Estrogen receptor ESR1 regulates the phospholipase C-inositol phosphate signaling in the hippocampus from rats in proestrous and estrous phases

Accepted Manuscript Estrogen receptor ESR1 regulates the phospholipase C-inositol phosphate sig‐ naling in the hippocampus from rats in proestrous and estrous phases Nadia O. Maruyama, Thaís F. G. Lucas, Catarina S. Porto, Fernando M. F. Abdalla PII: DOI: Reference:

S0039-128X(12)00276-0 http://dx.doi.org/10.1016/j.steroids.2012.10.005 STE 7289

To appear in:

Steroids

Received Date: Revised Date: Accepted Date:

29 April 2012 19 September 2012 2 October 2012

Please cite this article as: Maruyama, N.O., G. Lucas, T.F., Porto, C.S., F. Abdalla, F.M., Estrogen receptor ESR1 regulates the phospholipase C-inositol phosphate signaling in the hippocampus from rats in proestrous and estrous phases, Steroids (2012), doi: http://dx.doi.org/10.1016/j.steroids.2012.10.005

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Estrogen receptor ESR1 regulates the phospholipase C-inositol phosphate 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

signaling in the hippocampus from rats in proestrous and estrous phases.

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Nadia O. Maruyama, 2Thaís F. G. Lucas, 2Catarina S. Porto and 1Fernando M. F.

Abdalla.

1

Laboratory of Pharmacology, Instituto Butantan, Brazil; 2Section of Experimental

Endocrinology, Department of Pharmacology, Escola Paulista de Medicina Universidade Federal de São Paulo, Brazil.

Address all correspondence and requests for reprints to: Fernando M. F. Abdalla Laboratory of Pharmacology, Instituto Butantan Av. Vital Brazil 1500, São Paulo, SP, 05503-900, Brazil Tel/Fax 55 11 3726 7222 e-mail: [email protected]

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Abstract 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

The aim of the present study was to investigate the involvement of estrogen receptors in the activation of phospholipase C (PLC)-phosphoinositide hydrolysis in the hippocampus from rats in estrous and proestrous phases. 17β-estradiol (E2) and ESR1selective agonist PPT, but not ESR2-selective agonist DPN, induced a rapid increase on total [3H]-inositol phosphate accumulation in the hippocampus from both rats. These effects are mediated by PLC activation, since the inhibition of this protein decreased the total [3H]-inositol phosphate accumulation. The pretreatment with ESR1 and ESR2 antagonist ICI 182,780, but not with GPER antagonist G-15, blocked the total [3H]inositol phosphate accumulation induced by E2 and PPT, confirming that ESR1 is upstream component regulating this rapid effect. SRC family of protein tyrosine kinases inhibitor PP2 blocked the total [3H]-inositol phosphate accumulation induced by E2 and PPT in hippocampus, suggesting that ESR1 undergoes translocation from the nuclei to the plasma membrane region via SRC to activate rapid signaling pathways. Furthermore, the magnitude of the response to E2 and PPT was higher in hippocampus from rats in proestrus than in estrus. On the other hand, the expression of the ESR1 is higher in hippocampus from rats in estrous than in proestrous, indicating that the regulation of this receptor by estrous cycle does not play a role in the magnitude of the response to E2 and PPT in hippocampus. In conclusion, our results indicate that E2 activates SRC-mediated translocation of ESR1 to the plasma membrane, which results in the activation of PLC-inositol phosphate signaling pathway in rat hippocampus. Thus, these rapid estrogen actions in hippocampus might be a key step mediating cellular events important for learning and memory.

Keywords: Hippocampus, 17β-estradiol, PPT, inositol phosphate, estrogen receptors 2

