupted peptide corresponding to aa176-253 blocked ERα/Striatin interaction and prevented E2-induced MAPK, Akt and eNOS activation, but did not abolished ERE-dependent transcriptional activity in vitro [START_REF] Lu | Striatin assembles a membrane signaling complex necessary for rapid, nongenomic activation of endothelial no synthase by estrogen receptor alpha[END_REF] . Altogether C451A, DPM and R264A-ERα mice reveal the complexity of membrane ERα sub-signaling in the control of female fertility. Importantly, since deletion of aa250-274 has been 49 demonstrated to do not disturb plasma membrane localization of ERα contrary to the C451A mutation [START_REF] Acconcia | Palmitoylation-dependent estrogen receptor alpha membrane localization: Regulation by 17betaestradiol[END_REF][START_REF] Adlanmerini | Mutation of the palmitoylation site of estrogen receptor alpha in vivo reveals tissue-specific roles for membrane versus nuclear actions[END_REF] , this discrepancy could explain their respective fertility profiles. R264A-ERα mice clearly demonstrate that the R264 residue and thus, as discussed below, ERα methylation and/or ERα/ Gα i interaction, are not crucial for the phenotype of female and male fertility.

We explored the transcriptional regulation ability of the mutated R264A-ERα. Previous in vitro studies have shown that R264A mutation did not alter ERE-dependent transcriptional regulation, but affected the indirect AP1-dependent regulation of transcription . Using EDC to activate MISS effects or interrogating the mutant ERα-C451A in vitro, membrane initiated actions of estrogens were also reported to secondary modulate the nuclear transcriptional response in vitro [START_REF] Rosa | Palmitoylation regulates 17beta-estradiolinduced estrogen receptor-alpha degradation and transcriptional activity[END_REF][START_REF] Madak-Erdogan | Nuclear and extranuclear pathway inputs in the regulation of global gene expression by estrogen receptors[END_REF] . The large scale analysis performed on R264A uteri provided a broad overview of the E2 effect on gene transcription. Surprisingly, almost all gene transcriptional regulation by E2 is conserved in R264A-ERα mice as compared to WT mice. Even though E2 is a master regulator of gene transcription on the uterus (8800 differentially expressed genes), the R264A mutation has a minor impact on gene regulation (6 genes) as compared to the C451A mutation. Among these genes, it is interesting to note that the Gspm2 which encodes a G protein signaling modulator is down-regulated by E2 in R264A-ERα as compared to WT. This protein is also called LGN (Leu-Gly-Asn repeat enriched protein) and it has been shown to modulate the activity of the NO receptor soluble guanylate cyclase [START_REF] Chauhan | The g-protein regulator lgn modulates the activity of the no receptor soluble guanylate cyclase[END_REF] , a major actor of the downstream NO signaling pathway. Among the 3 other validated genes by RT-qPCR, one is the Il-11, the interleukin-11, reflecting a potential impact on immunity while the Kalrn encodes a Kalirin RhoGEF Kinase. It is interesting to note that even though rare genes are found to be dysregulated by E2 between WT an R264A-ERα, two of these genes encodes signaling proteins (Gpsm2 and Kalrn), probably pointing the importance of R264 on the rapid signaling pathway of ERα. As already mentioned in luciferase reporters assays in Hela cells [START_REF] Wu | Wound healing effects of porcine placental extracts on rats with thermal injury[END_REF] , our motif analysis further indicated that AP1 binding sites may be absent from promoters and putative ER binding regions controlling the activity of the genes specific for the R264A-ERα mouse uteri. Whether this observation is actually true could be stressed by determining the cistrome of AP1 in both mouse genotypes. Physiologically, the mutation R264A induces a slight but significant increase in uterine weight and epithelial proliferation following acute E2 treatment without any impact on uterine weights in intact mice. This transient increase in uterine weight at 24 hours might be due to edema induced by impacted vascular effects of E2 [START_REF] Rockwell | Inhibition of vascular endothelial growth factor/vascular permeability factor action blocks estrogen-induced uterine edema and implantation in rodents[END_REF] . But, this small increase in proliferation could have consequences in tumor cells, since the R264 motif has been shown to be involved in activation of Akt in breast cancers [START_REF] Poulard | Activation of rapid oestrogen signalling in aggressive human breast cancers[END_REF] . However, the functional importance of this hypothetical change in the balance of ERα activity through indirect/direct binding in uteri caused by the R264A seems at the end very low in this tissue in physiological conditions, as most transcriptional regulations are unchanged.

A c c e p t e d m a n u s c r i p t

In contrast to the uterus where 8800 genes are found regulated by E2, a large microarray analysis performed on the aorta of DPM mice has demonstrated that only 200 genes in average are regulated by E2 on WT mice following 4 hours treatment [START_REF] Moens | Rapid estrogen receptor signaling is essential for the protective effects of estrogen against vascular injury[END_REF] . However, in DPM mice, the transcriptional response to E2 was greatly altered. These results suggested that ERα /Striatin interaction and thus membrane ERα signaling plays a major role in vascular gene regulatory response to estrogen. Nevertheless, the disrupting strategy by overexpression of blocking peptide may induce off-target effects, as indicated by authors, especially on ERβ. In this present work, the R264A mutation does not significantly altered the gene transcriptional response on any of the tested genes. Altogether, these results demonstrated that the R264A mutation does not alter significantly the transcriptional activity of ERα neither in uterus nor in aorta.

