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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online August 2, 2004 as doi:10.1096/fj.04-1495fje. |
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Laboratories of
* Experimental Endocrinology,
Pharmacology,
Biochemistry, and
Pathology, University of Crete, School of Medicine, Heraklion, Greece
1Correspondence: Laboratory of Experimental Endocrinology, University of Crete, School of Medicine, P.O. Box 2208, Heraklion, GR-71003, Greece. E-mail: castanas{at}med.uoc.gr
SPECIFIC AIMS
The neuroprotective role of estrogen (E2) is supported by a multitude of experimental and epidemiological data. Nevertheless, their mode of action has not been clear until now. E2 actions were examined in various systems; mechanisms proposed to explain their action include 1) action through classical estrogen receptors (ER) either 
or ß; 2) antioxidant activity of E2, due primarily to the existence of an OH moiety on the C3 position of the A ring; and 3) action through a membrane binding component, either a true binding site, distinct from intracellular ER, or implying anchorage of ER at the plasma membrane level, either through post-translational modifications, or through an interaction with not yet fully characterized integrated membrane proteins. In the present study we provide new insight about the cytoprotective, antiapoptotic effect of E2. We used rat pheochromocytoma cell line PC12 and serum deprivation as an apoptotic stimulus. We investigated the antiapoptotic effect of E2 and its possible mediation through classical (intracellular) and alternative (membrane) steroid receptors. Our results indicate the presence of membrane binding sites for estrogen on PC12 cells. We provide evidence that E2 can prevent serum deprivation-induced PC12-cell apoptosis through interaction with these membrane sites and downstream activation of the Ca2+/Akt/NOS pathway.
PRINCIPAL FINDINGS
1. Estrogen protects PC12 cells against serum deprivation-induced apoptosis
Serum deprivation of PC12 cells induced apoptosis and incubation of cells with E2 completely reversed apoptosis within 24 h. This effect is shared by E2-BSA, but not by progesterone and testosterone, indicating its E2-specificity. The specific ER antagonist ICI 182780 had a minor effect on the antiapoptotic effect of E2 but did not affect that of E2-BSA. The IC50 of the antiapoptotic effect of E2 was 2.4 ± 0.5 and 18.7 ± 3.8 nM in the absence and presence of ICI 182780, respectively. The IC50 of the antiapoptotic effect of BSA-bound E2 was not significantly different in the absence (23.1±2.9 nM) or presence of anti-ER compound ICI 182780 (18.3±4.5 nM).
The antiapoptotic effect of E2 could not be explained by its antioxidant capacity. 10–7 M of testosterone and progesterone, which were both ineffective in protecting serum-starved PC12 cells, highly reduced ROS production, while cytoprotective estradiol at 10–7 M showed a much lower potency in reducing ROS production.
2. Detection of intracellular steroid receptors in PC12 cells
Saturation binding experiments in PC12 cells revealed presence of low concentrations of estrogen-selective receptors (52,500 sites/cell, KD 4.7±0.9 nM). No specific binding was observed for progesterone and androgens. Immunocytochemistry with an antibody directed against the N-terminal of the ER revealed cytoplasmic and nuclear staining for ER in PC12 cells. An alternative explanation of the antiapoptotic effect of estradiol in our system could be its binding to the Aryl-hydrocarbon receptor (AhR). We investigated the possible competition of radiolabeled 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by E2. Our results showed that TCDD could be completely displaced by estradiol, indicating that TCDD could bind to estrogen receptors in PC12 cells. Absence of AhR in this cell line was confirmed by absence of ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) activity of CYP1A1 and CYP1A2 (known targets of AhR activation). It is possible that TCDD binds to conventional intracellular ER present in these cells. This hypothesis is also supported by the fact that TCDD acts as an antagonist of ER and partially reversed the cytoprotective effect of both E2- or E2-BSA.
3. Detection of membrane steroid receptors
The effectiveness of E2-BSA to protect serum-starved PC12 cells from apoptosis supports the involvement of membrane binding of E2. We assayed the presence of membrane binding sites for estrogen using a variety of methods.
Membrane binding studies
Binding experiments of tritiated estradiol on acid-treated membrane preparations of PC12 cells reveal the presence of membrane binding sites. Progesterone and testosterone did not show any specific binding, indicating the absence of membrane sites for these classes of steroid hormones. Detected sites were of high affinity for homologous ligands (KD 10.55 nM, binding capacity 279.4 fmol/mg protein) and were selective for estrogen.
Flow cytometry-confocal microscopy
Existence of membrane sites for estradiol was further confirmed by use of fluorescent analogs of BSA-bound E2 and flow cytometry. This binding is specific, compared with the binding of fluorescent-labeled BSA and is saturable, since coincubation of cells with a 100-fold molar excess of the homologous ligand completely cleared the specific fluorescence. Membrane localization of estradiol- BSA binding was confirmed by confocal laser microscopy.
Calcium flow
An early effect of the activation of membrane steroid sites is the increase of intracellular calcium levels 120 s after application of estrogen, a substantial increase of intracellular calcium was observed. Addition of pure antiestrogen ICI 182780 did not produce any effect on Ca2+ flow in PC12 cells. It did not modify the rise of intracellular calcium induced by estrogen. It appears that intracellular stores of Ca2+ are also involved in the rapid increase of intracellular calcium induced by estrogen, since a comparable increase of Ca2+ is observed after E2 application in cells incubated in a Ca-free medium.