1. Introduction 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Endogenous and exogenous fluctuations in estrogens influence neuroplasticity and function of the hippocampus throughout the lifespan. In the female rodent estrous cycle lasts for 4-5 days, the 17β-estradiol levels increase slowly during the diestrous phase until the day of proestrous when 17β-estradiol levels rise and fall quickly. 17βestradiol levels are lowest during the estrous phase, which follows proestrous (reviewed in [1, 2]). This naturally occurring fluctuation in gonadal hormones influences neurogenesis and morphology of the hippocampus. Adult female rats have 50% more cells proliferating during proestrous compared to diestrous females, estrous females or intact male rats [3]. Female rats in late proestrous have 30% higher density of apical dendritic spines [4] and 32% more synapses [5] than rats in estrous. Furthermore, physiological levels of estrogens as seen during the different phases of the estrous cycle also alter spatial learning and memory (reviewed in [2, 6, 7]). Estrogen receptors (ERs) ESR1 and ESR2 (also known as ERα and ERβ) are present in the dendrites of hippocampal CA1 and CA3 neurons of the adult male and female rodents [8-11]. ERs may initiate both genomic and non-genomic (rapid) actions [12]. 17β-estradiol binds with nearly equal affinity to ESR1 and ESR2 [13-15]. In the rat hippocampus, ESR1 and ESR2 are differentially located at nuclear and extranuclear regions [16-18]. Furthermore, the amount of extranuclear ESR1-immunoreactivity in the rat hippocampus is sensitive to fluctuating hormone levels [9, 18]. Recent study has shown that ESR1 is more abundant during estrous in relation to other phases of the cycle in CA1 region, while it is more abundant during metestrous in CA3, indicating that there is a differential expression of ESR1 in CA1 and CA3 regions. Interestingly,

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this receptor is detected in both cytoplasmic and nuclear regions [11]. On the other 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

hand, ESR2 is not regulated in the rat hippocampus during the estrous cycle [11]. The extranuclear localization of ESR1 in the rat hippocampus and its sensitivity to fluctuating steroid levels suggests that rapid actions may be responsible for the effects of ovarian steroid hormone on hippocampal function. The rapid effects of 17βestradiol may be mediated by: 1) ESR1 and ESR2 localized at or near the plasma membrane after exposure to ligand (reviewed in [19-22]); 2) truncated variants of ESR1 called ER-46 [23] and ER-36 [24], and/or 3) G protein-coupled estrogen receptor (GPER or GPR30) (reviewed in [22, 25-27]). These rapid responses include activation of different downstream signaling pathways, for example, the mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI3K) pathways, endothelial nitric oxide synthase (eNOS) activation, cyclic adenosine monophosphate (cyclic AMP) production and intracellular calcium mobilization, which in turn can modulate nuclear transcriptional events (reviewed in [19-22, 27]). In fact, in the hippocampus, the 17β-estradiol induces activation of these different signaling pathways that play a role on cell excitability, synaptic transmission and neuroprotection (reviewed in [28, 29]). For exemple, 17β-estradiol through ESR1 and ESR2 and activation of extracellular signal-regulated kinase (ERK) increases the synaptic transmission [30]. Recent study has shown that 29% of cultured hippocampal neurons express membrane estrogen receptors. These receptors might be closely related to ESR1 and ESR2 as shown by selective ER agonists that are able to partially compete for binding. The membrane 17β-estradiol binding triggers an intracellular calcium rise that led to activation of ERK phosphorylation in individual rat hippocampal neurons [31]. Both calcium and ERK were previously reported to be crucial components in estrogen neuroprotection and neurotrophism [32, 33]. In immortalized hippocampal cell 4

lines from murine embryonic, 17β-estradiol activated both PI3K/AKT and signal 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

transducer and activator of transcription (STAT3) signaling pathways and GPER may be involved in protective effects [34]. The activation of the phosphoinositide hydrolysis may play an important role in contributing to various neuronal processes such as the changes in synaptic plasticity that underlies learning and memory (reviewed in [35]). Thus, the aim of the present study was to investigate the involvement of estrogen receptors in the activation of phospholipase C (PLC)-phosphoinositide hydrolysis in the hippocampus obtained from rats in proestrous and estrous phases.

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2. Experimental 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

2.1. Animals Female Wistar rats, 4 months-old, were maintained on a 12 h light, 12 h dark schedule, at 22ºC, with food and water ad libitum. The experimental procedures were conducted according to guidelines for the care and use of laboratory animals as approved by the Research Ethical Committee from Instituto Butantan (no. 711/10). Vaginal smears were obtained for histological determination of estrous cycle [36] between 08:00 a.m. to 10:00 a.m. Rats in proestrous and estrous were sacrificed by decapitation, and the hippocampi were isolated for in vitro studies. In rat, the plasma 17β-estradiol level reaches a peak at 6:00 a.m. on proestrous, whereas progesterone level is low. Both steroids levels are low on estrous [37]. Recent studies confirmed these results in the plasma and showed that the concentration of the 17β-estradiol in the hippocampus does not change during the estrous cycle, but is higher in hippocampus than in the plasma [1].

2.2. Measurement of total [3H]inositol phosphate Hippocampi were isolated from proestrous and estrous rats, and washed with a nutrient solution (mM): NaCl 118.00; KCl 4.78; CaCl2 2.43; MgSO4 1.16; NaHCO3 23.80; KH2PO4 1.17; glucose 2.92 (pH 7.4). The entire hippocampus from one animal of each experimental group was sliced and allowed to equilibrate for 10 min in nutrient solution at 37oC, under constant shaking. The tissues were incubated with 1 Ci of myo[3H]-inositol (specific activity 18.0 Ci/mmol; Amersham, Little Chalfont,

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Buckinghamshire, UK) for 80 min, and with lithium chloride (10 mM) for additional 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

min [38]. Afterwards, the tissues were incubated in the absence (vehicle, basal level) and presence of 17-estradiol (E2, 1 nM; Sigma Chemical Co., St Louis, MO) [39, 40], ESR1-selective agonist PPT (4,4’,4”-(4-propyl-(1H)-pyrazole-1,3,5-triyl)trisphenol, 10 nM; Tocris Bioscience, Ellisville, MI) or ESR2-selective agonist DPN (2,3-bis(4hydroxyphenyl)-propionitrile, 10 nM; Tocris Bioscience) [41] at the indicated time for each specific experiment, at 37oC. E2 was prepared in ethanol (10 M), PPT and DPN were prepared in dimethyl sulfoxide (100 mM) and diluted in phosphate-buffered saline (PBS, 137 mM NaCl, 2.68 mM KCl, 6.03 mM Na2HPO4, and 1.47 mM KH2PO4; pH 7.4). The treatment of hippocampus with each vehicle did not change the basal level of the total [3H]-inositol phosphate accumulation. In another series of experiments, the hippocampi were incubated in the absence (basal level) and presence of ESR1 and ESR2 antagonist ICI 182,780 (Fulvestrant, 1 nM, 30 min; AstraZenica, São Paulo, SP, Brazil) [39, 40], specific inhibitor of different isoforms of PLC U73122 (1-[6-{17β}-3-methoxyestra-1,3,5[10]-trien-17yl]amino)hexyl]1H-pyrole-2,5-dione, 10 nM, 20 min, Sigma) [40], selective inhibitor of the SRC family of protein tyrosine kinases PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d]pyrimidine, 10 nM, 30 min; Calbiochem, Darmstadt, Germany) [39, 40] or

GPER

antagonist

G-15

(3aS*,4R*,9bR8)-4(bromo-1,3-benzodioxol-5-yl)-

3a,4,5,9b,3H-cyclopenta[c]quinoline, 10 nM, 30 min; Tocris Bioscience) [41]. Afterwards, tissues were stimulated with 17-estradiol (1 nM, 1 min) or PPT (10 nM, 1 min) at 37oC. Tissues were washed three times with nutrient solution, transferred into 2 ml of methanol:chloroform (2:1 v/v) at 4oC and homogenized (Ultra-Turrax T25

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homogenizer, 9500 rpm). Chloroform (0.62 ml) and H2O (0.93 ml) were added, and the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

samples were centrifugated (2,000 x g, 10 min, 4oC) to separate the aqueous and organic phases [38, 42, 43]. Total [3H]-inositol phosphate was measured as previously described by Ascoli et al. (1986) with slight modification. The aqueous layer was mixed with 1 ml anion-exchange resin (Dowex AG-X8, formate form, 200-400 mesh), allowed to equilibrate for 30 min at room temperature, and centrifugated at 1,000 x g, for 5 min at 4oC. The resin was then washed sequentially with myoinositol (4 ml) and 5 mM sodium tetraborate/60 mM sodium formate (2 ml). The resin was incubated for 30 min at room temperature with 2 ml of 0.1 M formic acid/1 M ammonium formate. The total [ 3H]inositol phosphate was eluted and placed in scintillation vials containing OptiPhase HiSafe 3 (Perkin Elmer, Loughborough Leics, UK). The amount of radioactivity was determined in scintillation -counter (LS 6500 IC, Beckman Coulter, Fullerton, CA, USA). Total [3H]-inositol phosphate was expressed as % above basal level.

