Integration of steroid hormone initiated membrane action to genomic function in the brain
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.
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2020, PsychoneuroendocrinologyCitation Excerpt :E2-BSA was confirmed to be specific for mER but not for nER; also MIES activation induced an acute stimulation of CRH secretion while NIES caused a delayed upregulation of CRH (Fig. 1I, J). We applied columns to elute any free E2 (see Supplementary Methods) and confirmed that the ratio of free E2 to E2-BSA in E2-BSA is so low that the effect of free E2 dissociated from E2-BSA is negligible (Vasudevan et al., 2005). We confirmed that E2-BSA increased NOS1 mRNA but not NOS2 mRNA nor NOS3 mRNA (Gingerich and Krukoff, 2008).
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