Nandini Vasudevan

Non-genomic actions of estrogens and their interaction with genomic actions in the brain

Ligands for the nuclear receptor superfamily have at least two mechanisms of action: (a) classical transcriptional regulation of target genes (genomic mechanisms); and (b) non-genomic actions, which are initiated at the cell membrane, which could also impact transcription. Though transcriptional mechanisms are increasingly well understood, membrane-initiated actions of these ligands are incompletely understood. This has led to considerable debate over the physiological relevance of membrane-initiated actions of hormones versus genomic actions of hormones, with genomic actions predominating in the endocrine field. There is good evidence that the membrane-limited actions of hormones, particularly estrogens, involve the rapid activation of kinases and the release of calcium and that these are linked to physiologically relevant scenarios in the brain. We show evidence in this review, that membrane actions of estrogens, which activate these rapid signaling cascades, can also potentiate nuclear transcription in both the central nervous system and in non-neuronal cell lines. We present a theoretical scenario which can be used to understand this phenomenon. These signaling cascades may occur in parallel or in series but subsequently, converge at the modification of transcriptionally relevant molecules such as nuclear receptors and/or coactivators. In addition, other non-cognate hormones or neurotransmitters may also activate cascades to crosstalk with estrogen receptor-mediated transcription, though the relevance of this is less clear. The idea that coupling between membrane-initiated and genomic actions of hormones is a novel idea in neuroendocrinology and provides us with a unified view of hormone action in the central nervous system.

Early membrane estrogenic effects required for full expression of slower genomic actions in a nerve cell line

Interpretations of steroid hormone actions as slow, nuclear, transcriptional events have frequently been seen as competing against inferences of rapid membrane actions. We have discovered conditions where membrane-limited effects potentiate later transcriptional actions in a nerve cell line. Making use of a two-pulse hormonal schedule in a transfection system, early and brief administration of conjugated, membrane-limited estradiol was necessary but not sufficient for full transcriptional potency of the second estrogen pulse. Efficacy of the first pulse depended on intact signal transduction pathways. Surprisingly, the actions of both pulses were blocked by a classical estrogen receptor (ER) antagonist. Thus, two different modes of steroid hormone action can synergize.

Integration of steroid hormone initiated membrane action to genomic function in the brain

Estrogen is a ligand for the estrogen receptor (ER), which on binding 17beta-estradiol, functions as a ligand-activated transcription factor and regulates the transcription of target genes. This is the slow genomic mode of action. However, rapid non-genomic actions of estrogen also exist at the cell membrane. Using a novel two-pulse paradigm in which the first pulse rapidly initiates non-genomic actions using a membrane-limited estrogen conjugate (E-BSA), while the second pulse promotes genomic transcription from a consensus estrogen response element (ERE), we have demonstrated that rapid actions of estrogen potentiate the slower transcriptional response from an ERE-reporter in neuroblastoma cells. Since rapid actions of estrogen activate kinases, we used selective inhibitors in the two-pulse paradigm to determine the intracellular signaling cascades important in such potentiation. Inhibition of protein kinase A (PKA), PKC, mitogen activated protein kinase (MAPK) or phosphatidylinositol 3-OH kinase (PI-3K) in the first pulse decreases potentiation of transcription. Also, our data with both dominant negative and constitutive mutants of Galpha subunits show that Galpha(q) initiates the rapid signaling cascade at the membrane in SK-N-BE(2)C neuroblastoma cells. We discuss two models of multiple kinase activation at the membrane Pulses of estrogen induce lordosis behavior in female rats. Infusion of E-BSA into the ventromedial hypothalamus followed by 17beta-estradiol in the second pulse could induce lordosis behavior, demonstrating the applicability of this paradigm in vivo. A model where non-genomic actions of estrogen couple to genomic actions unites both aspects of hormone action.

