Fibronectin rescues estrogen receptor α from lysosomal degradation in breast cancer cells

Estrogen receptor α (ERα) is expressed in tissues as diverse as brains and mammary glands. In breast cancer, ERα is a key regulator of tumor progression. Therefore, understanding what activates ERα is critical for cancer treatment in particular and cell biology in general. Using biochemical approaches and superresolution microscopy, we show that estrogen drives membrane ERα into endosomes in breast cancer cells and that its fate is determined by the presence of fibronectin (FN) in the extracellular matrix; it is trafficked to lysosomes in the absence of FN and avoids the lysosomal compartment in its presence. In this context, FN prolongs ERα half-life and strengthens its transcriptional activity. We show that ERα is associated with β1-integrin at the membrane, and this integrin follows the same endocytosis and subcellular trafficking pathway triggered by estrogen. Moreover, ERα+ vesicles are present within human breast tissues, and colocalization with β1-integrin is detected primarily in tumors. Our work unravels a key, clinically relevant mechanism of microenvironmental regulation of ERα signaling.Fil: Sampayo, Rocío Guadalupe. Universidad Nacional de San Martin. Instituto de Nanosistemas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Toscani, Andrés Martin. Universidad Nacional de Luján; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Rubashkin, Matthew G.. University of California; Estados UnidosFil: Thi, Kate. Lawrence Berkeley National Laboratory; Estados UnidosFil: Masullo, Luciano Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; ArgentinaFil: Violi, Ianina Lucila. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones en Bionanociencias "Elizabeth Jares Erijman"; ArgentinaFil: Lakins, Jonathon N.. University of California; Estados UnidosFil: Caceres, Alfredo Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; ArgentinaFil: Hines, William C.. Lawrence Berkeley National Laboratory; Estados UnidosFil: Coluccio Leskow, Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad Nacional de Luján; ArgentinaFil: Stefani, Fernando Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; ArgentinaFil: Chialvo, Dante Renato. Universidad de Buenos Aires; Argentina. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro Internacional de Estudios Avanzados; ArgentinaFil: Bissell, Mina J.. Lawrence Berkeley National Laboratory; Estados UnidosFil: Weaver, Valerie M.. University of California; Estados UnidosFil: Simian, Marina. Universidad Nacional de San Martin. Instituto de Nanosistemas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentin

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

Estrogen receptor alpha (ERalpha) activation functions AF-1 and AF-2 classically mediate gene transcription in response to estradiol (E2). A fraction of ERalpha is targeted to plasma membrane and elicits membrane-initiated steroid signaling (MISS), but the physiological roles of MISS in vivo are poorly understood. We therefore generated a mouse with a point mutation of the palmitoylation site of ERalpha (C451A-ERalpha) to obtain membrane-specific loss of function of ERalpha. The abrogation of membrane localization of ERalpha in vivo was confirmed in primary hepatocytes, and it resulted in female infertility with abnormal ovaries lacking corpora lutea and increase in luteinizing hormone levels. In contrast, E2 action in the uterus was preserved in C451A-ERalpha mice and endometrial epithelial proliferation was similar to wild type. However, E2 vascular actions such as rapid dilatation, acceleration of endothelial repair, and endothelial NO synthase phosphorylation were abrogated in C451A-ERalpha mice. A complementary mutant mouse lacking the transactivation function AF-2 of ERalpha (ERalpha-AF2(0)) provided selective loss of function of nuclear ERalpha actions. In ERalpha-AF2(0), the acceleration of endothelial repair in response to estrogen-dendrimer conjugate, which is a membrane-selective ER ligand, was unaltered, demonstrating integrity of MISS actions. In genome-wide analysis of uterine gene expression, the vast majority of E2-dependent gene regulation was abrogated in ERalpha-AF2(0), whereas in C451A-ERalpha it was nearly fully preserved, indicating that membrane-to-nuclear receptor cross-talk in vivo is modest in the uterus. Thus, this work genetically segregated membrane versus nuclear actions of a steroid hormone receptor and demonstrated their in vivo tissue-specific roles

Transcriptional Regulation of T-Cell Lipid Metabolism: Implications for Plasma Membrane Lipid Rafts and T-Cell Function