Nevertheless, in contrast to this preservation of gene expression by E2 in aorta from R264A-ERα mice, the membrane-initiated vascular actions of ERα are abrogated in R264A-ERα mice. Contrary to WT mice, R264A-ERα mice do not show any accelerative effects of E2 on re-endothelialization of carotid after electric injury. This E2 vascular effect is undoubtly the most characterized in vivo model of membrane ERα signaling of estrogens. Indeed, EDC (Estrogen Dendrimer conjugate), which only activates membrane ER, is sufficient to induce acceleration of re-endothelialization [START_REF] Chambliss | Non-nuclear estrogen receptor alpha signaling promotes cardiovascular protection but not uterine or breast cancer growth in mice[END_REF] . Furthermore, we recently demonstrated that this effect is lost in membrane C451A-ERα mutant mice, whereas it is conserved in both ERα mutant mice mutated for either the AF2 or the AF1 transactivation function [START_REF] Adlanmerini | Mutation of the palmitoylation site of estrogen receptor alpha in vivo reveals tissue-specific roles for membrane versus nuclear actions[END_REF] . In addition, the female R264A-ERα mice lost the E2-dependent vasodilatory response in mesenteric arteries, as previously observed in C451A-ERα mice. No sex difference was observed in this E2 vascular effect, since this rapid E2-induced dilation was also observed in WT males, but also lost in male R264A-ERα mice. Interestingly, this E2-induced acute dilation in mesenteric arteries was completely abrogated by an NO synthesis inhibitor (L-NNA), highlighting the crucial role of eNOS activity and NO production in these membrane initiated vascular effects of E2. This is in agreement with previous works by our group [START_REF] Guivarc'h E | Predominant role of nuclear versus membrane estrogen receptor alpha in arterial protection: Implications for estrogen receptor alpha modulation in cardiovascular prevention/safety[END_REF] and others (review of the literature in [START_REF] Arnal | Membrane and nuclear estrogen receptor alpha actions: From tissue specificity to medical implications[END_REF] ). In contrast, previous data [START_REF] Billon | The estrogen effects on endothelial repair and mitogen-activated protein kinase activation are abolished in endothelial nitric-oxide (no) synthase knockout mice, but not by no synthase inhibition by n-nitro-l-arginine methyl ester[END_REF] have demonstrated that the presence of eNOS but not its enzymatic activity is determinant for estrogen signaling in the the endothelium for re-endothelialization. These studies concur to underline that, according to the model and the vascular function explored, membrane ERα may have different interactions and relationships involving either the eNOS activity or the presence of eNOS protein as a scaffolder. Our present data further demonstrate no involvement of R264-ERa signaling in the E2 atheroprotective effect and vascular remodeling even though this mutation abrogates NOdependent rapid dilation. This result is consistent with the literature as the E2-induced 54 atheroprotective effect was reported to be conserved after NOS inhibition and in hypercholesterolemic eNOS -/-mice 55 even though the eNOS-KO mice have been reported to exhibit accelerated atherosclerosis. Altogether, these results highlight the complexity of E2/ERα/eNOS pathways depending of the experimental model used and the engagement of membrane versus nuclear ERα.

Therefore, it is clear that R264-ERα is crucial for two membrane-initiated vascular effects of E2, namely eNOS acute activation during E2-mediated dilation of mesenteric arteries and acceleration of endothelial healing.while it is dispensable for the prevention of atherosclerosis and arterial remodeling, known to be mediated by nuclear/genomic actions of E2, extending previous observations done with C451A-ERα mice, impeded for ERα membrane location [START_REF] Guivarc'h E | Predominant role of nuclear versus membrane estrogen receptor alpha in arterial protection: Implications for estrogen receptor alpha modulation in cardiovascular prevention/safety[END_REF] . More recently, G protein-coupled estrogen receptor 1 (GPER) has been proposed as a third Estrogen receptor present at the membrane and been reported to mediate some beneficial cardiovascular effects of estrogens, as endothelium-dependent and nitric oxide-dependent effects of E2 and prevention of atherosclerosis [START_REF] Fredette | Role of gper in estrogen-dependent nitric oxide formation and vasodilation[END_REF][START_REF] Barton | Emerging roles of gper in diabetes and atherosclerosis[END_REF] . GPER could concur to the protective action of E2, but nuclear ERα is absolutely required as this E2 action if lost in the AF2°-ERα mice [START_REF] Billon-Gales | Activation function 2 (af2) of estrogen receptor-alpha is required for the atheroprotective action of estradiol but not to accelerate endothelial healing[END_REF] .

Finally, a question remains open concerning the respective roles of ERα methylation and ERα/Gα i interaction in the observed R264A phenotype. Indeed, R260 have been studied from two different points of view. On one hand, R260 has been seen as a methylation site A c c e p t e d m a n u s c r i p t necessary for ERα/Src/PI3K complex formation in response to E2 that mediates the Akt induced proliferation of the epithelium [START_REF] Romancer | Regulation of estrogen rapid signaling through arginine methylation by prmt1[END_REF] . On the other hand, Shaul and coll. have shown that R260 is also part of a domain interacting with Gα i and this ERα/Gαi interaction is further enhanced by E2 and necessary for eNOS activation [START_REF] Chambliss | Non-nuclear estrogen receptor alpha signaling promotes cardiovascular protection but not uterine or breast cancer growth in mice[END_REF][START_REF] Romancer | Regulation of estrogen rapid signaling through arginine methylation by prmt1[END_REF][START_REF] Kumar | Direct interactions with g alpha i and g betagamma mediate nongenomic signaling by estrogen receptor alpha[END_REF] . The in vivo results on the microarray analysis revealed the down-regulation of a G protein modulator that is in agreement with the interaction with the Gαi protein. Moreover, pretreatment in vitro of mesenteric arteries with pertussis toxin abrogated the acute E2-induced dilation, highlighting the involvement of protein G activation in this E2-induced vasodilatory action which is lost in R264A-ERα mice. Wu and his collaborators [START_REF] Wu | Point mutations in the eralpha galphai binding domain segregate nonnuclear from nuclear receptor function[END_REF] have previously shown that the aspartic acid D258A-ERα was unable to associate with Gi, preventing eNOS activation by E2. We further demonstrate here that the arginine 264 residue (counterpart of 260 in human ERα) is necessary and sufficient to mediate eNOS activation by E2, likely through interaction with G protein.

A question remains open: is methylation of ERα in response to E2 involved in the conformational change of ERα implicated in the ERα /Gαi interaction? In that case, methylation of ERα could be a requirement for Gαi interaction that will further activate the downstream signaling. However, this model and kinetics of interaction are out of reach and beyond the scope of in vivo studies, in particular as this methylated form appeared very transient. In consequent, in vitro studies in appropriate models should be done to further understand the molecular mechanism(s) involved.