2.3. Western blot for ESR1 detection Western blot assays were performed as described by Lucas et al. [39]. Results were normalized to the respective actin expression.

2.4. Protein assays Protein concentration was determined with the Bio Rad protein assays, using BSA as standard (Bio Rad Laboratories, Hercules, CA).

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2.5. Drugs and reagents 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

All other drugs and reagents were obtained from Sigma Chemical Co., Bio Rad Laboratories or Merck (Darmstadt, Germany).

2.5. Statistical analysis Data are expressed as mean  S.E.M. Statistical analysis was carried out by ANOVA followed by Newman-Keuls test for multiple comparisons, or by Student t-test to compare the differences between two data [44]. P  0.05 was considered as significant.

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3. Results 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

3.1. 17β-Estradiol induces total [3H]-inositol phosphate accumulation in the hippocampus

17β-Estradiol induced a rapid time-dependent increase of total [3H]-inositol phosphate in the hippocampus from rats in estrous (Fig. 1 A) and proestrous (Fig. 1B). The maximum effect was observed at 1 min in both hippocampi (Fig. 1). The magnitude of the response to 17β-estradiol was higher in hippocampus from rats in proestrous than in estrous (54.59 ± 7.22%, n=6 and 31.50 ± 3.15% above basal levels, n=5, respectively; P  0.05) (Fig. 1C). Treatment of hippocampus with vehicle did not change the basal level of the total [3H]-inositol phosphate accumulation among the different periods of incubation (data not shown).

3.2. 17β-Estradiol and PPT induce total [3H]-inositol phosphate accumulation in the hippocampus through of the classical estrogen receptor ESR1

The ESR1-selective agonist PPT also increased of total [3H]-inositol phosphate accumulation in the hippocampus obtained from rats in estrous (Fig. 2A) and proestrous (Fig 2B). On the other hand, the ESR2-selective agonist DPN did not change the basal level of total [3H]-inositol phosphate in hippocampi from both animals (Fig 2). 10

The magnitude of the response to PPT was higher in hippocampus from rats in 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

proestrous than in estrous (47.21 ± 1.79%, n=4 and 30.65 ± 3.15% above basal levels, n=4, respectively; P  0.05) (Fig 2C). The total [3H]-inositol phosphate accumulation induced by 1-min treatment with PPT in the hippocampus obtained from rats in estrous (Fig. 3A) and proestrous (Fig 3B) was blocked by pretreatment with ICI 182,780, confirming that ESR1 is upstream component regulating this rapid effect. On the other hand, the GPER-selective antagonist G-15 did not block the effects induced by PPT. Similar results were observed when hippocampi were pretreated with these antagonists followed by 17β-estradiol (Supplemental Fig. S1A). The treatment with ICI 182,780 or G-15 in the absence of PPT did not have any effects on basal level of total [3H]-inositol phosphate accumulation (Fig. 3A and 3B).

3.3. PPT induces total [3H]-inositol phosphate accumulation in the hippocampus through nuclear export of the classical estrogen receptor and activation of PLC

To examine whether the effect of 17β-estradiol or PPT would be mediated by the classic estrogen receptor ESR1 that migrated from the nucleus to the plasma membrane after hormonal stimulation, the hippocampus was pretreated with the selective inhibitor of the SRC family of protein tyrosine kinases PP2. SRC is involved in 17β-estradiolinduced translocation of ESR1 and ESR2 to the plasma membrane region of different cells ([39, 40] reviewed in [19, 45]). PP2 blocked the total [ 3H]-inositol phosphate accumulation induced by PPT in hippocampus from rats in estrous (Fig. 4A) and

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proestrous (Fig. 4B), suggesting that ESR1 undergoes nuclear export to induce total 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

[3H]-inositol phosphate accumulation. The treatment with PP2 in the absence of PPT did not have any effects on basal level of total [3H]-inositol phosphate accumulation (Fig. 4A and 4B). The PLC inhibitor U73122 decreased the total [3H]-inositol phosphate accumulation induced by PPT in hippocampus from rats in estrous (Fig. 5A) and proestrous (Fig. 5B), indicating that PLC is involved in this intracellular signaling pathway. The treatment with U73122 in the absence of PPT did not have any effects on basal level of total [3H]-inositol phosphate accumulation (Fig. 5A and 5B). Similar results were observed when hippocampi were pretreated with these inhibitors (PP2 and U73122) followed by 17β-estradiol (Supplemental Fig. S1B).

