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Lambda-cyhalothrin disrupts the up-regulation effect of17β-estradiol on post-synaptic density 95 protein expressionvia estrogen receptor α-dependent Akt pathway

Qunan Wang1,2, Xin Xia2, Xiaomei Deng3, Nian Li2, Daji Wu2, Long Zhang2,Chengwei Yang2, Fangbiao Tao4, Jiangning Zhou1,⁎

1. Chinese Academy of Science Key Laboratory of Brain Function and Diseases, School of Life Sciences, University of Science and Technology ofChina, Hefei 230027, Anhui, China. E-mail: wqn@ahmu.edu.cn2. Department of Toxicology, College of Public Health, Anhui Medical University, Hefei 230032, China3. Department of Pharmacy, Affiliated Anhui Provincial Hospital, Anhui Medical University, Hefei 230001, China4. Department of Maternal and Child health, College of Public Health, Anhui Medical University, Hefei 230032, China

A R T I C L E I N F O

⁎ Corresponding author. E-mail: jnzhou@ustc

http://dx.doi.org/10.1016/j.jes.2015.04.0371001-0742/© 2015 The Research Center for Ec

A B S T R A C T

Article history:Received 13 March 2015Revised 30 March 2015Accepted 2 April 2015Available online 11 November 2015

Lambda-cyhalothrin (LCT), one of the type II pyrethroids, has been widely used throughoutthe world. The estrogenic effect of LCT to increase cell proliferation has been wellestablished. However, whether the estrogenic effect of LCT will influence neuro-development has not been investigated. In addition, 17β-Estradiol (E2) plays a crucial rolein neurodevelopment and induces an increase in synaptic proteins. The post-synapticdensity 95 (PSD95) protein, which is involved in the development of the structure andfunction of new spines and localized with estrogen receptor α (ERα) at the post-synapticdensity (PSD), was detected in our study by using hippocampal neuron cell line HT22. Wefound that LCT up-regulated PSD95 and ERα expression, estrogen receptor (ER) antagonistICI182,780 and phosphatidylinositol-4; 5-bisphosphate 3-kinase (PI3K) inhibitor LY294,002blocked this effect. In addition, LCT disrupted the promotion effect of E2 on PSD95. Toinvestigate whether the observed changes are caused by ERα-dependent signalingactivation, we next detected the effects of LCT on the ERα-mediated PI3K-Protein kinase B(PKB/Akt)-eukaryotic initiation factor (eIF) 4E-binding protein 1 (4E-BP1) pathway. Thereexisted an activation of Akt and the downstream factor 4E-BP1 after LCT treatment. Inaddition, LCT could disrupt the activation effect of E2 on the Akt pathway. However, nochanges in cAMP response element-binding protein (CREB) activation and PSD95messengerribonucleic acid (mRNA) were observed. Our findings demonstrated that LCT could increasethe PSD95 protein level via the ERα-dependent Akt pathway, and LCT might disrupt theup-regulation effect of E2 on PSD95 protein expression via this signaling pathway.© 2015 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences.

Published by Elsevier B.V.

Keywords:17β-EstradiolAkt pathwayInterferenceLambda-cyhalothrinPost-synaptic density 95

.edu.cn (Jiangning Zhou).

o-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V.

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Introduction

Pyrethroids have been widely used in the last five decades inagriculture, household insect control and public health all overthe world, due to their broad spectrum and high-efficiencyinsecticidal effect (Butenhoff et al., 2006; Elliott et al., 1978;Hirano, 1989). This extensive usage has led to an increasedexposure of the general population to pyrethroids and causeddamage to human health (Alavanja et al., 2004). Themain effectof pyrethroids is to disrupt the voltage-gated sodium channelsand voltage-gated calciumchannels, causing thedepolarizationof nerve membranes and the release of neurotoxin (Kadala etal., 2014; Shafer & Meyer, 2004; Soderlund, 2012; Wang &Wang,2003). Othermechanisms of pyrethroids, including their actionson voltage-gate chloride channels and GABAA receptors, havealso been proposed (Soderlund, 2012). Massive evidence hasdemonstrated that the nervous system of mammals could alsobe affected by pyrethroids (Casida & Durkin, 2013; Casida et al.,1983). Impairments in memory and cognition with a loss ofhippocampal neuronswere observedwhen treating prenatal andearly postnatal rats with pyrethroids (Sinha et al., 2006). In an invivo study, pyrethroids also induced spatial learning andmemorydeficits with a decrease in presynaptic proteins, includingN-methyl-D-aspartate receptor 1 (NMDAR1), synaptophysin,and 6synapsin I (Chen et al., 2012). However, contemporaneousstudies demonstrated an opposite effect of pyrethroids onneurodevelopment. Neurotrophic effects of pyrethroids onneurodevelopment, especially on the increase of spine densityand length, have also been reported (Ihara et al., 2012; Ihara et al.,2009; Matsuya et al., 2012; Takasaki et al., 2013).

The formation and function of new spines require thesynthesis of new proteins at the postsynaptic density (PSD)(Steward & Schuman, 2001). One fundamental structural proteinis the postsynaptic density 95 (PSD95) protein, which is involvedin the maturation of excitatory synapses and localized withthe estrogen receptor α (ERα) at PSD of the hippocampal neurons(McEwen et al., 2001). After exposure to pyrethroids, thePSD95 expression increased unexpectedly (Chen, et al., 2012;El-Husseini et al., 2000). However, the promoting mechanisms ofthese pyrethroids on neurodevelopment have not been clearlyexplored.

It has been well documented that 17β-Estradiol (E2) promotesneurodevelopment and enhances synaptic protein expression(Inagaki et al., 2012; Tange et al., 2014). E2 binds to estrogenreceptor (ER) to activatenuclear- ormembrane-initiated signalingto regulate synaptogenesis (Akama & McEwen, 2003; Roepke etal., 2009; Yang et al., 2010). E2 binding to estrogen nuclearreceptors and inducing messenger ribonucleic acid (mRNA)transcription is the classical estrogen signaling (Jin et al., 2010),which promotes neurodevelopment (Mamounis et al., 2014;Sawyer et al., 2006); and in ER-mediated rapid signaling, thephosphatidylinositol-4, 5-bisphosphate 3-kinase (PI3K)-Proteinkinase B (PKB/Akt) signaling pathway plays an important role inpromoting PSD95 expression, which could be inhibited by ICI182,780, an ER antagonist (Akama & McEwen, 2003; Vasudevan& Pfaff, 2007). Akt is one of the E2-responsive signalingintermediates which can regulate protein translation viamammalian targeting of the rapamycin (mTOR) signalingpathway (LoPiccolo et al., 2008), and phosphorylated Akt results

in the activation of eukaryotic initiation factor (eIF) 4E-bindingprotein 1 (4E-BP1), which relieves the translational repression(She et al., 2010).

Lambda-cyhalothrin (LCT), a type II synthetic pyrethroid, hasbeen extensively used in public health. Learning and memoryimpairments have been detected when rats are exposed to LCT(Ansari et al., 2012). In addition, LCT also displayed an estrogeniceffect and promoted MCF-7 cell proliferation, but ICI182,780could block this effect (H. Chen et al., 2002; Zhao et al., 2008).Furthermore, the reporter gene assays in CHO cells illustratedthat LCT affected ERα-dependent signaling (Du et al., 2010).However, the effects of LCT on neurodevelopment have notbeen explored and themechanisms have not been demonstrat-ed. We hypothesized that LCT influences the cytoskeletonprotein PSD95 expression via ERα dependent signaling in HT22cells.

1. Materials and methods

1.1. