Membrane estrogen receptor-α contributes to female protection against high-fat diet-induced metabolic disorders

BackgroundEstrogen Receptor α (ERα) is a significant modulator of energy balance and lipid/glucose metabolisms. Beyond the classical nuclear actions of the receptor, rapid activation of intracellular signaling pathways is mediated by a sub-fraction of ERα localized to the plasma membrane, known as Membrane Initiated Steroid Signaling (MISS). However, whether membrane ERα is involved in the protective metabolic actions of endogenous estrogens in conditions of nutritional challenge, and thus contributes to sex differences in the susceptibility to metabolic diseases, remains to be clarified.MethodsMale and female C451A-ERα mice, harboring a point mutation which results in the abolition of membrane localization and MISS-related effects of the receptor, and their wild-type littermates (WT-ERα) were maintained on a normal chow diet (NCD) or fed a high-fat diet (HFD). Body weight gain, body composition and glucose tolerance were monitored. Insulin sensitivity and energy balance regulation were further investigated in HFD-fed female mice.ResultsC451A-ERα genotype had no influence on body weight gain, adipose tissue accumulation and glucose tolerance in NCD-fed mice of both sexes followed up to 7 months of age, nor male mice fed a HFD for 12 weeks. In contrast, compared to WT-ERα littermates, HFD-fed C451A-ERα female mice exhibited: 1) accelerated fat mass accumulation, liver steatosis and impaired glucose tolerance; 2) whole-body insulin resistance, assessed by hyperinsulinemic-euglycemic clamps, and altered insulin-induced signaling in skeletal muscle and liver; 3) significant decrease in energy expenditure associated with histological and functional abnormalities of brown adipose tissue and a defect in thermogenesis regulation in response to cold exposure.ConclusionBesides the well-characterized role of ERα nuclear actions, membrane-initiated ERα extra-nuclear signaling contributes to female, but not to male, protection against HFD-induced obesity and associated metabolic disorders in mouse

Reprogramming of endothelial gene expression by tamoxifen inhibits angiogenesis and ERα-negative tumor growth.

peer reviewedRationale: 17β-estradiol (E2) can directly promote the growth of ERα-negative cancer cells through activation of endothelial ERα in the tumor microenvironment, thereby increasing a normalized tumor angiogenesis. ERα acts as a transcription factor through its nuclear transcriptional AF-1 and AF-2 transactivation functions, but membrane ERα plays also an important role in endothelium. The present study aims to decipher the respective roles of these two pathways in ERα-negative tumor growth. Moreover, we delineate the actions of tamoxifen, a Selective Estrogen Receptor Modulator (SERM) in ERα-negative tumors growth and angiogenesis, since we recently demonstrated that tamoxifen impacts vasculature functions through complex modulation of ERα activity. Methods: ERα-negative B16K1 cancer cells were grafted into immunocompetent mice mutated for ERα-subfunctions and tumor growths were analyzed in these different models in response to E2 and/or tamoxifen treatment. Furthermore, RNA sequencings were analyzed in endothelial cells in response to these different treatments and validated by RT-qPCR and western blot. Results: We demonstrate that both nuclear and membrane ERα actions are required for the pro-tumoral effects of E2, while tamoxifen totally abrogates the E2-induced in vivo tumor growth, through inhibition of angiogenesis but promotion of vessel normalization. RNA sequencing indicates that tamoxifen inhibits the E2-induced genes, but also initiates a specific transcriptional program that especially regulates angiogenic genes and differentially regulates glycolysis, oxidative phosphorylation and inflammatory responses in endothelial cells. Conclusion: These findings provide evidence that tamoxifen specifically inhibits angiogenesis through a reprogramming of endothelial gene expression via regulation of some transcription factors, that could open new promising strategies to manage cancer therapies affecting the tumor microenvironment of ERα-negative tumors

Dissection des effets membranaires et nucléaires du récepteur aux oestrogènes ERα in vivo

Les œstrogènes sont impliqués dans le développement et l'homéostasie de nombreux tissus reproducteurs et extra-reproducteurs et influencent de nombreux processus physiologiques et pathologiques. L'action des œstrogènes est relayée majoritairement par le récepteur des œstrogènes ERα dont l'action nucléaire conduit à la régulation transcriptionnelle de ses gènes cibles alors que son activation membranaire induit différentes voies de signalisations cytoplasmiques. Le but de ce travail de thèse a été d'évaluer in vivo les rôles respectifs des effets nucléaires et membranaires dans différents processus physiologiques (modulation de la fertilité, vasculo-protection, prolifération endométriale) ou pathologiques (angiogenèse tumorale) en réponse au 17β-œstradiol (E2). Différents modèles de souris transgéniques ont été utilisés, notamment les souris C451A-ERα présentant une mutation du site de palmitoylation nécessaire à l'adressage membranaire de ERα. Nous avons ainsi pu mettre en évidence la part respective des effets membranaires et nucléaires de ERα dans différents tissus.Estrogen Receptor ERα is a nuclear receptor, which regulates many physiological functions through estradiol (E2) binding on two cellular sub-localizations: Nuclear ERa is implicated in the regulation of gene expression while membrane ERα (targeting through Cys451 palmitoylation) activates kinase signaling. The main objective of my PhD thesis was to investigate the respective roles of membrane and nuclear ERα signaling in physiological functions (fertility, vascular protection, uterine proliferation) or pathological functions (tumoral angiogenesis) in response to 17β-estradiol, pharmacological tools (EDC, Estrogen Dendrimer Conjugate) or selective ligand as tamoxifen or Estetrol (E4). Two complementary mouse models were used to delineate their respective functions of membrane and nuclear ERa signaling in vivo: ERα-AF20 mice with specific deletion of the AF2 transactivation function necessary to recruit transcriptional coactivators; C451A-ERα mice with specific mutation of the palmitoylation site of ERα necessary for membrane targeting. Furthermore, the specific role of ERα methylation, occurring on Arg264 and essential for ERα/Src/PI3K complex formation, has been evaluated in vivo using mice R264A-ERα. This work demonstrates for the first time the respective role of membrane and nuclear ERα signaling in vivo. We have highlighted some tissue-specific roles of nuclear and membrane signaling in uterus and vascular protection respectively, but also in fertility. These findings contribute to a better understanding of the molecular ERα signaling in vivo which is of major importance for the design of new SERMs (Selective ER Modulators)