3.4. Differential expression of ESR1 in the hippocampus by estrous cycle

ESR1 was detected in hippocampus from rats in estrous and proestrous (Fig. 6). The expression of this receptor was higher in hippocampus from rats in estrous than in proestrous (P  0.05) (Fig. 6).

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4. Discussion 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

The present study indicates that 17β-estradiol promotes rapid effects on the rat hippocampus through activation of PLC-mediated phosphoinositide hydrolysis and requires nucleocytoplasmic shuttle of the ESR1. PLC is a key enzyme, which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) into two second messengers, inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and diacylglycerol (DAG). Ins(1,4,5)P3 triggers the release of calcium from intracellular stores, and DAG mediates the activation of protein kinase C (PKC). In parallel, PI(4,5)P2 also directly regulates a variety of cellular functions, including cytoskeletal remodeling, cytokinesis, phagocytosis, membrane dynamics, and channel activity, in addition to its role as a substrate for PLC and PI3K, which generates PI(3,4,5)P3 (reviewed in [46]). PLCβ1 is expressed at high levels in the cerebral cortex and hippocampus, and it is involved in postnatal-cortical development and neuronal plasticity [47, 48], participating in neuronal function via regulation of calcium mobilization (reviewed in [46]). 17β-estradiol and the ESR1-selective agonist PPT induced a rapid increase on total [3H]-inositol phosphate accumulation in the hippocampus from rat in estrus and proestrus. These effects are mediated by PLC activation, since pharmacological inhibition of this protein with U73122 decreased the total [3H]-inositol phosphate accumulation. The pretreatment with ESR1 and ESR2 antagonist ICI 182,780 also blocked the total [3H]-inositol phosphate accumulation induced by 17β-estradiol and PPT, confirming that ESR1 is upstream component regulating this rapid effect. The ESR213

selective agonist DPN did not change the basal level of total [3H]-inositol phosphate in 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

hippocampi from both animals. It is important to emphasize that ESR1 and ESR2 also differentially regulate intracellular calcium dynamics leading to ERK phosphorylation and estrogen neuroprotection in hippocampal neurons [49-52]. GPER, which belongs to the family of seven-transmembrane G protein-coupled receptors, has been detected in hippocampal neurons [26]. The expression of this receptor (mRNA and protein) did not differ in the hippocampus during the estrous cycle [53]. GPER mediates extra-nuclear actions of 17β-estradiol, for example, the rapid mobilization of intracellular calcium stores in transfected HEK-293 cells [54]. In the present study, the antagonist of GPER (G-15) did not block 17β-estradiol- or PPTmediated total [3H]-inositol phosphate accumulation in the hippocampus from rats in proestrous and estrous, suggesting that GPER is not involve in this rapid action of 17βestradiol. It is important to emphasize that the relative binding affinity of 17β-estradiol to classical estrogen receptors is higher than to GPER [55, 56]. GPER, ESR1 and ESR2 are present in hippocampus and the pattern of the expression of these receptors in this tissue may be important for activation of PLC pathway. This aspect remains to be explored in hippocampus. Despite advances in our understanding of rapid extra-nuclear actions of ESR1 in recent years, the molecular mechanisms by which ESR1 initiates and participates in extra-nuclear signaling remains unclear. The mechanisms responsible for ERs cytoplasm/membrane localization include lipid modification of the receptor, palmitoylation, phosphorylation and interactions with membrane and cytoplasmic adaptor proteins including, caveolins, striatin, p130 Cas (Cas-associated substrate), SHC (SRC homology and collagen homology), HPIP (hematopoietic PBX interaction protein), MTA1 (metastasis-associated protein-1), MNAR (modulator of non-genomic 14

action of estrogen receptor)/PELP1 (proline-glutamic acid-, leucine-rich protein) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

