Signaling from the membrane via membrane estrogen receptor-alpha: estrogens, xenoestrogens, and phytoestrogens

Estrogen mimetics in the environment and in foods can have important consequences for endocrine functions. When previously examined for action via genomic steroid signaling mechanisms, most of these compounds were found to be very weak agonists. We have instead tested their actions via several membrane-initiated signaling mechanisms in GH3/B6 pituitary tumor cells extensively selected for high (responsive) or low (nonresponsive) expression of the membrane version of estrogen receptor-alpha (mERalpha). We found many estrogen mimetic compounds to be potently active in our quantitative extracellular-regulated kinase (ERK) activation assays, to increase cellular Ca++ levels, and to cause rapid prolactin release. However, these compounds may activate one or both mechanisms with different potencies. For instance, some compounds activate ERKs in both pM and nM concentration ranges, while others are only active at nM and higher concentrations. Compounds also show great differences in their temporal activation patterns. While estradiol causes a bimodal time-dependent ERK activation (peaking at both 3 and 30 min), most estrogen mimetics cause either an early phase activation, a late phase activation, or an early sustained activation. One xenoestrogen known to be a relatively potent activator of estrogen response element-mediated actions (bisphenol A) is inactive as an ERK activator, and only a modest inducer of Ca++ levels and prolactin release. Many different signaling machineries culminate in ERK activation, and xenoestrogens differentially affect various pathways. Clearly individual xenoestrogens must be individually investigated for their differing abilities to activate distinct membrane-initiated signal cascades that lead to a variety of cellular functions.     

Estrogen- and xenoestrogen-induced ERK signaling in pituitary tumor cells involves estrogen receptor-α interactions with G protein-αi and caveolin I

Multiple physiologic estrogens (estradiol, estriol, and estrone), as well as xenoestrogenic compounds (including alkylphenols and bisphenol A), can act via nongenomic signaling initiated by liganding of the plasma membrane estrogen receptor-α (mERα). We examined heterotrimeric G protein involvement leading to extracellular-regulated kinase (ERK) activation in GH3/B6/F10 rat anterior pituitary tumor cells that express abundant mERα, and smaller amounts of mERβ and GPR30. A combination of microarrays, immunoblots, and quantitative immunoassays demonstrated the expression of members of all α, β, and γ G protein classes in these cells. Use of selective inhibitors showed that the G(αi) subtype was the primary initiator of downstream ERK signaling. Using antibodies against the GTP-bound form of G(α) protein subtypes i and s, we showed that xenoestrogens (bisphenol A, nonylphenol) activated G(αi) at 15-30s; all alkylphenols examined subsequently suppressed activation by 5min. GTP-activation of G(αi) for all estrogens was enhanced by irreversible cumulative binding to GTPγS. In contrast, G(αs) was neither activated nor deactivated by these treatments with estrogens. ERα and G(αi) co-localized outside nuclei and could be immuno-captured together. Interactions of ERα with G(αi) and caveolin I were demonstrated by epitope proximity ligation assays. An ERα/β antagonist (ICI182780) and a selective disruptor of caveolar structures (nystatin) blocked estrogen-induced ERK activation. Conclusions: Xenoestrogens, like physiologic estrogens, can evoke downstream kinase signaling involving selective interactions of ERα with G(αi) and caveolin I, but with some different characteristics, which could explain their disruptive actions.     