In conclusion, steroid receptors are capable to elicit rapid, nongenomic actions, but mouse models which allow to study the physiological role of these original actions so far called nuclear receptor remains limited to estrogen receptor. Indeed, after the palmitoylable C451 residue, the arginine 264 represents the second major amino acid absolutely necessary for membrane ERα signaling in the vasculature. In contrast, the transcriptional gene regulation by E2 is highly conserved in aorta and uterus of these mice, demonstrating that R264 is essentially dispensable for these gene expression changes and supporting the idea that MISS and nuclear actions are independent in the arteries. Finally, these findings support and extent the conclusions previously established with AF2 0 -ERα and C451A-ERα mice [START_REF] Guivarc'h E | Predominant role of nuclear versus membrane estrogen receptor alpha in arterial protection: Implications for estrogen receptor alpha modulation in cardiovascular prevention/safety[END_REF] , and will help to further delineate the medical potential of the selective activation/inhibition of MISS and nuclear ERα actions. The staff of the animal facilities (in particular P. Espagnolle and G. Carcassès) and of the "Plateforme d'expérimentation fonctionnelle" is acknowledged for their skillful technical assistance. We also thank J. Guillermet and B. Masri for helpful discussions. We are also grateful to M. Garcia. and C. Proux (Angers) for their technical helps for experiments in E2acute dilation, I. Bleuart and I. Pardo for providing excellent technical support and advice regarding the histology (ENVT). We are also grateful to Y. Lippi for its excellent contribution to the microarray analysis performed at the GeT-TQ Genopole Toulouse Facility and to Isabelle Bleuart and Isabelle Pardo for providing excellent technical support and advice regarding the histology (ENVT).

Sources of Funding

This work was also supported by the INSERM, the Université de Toulouse III, the Fondation pour la Recherche Médicale), The Région Midi-Pyrénées and FEDER (Fonds européen de développement régional (REGEN-EVE), the ANR (Agence Nationale de la Recherche, Estroshear, contract # ANR-18-CE14-0016-01), the CNRS, the University of Rennes1. M.A. was supported by grant from the Ministere de la Recherche. C.F was supported by ANR-18-CE14-0016-01.

Disclosures:

Nothing to disclose All data and supporting materials have been provided with the published article Uteri from WT mice was loaded as positive control and stain free staining of the membrane after transfer was performed as loading control of total protein. MW: Molecular weight. B)Quantitative analysis of expression level of ERα in primary endothelial cells at passage P0 from aorta from WT and R264A-ERα mice , expressed as a ratio to the total loaded protein as detected using stain free of the membrane (n=3 for each genotype). C) Typical tracings obtained for dilation of mesenteric arteries cannulated in vitro under a pressure of 75 mmHg) in a arteriograph from WT and R264A-ERα female mice following increased concentration of E2 (0.01 to 1µM) diluted in ethanol (EtOH, 1/10000, 1/10000 and 1/1000 respectively), added to the bath and after precontraction with phenylephrine D) Percentages of dilation of mesenteric arteries from female WT and R264A-ERα mice following addition of E2 (0.01 to 1µM )-Results of 2-way Anova was shown, explaining the effect of both variables (Treat.= E2 treatment ; Gen.=Genotype) and their interaction (Inter.)) . As an interaction was observed between the 2 factors (P<0.0001), Bonferroni post-tests were then performed. A significant effect of E2 was found in the WT-ERα at 10-7 and 10 -6 (* (P<0.05) and *** (P<0.001) respectively), but not in R264A-ERα. A significant difference was also found between WT-E2 with R264A-ERα-E2 at 10-7 and 10 -6 ($$ (P<0.01) and $$$ (P<0.001) respectively). E) Percentages of E2-mediated dilation of mesenteric arteries from female WT and R264A-ERα mice previously incubated with L-NNA (100 µM) during 30 min. These results were compared to the results obtained for ERα-WT-E2 and R264A-ERα-E2, shown in panel D. A 2-way Anova was performed between these 4 groups and an interaction was observed between the 2 factors (P inter : *** ). The effect of treatment then was studied in each genotype using Bonferroni post-test. WT-E2 versus WT-LNNA-E2 at 10-

D) Schematic representation of the protocol used to induce the flow-mediated remodeling of mesenteric arteries in vivo (see Materials and Methods

). Following ligation of some 2 nd order mesenteric arteries downstream the 1 st order mesenteric arteries, 1 st order arteries with ligation are submitted chronically to high flow (HF) and compared to the 1 st order mesenteric arteries without ligation (normal flow, NF) E) Flow-mediated remodeling was then evaluated in mesenteric arteries isolated from WT and R264A mice in response to stepwise increases in pressure over 2 weeks (n= 4 for WT-ERα and 5 for R264A-ERα). Arterial diameter was measured ex vivo in response to stepwise increases in pressure in mesenteric arteries initially submitted chronically to high flow (HF) or normal flow (NF). ). As the 2-way Anova revealed an interaction between E2 treatment and Genotype (Interaction: ****, P<0.001), Bonferroni post-test was performed. A significant effect of E2 was found in the WT-ERα (****, P<0.001). A significant effect in A c c e p t e d m a n u s c r i p t remodeling was found between NF and HF in both WT and R264A-ERα mice at 50 and 75 mmHg (* and $, P<0.05) and at 100 and 125 mmHg (** , P<0.01 and $, P<0.05, respectively) . No significant difference was found between HF-WT and HF-R264A-ERα and NF-WT and NF-R264A-ERα .

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 19

Figure 1 :

 1 Figure legends:

Figure 2 :

 2 Figure 2: Uterine weight is maintained in R264A-ERα mice while acute proliferative response to E2 is increased at 24 hours.A) Representative images of intact uteri from WT and R264A-ERα mice B) Uterine wet weight (mean ± SEM, n=8 for each genotype). Unpaired t-test with Welch's correction was used. ns: not significant between WT-ERα versus R264-ERα mice C) Expression of ERα in uteri from WT and R264A-ERα mice, as detected by Western blot using anti-ERα antibody (60C) and β-actin as a loading control. D) Relative expression of ERα in uteri of WT and R264A-ERα to β-actin as loading control. (n=6 for each genotype). Unpaired t-test with Welch's correction was used. ns: not significant between WT-ERα versus R264-ERα mice E) ERα immunodetection in stromal and epithelial compartments from transverse uterus sections in WT and R264A-ERα mice with higher magnification on the middle panels. Scale bar= 50µm. F) Uterine wet weights in ovariectomized WT and R264A-ERα mice injected s.c with vehicle (Veh, castor oil) or 17β-estradiol (E2, 8μg/kg) and euthanized 24 hours after treatment. The number of mice are mentionned on the botton of the bar. Two-way ANOVA followed by Bonferroni post-test was performed (Interaction: *) -A significant effect of E2 was found between WT and R264A-ERα (*, P<0.05). G) Representative Ki-67 immmunodetection. H) Percentages of Ki-67 positive cells in epithelium 24 hours after s.c E2 injection in uteri of R264A-ERα and WT mice as compared to placebo treated mice. The number of mice are mentionned on the botton of the bar. Two-way ANOVA followed by Bonferroni post-test was performed (Interaction: *) -A significant effect of E2 was found between WT and R264A-ERα (**, P<0.01).

