Elsevier

Steroids

Volume 70, Issues 5–7, May–June 2005, Pages 388-396
Steroids

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

https://doi.org/10.1016/j.steroids.2005.02.007Get rights and content

Abstract

Estrogen is a ligand for the estrogen receptor (ER), which on binding 17β-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 Gα subunits show that Gαq 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 17β-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.

Introduction

Estrogen is a central effector of reproduction [1]. Two isoforms of the estrogen receptor (ER), α and β, are products of different genes and bind 17β-estradiol with equal affinity but have different tissue distribution [2]. Estrogen binds the ER, transforming it into a ligand-dependent transcription factor, which can regulate target genes and allow the cell to respond to various stimuli [3], [4]. This classical mode of genomic action is also complemented by an alternative non-genomic mode of steroid action, whereby estrogens acting at the membrane can rapidly elicit the activation of kinases and increase calcium within cells [5], [6] and references therein.

Lordosis, the primary female reproductive behavior in quadrupeds is exquisitely dependent on estrogen [7]. In the ventromedial hypothalamus (VMH), estrogen imposes hormonal dependence on neurons that control descending efferents to the muscles in the lower back that allow for the arching of the back, a characteristic of lordosis [7], [8], [9]. Mice that lack estrogen receptor α do not show lordosis behavior, demonstrating the importance of this receptor in lordosis [10]. Estrogen may influence lordosis through direct (gene activation) and indirect (neuronal growth) mechanisms. Some of the genes transcriptionally regulated by estrogen, which may play a role in approach, and sex behaviors in rodents include the progesterone receptor, the oxytocin receptor and its ligand, oxytocin, the preproenkephalin peptides and the α1b-adrenergic and muscarinic receptors [11], [12], [13] and references therein.

If genomic actions of estrogen are important for lordosis, do non-genomic actions of estrogen influence lordosis? Conceivably, estrogen could activate second messengers, which in turn can potentiate lordosis. In support of this idea, second messengers in the VMH facilitate lordosis. Intra-VMH infusion of a protein kinase C (PKC) agonist increased lordosis while inhibitors to PKC could reduce or inhibit lordosis [9]. Dibutryl cAMP and 8-bromo cAMP infused into the VMH could reduce the inhibition mediated by serotonin [14] suggesting that PKA activation can overcome inhibitory effects. Similarly, phorbol esters infused into the midbrain central gray could facilitate lordosis, suggesting the involvement of PKC [15]. Delta-opioid receptor agonists infused into the VMH, but not into the medial preoptic area, can increase lordosis in female rats already primed with estrogen and progesterone, demonstrating that activation of a G-protein coupled receptor (GPCR) is important in lordosis [16]. The importance of activation of kinases is underscored by the use of antagonists that reduce lordosis behavior in rodents. For example, insulin growth factor antagonists block lordosis induction by estrogen in female rats [17], [18]. Infusion of PI-3K or MAPK inhibitors into the VMH during estrogen priming attenuate lordosis [17]. This suggests that signaling cascades initiated at the membrane can influence lordosis in female rodents.

Since both genomic and non-genomic actions of estrogen are possibly important in behavior, we initially hypothesized that rapid actions of estrogen could possibly influence slower, genomic actions of estrogen to result in a unified neuroendocrine effect in the brain [19]. In order to first demonstrate a “proof of concept” result for this hypothesis ex vivo, we used transient transfections in a neuroblastoma cell line (SK-N-BE(2)C) which is devoid of endogenous ERα or ERβ isoforms. This cell line has both μ and δ-opioid receptors demonstrating that GPCR mediated receptor effects are possible in this cell line. This cell line has been used previously as a model for estrogen action in the brain [20], [21]. We adopted a novel two-pulse paradigm (see below) on transiently transfected neuroblastoma cells to mimic and separate the rapid actions of estrogen from the genomic slower actions of estrogen.