(reviewed in [21, 57]). P130 Cas is a major substrate of the SRC tyrosine kinase [58, 59]. Estrogens treatment induces SHC membrane translocation, phosphorylation and binding to ESR1 ([60] reviewed in [19]). SHC also binds directly to the c-SRC kinase domain activating loop, amino acid 401-435, and activates its kinase activity [61], suggesting an alternative mechanism for ESR1-SHC complexes to activate SRC/MAPK pathway. In fact, PP2, a selective inhibitor of SRC, blocked the translocation of the classical estrogen receptors from the nuclei to the plasma membrane region in different cells [39, 40, 60]. Our study also showed that PP2 blocked the total [3H]-inositol phosphate accumulation induced by 17β-estradiol and PPT in hippocampus from rats in estrous and proestrous. Taking together, these results indicate that ESR1 undergoes translocation from the nuclei to the plasma membrane region via SRC to activate rapid signaling pathways. Although the concentration of 17β-estradiol within hippocampus is similar between rats in proestrous and estrous [1, 62] and we used the same concentration of 17β-estradiol (1 nM) to induce the total [3H]-inositol phosphate accumulation in both hippocampi, the response to 17β-estradiol or PPT was higher in hippocampus from rats in proestrous than in estrous. The regulation of the ESR1 expression by estrous cycle does not play a role in this effect, since in our study the expression of this receptor is higher in hippocampus from rats in estrous than in proestrous, confirming previous results [11]. The exact mechanisms involved in the high response to 17β-estradiol or PPT in hippocampus from rats in proestrous were not explored in the present study. Whether the affinity of the ESR1 and/or the expression of proteins involved with PLCphosphoinositide signaling pathway change during the estrous cycle, as previously shown in rat uterus during pregnancy [63-64], remain to be explored in hippocampus. 15

It is important to emphasize that the high level of total [3H]-inositol phosphate 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

induced by 17β-estradiol in hippocampus from rats in proestrous may play a role in the hippocampal plasticity and function via regulation of calcium mobilization (reviewed in [35, 46]). In mice [65] and rats [66] spatial memory is enhanced in proestrous in relation to estrous. 17β-estradiol rapidly modulates cell signaling, synaptic transmission and dendritic spine density within 1 hour of administration. Activation of signaling cascades [31, 32, 67, 68] and excitatory transmission [67, 69] are enhanced in cultured neurons or hippocampal sections within 30 min of 17β-estradiol or estradiol benzoate administration. 17β-estradiol facilitates long-term potentiation, affects long-term depression, and rapidly increases dendritic spine density and synapse number 15 min after administration, thereby enhancing neuronal connections in brain regions important for learning and memory [70 - 72]. Recent study demonstrates in vivo effects of estrogen receptor agonists on CA1 dendritic spines and learning 40 min after agonist injection. ESR1 seems to have a greater role in promoting estrogen-mediated enhancements in learning and memory processes [73]. In conclusion, our results indicate that 17β-estradiol activates SRC-mediated translocation of ESR1 to the plasma membrane, which results in the activation of PLCinositol phosphate signaling pathway in rat hippocampus. Thus, these rapid estrogen actions in hippocampus might be a key step mediating cellular events important for learning and memory.

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Acknowledgments 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grant 08/56564-1), Brazil. Research fellowship (C.S.P.) supported by Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq). Postdoctoral fellowship supported by FAPESP (T.F.G.L.). Master fellowship supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (N.O.M.).

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Figure Legends: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Fig. 1. 17β-estradiol induces total [3H]-inositol phosphate accumulation in the hippocampus from rats in estrous (A) and proestrous (B). Tissues were incubated in the absence (vehicle-treated, basal level) and presence of 17β-estradiol (1 nM) for 0.5 to 30 min. Total [3H]-inositol phosphate was measured. (C) Effects of 17β-estradiol (1 nM, 1 min) in hippocampus from rats in estrous and proestrous. The data shown are expressed as mean ± S.E.M. of 5-6 independent experiments. Different letters indicate statistical significance (P < 0.05, Newman-Keuls test). * Significantly different from estrous (P < 0.05, Student t-test).