Figure 3 :

 3 Figure 3: Large scale analysis on response to E2 in the uterus of R264A-ERα mice as compared to its control littermates. Gene expression profiles were obtained by microarray

Figure 4 :

 4 Figure 4: Rapid vascular effects of E2 such as rapid dilation are lost with the mutation R264A of ERα in female mice.A) Expression of ERα in primary endothelial cells (ECs) at passage PO isolated from aorta of WT and R264A mice, as detected by Western blot using anti-ERα antibody (60C) . Uteri from WT mice was loaded as positive control and stain free staining of the membrane after transfer was performed as loading control of total protein. MW: Molecular weight. B)Quantitative analysis of expression level of ERα in primary endothelial cells at passage P0 from aorta from WT and R264A-ERα mice , expressed as a ratio to the total loaded protein as detected using stain free of the membrane (n=3 for each genotype). C) Typical tracings obtained for dilation of mesenteric arteries cannulated in vitro under a pressure of 75 mmHg) in a arteriograph from WT and R264A-ERα female mice following increased concentration of E2 (0.01 to 1µM) diluted in ethanol (EtOH, 1/10000, 1/10000 and 1/1000 respectively), added to the bath and after precontraction with phenylephrine D) Percentages of dilation of mesenteric arteries from female WT and R264A-ERα mice following addition of E2 (0.01 to 1µM )-Results of 2-way Anova was shown, explaining the effect of both variables (Treat.= E2 treatment ; Gen.=Genotype) and their interaction (Inter.)) . As an interaction was observed between the 2 factors (P<0.0001), Bonferroni post-tests were then performed. A significant effect of E2 was found in the WT-ERα at 10-7 and 10 -6 (* (P<0.05) and *** (P<0.001) respectively), but not in R264A-ERα. A significant difference was also found between WT-E2 with R264A-ERα-E2 at 10-7 and 10 -6 ($$ (P<0.01) and $$$ (P<0.001) respectively). E) Percentages of E2-mediated dilation of mesenteric arteries from female WT and R264A-ERα mice previously incubated with L-NNA (100 µM) during 30 min. These results were compared to the results obtained for ERα-WT-E2 and R264A-ERα-E2, shown in panel D. A 2-way Anova was performed between these 4 groups and an interaction was observed between the 2 factors (P inter : *** ). The effect of treatment then was studied in each genotype using Bonferroni post-test. WT-E2 versus WT-LNNA-E2 at 10-7 and 10-6 were significantly different (**, P<0.01) and (***, P<0.001) respectively). F) Percentages of acetylcholine (1 µM)-mediated dilation of mesenteric resistance arteries in vitro from female WT-ERα or R264A-ERα mice measured in the presence and

  Figure 4: Rapid vascular effects of E2 such as rapid dilation are lost with the mutation R264A of ERα in female mice.A) Expression of ERα in primary endothelial cells (ECs) at passage PO isolated from aorta of WT and R264A mice, as detected by Western blot using anti-ERα antibody (60C) . Uteri from WT mice was loaded as positive control and stain free staining of the membrane after transfer was performed as loading control of total protein. MW: Molecular weight. B)Quantitative analysis of expression level of ERα in primary endothelial cells at passage P0 from aorta from WT and R264A-ERα mice , expressed as a ratio to the total loaded protein as detected using stain free of the membrane (n=3 for each genotype). C) Typical tracings obtained for dilation of mesenteric arteries cannulated in vitro under a pressure of 75 mmHg) in a arteriograph from WT and R264A-ERα female mice following increased concentration of E2 (0.01 to 1µM) diluted in ethanol (EtOH, 1/10000, 1/10000 and 1/1000 respectively), added to the bath and after precontraction with phenylephrine D) Percentages of dilation of mesenteric arteries from female WT and R264A-ERα mice following addition of E2 (0.01 to 1µM )-Results of 2-way Anova was shown, explaining the effect of both variables (Treat.= E2 treatment ; Gen.=Genotype) and their interaction (Inter.)) . As an interaction was observed between the 2 factors (P<0.0001), Bonferroni post-tests were then performed. A significant effect of E2 was found in the WT-ERα at 10-7 and 10 -6 (* (P<0.05) and *** (P<0.001) respectively), but not in R264A-ERα. A significant difference was also found between WT-E2 with R264A-ERα-E2 at 10-7 and 10 -6 ($$ (P<0.01) and $$$ (P<0.001) respectively). E) Percentages of E2-mediated dilation of mesenteric arteries from female WT and R264A-ERα mice previously incubated with L-NNA (100 µM) during 30 min. These results were compared to the results obtained for ERα-WT-E2 and R264A-ERα-E2, shown in panel D. A 2-way Anova was performed between these 4 groups and an interaction was observed between the 2 factors (P inter : *** ). The effect of treatment then was studied in each genotype using Bonferroni post-test. WT-E2 versus WT-LNNA-E2 at 10-7 and 10-6 were significantly different (**, P<0.01) and (***, P<0.001) respectively). F) Percentages of acetylcholine (1 µM)-mediated dilation of mesenteric resistance arteries in vitro from female WT-ERα or R264A-ERα mice measured in the presence andA c c e p t e d m a n u s c r i p t

Figure 5 :

 5 Figure 5: E2-induced acceleration of reendothelialization are lost with the mutation R264A of ERα in female mice. A) Carotid artery reendothelialization in ovariectomized WT and R264-ERα mice treated or not with E2 (80 μg/Kg/d, 2 wk). Representative photographs of blue evans staining of reendothelialization are shown (right panel) and corresponding quantification (left panel). (n=9 and 10 for WT-PLB and WT-E2, respectively and n=7 and 8, for R264-ERα PLB and E2, respectively). As the 2-way Anova revealed an interaction between E2 treatment and genotype (Interaction: *), Bonferroni post-test was performed. A significant effect of E2 was found in the WT-ERα (***, P<0.001).B)Carotid artery reendothelialization in ovariectomized WT reconstituted with hematopoietic cells from WT or R264-ERα mice treated or not with E2 (80 μg/Kg/d, 2 wk). (n=6 and 7 for WT-PLB and WT-E2, respectively and n=7 and 5, for R264-ERα PLB and E2, respectively). (2-way Anova: interaction (Inter.) E2 treatment (Treat); Genotype (Gen).