Section snippets

Physiological significance of membrane effects of estrogen potentiating later genomic effects: the choice of a two-pulse paradigm

Neuronal activity is important in the display of reproductive behavior by mammals. Roy et al. showed that if the ovariectomized rat was anaesthetized at the time of estrogen administration, mating behavior 48 h later was abolished [22]. A possible consequence of rapid membrane actions of estrogen, the loss of neuronal activity, could be a reason why anesthesia abolished mating behavior. Also, continuous estrogen exposure is not needed for estrogen-regulated physiological functions. And

The signal transduction pathways that potentiate transcription are rapid and involve kinases

The rapid membrane-limited actions of estrogen have been documented both in neurons and in non-neuronal cells and are implicated in different functions. In neurons, estrogens stimulate protein kinase C (PKC) and mitogen activated protein kinase (MAPK) activities in both cerebrocortical neurons and hippocampal neurons. This has been thought to play a role in protection against injury by beta amyloid and could protect against the pathogenic process of Alzheimer's disease [26], [27], [28]. In the

Possible models of estrogen action at the neuroblastoma cell membrane

Our data with specific inhibitors to various kinases has led to two possible models of the events that may occur when E-BSA contacts the membrane in neuroblastoma cells (Fig. 4; Models 1 and 2). These models differ in the manner of activation of MAPK. The Gβγ activated PI-3K, which in turn activates MAPK has been shown to be relevant in signaling by carbachol through the M2 receptor in this neuroblastoma cell line [40]. The activation of MAPK by c-src is possible (Model 2) and is under

The membrane estrogen receptor in neurons: is it a G-protein coupled receptor (GPCR)?

Understanding estrogen action at the membrane has been severely hampered by a lack of knowledge of the nature of the membrane ER (mER). In the absence of a cloned receptor specifically mediating membrane actions of estrogen in mammals, there have been conflicting reports on the nature of this ER, mostly based on studies involving an ER-antagonist (ICI 182 and 780). The ICI compound was unable to block E-BSA's ability to activate PKC in chrondocytes [43] or MAPK activation by E-BSA in rat

Integration of rapid membrane-initiated actions to transcription in the nucleus

How does the transduction of a signal at the membrane to transcription in the nucleus occur? This is especially unclear but interesting if the same ligand, i.e. 17β-estradiol has the ability to signal at both membrane and nucleus. For estrogen-regulated genes, such coupling from membrane to nucleus may occur at various levels such as (a) destabilization of heat shock proteins and subsequent release of the ER, (b) nuclear translocation of the ERα (c) modification of proteins other than the ERα

Lordosis in rats is possibly dependent on both genomic and non-genomic actions of estrogen in the VMH

Since the two-pulse paradigm in neuroblastoma cells directly speaks to possibly synergistic modes of estrogen action in the brain, we also tested the ability of E-BSA in the first pulse to potentiate the action of estradiol in the second pulse using cannulae into the VMH of ovariectomized female Sprague Dawley rats [75]. Different hormonal regimens administered through the cannulae are given two days before lordosis testing with the two pulses separated by 5 h. Lordosis testing consisted of

Acknowledgements

We are grateful to Dr. Pierre Chambon (Strasbourg, France) and Dr. Donald McDonnell (Duke University) for their kind gifts of the ERα expression and ERE-reporter plasmids, respectively. We also thank Dr. Benita Katzenellenbogen for helpful discussions and for phospho-mutants of the ERα.

References (81)

  • Z. Li et al.

    Calmodulin enhances the stability of the estrogen receptor

    J Biol Chem

    (2001)
  • M.J. Kelly et al.

    Rapid effects of estrogen on G protein-coupled receptor activation of potassium channels in the central nervous system (CNS)

    J Steroid Biochem Mol Biol

    (2002)
  • Y. Kuroki et al.

    Putative membrane-bound estrogen receptors possibly stimulate mitogen-activated protein kinase in the rat hippocampus

    Eur J Pharmacol

    (2000)
  • A. Nadal et al.

    The plasma membrane estrogen receptor: nuclear or unclear?

    Trends Pharmacol Sci

    (2001)
  • M.H. Wyckoff et al.