Estrogens, brain and behavior: studies in fundamental neurobiology and observations related to women's health

Mechanisms and consequences of the effects of estrogen on the brain have been studied both at the fundamental level and with therapeutic applications in mind. Estrogenic hormones binding in particular neurons in a limbic-hypothalamic system and their effects on the electrophysiology and molecular biology of medial hypothalamic neurons were central in establishing the first circuit for a mammalian behavior, the female-typical mating behavior, lordosis. Notably, the ability of estradiol to facilitate transcription from six genes whose products are important for lordosis behavior proved that hormones can turn on genes in specific neurons at specific times, with sensible behavioral consequences. The use of a gene knockout for estrogen receptor alpha (ERalpha) revealed that homozygous mutant females simply would not do lordosis behavior and instead were extremely aggressive, thus identifying a specific gene as essential for a mammalian social behavior. In dramatic contrast, ERbeta knockout females can exhibit normal lordosis behavior. With the understanding, in considerable mechanistic detail, of how the behavior is produced, now we are also studying brain mechanisms for the biologically adaptive influences which constrain reproductive behavior. With respect to cold temperatures and other environmental or metabolic circumstances which are not consistent with successful reproduction, we are interested in thyroid hormone effects in the brain. Competitive relations between two types of transcription factors - thyroid hormone receptors and estrogen receptors have the potential of subserving the blocking effects of inappropriate environmental circumstances on female reproductive behaviors. TRs can compete with ERalpha both for DNA binding to consensus and physiological EREs and for nuclear coactivators. In the presence of both TRs and ERs, in transfection studies, thyroid hormone coadministration can reduce estrogen-stimulated transcription. These competitive relations apparently have behavioral consequences, as thyroid hormones will reduce lordosis, and a TRbeta gene knockout will increase it. In sum, we not only know several genes that participate in the selective control of this sex behavior, but also, for two genes, we know the causal routes. Estrogenic hormones are also the foci of widespread attention for their potential therapeutic effects improving, for example, certain aspects of mood and cognition. The former has an efficient animal analog, demonstrated by the positive effects of estrogen in the Porsolt forced swim test. The latter almost certainly depends upon trophic actions of estrogen on several fundamental features of nerve cell survival and growth. The hypothesis is raised that the synaptic effects of estrogens are secondary to the trophic actions of this type of hormone in the nucleus and nerve cell body.

Differential crosstalk between estrogen receptor (ER)α and ERβ and the thyroid hormone receptor isoforms results in flexible regulation of the consensus ERE

Abstract Crosstalk between nuclear receptors is important for conversion of external and internal stimuli to a physiologically meaningful response by cells. Previous studies from this laboratory have demonstrated crosstalk between the estrogen (ER) and thyroid hormone receptors (TR) on two estrogen responsive physiological promoters, the preproenkephalin and oxytocin receptor gene promoter. Since ERα and ERβ are isoforms possessing overlapping and distinct transactivation properties, we hypothesized that the interaction of ERα and β with the various TR isoforms would not be equivalent. To explore this hypothesis, the consensus estrogen response element (ERE) derived from the Xenopus vitellogenin gene is used to investigate the differences in interaction between ERα and β isoforms and the different TR isoforms in fibroblast cells. Both the ER isoforms transactivate from the consensus ERE, though ERα transactivates to a greater extent than ERβ. Although neither of the TRβ isoforms have an effect on ERα transactivation from the consensus ERE, the liganded TRα1 inhibits the ERα transactivation from the consensus ERE. In contrast, the liganded TRα1 facilitates ERβ-mediated transactivation. The crosstalk between the TRβ isoforms with the ERα isoform, on the consensus ERE, is different from that with the ERβ isoform. The use of a TRα1 mutant, which is unable to bind DNA, abolishes the ability of the TRα1 isoform to interact with either of the ER isoforms. These differences in nuclear receptor crosstalk reveal an important functional difference between isoforms, which provides a novel mechanism for neuroendocrine integration.

