Dichotomous engagement of HDAC3 activity governs inflammatory responses.

The histone deacetylases (HDACs) are a superfamily of chromatin-modifying enzymes that silence transcription through the modification of histones. Among them, HDAC3 is unique in that interaction with nuclear receptor corepressors 1 and 2 (NCoR1/2) is required to engage its catalytic activity1-3. However, global loss of HDAC3 also results in the repression of transcription, the mechanism of which is currently unclear4-8. Here we report that, during the activation of macrophages by lipopolysaccharides, HDAC3 is recruited to activating transcription factor 2 (ATF2)-bound sites without NCoR1/2 and activates the expression of inflammatory genes through a non-canonical mechanism. By contrast, the deacetylase activity of HDAC3 is selectively engaged at ATF3-bound sites that suppress Toll-like receptor signalling. Loss of HDAC3 in macrophages safeguards mice from lethal exposure to lipopolysaccharides, but this protection is not conferred upon genetic or pharmacological abolition of the catalytic activity of HDAC3. Our findings show that HDAC3 is a dichotomous transcriptional activator and repressor, with a non-canonical deacetylase-independent function that is vital for the innate immune system.

Membrane and Nuclear Estrogen Receptor Alpha Actions: From Tissue Specificity to Medical Implications.

Estrogen receptor alpha (ERα) has been recognized now for several decades as playing a key role in reproduction and exerting functions in numerous nonreproductive tissues. In this review, we attempt to summarize the in vitro studies that are the basis of our current understanding of the mechanisms of action of ERα as a nuclear receptor and the key roles played by its two activation functions (AFs) in its transcriptional activities. We then depict the consequences of the selective inactivation of these AFs in mouse models, focusing on the prominent roles played by ERα in the reproductive tract and in the vascular system. Evidence has accumulated over the two last decades that ERα is also associated with the plasma membrane and activates non-nuclear signaling from this site. These rapid/nongenomic/membrane-initiated steroid signals (MISS) have been characterized in a variety of cell lines, and in particular in endothelial cells. The development of selective pharmacological tools that specifically activate MISS and the generation of mice expressing an ERα protein impeded for membrane localization have begun to unravel the physiological role of MISS in vivo. Finally, we discuss novel perspectives for the design of tissue-selective ER modulators based on the integration of the physiological and pathophysiological roles of MISS actions of estrogens.

Membrane expression of the estrogen receptor ERα is required for intercellular communications in the mammary epithelium.

17ß-Estradiol induces the postnatal development of mammary gland and influences breast carcinogenesis by binding to the estrogen receptor ERα. ERα acts as a transcription factor but also elicits rapid signaling through a fraction of ERα expressed at the membrane. Here, we have used the C451A-ERα mouse model mutated for the palmitoylation site to understand how ERα 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-ERα 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-ERα mice did not produce normal outgrowths. Ex vivo, C451A-ERα basal cells displayed reduced matrix degradation capacities, suggesting altered migration properties. More importantly, C451A-ERα basal cells recovered in vivo repopulating ability when co-transplanted with wild-type luminal cells and specifically with ERα-positive luminal cells. Transcriptional profiling identified crucial paracrine luminal-to-basal signals. Altogether, our findings uncover an important role for membrane ERα expression in promoting intercellular communications that are essential for mammary gland development.

Histone deacetylase 3 prepares brown adipose tissue for acute thermogenic challenge.

Brown adipose tissue is a thermogenic organ that dissipates chemical energy as heat to protect animals against hypothermia and to counteract metabolic disease. However, the transcriptional mechanisms that determine the thermogenic capacity of brown adipose tissue before environmental cold are unknown. Here we show that histone deacetylase 3 (HDAC3) is required to activate brown adipose tissue enhancers to ensure thermogenic aptitude. Mice with brown adipose tissue-specific genetic ablation of HDAC3 become severely hypothermic and succumb to acute cold exposure. Uncoupling protein 1 (UCP1) is nearly absent in brown adipose tissue lacking HDAC3, and there is also marked downregulation of mitochondrial oxidative phosphorylation genes resulting in diminished mitochondrial respiration. Remarkably, although HDAC3 acts canonically as a transcriptional corepressor, it functions as a coactivator of oestrogen-related receptor α (ERRα) in brown adipose tissue. HDAC3 coactivation of ERRα is mediated by deacetylation of PGC-1α and is required for the transcription of Ucp1, Ppargc1a (encoding PGC-1α), and oxidative phosphorylation genes. Importantly, HDAC3 promotes the basal transcription of these genes independently of adrenergic stimulation. Thus, HDAC3 uniquely primes Ucp1 and the thermogenic transcriptional program to maintain a critical capacity for thermogenesis in brown adipose tissue that can be rapidly engaged upon exposure to dangerously cold temperature.

Estrogen receptor-α signaling in post-natal mammary development and breast cancers.