It is well established that cholesterol and glycosphingolipids are enriched in the plasma membrane (PM) and form signalling platforms called lipid rafts, essential for T-cell activation and function. Moreover, changes in PM lipid composition affect the biophysical properties of lipid rafts and have a role in defining functional T-cell phenotypes. Here we review the role of transcriptional regulators of lipid metabolism including liver X receptors α/β (LXR/LXRβ), peroxisome proliferator-activated receptor γ (PPAR-γ), estrogen receptors α/β (ERα/β) and sterol regulatory element-binding proteins (SREBPs) in T-cells. These receptors lie at the interface between lipid metabolism and immune cell function and are endogenously activated by lipids and/or hormones. Importantly, they regulate cellular cholesterol, fatty acid, glycosphingolipid and phospholipid levels but are also known to modulate a broad spectrum of immune responses. The current evidence supporting a role for lipid metabolism pathways in controlling immune cell activation by influencing PM lipid raft composition in health and disease, and the potential for targeting lipid biosynthesis pathways to control unwanted T-cell activation in autoimmunity is reviewed

Testosterone Prevents Cutaneous Ischemia and Necrosis in Males Through Complementary Estrogenic and Androgenic Actions

OBJECTIVE: Chronic nonhealing wounds are a substantial medical concern and are associated with morbidity and mortality; thus, new treatment strategies are required. The first step toward personalized/precision medicine in this field is probably in taking sex differences into account. Impaired wound healing is augmented by ischemia, and we previously demonstrated that 17β-estradiol exerts a major preventive effect against ischemia-induced skin flap necrosis in female mice. However, the equivalent effects of testosterone in male mice have not yet been reported. We then investigated the role of steroid hormones in male mice using a skin flap ischemia model. APPROACH AND RESULTS: Castrated male mice developed skin necrosis after ischemia, whereas intact or castrated males treated with testosterone were equally protected. Testosterone can (1) activate the estrogen receptor after its aromatization into 17β-estradiol or (2) be reduced into dihydrotestosterone, a nonaromatizable androgen that activates the androgen receptor. We found that dihydrotestosterone protected castrated wild-type mice by promoting skin revascularization, probably through a direct action on resistance arteries, as evidenced using a complementary model of flow-mediated outward remodeling. 17β-estradiol treatment of castrated male mice also strongly protected them from ischemic necrosis through the activation of estrogen receptor-α by increasing skin revascularization and skin survival. Remarkably, 17β-estradiol improved skin survival with a greater efficiency than dihydrotestosterone. CONCLUSIONS: Testosterone provides males with a strong protection against cutaneous necrosis and acts through both its estrogenic and androgenic derivatives, which have complementary effects on skin survival and revascularization

Predominant Role of Nuclear Versus Membrane Estrogen Receptor α in Arterial Protection: Implications for Estrogen Receptor α Modulation in Cardiovascular Prevention/Safety

BACKGROUND: Although estrogen receptor α (ERα) acts primarily as a transcription factor, it can also elicit membrane-initiated steroid signaling. Pharmacological tools and transgenic mouse models previously highlighted the key role of ERα membrane-initiated steroid signaling in 2 actions of estrogens in the endothelium: increase in NO production and acceleration of reendothelialization. METHODS AND RESULTS: Using mice with ERα mutated at cysteine 451 (ERaC451A), recognized as the key palmitoylation site required for ERα plasma membrane location, and mice with disruption of nuclear actions because of inactivation of activation function 2 (ERaAF20 = ERaAF2°), we sought to fully characterize the respective roles of nuclear membrane-initiated steroid signaling in the arterial protection conferred by ERα. ERaC451A mice were fully responsive to estrogens to prevent atheroma and angiotensin II-induced hypertension as well as to allow flow-mediated arteriolar remodeling. By contrast, ERαAF20 mice were unresponsive to estrogens for these beneficial vascular effects. Accordingly, selective activation of nuclear ERα with estetrol was able to prevent hypertension and to restore flow-mediated arteriolar remodeling. CONCLUSIONS: Altogether, these results reveal an unexpected prominent role of nuclear ERα in the vasculoprotective action of estrogens with major implications in medicine, particularly for selective nuclear ERα agonist, such as estetrol, which is currently under development as a new oral contraceptive and for hormone replacement therapy in menopausal women

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)