Environmental estrogens differentially engage the histone methyltransferase EZH2 to increase risk of uterine tumorigenesis

Environmental exposures during sensitive windows of development can reprogram normal physiologic responses and alter disease susceptibility later in life in a process known as developmental reprogramming. For example, exposure to the xenoestrogen diethylstilbestrol during reproductive tract development can reprogram estrogen-responsive gene expression in the myometrium, resulting in hyperresponsiveness to hormone in the adult uterus and promotion of hormone-dependent uterine leiomyoma. We show here that the environmental estrogens genistein, a soy phytoestrogen, and the plasticizer bisphenol A, differ in their pattern of developmental reprogramming and promotion of tumorigenesis (leiomyomas) in the uterus. Whereas both genistein and bisphenol A induce genomic estrogen receptor (ER) signaling in the developing uterus, only genistein induced phosphoinositide 3-kinase (PI3K)/AKT nongenomic ER signaling to the histone methyltransferase enhancer of zeste homolog 2 (EZH2). As a result, this pregenomic signaling phosphorylates and represses EZH2 and reduces levels of H3K27me3 repressive mark in chromatin. Furthermore, only genistein caused estrogen-responsive genes in the adult myometrium to become hyperresponsive to hormone; estrogen-responsive genes were repressed in bisphenol A-exposed uteri. Importantly, this pattern of EZH2 engagement to decrease versus increase H3K27 methylation correlated with the effect of these xenoestrogens on tumorigenesis. Developmental reprogramming by genistein promoted development of uterine leiomyomas, increasing tumor incidence and multiplicity, whereas bisphenol A did not. These data show that environmental estrogens have distinct nongenomic effects in the developing uterus that determines their ability to engage the epigenetic regulator EZH2, decrease levels of the repressive epigenetic histone H3K27 methyl mark in chromatin during developmental reprogramming, and promote uterine tumorigenesis.     

Rapid extranuclear signaling by the estrogen receptor (ER): MNAR couples ER and Src to the MAP kinase signaling pathway

In addition to their well-studied ability to transactivate the expression of many genes, estrogen receptors (ERs) also effect cytoplasmic changes occurring too quickly to be accounted for by gene expression. Indeed, these immediate, "nongenomic" effects have been intensely studied, but the identification of important protein partners in quick ER-mediated signaling has lagged behind. Now, Wong et al. have identified MNAR (modulator of nongenomic activity of estrogen receptor) as an adaptor protein that allows the ER to bridge the signaling pathways of tyrosine kinases (i.e., Src) and the mitogen-activated protein kinase (MAPK) cascade. The MNAR-ER complex also appears to positively influence ER-mediated gene expression.     

Estradiol-induced nongenomic calcium signaling regulates genotropic signaling in macrophages.

Estradiol (E(2)) exerts not only genotropic but also nongenomic actions through nuclear estrogen receptors (ER). Here, we provide a novel paradigm for nongenomic E(2) signaling independent of nuclear ER. E(2) induces a rapid rise in the intracellular free Ca(2+) concentration ([Ca(2+)](i)) through membrane estrogen receptors in murine RAW 264.7 macrophages. This E(2)-induced Ca(2+) signaling is not prevented by different ER blockers and cannot directly activate stably transfected c-fos promoter or the mitogen-activated protein kinases p38, ERK1/2, and SAPK/JNK, or NO production. However, the E(2)-induced rise in [Ca(2+)](i) specifically down-regulates the serum-stimulated activation of c-fos promoter and ERK1/2, and conversely, it specifically up-regulates lipopolysaccharide-stimulated activation of c-fos promoter, p38, and NO production. The E(2)-changed activation of c-fos promoter can be prevented by an intracellular Ca(2+) chelator. Our data indicate that E(2)-induced nongenomic Ca(2+) signaling through membrane ER is able to specifically modulate genotropic signaling pathways with impact on macrophage activation.     