17ß-estradiol controls post-natal mammary gland development and exerts its effects through Estrogen Receptor ERα, a member of the nuclear receptor family. ERα is also critical for breast cancer progression and remains a central therapeutic target for hormone-dependent breast cancers. In this review, we summarize the current understanding of the complex ERα signaling pathways that involve either classical nuclear "genomic" or membrane "non-genomic" actions and regulate in concert with other hormones the different stages of mammary development. We describe the cellular and molecular features of the luminal cell lineage expressing ERα and provide an overview of the transgenic mouse models impacting ERα signaling, highlighting the pivotal role of ERα in mammary gland morphogenesis and function and its implication in the tumorigenic processes. Finally, we describe the main features of the ERα-positive luminal breast cancers and their modeling in mice.

SR9009 has REV-ERB-independent effects on cell proliferation and metabolism.

The nuclear receptors REV-ERBα and -ß link circadian rhythms and metabolism. Like other nuclear receptors, REV-ERB activity can be regulated by ligands, including naturally occurring heme. A putative ligand, SR9009, has been reported to elicit a range of beneficial effects in healthy as well as diseased animal models and cell systems. However, the direct involvement of REV-ERBs in these effects of SR9009 has not been thoroughly assessed, as experiments were not performed in the complete absence of both proteins. Here, we report the generation of a mouse model for conditional genetic deletion of REV-ERBα and -ß. We show that SR9009 can decrease cell viability, rewire cellular metabolism, and alter gene transcription in hepatocytes and embryonic stem cells lacking both REV-ERBα and -ß. Thus, the effects of SR9009 cannot be used solely as surrogate for REV-ERB activity.

Circadian lipid synthesis in brown fat maintains murine body temperature during chronic cold.

Ambient temperature influences the molecular clock and lipid metabolism, but the impact of chronic cold exposure on circadian lipid metabolism in thermogenic brown adipose tissue (BAT) has not been studied. Here we show that during chronic cold exposure (1 wk at 4 °C), genes controlling de novo lipogenesis (DNL) including Srebp1, the master transcriptional regulator of DNL, acquired high-amplitude circadian rhythms in thermogenic BAT. These conditions activated mechanistic target of rapamycin 1 (mTORC1), an inducer of Srebp1 expression, and engaged circadian transcriptional repressors REV-ERBα and ß as rhythmic regulators of Srebp1 in BAT. SREBP was required in BAT for the thermogenic response to norepinephrine, and depletion of SREBP prevented maintenance of body temperature both during circadian cycles as well as during fasting of chronically cold mice. By contrast, deletion of REV-ERBα and ß in BAT allowed mice to maintain their body temperature in chronic cold. Thus, the environmental challenge of prolonged noncircadian exposure to cold temperature induces circadian induction of SREBP1 that drives fuel synthesis in BAT and is necessary to maintain circadian body temperature during chronic cold exposure. The requirement for BAT fatty acid synthesis has broad implications for adaptation to cold.

Estetrol prevents Western diet-induced obesity and atheroma independently of hepatic estrogen receptor α.

Estetrol (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 with 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 estrogen receptor (ER) α in the preventive effect of E4 against obesity and associated disorders such as atherosclerosis and steatosis, mice harboring 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.NEW & NOTEWORTHY Estetrol prevents obesity, steatosis, and atherosclerosis in mice fed a Western diet. Hepatic ERα is necessary for the prevention of steatosis, but not of obesity and atherosclerosis.

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

OBJECTIVE: 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 17ß-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 C451A-ERα, the R264A-ERα 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 R264A-ERα 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.

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 17ß-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 17ß-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. 17ß-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, 17ß-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.

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

Estrogen receptor alpha (ERα) activation functions AF-1 and AF-2 classically mediate gene transcription in response to estradiol (E2). A fraction of ERα 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 ERα (C451A-ERα) to obtain membrane-specific loss of function of ERα. The abrogation of membrane localization of ERα 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-ERα 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-ERα mice. A complementary mutant mouse lacking the transactivation function AF-2 of ERα (ERα-AF2(0)) provided selective loss of function of nuclear ERα actions. In ERα-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 ERα-AF2(0), whereas in C451A-ERα 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.

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

Rationale: 17ß-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.

The REV-ERB Nuclear Receptors: Timekeepers for the Core Clock Period and Metabolism.

REV-ERB nuclear receptors are potent transcriptional repressors that play an important role in the core mammalian molecular clock and metabolism. Deletion of both REV-ERBα and its largely redundant isoform REV-ERBß in a murine tissue-specific manner have shed light on their specific functions in clock mechanisms and circadian metabolism. This review highlights recent findings that establish REV-ERBs as crucial circadian timekeepers in a variety of tissues, regulating overlapping and distinct processes that maintain normal physiology and protect from metabolic dysfunction.

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

Background: Estrogen 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. Methods: Male 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. Results: C451A-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. Conclusion: Besides 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.

The different natural estrogens promote endothelial healing through distinct cell targets.

The main estrogen, 17ß-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.

Segregation of nuclear and membrane-initiated actions of estrogen receptor using genetically modified animals and pharmacological tools.