    Plasma membrane estrogen receptors are coupled to eNOS through Galphai

    J Biol Chem

    (2001)
  • P. LeGoff et al.

    Phosphorylation of the human estrogen receptor. Identification of hormone-regulated sites and examination of their influence on transcriptional activity

    J Biol Chem

    (1994)
  • G. Lazennec et al.

    Involvement of cyclic AMP response element binding protein (CREB) and estrogen receptor phosphorylation in the synergistic activation of the estrogen receptor by estradiol and protein kinase activators

    J Steroid Biochem Mol Biol

    (2001)
  • R.A. Campbell et al.

    Phosphatidylinositol 3-kinase/AKT-mediated activation of estrogen receptor alpha: a new model for anti-estrogen resistance

    J Biol Chem

    (2001)
  • A. Tremblay et al.

    Ligand-independent recruitment of SRC-1 to estrogen receptor beta through phosphorylation of activation function AF-1

    Mol Cell

    (1999)
  • P.B. Joel et al.

    Estradiol-induced phosphorylation of serine 118 in the estrogen receptor is independent of p42/p44 mitogen-activated protein kinase

    J Biol Chem

    (1998)
  • A. Pedram et al.

    Integration of the non-genomic and genomic actions of estrogen. Membrane-initiated signaling by steroid to transcription and cell biology

    J Biol Chem

    (2002)
  • E. Knobil et al.

    Encyclopedia of reproduction

    (1999)
  • G.G. Kuiper et al.

    Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors alpha and beta

    Endocrinology

    (1997)
  • S. Nilsson et al.

    Mechanisms of estrogen action

    Physiol Rev

    (2001)
  • J.A. Gustafsson

    Estrogen receptor beta—a new dimension in estrogen mechanism of action

    J Endocrinol

    (1999)
  • E. Falkenstein et al.

    Nongenomically initiated steroid actions

    Eur J Clin Invest

    (2000)
  • P.J. Davis et al.

    Comparison of the mechanisms of nongenomic actions of thyroid hormone and steroid hormones

    J Endocrinol Invest

    (2002)
  • D. Lordosis Pfaff
  • D. Pfaff

    Hormonal and environmental control of lordosis behavior: neural and molecular mechanisms

    Eur J Neurosci

    (1998)
  • S. Ogawa et al.

    Roles of estrogen receptor-alpha gene expression in reproduction-related behaviors in female mice

    Endocrinology

    (1998)
  • D.W. Pfaff et al.

    Genetic mechanisms in neural and hormonal controls over female reproductive behaviors.

  • D. Pfaff et al.

    Genetic influences on neural and behavioral functions

    (1999)
  • A.M. Etgen et al.

    Participation of growth factor signal transduction pathways in estradiol facilitation of female reproductive behavior

    Endocrinology

    (2003)
  • E.M. Apostolakis et al.

    Epidermal growth factor activates reproductive behavior independent of ovarian steroids in female rodents

    Mol Endocrinol

    (2000)
  • N. Vasudevan et al.

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

    Proc Natl Acad Sci USA

    (2001)
  • C. Patrone et al.

    Divergent pathways regulate ligand-independent activation of ER alpha in SK-N-BE2C neuroblastoma and COS-1 renal carcinoma cells

    Mol Endocrinol

    (1998)
  • J. Harris et al.

    Evidence for a discontinuous requirement for estrogen in stimulation of deoxyribonucleic acid synthesis in the immature rat uterus

    Endocrinology

    (1978)
  • B. Parsons et al.

    A discontinuous schedule of estradiol treatment is sufficient to activate progesterone-facilitated feminine sexual behavior and to increase cytosol receptors for progestins in the hypothalamus of the rat

    Endocrinology

    (1982)
  • Y. Taguchi et al.

    Binding of estrogen receptor with estrogen conjugated to bovine serum albumin (BSA)

    Nucl Recept

    (2004)
  • N. Linford et al.

    The rapid effects of estrogen are implicated in estrogen-mediated neuroprotection

    J Neurocytol

    (2000)
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