Découplage de l’activation nucléaire et membranaire du ERα dans la protection artérielle et métabolique: implication clinique pour un nouveau traitement substitutif de la ménopause

Les femmes occidentales passent maintenant plus d’un tiers de leur vie ménopausées. Le déclin de la production d’œstrogènes endogènes au cours de cette période s’accompagne de troubles fonctionnels qui affectent la qualité de vie. La ménopause est également associée à un risque de fractures élevé, ainsi qu’à une augmentation des événements cardiovasculaires et des troubles métaboliques. Ces désordres fonctionnels peuvent être limités par l’administration d’un traitement hormonal de la ménopause (THM) à base d’œstrogènes. Cependant ces traitements ont des effets délétères, comme l’augmentation du risque de thrombose veineuse et de cancer du sein. L’amélioration du THM représente donc un enjeu majeur pour la santé des femmes. Une des stratégies d’optimisation du THM repose sur la modulation du récepteur alpha des œstrogènes (ERα) qui appartient à la famille des récepteurs nucléaires. En plus de son action classique comme facteur de transcription, il peut également agir à la membrane en induisant des voies de signalisation cytoplasmique. Le découplage des effets membranaire/nucléaire pourrait améliorer la balance bénéfice/risque dans l’action de ce récepteur. Le but de mon projet a été d’évaluer l’impact de ce découplage dans le développement des maladies cardiovasculaires et métaboliques. En combinant des modèles d’animaux invalidés pour les fonctions membranaires ou nucléaires de ERα et des approches pharmacologiques, ce travail a permis de montrer que la majorité des effets protecteurs des œstrogènes sur le plan vasculaire étaient relayés par le ERα nucléaire, avec un rôle minime des effets membranaires. Dans ce contexte, nous avons pu mettre en évidence que l’Estétrol (E4), un œstrogène naturel et endogène ciblant spécifiquement le ERα nucléaire, était capable de prévenir l’obésité en réponse à un régime gras chez la souris ovariectomisée et les désordres vasculaires et métaboliques qui lui sont associés. Cette étude s’inscrit dans une démarche plus globale de caractérisation des effets de l’Estétrol puisque cette molécule est développée en clinique comme nouveau THM. Contrairement aux œstrogènes utilisés actuellement, l’E4 n’induit pas les facteurs de la coagulation hépatique aux doses utilisées, ce qui pourrait limiter le risque thrombotique. Mes résultats montrent un effet bénéfique à la fois sur le plan vasculaire et métabolique de cet œstrogène. Une meilleure compréhension des mécanismes cellulaires et moléculaires impliqués dans ces effets pourrait permettre de positionner l’E4 comme le premier œstrogène naturel agissant comme un modulateur sélectif du ERα

Vers une optimisation de la modulation du récepteur des œstrogènes dans le traitement hormonal de la ménopause

Les femmes vivent désormais plus d’un tiers de leur vie après la survenue de la ménopause. Le déclin de la production d’œstrogènes endogènes au cours de cette période s’accompagne fréquemment de troubles fonctionnels qui affectent la qualité de vie. Ces symptômes peuvent être soulagés par un traitement hormonal (THM) initialement fondé sur l’administration d’œstrogènes conjugués équins (principalement aux États-Unis, par voie orale) ou d’un œstrogène naturel, le 17β-estradiol (en Europe, notamment par voie transdermique). Le récepteur des œstrogènes α (REα) relaye la majorité des effets physiologiques des œstrogènes. REα appartient à la superfamille des récepteurs nucléaires. Il régule la transcription de gènes via ses fonctions activatrices (AF1 et AF2). Outre ces actions génomiques classiques, les œstrogènes peuvent aussi activer une sous-population de récepteurs REα présents à la membrane des cellules et ainsi induire des signaux rapides. Dans cette revue, nous résumerons l’évolution des THM depuis les débuts de la substitution hormonale jusqu’aux nouvelles molécules émergentes fondées sur une modulation sélective du REα. Nous décrirons également les progrès récents sur la compréhension des mécanismes d’action des œstrogènes, en détaillant les rôles respectifs des REα nucléaire et membranaire et les développements thérapeutiques possibles qui pourraient en découler

The AF-1 activation function of estrogen receptor α is necessary and sufficient for uterine epithelial cell proliferation in vivo.