Differential Interaction of Estrogen Receptor and Thyroid Hormone Receptor Isoforms on the Rat Oxytocin Receptor Promoter Leads to Differences in Transcriptional Regulation

Both the estrogen receptor (ER) and thyroid hormone receptor (TR) are members of the nuclear receptor superfamily. Two isoforms of the ER, α and β, exist. The TRα and β isoforms are products of two distinct genes that are further differentially spliced to give TRα1 and α2, TRβ1 and β2. The TRs have been shown to interfere with ER-mediated transcription from both the consensus estrogen response element (ERE) and the rat preproenkephalin (PPE) promoter, possibly by competing with ER binding to the ERE or by squelching coactivators essential for ER-mediated transcription. The rat oxytocin receptor (OTR) gene is thought to be involved in several facets of reproductive and affiliative behaviors. 17β-Estradiol-bound ERs upregulate the OTR gene in the ventromedial hypothalamus, a region critical for the induction of lordosis behavior in several species. We investigated the effects of the ligand-binding TR isoforms on the ER-mediated transcription from a physiological promoter of a behaviorally relevant gene such as the OTR. Only ERα could induce the OTR gene in two cell lines tested, the CV-1 and the SK-N-BE2C neuroblastoma cell lines. ERβ was incapable of inducing the gene in either cell line. ERα is therefore not equivalent to ERβ on this physiological promoter. Indeed, in the neural cell line, ERβ can inhibit ERα-mediated induction from the OTR promoter. While the TRα1 isoform inhibited ERα-mediated induction in the neural cell line, the TRβ1 isoform stimulated induction, thus demonstrating isoform specificity in the interaction. The use of a DNA-binding mutant, the TR P box mutant, showed that inhibition of ERα-mediated induction of the rat OTR gene promoter by the TRα1 isoform does not require DNA-binding ability. SRC-1 overexpression relieved TRα1-mediated inhibition in both cell lines, suggesting that squelching for coactivators is an important molecular mechanism in TRα-mediated inhibition. Such interactions between TR and ER isoforms on the rat OTR promoter provide a mechanism to achieve neuroendocrine integration.

Crosstalk Between Oestrogen Receptors and Thyroid Hormone Receptor Isoforms Results in Differential Regulation of the Preproenkephalin Gene

Nuclear receptors are ligand‐activated transcription factors, which have the potential to integrate internal metabolic events in an organism, with consequences for control of behaviour. Previous studies from this laboratory have shown that thyroid hormone receptor (TR) isoforms can inhibit oestrogen receptor (ER)α‐mediated induction of preproenkephalin (PPE) gene expression in the hypothalamus. Also, thyroid hormone administration inhibits lordosis, a behaviour facilitated by PPE expression. We have examined the effect of multiple ligand‐binding TR isoforms on the ER‐mediated induction of the PPE gene in transient transfection assays in CV‐1 cells. On a natural PPE gene promoter fragment containing two putative oestrogen response elements (EREs), both ERα and β isoforms mediate a four to five‐fold induction by oestrogen. Cotransfection of TRα1 along with ERα inhibited the ERα transactivation of PPE by approximately 50%. However, cotransfection with either TRβ1 or TRβ2 expression plasmids produced no effect on the ERα or ERβ mediated induction of PPE. Therefore, under these experimental conditions, interactions with a single ER isoform are specific to an individual TR isoform. Transfection with a TRα1 DNA‐binding mutant could also inhibit ERα transactivation, suggesting that competition for binding on the ERE may not be the exclusive mechanism for inhibition. Data with the coactivator, SRC‐1, suggested that coactivator squelching may participate in the inhibition. In dramatic contrast, when ERβ is cotransfected, TRα1 stimulated ERβ‐mediated transactivation of PPE by approximately eight‐fold over control levels. This is the first study revealing specific interactions among nuclear receptor isoforms on a neuroendocrine promoter. These data also suggest that the combinatorics of ER and TR isoforms allow multiple forms of flexible gene regulations in the service of neuroendocrine integration.