Figure 6 :

 6 Figure 6: R264A-ERα female mice conserve nuclear protective vascular actions of E2 in aorta preventing atherosclerosis in LDLR -/-mice and maintained arterial remodeling in mesenteric arteries. A) Normalized relative mRNA levels in aorta from ovariectomized WT and R264A mice treated acutely (6h) with E2 (8 μg/kg) or with vehicle (Veh). Statistical analyses (2-way Anova) were performed on Cq (log2) data. The P values for the 2 main effects and their interaction are given in the box. The data shown are geometrical means of relative quantities (2 -Cq +/-linearized log2-based SEM) n=6/group. B) Representative images and C) quantification of lipid deposition at the aortic sinus in WT LDLR-/-and R264-ERα LDLR-/-mice after red oil staining. (n=9 and 6 for WT-PLB and WT-E2, respectively and n=15 and 9, for R264-ERα PLB and E2, respectively). Results of 2way anova was shown.D) Schematic representation of the protocol used to induce the flow-mediated remodeling of mesenteric arteries in vivo (see Materials and Methods). Following ligation of some 2 nd order mesenteric arteries downstream the 1 st order mesenteric arteries, 1 st order arteries with ligation are submitted chronically to high flow (HF) and compared to the 1 st order mesenteric arteries without ligation (normal flow, NF) E) Flow-mediated remodeling was then evaluated in mesenteric arteries isolated from WT and R264A mice in response to stepwise increases in pressure over 2 weeks (n= 4 for WT-ERα and 5 for R264A-ERα). Arterial diameter was measured ex vivo in response to stepwise increases in pressure in mesenteric arteries initially submitted chronically to high flow (HF) or normal flow (NF). ). As the 2-way Anova revealed an interaction between E2 treatment and Genotype (Interaction: ****, P<0.001), Bonferroni post-test was performed. A significant effect of E2 was found in the WT-ERα (****, P<0.001). A significant effect in

Table 1 :

 1 Serum levels of Estradiol, Testosterone and Progesterone (n=18) and Luteinezing hormone (LH) and Follicle-Stimulating Hormone (FSH) in WT and R264A-ERα mice. LH and FSH measurements were performed in estrus phase (n=8). ns: nonsignificantly different from WT (Mann Whitney test was used and no significant difference was found between genotypes)..

Table 2 :

 2 Body and uterine weights with plasmatic lipid (total cholesterol and HDL cholesterol) profiles and Oil Red O area (ORO) area in representative sections of aortic sinus of WT LDLR -/-and R264A-ERα LDLR -/-mice fed for 12 weeks with a hypercholesterolemic-atherogenic diet (mean± SEM, n=X). Results of 2-way Anova between the genotype and treatment are indicated on the right column of the Table.

  A c c e p t e d m a n u s c r i p t Highlights:  Mutation of Arginine 264 of Estrogen receptor ERα abrogated acceleration of reendothelialization and rapid arteriolar remodeling in response to E2  In contrast, vasculoprotective effects of E2 such as prevention of atheroma or arterial remodeling, known to be mediated by nuclear actions of ERα are conserved  R264A-ERα mouse is the first fertile mouse model harboring a single mutation of ERα with specific loss-of function of rapid ERα signaling in vascular functions.

  Figure1

PG r e m 2 R

 2 Figure 2

Acknowledgments


Xia-Qing Shi

Giordano Fausti

Hugues Chaté

Cesare Nardini

Alexandre Solon

Self-Organized Critical Coexistence Phase in Repulsive Active Particles

We revisit motility-induced phase separation in two models of active particles interacting by pairwise repulsion. We show that the resulting dense phase contains gas bubbles distributed algebraically up to a typically large cutoff scale. At large enough system size and/or global density, all the gas may be contained inside the bubbles, at which point the system is microphase-separated with a finite cut-off bubble scale. We observe that the ordering is anomalous, with different dynamics for the coarsening of the dense phase and of the gas bubbles. This phenomenology is reproduced by a "reduced bubble model" that implements the basic idea of reverse Ostwald ripening put forward in Tjhung et al. [Phys. Rev. X 8, 031080 (2018)].

Self-propelled particles interacting solely with steric repulsion are well known to be able to spontaneously separate into a macroscopic dense cluster and a residual gas, in spite of the absence of explicit attraction forces. This motility-induced phase separation (MIPS) [START_REF] Cates | Motility-Induced Phase Separation[END_REF] of active particles has become a cornerstone of the physics of dry active matter (in which the fluid surrounding particles is neglected). As such, it has driven many theoretical works [START_REF] Tailleur | Statistical mechanics of interacting run-and-tumble bacteria[END_REF][START_REF] Stenhammar | Continuum theory of phase separation kinetics for active brownian particles[END_REF][START_REF] Wittkowski | Scalar phi4 field theory for active-particle phase separation[END_REF][START_REF] Solon | Pressure and Phase Equilibria in Interacting Active Brownian Spheres[END_REF][START_REF] Solon | Generalized thermodynamics of phase equilibria in scalar active matter[END_REF][START_REF] Tjhung | Cluster phases and bubbly phase separation in active fluids: Reversal of the ostwald process[END_REF] as well as countless numerical studies (see, e.g. [START_REF] Fily | Athermal phase separation of self-propelled particles with no alignment[END_REF][START_REF] Redner | Structure and dynamics of a phase-separating active colloidal fluid[END_REF][START_REF] Speck | Effective Cahn-Hilliard equation for the phase separation of active Brownian particles[END_REF][START_REF] Stenhammar | Phase behaviour of active Brownian particles: the role of dimensionality[END_REF][START_REF] Digregorio | Full phase diagram of active Brownian disks: From melting to motilityinduced phase separation[END_REF][START_REF] Klamser | Thermodynamic phases in two-dimensional active matter[END_REF][START_REF] Mandal | Motilityinduced temperature difference in coexisting phases[END_REF][START_REF] Caprini | Spontaneous velocity alignment in motility-induced phase separation[END_REF] to name a few prominent ones). The motility reduction resulting from persistent collisions, which leads to MIPS, is a generic ingredient encountered both in living and synthetic active matter [START_REF] Liu | Self-Driven Phase Transitions Drive Myxococcus xanthus Fruiting Body Formation[END_REF][START_REF] Buttinoni | Dynamical clustering and phase separation in suspensions of self-propelled colloidal particles[END_REF][START_REF] Geyer | Freezing a flock: Motility-induced phase separation in polar active liquids[END_REF][START_REF] Van Der Linden | Interrupted motility induced phase separation in aligning active colloids[END_REF].