Fig. 2. 17β-estradiol (E2) and ESR1-selective agonist PPT, but not ESR2-selective agonist DPN, induce total [3H]-inositol phosphate accumulation in the hippocampus from rats in estrous (A) and proestrous (B). Tissues were incubated in the absence (vehicle-treated, basal level) and presence of 17β-estradiol (E2, 1 nM), PPT (10 nM) or DPN (10 nM) for 1 min. Total [3H]-inositol phosphate was measured. (C) Effects of PPT (10 nM, 1 min) in hippocampus from rats in estrous and proestrous. The data shown are expressed as mean ± S.E.M. of 4-6 independent experiments. Different letters indicate statistical significance (P < 0.05, Newman-Keuls test). * Significantly different from estrous (P < 0.05, Student t-test).

Fig. 3. The classic estrogen receptor ESR1 plays a role on total [ 3H]-inositol phosphate accumulation in the hippocampus from rats in estrous (A) and proestrous (B). Tissues

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were untreated or pre-treated with ESR1 and ESR2 antagonist ICI 182,780 (ICI, 1 nM) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

or GPER antagonist G-15 (10 nM) for 30 min. Afterwards, tissues were stimulated with ESR1-selective agonist PPT (10 nM, 1 min). Total [3H]-inositol phosphate was measured. The data shown are expressed as mean ± S.E.M. of 3-5 independent experiments. Different letters indicate statistical significance (P < 0.05, Newman-Keuls test).

Fig. 4. SRC plays a role in the translocation of ESR1 from the nucleus to cell membrane, which induces total [3H]-inositol phosphate accumulation in the hippocampus from rats in estrous (A) and proestrous (B). Tissues were untreated or pretreated with inhibitor of the SRC family of protein tyrosine kinases PP2 (10 nM, 30 min). Afterwards, tissues were stimulated with PPT (10 nM, 1 min). Total [3H]-inositol phosphate was measured. The data shown are expressed as mean ± S.E.M. of 3-5 independent experiments. Different letters indicate statistical significance (P < 0.05, Newman-Keuls test).

Fig. 5. Involvement of PLC pathway on total [3H]-inositol phosphate accumulation in the hippocampus from rats in estrus (A) and proestrus (B) induced by ESR1-selective agonist PPT. Tissues were untreated or pre-treated with PLC inhibitor U73122 (10 nM, 20 min). Afterwards, tissues were stimulated with PPT (10 nM, 1 min). Total [3H]inositol phosphate was measured. The data shown are expressed as mean ± S.E.M. of 35 independent experiments. Different letters indicate statistical significance (P < 0.05, Newman-Keuls test).

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Fig. 6. Expression of the ESR1 in the hippocampus from rats in estrous and proestrous. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

(A) Total protein extracts (40 g protein/lane) obtained from hippocampi were subjected to 7.5% SDS-PAGE, transferred to PVDF membrane, and probed with rabbit anti-ESR1 antibodies (top panel). Negative controls were performed using the primary antibody preadsorbed with the respective blocking peptide (BP) (middle panel). Actin levels were monitored on the same blot to ensure equal protein loading (bottom panel). The relative positions of ESR1 and actin proteins were determined from molecular mass standards. The data shown are representative of 3 independent experiments. (B) The bars represent the densitometric analysis of the Western blot. Results were normalized to actin expression in each sample and plotted (mean  S.E.M.). * Significantly different from estrous (P < 0.05, Student t-test).

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SUPPLEMENTAL FIGURE LEGEND 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Supplemental Fig. S1. ESR1, SRC and PLC plays a role on total [3H]-inositol phosphate accumulation in the hippocampus from rats in estrous and proestrous. Tissues were untreated or pre-treated with ESR1 and ESR2 antagonist ICI 182,780 (ICI, 1 nM), GPER antagonist G-15 (10 nM) for 30 min (A), inhibitor of the SRC family of protein tyrosine kinases PP2 (10 nM, 30 min) or PLC inhibitor U73122 (10 nM, 20 min) (B). Afterwards, tissues were stimulated with 17β-estradiol (E2, 1 nM, 1 min). Total [3H]inositol phosphate was measured. The data shown are expressed as mean ± S.E.M. of 35 independent experiments. Different letters indicate statistical significance (P < 0.05, Newman-Keuls test).

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Figure

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Highlights:

17β-estradiol (E2) induce activation of the phosphoinositide hydrolysis in rat hippocampus Classic ESR1, that underwent nuclear export, are involved in these actions Regulation of ESR1 by estrous cycle does not play a role in the magnitude of the response to E2 The rapid actions might be a key step mediating cellular events important for learning and memory