Dissection of membrane and nuclear estrogen receptor alpha actions in vivo

Les œstrogènes sont impliqués dans le développement et l'homéostasie de nombreux tissus reproducteurs et extra-reproducteurs et influencent de nombreux processus physiologiques et pathologiques. L'action des œstrogènes est relayée majoritairement par le récepteur des œstrogènes ERα dont l'action nucléaire conduit à la régulation transcriptionnelle de ses gènes cibles alors que son activation membranaire induit différentes voies de signalisations cytoplasmiques. Le but de ce travail de thèse a été d'évaluer in vivo les rôles respectifs des effets nucléaires et membranaires dans différents processus physiologiques (modulation de la fertilité, vasculo-protection, prolifération endométriale) ou pathologiques (angiogenèse tumorale) en réponse au 17β-œstradiol (E2). Différents modèles de souris transgéniques ont été utilisés, notamment les souris C451A-ERα présentant une mutation du site de palmitoylation nécessaire à l'adressage membranaire de ERα. Nous avons ainsi pu mettre en évidence la part respective des effets membranaires et nucléaires de ERα dans différents tissus.Estrogen Receptor ERα is a nuclear receptor, which regulates many physiological functions through estradiol (E2) binding on two cellular sub-localizations: Nuclear ERa is implicated in the regulation of gene expression while membrane ERα (targeting through Cys451 palmitoylation) activates kinase signaling. The main objective of my PhD thesis was to investigate the respective roles of membrane and nuclear ERα signaling in physiological functions (fertility, vascular protection, uterine proliferation) or pathological functions (tumoral angiogenesis) in response to 17β-estradiol, pharmacological tools (EDC, Estrogen Dendrimer Conjugate) or selective ligand as tamoxifen or Estetrol (E4). Two complementary mouse models were used to delineate their respective functions of membrane and nuclear ERa signaling in vivo: ERα-AF20 mice with specific deletion of the AF2 transactivation function necessary to recruit transcriptional coactivators; C451A-ERα mice with specific mutation of the palmitoylation site of ERα necessary for membrane targeting. Furthermore, the specific role of ERα methylation, occurring on Arg264 and essential for ERα/Src/PI3K complex formation, has been evaluated in vivo using mice R264A-ERα. This work demonstrates for the first time the respective role of membrane and nuclear ERα signaling in vivo. We have highlighted some tissue-specific roles of nuclear and membrane signaling in uterus and vascular protection respectively, but also in fertility. These findings contribute to a better understanding of the molecular ERα signaling in vivo which is of major importance for the design of new SERMs (Selective ER Modulators)

Effets membranaires du récepteur alpha des œstrogènes

Les récepteurs des œstrogènes (ER) sont utilisés comme cibles thérapeutiques soit en les activant (par exemple dans le cas de la contraception ou de la ménopause), soit en les inhibant (comme pour le cancer du sein). Cependant, l’idéal serait de cibler chaque tissu spécifiquement. Certains modulateurs (les SERM, selective estrogen receptor modulator), spécifiques du récepteur alpha des œstrogènes (ERα), sont capables partiellement de réaliser ce ciblage spécifique. Il est donc important de comprendre la spécificité tissulaire de l’action de ERα afin d’optimiser le rapport bénéfice/risque de ces modulateurs. À côté de sa fonction nucléaire classique comme facteur de transcription, une fraction extranucléaire d’ERα relaye des actions rapides membranaires des œstrogènes. De nouveaux modèles de souris transgéniques ont récemment permis de dévoiler, dans chaque tissu, les rôles physiologiques et spécifiques des effets nucléaires et des effets membranaires d’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

Membrane expression of the estrogen receptor ER alpha is required for intercellular communications in the mammary epithelium

17 beta-Estradiol induces the postnatal development of mammary gland and influences breast carcinogenesis by binding to the estrogen receptor ER alpha. ER alpha acts as a transcription factor but also elicits rapid signaling through a fraction of ER alpha expressed at the membrane. Here, we have used the C451A-ER alpha mouse model mutated for the palmitoylation site to understand how ER alpha membrane signaling affects mammary gland development. Although the overall structure of physiological mammary gland development is slightly affected, both epithelial fragments and basal cells isolated from C451A-ER alpha mammary glands failed to grow when engrafted into cleared wild-type fat pads, even in pregnant hosts. Similarly, basal cells purified from hormone-stimulated ovariectomized C451A-ER alpha mice did not produce normal outgrowths. Ex vivo, C451A-ER alpha basal cells displayed reduced matrix degradation capacities, suggesting altered migration properties. More importantly, C451A-ER alpha basal cells recovered in vivo repopulating ability when co-transplanted with wild-type luminal cells and specifically with ER alpha-positive luminal cells. Transcriptional profiling identified crucial paracrine luminal-to-basal signals. Altogether, our findings uncover an important role for membrane ER alpha expression in promoting intercellular communications that are essential for mammary gland development

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