Despite its purely non-equilibrium origin, MIPS was initially described as a conventional phase separation between two homogeneous macroscopic phases. It was first predicted in models of quorum-sensing particles [START_REF] Tailleur | Statistical mechanics of interacting run-and-tumble bacteria[END_REF] where particle speed decreases with the local density, without two-body interactions. In this case, equilibrium-like thermodynamics can be constructed to account quantitatively for phase coexistence [START_REF] Solon | Generalized thermodynamics of motilityinduced phase separation: phase equilibria, Laplace pressure, and change of ensembles[END_REF]. For systems of repulsive disks, attempts were made to model the speed reduction due to collisions by a quorum-sensing interaction [START_REF] Stenhammar | Continuum theory of phase separation kinetics for active brownian particles[END_REF][START_REF] Fily | Athermal phase separation of self-propelled particles with no alignment[END_REF][START_REF] Speck | Effective Cahn-Hilliard equation for the phase separation of active Brownian particles[END_REF], but the results are not satisfactory, due to fundamental differences between the two cases [START_REF] Solon | Pressure and Phase Equilibria in Interacting Active Brownian Spheres[END_REF][START_REF] Solon | Pressure is not a state function for generic active fluids[END_REF].

There is indeed mounting evidence that more complex physics is at play in systems of repulsive disks. In particular, the surface tension between the dense phase and the gas, defined via the Laplace law, has been measured to be negative [START_REF] Solon | Generalized thermodynamics of motilityinduced phase separation: phase equilibria, Laplace pressure, and change of ensembles[END_REF][START_REF] Bialké | Negative interfacial tension in phase-separated active brownian particles[END_REF][START_REF] Patch | Curvature-dependent tension and tangential flows at the interface of motility-induced phases[END_REF], triggering a spate of controversy [24]. This was rationalized at field theoretical level by including terms that break detailed balance in the classical theory for equilibrium liquid-gas phase separation. In this active Model B+ (AMB+), surface tension can become negative for some parameter values, in which case Ostwald ripening is reversed for vapor bubbles while still remaining normal for liquid droplets. This means that small vapor bubbles, contrary to the standard scenario, grow at the expense of larger ones due to a diffusion flux.

When this happens, simulations of AMB+ lead to either a bubbly fluid interpreted as microphase separation, or to the coexistence of a dense phase populated of bubbles with an outer gas [START_REF] Tjhung | Cluster phases and bubbly phase separation in active fluids: Reversal of the ostwald process[END_REF]. There is in fact incidental evidence for such a bubbly liquid at particle level [START_REF] Redner | Structure and dynamics of a phase-separating active colloidal fluid[END_REF][START_REF] Stenhammar | Phase behaviour of active Brownian particles: the role of dimensionality[END_REF][START_REF] Digregorio | Full phase diagram of active Brownian disks: From melting to motilityinduced phase separation[END_REF][START_REF] Solon | Generalized thermodynamics of motilityinduced phase separation: phase equilibria, Laplace pressure, and change of ensembles[END_REF][START_REF] Bialké | Negative interfacial tension in phase-separated active brownian particles[END_REF], but it has not yet been studied per se. Very recently, Caporusso et al [START_REF] Caporusso | Micro and macro motilityinduced phase separation in a two-dimensional active brownian particle system[END_REF] have shown more clearly that in systems with hard-core interactions, the dense phase is made of hexatic subdomains and interstitial gas regions.

In this Letter we show, within two standard particle models displaying MIPS, that not only the dense phase is endowed with bubbles, but also that these are distributed algebraically up to some cutoff scale that we observe to grow with system size. Finite-size scaling based on this observation suggests that, as system size increases, more and more of the gas is contained in bubbles. At large densities, we are able to observe the vanishing of the macroscopic gas reservoir, and the system is then microphase-separated with bubbles of all sizes up to a maximal bubble size that depends on the average density. Moreover, the coarsening of bubbles is anomalous with the typical length scale growing as t 0.22 . We elucidate the basic mechanisms at play, and show, within a reduced model implementing reversed Ostwald ripening for gas bubbles, that they indeed lead to a self-organized critical dynamics.

Self-organized critical phase coexistence. We first consider the paradigmatic active brownian particles (ABPs) introduced in [START_REF] Fily | Athermal phase separation of self-propelled particles with no alignment[END_REF]. Self-propelled by a force of constant magnitude F 0 along its internal polarity u i = (cos θ i , sin θ i ), particle i evolves according to the overdamped Langevin equations governing its position r i and polar angle θ i :

ṙi = µ i (F 0 u i + F i ) + η i ; θi = η i (1)
where F i =j =i ∇V (r ir j ) is the force exerted on particle i by the other particles. We choose the pair potential to be a short-range harmonic repulsion repulsive strength and σ = 1 the interaction radius. In contrast to previous studies, we allow the mobility tensor µ i and the translational noise η i to be anisotropic, as expected generically for active particles:

V (r) = k 2 (σ -r) 2 if
µ i = µ u i u i + µ ⊥ (I -u i u i ), η i = 2τ µ ξ i u i + √ 2τ µ ⊥ ξ ⊥ i (u i × z)
, and η i = √ 2τ µ θ ξ θ i with τ a parameter controlling the noise strength, z the unit vector perpendicular to the plane of motion, and the ξ i 's Gaussian white noises with unit variance.

All simulations below are of large two-dimensional domains with periodic boundary conditions. Numerical details are given in [26]. At phase coexistence, we observe, inside the macroscopic dense domain, persistent bubbles with a range of sizes, surrounded by a liquid. Bubbles are more prominent when the mobility is anisotropic [27]. Typical snapshots for µ = 4µ ⊥ and µ θ = 6µ ⊥ are shown in Fig. 1(a,b) for systems only differing by their size. Clearly, doubling system size increases the size of the bubbles. Fig. 1(c) shows, at different system sizes, n(a), the average number of bubbles of area a, normalized by S , the total area of the liquid in which bubbles live. The distributions collapse on an increasing range, span several orders of magnitude, and decay approximately as a power law n(a) ∼ a -α with α ≈ 1.75 terminated by a cutoff that increases with system size.