Estrogen receptor alpha (ERα) and beta (ERß) are members of the nuclear receptor superfamily, playing widespread functions in reproductive and non-reproductive tissues. Beside the canonical function of ERs as nuclear receptors, in this review, we summarize our current understanding of extra-nuclear, membrane-initiated functions of ERs with a specific focus on ERα. Over the last decade, in vivo evidence has accumulated to demonstrate the physiological relevance of this ERα membrane-initiated-signaling from mouse models to selective pharmacological tools. Finally, we discuss the perspectives and future challenges opened by the integration of extra-nuclear ERα signaling in physiology and pathology of estrogens.

A historical view of estrogen effect on arterial endothelial healing: From animal models to medical implication.

Endothelial barrier integrity is required for maintaining vascular homeostasis and fluid balance between the circulation and surrounding tissues. In contrast, abnormalities of endothelial cell function and loss of the integrity of the endothelial monolayer constitute a key step in the onset of atherosclerosis. Endothelial erosion is directly responsible for thrombus formation and cardiovascular events in about one-third of the cases of acute coronary syndromes. Thus, after endothelial injury, the vascular repair process is crucial to restore endothelial junctions and rehabilitate a semipermeable barrier, preventing the development of vascular diseases. Endothelial healing can be modulated by several factors. In particular, 17ß-estradiol (E2), the main estrogen, improves endothelial healing, reduces neointimal accumulation of smooth muscle cells and atherosclerosis in several animal models. The aim of this review is to highlight how various experimental models enabled the progress in the cellular and molecular mechanisms underlying the accelerative E2 effect on arterial endothelial healing through the estrogen receptor (ER) α, the main receptor mediating the physiological effects of estrogens. We first summarize the different experimental procedures used to reproduce vascular injury. We then provide an overview of how the combination of transgenic mouse models impacting ERα signalling with pharmacological tools demonstrated the pivotal role of non-genomic actions of ERα in E2-induced endothelial repair. Finally, we describe recent advances in the action of selective estrogen receptor modulators (SERMs) on this beneficial vascular effect, which surprisingly involves different cell types and activates different ERα subfunctions compared to E2.

Hypothalamic REV-ERB nuclear receptors control diurnal food intake and leptin sensitivity in diet-induced obese mice.

Obesity occurs when energy expenditure is outweighed by energy intake. Tuberal hypothalamic nuclei, including the arcuate nucleus (ARC), ventromedial nucleus (VMH), and dorsomedial nucleus (DMH), control food intake and energy expenditure. Here we report that, in contrast with females, male mice lacking circadian nuclear receptors REV-ERBα and -ß in the tuberal hypothalamus (HDKO mice) gained excessive weight on an obesogenic high-fat diet due to both decreased energy expenditure and increased food intake during the light phase. Moreover, rebound food intake after fasting was markedly increased in HDKO mice. Integrative transcriptomic and cistromic analyses revealed that such disruption in feeding behavior was due to perturbed REV-ERB-dependent leptin signaling in the ARC. Indeed, in vivo leptin sensitivity was impaired in HDKO mice on an obesogenic diet in a diurnal manner. Thus, REV-ERBs play a crucial role in hypothalamic control of food intake and diurnal leptin sensitivity in diet-induced obesity.

REV-ERB nuclear receptors in the suprachiasmatic nucleus control circadian period and restrict diet-induced obesity.

Circadian disruption, as occurs in shift work, is associated with metabolic diseases often attributed to a discordance between internal clocks and environmental timekeepers. REV-ERB nuclear receptors are key components of the molecular clock, but their specific role in the SCN master clock is unknown. We report here that mice lacking circadian REV-ERB nuclear receptors in the SCN maintain free-running locomotor and metabolic rhythms, but these rhythms are notably shortened by 3 hours. When housed under a 24-hour light:dark cycle and fed an obesogenic diet, these mice gained excess weight and accrued more liver fat than controls. These metabolic disturbances were corrected by matching environmental lighting to the shortened endogenous 21-hour clock period, which decreased food consumption. Thus, SCN REV-ERBs are not required for rhythmicity but determine the free-running period length. Moreover, these results support the concept that dissonance between environmental conditions and endogenous time periods causes metabolic disruption.

Membrane estrogen receptor alpha (ERα) participates in flow-mediated dilation in a ligand-independent manner.

Estrogen receptor alpha (ERα) activation by estrogens prevents atheroma through its nuclear action, whereas plasma membrane-located ERα accelerates endothelial healing. The genetic deficiency of ERα was associated with a reduction in flow-mediated dilation (FMD) in one man. Here, we evaluated ex vivo the role of ERα on FMD of resistance arteries. FMD, but not agonist (acetylcholine, insulin)-mediated dilation, was reduced in male and female mice lacking ERα (Esr1-/- mice) compared to wild-type mice and was not dependent on the presence of estrogens. In C451A-ERα mice lacking membrane ERα, not in mice lacking AF2-dependent nuclear ERα actions, FMD was reduced, and restored by antioxidant treatments. Compared to wild-type mice, isolated perfused kidneys of C451A-ERα mice revealed a decreased flow-mediated nitrate production and an increased H2O2 production. Thus, endothelial membrane ERα promotes NO bioavailability through inhibition of oxidative stress and thereby participates in FMD in a ligand-independent manner.