4. Early signaling effects occurring after membrane estradiol binding
We also tested the effect of estradiol on Ca2+-related signaling molecules associated with cell apoptosis and survival. Our findings show that the rise of intracellular Ca2+ at 2 min, was followed by a transient increase of phosphorylated PI3K at 5 min. This rise was followed (at 10 min) by an increased phosphorylation of PKB/Akt which returned to basal levels after 30 min. Antiestrogen ICI 182780 had no effect in these early effects, indicating that at least in early stages of non-genomic E2 actions, its effect is not antagonized by antiestrogens and is reverted by addition of specific antagonists (wortmannin, L-NAME). Akt phosphorylation/activation results in NOS activation. Based on these findings, we hypothesize that the following sequence of events is taking place in serum-deprived PC12 cells: activation by E2 of specific membrane binding sites
Ca2+ mobilizationPI3K activationAkt phosphorylationNOS activation (Fig. 1
).
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CONCLUSIONS AND SIGNIFICANCE
Estrogens are pleiotropic factors for the brain. Estrogens and ER have been discovered in a number of CNS structures. Because the majority of E2 effects in the CNS are stimulatory and beneficial for cell survival, several studies have suggested that estrogens are neuroprotective factors. Although much work has been conducted in order to elucidate the E2 mode of action in neuronal cells, its mechanism(s) of action remain speculative, which is most probably due to the complexity of the CNS. For this reason, simpler models have been proposed to identify and characterize the underlying mechanisms of its neuroprotective properties. These systems include established cell lines from a number of brain regions and species as well as the PC12 adrenomedullary cell line. Adrenal medulla derives from neuronal crest and is an integral component of the sympathetic nervous system, expressing a mixed neuronal-epithelioid phenotype. It has been extensively used as a model in the study of sympathetic nervous system physiology. We used this model to study the neuroprotective role of estrogen and used serum-deprivation manipulation to challenge cell survival.
Serum-deprivation results in induction of apoptosis. E2 specifically reversed apoptosis in a receptor-mediated manner, since other steroids were ineffective. Testosterone and progesterone were both ineffective in rescuing serum-deprived PC12 cells. The cytoprotective effect of E2 was very effective compared with that of serum supplementation and was not affected by the pure ER antagonist ICI 182870, suggesting that conventional intracellular ER does not mediate this action. In the present study we reported expression of estrogen-specific membrane binding sites on PC12 cells. Accumulating evidence suggests a complementary mechanism of steroid action occurring rapidly via steroid receptors located on the cell membrane. Steroid hormones are shown to elicit rapid induction of intracellular Ca2+ levels within seconds, due to a significant release of Ca2+ from intracellular Ca2+ stores or to the import of extracellular Ca2+ through Ca2+ channels. Estrogen membrane binding was shown by means of radioligand binding assays, flow cytometry, and confocal laser microscopy of E2-BSA-FITC. This site is specific for estrogen, since progesterone and testosterone did not displace radiolabeled E2. Our findings reveal for the first time the presence of specific E2 membrane binding on PC12 cells. This observation is supported by a recent report showing guanylate cyclase stimulation by E2, implicating a GPCR mechanism of action. According to the Mannheim criteria, a non-genomic effect may occur within minutes, being present in cells not expressing classical steroid receptors, and being insensitive to inhibitors of transcription and translation. Activation of these receptors might be triggered by nonpermeable steroids (e.g., BSA-coupled) and has to be insensitive to steroid antagonists. The identified estrogen membrane binding component on PC12 cells fulfils the criteria and can be suggested as a true binding site that mediates early effects of E2.
We provide evidence that E2 induces Ca2+ influx in the cytosol in an antiestrogen-independent manner and induces a subsequent rapid activation of PI3K/Akt/NOS system. Ca2+ activation induced PI3K activation, leading to an increase of Akt/NOS activity in an antiestrogen-independent manner. The early effect of E2 on Ca2+ mobilization includes, in addition to the influx of extracellular calcium, an intracellular component since it is reproduced in a Ca2+-free medium. The crucial role of NOS in early effects of E2 is supported by the fact that NOS inhibitors almost completely reversed the cytoprotective effect of E2. PI3K activation after E2 stimulation has been previously reported in arterial endothelial cells and is implicated as a protective factor in the ischemia-reperfusion model. Ca2+ flux and PI3K activation have also been considered as pivotal for integration of nongenomic actions of steroids. Various authors have reported interaction of E2 with the Akt/NOS pathway in different systems (including the CNS) as an explanation of the rapid effects of E2 and have suggested it as a possible mechanism of endocrine therapy for human tumors. Rapid activation of eNOS after E2 application through genomic and nongenomic mechanisms has also been reported. It has been proposed that this action is mediated in caveolae formed after E2 rapid action and through classic intracellular ER since it is inhibited by pure antiestrogens. Our data point toward a mechanism of action. ICI 182780 did not prevent activation of this pathway, suggesting that antiestrogen insensitive membrane receptors may be implicated in the phenomenon.
Elucidation of the mode of action of estrogens in neural cells presents a major challenge in endocrine pathophysiology. E2 promotes cell survival and differentiation of fetal and adult brain tissue. A number of clinical and epidemiological studies characterize this hormone as a protective factor in a number of acute (stroke) and chronic neurodegenerative disorders (Alzheimer’s disease) and schizophrenia. We report here for the first time the existence of E2 specific membrane binding sites on neuronal-crest-derived PC12 cells that are strongly related to the cytoprotective action of E2 in this system. Presence of E2 membrane receptors in the rat brain, especially in the hippocampal region which is strongly related to memory and senescence, indicates that membrane estrogen binding may also contribute to the protective effects of E2 in the nervous system.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1495fje;
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