The ABP simulations reported above only show a rather short scaling range. Numerically, the main limitation is not so much system size than the huge times needed to obtain clean averages [28]. We thus implemented an active lattice gas [START_REF] Thompson | Lattice models of nonequilibrium bacterial dynamics[END_REF][START_REF] Soto | Self-assembly of catalytically active colloidal molecules: Tailoring activity through surface chemistry[END_REF][START_REF] Sepúlveda | Wetting transitions displayed by persistent active particles[END_REF][START_REF] Whitelam | Phase separation and large deviations of lattice active matter[END_REF][START_REF] Partridge | Critical motility-induced phase separation belongs to the Ising universality class[END_REF]: on an hexagonal lattice [START_REF] Partridge | Critical motility-induced phase separation belongs to the Ising universality class[END_REF], particles carrying an internal polarity pointing to one of the 6 lattice directions attempt to perform one of 3 moves (see [26] for details). (i) With a rate r P they perform a 'self-propelled' jump to the nearest site along their internal polarity direction. (ii) They undergo spatial diffusion to any neighboring site at rate r D , and (iii) rotational diffusion (changing their polarity to one of its two neighboring orientations) at rate r R . For optimal efficiency, we impose strict exclusion and parallel updating.

In the following, we use r P = 1, r D = 2 and r R = 0.032, typical values leading to phase separation (a study of the phase diagram will be presented elsewhere). Persistent bubbles are clearly visible (Fig. 2(a-d)). The bubble area distribution in the globally phase-separated regime is similar to that observed for ABPs but with a much larger scaling region (Fig. 2(e)): n(a) ∝ a -α with α = 1.75 [START_REF] Solon | Pressure and Phase Equilibria in Interacting Active Brownian Spheres[END_REF]. This region extends to a cut-off size a c that grows with the total liquid area as a c ∝ S γ with γ = 1.40 [START_REF] Solon | Pressure and Phase Equilibria in Interacting Active Brownian Spheres[END_REF]. The distributions can thus be collapsed on a master curve using these two exponents (Fig. 2(f)).

In both models presented, we find that whenever the system is globally phase separated, the dense phase contains bubbles. This phase bears the hallmarks of selforganized criticality (SOC) (for recent overviews, see [START_REF] Pruessner | Self-Organised Criticality[END_REF][START_REF] Buendía | Feedback mechanisms for self-organization to the edge of a phase transition[END_REF]). Small bubbles are nucleated inside the liquid, diffuse and grow by merging with other bubbles. This process get slower and slower with increasing bubble size. Bubbles are eventually expelled into the reservoir of outside gas upon touching the boundary, in sudden, avalanche-like events, providing separation of timescales (see movie in [26]). As in typical SOC systems, avalanches occur at all accessible scales.

The SOC-like mechanisms leading to an algebraic distribution of bubbles do not invalidate the global picture of a phase separation between gas and liquid with fixed densities ρ g and ρ independent of system size up to small finite-size corrections. However, the gaseous part of the system is formed here of the outside gas reservoir and of the bubbles. With this definition, the gas fraction x g fluctuates very little and is independent of system size to a good approximation (Fig. 3(a)). Moreover, x g varies linearly with the average density ρ 0 (Fig. 3(b)) so that the lever rule still applies: For ρ g < ρ 0 < ρ , the average density sets the fraction of liquid and gas in the system x g = (ρ -ρ 0 )/(ρ -ρ g ) and x = 1-x g . The fluctuations of x b , the fraction of the system occupied by bubbles, in contrast to the gentle ones of x g , are large, intermittent, and increase with system size. (Note the huge timescales over which fluctuations of x b occur even at the modest sizes shown in Fig. 3(a).) Their stronger and stronger peaks reflect the larger and larger avalanches (expulsion of bubbles) (Fig. 3

(a), insets).

Microphase-separated bubbly liquid. The lever rule immediately tells us that the SOC scaling evidenced above cannot continue asymptotically when system size S → ∞. Indeed, the bubble area fraction grows with system size:

x b ≡ 1 S ∞ 0 a n(a)da ≈ S S ac 0 a 1-α da ∝ x S γ(2-α) , (2)
where we used the scalings of n(a) and a c (S ). Our numerical data confirm this (Fig. 3(c)). Surely, Eq. ( 2)

384

× 256 ceases to be possible once all the gas is contained in the bubbles, x b = x g , which happens at a typical crossover size S * ∝ (x g /x ) 1/[γ(2-α)] /x . The cutoff on bubble size then reads

a * c ≡ a c (S * ) ∝ (x g /x ) 1/(2-α) . (3)
Equation ( 3) implies that S * and a * c depend on the average density ρ 0 through x g and x and that they diverge near the gas binodal ρ 0 → ρ g . On the other hand they get smaller when approaching the liquid binodal. Beyond S * the system settles in a micro-phase separated state, a homogeneous liquid with bubbles of all sizes up to a * c , SOC scaling then breaks down, and n(a) becomes independent of system size.

Using our lattice gas model at high enough ρ 0 , we are able to reach system sizes where all the gas is contained in bubbles, and the system settles in the micro-phase separated state (Fig. 2). Our data are in agreement with our scaling arguments: n(a) is then independent of system size and is cut off at some scale that depends only on the average density (Fig. 2(g)). Plotting n(a/a * c ) collapses the distributions for different ρ 0 , confirming the validity of Eq. (3) (Fig. 2(h)), at least close to the liquid binodal.

Reduced bubble model. The AMB+ field theory of Ref. [START_REF] Tjhung | Cluster phases and bubbly phase separation in active fluids: Reversal of the ostwald process[END_REF] suggests that bubbles exist because of reverse Ostwald ripening, which causes large bubbles to shrink at the advantage of small ones, thus competing with coalescence. To test whether these ingredients are sufficient to reproduce the phenomenology described above, we implemented them in a reduced model whose degrees of freedom are the positions and radii of bubbles that we assume to be perfectly circular [START_REF]For a similar approach in a different context[END_REF] The bubble-particles evolve in continuous time in a continuous domain. New bubbles with radius r 0 = 1 are nucleated in the liquid at a small rate k n per unit area. In line with the reverse Ostwald scenario, the new bubbles are nucleated at the expense of the larger ones: all other bubbles shrink by an amount κr(1r 0 /r) (where r is their current radius), with κ chosen such that the total area of gas is conserved. (Note that this neglects spatial effects: In principle, bubbles would equilibrate in priority with neighboring ones.) Bubbles chosen randomly among the current n(t) existing ones diffuse with a coefficient D, that for simplicity we assume constant. If the move brings the bubble into contact with another, they merge into a single one located at their "center of mass", conserving total area. To have the same geometry as in globally phase-separated microscopic models, we also add the possibility to have a gas reservoir outside two parallel interfaces that move along the dynamics to insure that x remains constant. While a complete presentation of the behavior of this reduced model and some variants will be reported elsewhere, here we show that it typically yields a phenomenology remarkably similar to that described above. Fixing D = 1 and k n = 10 -4 , we vary x g the total gas fraction and the system size S. At high x g or small system size S, we observe the SOC coexistence between a bubbly liquid and a gas reservoir (Fig. 4(a,b)). The bubble size distribution scales as in Fig. 2(f) with exponent values close to those of the lattice gas (α = 1.77 [START_REF] Tailleur | Statistical mechanics of interacting run-and-tumble bacteria[END_REF] and γ = 1.48(5)), but preliminary results (not shown) suggest that they are not universal. Decreasing x g or increasing S, the gas reservoir becomes smaller and smaller until it disappears, at which point we have a microphase separated regime with n(a) independent of system size (Fig. 4(c,d)). Despite its simplicity, our bubble model thus captures the essential phenomenology described here.