International audienceEstrogen receptor-α (ERα) regulates gene transcription through the 2 activation functions (AFs) AF-1 and AF-2. The crucial role of ERαAF-2 was previously demonstrated for endometrial proliferative action of 17β-estradiol (E2). Here, we investigated the role of ERαAF-1 in the regulation of gene transcription and cell proliferation in the uterus. We show that acute treatment with E2 or tamoxifen, which selectively activates ERαAF-1, similarly regulate the expression of a uterine set of estrogen-dependent genes as well as epithelial cell proliferation in the uterus of wild-type mice. These effects were abrogated in mice lacking ERαAF-1 (ERαAF-1(0)). Four weeks of E2 treatment led to uterine hypertrophy and sustained luminal epithelial and stromal cell proliferation in wild-type mice, but not in ERαAF-1(0) mice. However, ERαAF-1(0) mice still presented a moderate uterine hypertrophy essentially due to a stromal edema, potentially due to the persistence of Vegf-a induction. Epithelial apoptosis is largely decreased in these ERαAF-1(0) uteri, and response to progesterone is also altered. Finally, E2-induced proliferation of an ERα-positive epithelial cancer cell line was also inhibited by overexpression of an inducible ERα isoform lacking AF-1. Altogether, these data highlight the crucial role of ERαAF-1 in the E2-induced proliferative response in vitro and in vivo. Because ERαAF-1 was previously reported to be dispensable for several E2 extrareproductive protective effects, an optimal ERα modulation could be obtained using molecules activating ERα with a minimal ERαAF-1 action

Estetrol prevents western diet-induced obesity and atheroma independently of hepatic estrogen receptor (ER)α

International audienceEstetrol (E4), a natural estrogen synthesized by the human fetal liver, is currently evaluated in phase III clinical studies as a new menopause hormone therapy. Indeed, E4 significantly improves vasomotor and genito-urinary menopausal symptoms and prevents bone demineralization. Compared to other estrogens, E4 was found to have limited effects on coagulation factors in the liver of women allowing to expect less thrombotic events. To fully delineate its clinical potential, the aim of this study was to assess the effect of E4 on metabolic disorders. Here, we studied the pathophysiological consequences of a western diet (42% kcal fat, 0.2% cholesterol) in ovariectomized female mice under chronic E4 treatment. We showed that E4 reduces body weight gain and improves glucose tolerance in both C57Bl/6 and LDLR(-/-) mice. To evaluate the role of hepatic ERα in the preventive effect of E4 against obesity and associated disorders such as atherosclerosis and steatosis, mice harbouring a hepatocyte-specific ERα deletion (LERKO) were crossed with LDLR(-/-) mice. Our results demonstrated that, whereas liver ERα is dispensable for the E4 beneficial actions on obesity and atheroma, it is necessary to prevent steatosis in mice. Overall, these findings suggest that E4 could prevent metabolic, hepatic and vascular disorders occurring at menopause, extending the potential medical interest of this natural estrogen as a new hormonal treatment

The tissue-specific effects of different 17β-estradiol doses reveal the key sensitizing role of AF1 domain in ERα activity

International audience17β-Estradiol (E2) action can be mediated by the full-length estrogen receptor alpha (ERα66), and also by the AF1 domain-deficient ERα (ERα46) isoform, but their respective sensitivity to E2 is essentially unknown. We first performed a dose response study using subcutaneous home-made pellets mimicking either metestrus, proestrus or a pharmacological doses of E2, which resulted in plasma concentrations around 3, 30 and 600 pM, respectively. Analysis of the uterus, vagina and bone after chronic exposure to E2 demonstrated dose-dependent effects, with a maximal response reached at the proestrus-dose in wild type mice expressing mainly ERα66. In contrast, in transgenic mice harbouring only an ERα deleted in AF1, these effects of E2 were either strongly shifted rightward (10-100-fold) and/or attenuated, depending on the tissue studied. Finally, experiments in different cell lines transfected with ERα66 or ERα46 also delineated varying profiles of ERα AF1 sensitivity to E2. Altogether, this work emphasizes the importance of dose in the tissue-specific actions of E2 and demonstrates the key sensitizing role of AF1 in ERα activity

The different natural estrogens promote endothelial healing through distinct cell targets

International audienceThe main estrogen, 17β-estradiol (E2), exerts several beneficial vascular actions through estrogen receptor α (ERα) in endothelial cells. However, the impact of other natural estrogens such as estriol (E3) and estetrol (E4) on arteries remains poorly described. In the present study, we report the effects of E3 and E4 on endothelial healing after carotid artery injuries in vivo. After endovascular injury, which preserves smooth muscle cells (SMCs), E2, E3, and E4 equally stimulated reendothelialization. By contrast, only E2 and E3 accelerated endothelial healing after perivascular injury that destroys both endothelial cells and SMCs, suggesting an important role of this latter cell type in E4’s action, which was confirmed using Cre/lox mice inactivating ERα in SMCs. In addition, E4 mediated its effects independently of ERα membrane-initiated signaling, in contrast with E2. Consistently, RNA sequencing analysis revealed that transcriptomic and cellular signatures in response to E4 profoundly differed from those of E2. Thus, whereas acceleration of endothelial healing by estrogens had been viewed as entirely dependent on endothelial ERα, these results highlight the very specific pharmacological profile of the natural estrogen E4, revealing the importance of dialogue between SMCs and endothelial cells in its arterial protection