Nongenomic, endothelium-independent effects of estrogen on human coronary smooth muscle are mediated by type I (neuronal) NOS and PI3-kinase-Akt signaling

Sex steroids exert profound and controversial effects on cardiovascular function. For example, estrogens have been reported to either ameliorate or exacerbate coronary heart disease. Although estrogen dilates coronary arteries from a variety of species, the molecular basis for this acute, nongenomic effect is unclear. Moreover, we know very little of how estrogen affects human coronary artery smooth muscle cells (HCASMC). The purpose of this study was to elucidate nongenomic estrogen signal transduction in HCASMC. We have used tissue (arterial tension studies), cellular (single-channel patch clamp, fluorescence), and molecular (protein expression) techniques to now identify novel targets of estrogen action in HCASMC: type I (neuronal) nitric oxide synthase (nNOS) and phosphatidylinositol 3-kinase (PI3-kinase)Akt. 17beta-Estradiol (E(2)) increased NO-stimulated fluorescence in HCASMC, and cell-attached patch-clamp experiments revealed that stimulation of nNOS leads to increased activity of calcium-activated potassium (BK(Ca)) channels in these cells. Furthermore, overexpression of nNOS protein in HCASMC greatly enhanced BK(Ca) channel activity. Immunoblot studies demonstrated that E(2) enhances Akt phosphorylation in HCASMC and that wortmannin, an inhibitor of PI3-kinase, attenuated E(2)-stimulated channel activity, NO production, Akt phosphorylation, and estrogen-stimulated coronary relaxation. These studies implicate the PI3-kinase/Akt signaling axis as an estrogen transduction component in vascular smooth muscle cells. We conclude, therefore, that estrogen opens BK(Ca) channels in HCASMC by stimulating nNOS via a transduction sequence involving PI3-kinase and Akt. These findings now provide a molecular mechanism that can explain the clinical observation that estrogen enhances coronary blood flow in patients with diseased or damaged coronary arteries.     

Position paper: The membrane estrogen receptor GPER - Clues and questions

Rapid signaling of estrogen involves membrane estrogen receptors (ERs), including membrane subpopulations of ERα and ERβ. In the mid-1990s, several laboratories independently reported the cloning of an orphan G protein-coupled receptor from vascular and cancer cells that was named GPR30. Research published between 2000 and 2005 provided evidence that GPR30 binds and signals via estrogen indicating that this intracellular receptor is involved in rapid, non-genomic estrogen signaling. The receptor has since been designated as the G protein-coupled estrogen receptor (GPER) by the International Union of Pharmacology. The availability of genetic tools such as different lines of GPER knock-out mice, as well as GPER-selective agonists and antagonists has advanced our understanding, but also added some confusion about the new function of this receptor. GPER not only binds estrogens but also other substances, including SERMs, SERDs, and environmental ER activators (endocrine disruptors; xenoestrogens) and also interacts with other proteins. This article represents a summary of a lecture given at the 7(th) International Meeting on Rapid Responses to Steroid Hormones in September 2011 in Axos, Crete, and reviews the current knowledge and questions about GPER-dependent signaling and function. Controversies that have complicated our understanding of GPER, including interactions with human ERα-36 and aldosterone as a potential ligand, will also be discussed.     

Signaling by estrogens

By regulating activities and expression levels of key signaling molecules, estrogens control mechanisms that are responsible for crucial cellular functions. Ligand binding to estrogen receptor (ER) leads to conformational changes that regulate the receptor activity, its interaction with other proteins and DNA. In the cytoplasm, receptor interactions with kinases and scaffolding molecules regulate cell signaling cascades (extranuclear/nongenomic action). In the nucleus, estrogens control a repertoire of coregulators and other auxiliary proteins that are associated with ER, which in turn determines the nature of regulated genes and level of their expression (genomic action). The combination of genomic and nongenomic actions of estrogens ultimately confers the cell-type and tissue-type selectivity. Recent studies have revealed some important new insights into the molecular mechanisms underlying ER action, which may help to explain the functional basis of existing selective ER modulators (SERMs) and provide evidence into how ER might be selectively targeted to achieve specific therapeutic goals. In this review, we will summarize some new molecular details that relate to estrogen signaling. We will also discuss some new strategies that may potentially lead to the development of functionally selective ER modulators that can separate between the beneficial, prodifferentiative effects in bone, the cardiovascular system and the CNS as well as the "detrimental," proliferative effects in reproductive tissues and organs.