Coarsening process. We finally study the growth of order following random initial conditions, considering the characteristic length extracted from the structure factor [26]. We only present results for our active lattice gas (Fig. 5), but similar ones, albeit of lesser quality, were obtained for ABPs. When the liquid is the majority phase, after an initial transient, the coarsening is dominated by vapor bubbles and follows an anomalous t 0.22 law. We currently lack an analytical explanation of such law. Instead, when the liquid is the minority phase, it is dominated by liquid droplets and coarsening is normal t 1/3 as expected both from Ostwald ripening and coalescence in models with conserved order parameter [START_REF] Bray | Theory of phase-ordering kinetics[END_REF].

In fact, within the liquid droplets we expect the bubbles to coarsen as well with the anomalous law above. However, being slower than the liquid coarsening, it is not surprising that this is not visible in our data.

Conclusion.

We have shown, using two very different models of active particles interacting strictly by pairwise repulsion, that the dense phase resulting from MIPS is critical, containing bubbles of gas distributed algebraically up to some cutoff scale. We observe at high density that, as long as an outer gas phase is present, this cutoff increases as a power of system size. At large enough system size and/or global density, the gas reservoir may disappear and the cutoff scale becomes independent of system size. This asymptotic regime is thus microphase separated. A "reduced bubble model" captures this essential phenomenology within a minimal framework that implements the basic idea of reverse Ostwald ripening put forward in Ref. [START_REF] Tjhung | Cluster phases and bubbly phase separation in active fluids: Reversal of the ostwald process[END_REF].

In the models presented here, the asymptotic cutoff scale a * c grows very fast with ρ 0 . Numerically, we are only able to access the asymptotic regime at rather high density (Fig. 2). What happens asymptotically at low densities thus remains unknown, but extrapolating the scaling laws uncovered here lead us to speculate that our scenario remains valid in the whole phase coexistence region ρ g < ρ 0 < ρ . This Letter leaves several important open questions. In particular, whether the scenario described here is observed whenever pairwise repulsion is present, and whether the critical exponents are universal could be addressed numerically by considering other models showing MIPS, the AMB+ field theory, and reduced bubble models with different parameters. In this context, the very recent work of Caporusso et al [START_REF] Caporusso | Micro and macro motilityinduced phase separation in a two-dimensional active brownian particle system[END_REF], where a very hard potential between ABPs leads to crystalline clus-ters that aggregate to form a dense phase with interstitial gas, might be understood within our scenario. Finally, in regards to the current controversy about the nature of the critical point of MIPS [START_REF] Partridge | Critical motility-induced phase separation belongs to the Ising universality class[END_REF][START_REF] Siebert | Critical behavior of active Brownian particles[END_REF], our results make it unlikely that it belongs to the Ising universality class, but this hard problem remains thus unsettled.

FIG. 1 .

 1 FIG. 1. Active Brownian particles. Typical snapshots in steady state at system size S = 1440 × 360 (a) and S = 2880 × 720 (b). (Colors represent the packing fraction calculated over 2 × 2 boxes, from 0 (dark blue) to 1 (red).) (c): Rescaled distribution of bubble area n(a)/S at various system sizes (indicated in legends). Parameters: ρ0 = N π 4S = 0.6, τ = 0.01, µ ⊥ = 1/4, µ = 1, µ θ = 3/2, F0 = 1 and k = 20. Typical averaging time is 4 × 10 6 after a transient of 10 6 .

FIG. 2 .FIG. 3 .

 23 FIG. 2. Active lattice gas. (a-d) Snapshots in the steady state at ρ0 = 0.8 in systems of different sizes from S = 384 × 256 to S = 1532 × 1024 (colors as in Fig. 1(a,b)). For the two biggest sizes (c,d), the system is in the microphase-separated regime. (e-h) bubble area distribution in the SOC scaling regime (e,f: ρ0 = 0.6) and in the microphase-separated regime (g,h). (e) n(a)/S at various system sizes (indicated by the legends in (f)). Typical averaging time is 10 10 timesteps after discarding a transient of 10 8 . (f): same as (e), but as function of a/S γ with γ = 1.40. (g): n(a)/S at different ρ0 values for S = 768 × 1024 (dashed lines) and S = 1536 × 2048 (solid lines). (h): same as (g), but rescaled according to Eq. (3). The grey dashed lines have slope -α = -1.75.

FIG. 4 .

 4 FIG. 4. Reduced bubble model. (a,c): Typical snapshots at xg = 0.7 in the SOC coexistence regime (a) and at xg = 0.05 in the microphase regime (c). System size S = 600×400. The liquid and gas are represented in red and black respectively. (b,d) Bubble area distribution in the two regimes, rescaled using α = 1.77, γ = 1.48. For all panels D = 1, kn = 10 -4 .

FIG. 5 .

 5 FIG. 5. Coarsening process in the active lattice gas (S = 8192×6144). (a) Timeseries of the typical lengthscale L starting from random initial conditions at different ρ0 values. (b,c) Snapshots of the system taken at t = 10 7 for ρ0 = 0.5 (b) and 0.7 (c). At all times shown here, L √ S.

We thank M.E. Cates, A. Patelli and J. Tailleur for interesting discussions related to this work. This work is supported by the National Natural Science Foundation of China (Grant No. 11635002 to X.-q.S. and H.C., Grants No. 11922506 and No. 11674236 to X.-q.S.). C.N. acknowledges the support of an Aide Investissements d'Avenir du LabEx PALM (ANR-10-LABX-0039-PALM).