Effect of chronic estradiol plus progesterone treatment on experimental arterial and venous thrombosis in mouse

<div><p>Postmenopausal hormone replacement therapy (HRT) with estrogen plus progestogens is the first line therapy to treat menopausal symptoms. The progestogen is added to estrogen to reduce or eliminate the excess risk of endometrial cancer due to the unopposed effect of estrogen. Whereas progestin clearly opposes the proliferative and deleterious long-term actions of estrogen on the endometrium, the interference of progestin on the other estrogen action remains unclear. We previously reported that chronic subcutaneous 17α-estradiol (E2) in mice decreases platelet responsiveness, prolongs the tail-bleeding time and protects against acute thromboembolism. Here, we report the tissue-specific interference of progesterone (P4) on the action of E2 in ovariectomized mice. We first confirm that, in our experimental conditions, P4 attenuates the proliferative action of E2 on the uterus and the effects of E2 on vagina weight and lubrication. We then studied the effect of E2 combined with P4 on hemostasis and thrombosis <i>in vivo</i> in mice and found that P4 did not interfere with the main actions of E2 on platelets, bleeding time and arterial and venous thrombosis. Thus, whereas activation of progesterone receptor interferes with the action of E2 on its classic sex targets, P4 appears to have minimal effect on the hemostasis and thrombosis actions of E2, supporting the prominent role of estrogens and the accessory role of natural progestin on the extra-reproductive cells and tissues involved in thrombosis.</p></div

Selective Liver Estrogen Receptor Modulation Prevents Steatosis, Diabetes, and Obesity Through the Anorectic Growth Differentiation Factor 15 Hepatokine in Mice

International audienceHepatocyte estrogen receptor α (ERα) was recently recognized as a relevant molecular target for nonalcoholic fatty liver disease (NAFLD) prevention. The present study defined to what extent hepatocyte ERα could be involved in preserving metabolic homeostasis in response to a full (17β-estradiol [E2]) or selective (selective estrogen receptor modulator [SERM]) activation. Ovariectomized mice harboring a hepatocyte-specific deletion ( mice) and their wild-type (WT) littermates were fed a high-fat diet (HFD) and concomitantly treated with E2, tamoxifen (TAM; the most used SERM), or vehicle. As expected, both E2 and TAM prevented all HFD-induced metabolic disorders in WT mice, and their protective effects against steatosis were abolished in mice. However, while E2 still prevented obesity and glucose intolerance in mice, hepatocyte deletion also abrogated TAM-mediated control of food intake as well as its beneficial actions on adiposity, insulin sensitivity, and glucose homeostasis, suggesting a whole-body protective role for liver-derived circulating factors. Moreover, unlike E2, TAM induced a rise in plasma concentration of the anorectic hepatokine growth differentiation factor 15 (Gdf15) through a transcriptional mechanism dependent on hepatocyte ERα activation. Accordingly, ERα was associated with specific binding sites in the regulatory region in hepatocytes from TAM-treated mice but not under E2 treatment due to specific epigenetic modifications. Finally, all the protective effects of TAM were abolished in HFD-fed knockout mice. We identified the selective modulation of hepatocyte ERα as a pharmacologic strategy to induce sufficient anorectic hepatokine Gdf15 to prevent experimental obesity, type 2 diabetes, and NAFLD

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

International audienceObjective - ERα (estrogen receptor alpha) exerts nuclear genomic actions and also rapid membrane-initiated steroid signaling. The mutation of the cysteine 451 into alanine in vivo has recently revealed the key role of this ERα palmitoylation site on some vasculoprotective actions of 17β-estradiol (E2) and fertility. Here, we studied the in vivo role of the arginine 260 of ERα which has also been described to be involved in its E2-induced rapid signaling with PI-3K (phosphoinositide 3-kinase) as well as G protein in cultured cell lines. Approach and Results: We generated a mouse model harboring a point mutation of the murine counterpart of this arginine into alanine (R264A-ERα). In contrast to the , the females are fertile with standard hormonal serum levels and normal control of hypothalamus-pituitary ovarian axis. Although R264A-ERα protein abundance was normal, the well-described membrane ERα-dependent actions of estradiol, such as the rapid dilation of mesenteric arteries and the acceleration of endothelial repair of carotid, were abrogated in mice. In striking contrast, E2-regulated gene expression was highly preserved in the uterus and the aorta, revealing intact nuclear/genomic actions in response to E2. Consistently, 2 recognized nuclear ERα-dependent actions of E2, namely atheroma prevention and flow-mediated arterial remodeling were totally preserved. Conclusions - These data underline the exquisite role of arginine 264 of ERα for endothelial membrane-initiated steroid signaling effects of E2 but not for nuclear/genomic actions. This provides the first model of fertile mouse with no overt endocrine abnormalities with specific loss-of-function of rapid ERα signaling in vascular functions