GPR30 activation decreases anxiety in the open field test but not in the elevated plus maze test in female mice

The GPR30 is a novel estrogen receptor (ER) that is a candidate membrane ER based on its binding to 17β estradiol and its rapid signaling properties such as activation of the extracellular‐regulated kinase (ERK) pathway. Its distribution in the mouse limbic system predicts a role for this receptor in the estrogenic modulation of anxiety behaviors in the mouse. A previous study showed that chronic administration of a selective agonist to the GPR30 receptor, G‐1, in the female rat can improve spatial memory, suggesting that GPR30 plays a role in hippocampal‐dependent cognition. In this study, we investigated the effect of a similar chronic administration of G‐1 on behaviors that denote anxiety in adult ovariectomized female mice, using the elevated plus maze (EPM) and the open field test as well as the activation of the ERK pathway in the hippocampus. Although estradiol benzoate had no effect on behaviors in the EPM or the open field, G‐1 had an anxiolytic effect solely in the open field that was independent of ERK signaling in either the ventral or dorsal hippocampus. Such an anxiolytic effect may underlie the ability of G‐1 to increase spatial memory, by acting on the hippocampus.

Activation of G-protein-coupled receptor 30 is sufficient to enhance spatial recognition memory in ovariectomized rats.

In ovariectomized rats, administration of estradiol, or selective estrogen receptor agonists that activate either the α or β isoforms, have been shown to enhance spatial cognition on a variety of learning and memory tasks, including those that capitalize on the preference of rats to seek out novelty. Although the effects of the putative estrogen G-protein-coupled receptor 30 (GPR30) on hippocampus-based tasks have been reported using food-motivated tasks, the effects of activation of GPR30 receptors on tasks that depend on the preference of rats to seek out spatial novelty remain to be determined. Therefore, the aim of the current study was to determine if short-term treatment of ovariectomized rats with G-1, an agonist for GPR30, would mimic the effects on spatial recognition memory observed following short-term estradiol treatment. In Experiment 1, ovariectomized rats treated with a low dose (1 μg) of estradiol 48 h and 24 h prior to the information trial of a Y-maze task exhibited a preference for the arm associated with the novel environment on the retention trial conducted 48 h later. In Experiment 2, treatment of ovariectomized rats with G-1 (25 μg) 48 h and 24 h prior to the information trial of a Y-maze task resulted in a greater preference for the arm associated with the novel environment on the retention trial. Collectively, the results indicated that short-term treatment of ovariectomized rats with a GPR30 agonist was sufficient to enhance spatial recognition memory, an effect that also occurred following short-term treatment with a low dose of estradiol.

Activation of the G-protein coupled receptor 30 (GPR30) has different effects on anxiety in male and female mice.

The GPR30, a former orphan GPCR, is a putative membrane estrogen receptor that can activate rapid signaling pathways such as extracellular regulated kinase (ERK) in a variety of cells and may contribute to estrogens effects in the central nervous system. The distribution of GPR30 in the limbic system predicts a role for this receptor in the regulation of learning and memory and anxiety by estrogens. Though acute G-1 treatment is reported to be anxiogenic in ovariectomised female mice and in gonadally intact male mice, the effect of GPR30 activation is unknown in gonadectomised male mice. In this study, we show that an acute administration of G-1 to gonadectomised male mice, but not female mice, was anxiolytic on an elevated plus maze task, without affecting locomotor activity. In addition, though G-1 treatment did not regulate ERK, it was associated with increased estrogen receptor (ER)α phosphorylation in the ventral, but not dorsal, hippocampus of males. In the female, G-1 increased the ERK activation solely in the dorsal hippocampus, independent of state anxiety. This is the first study to report an anxiolytic effect of GPR30 activation in male mice, in a rapid time frame that is commensurate with non-genomic signaling by estrogen.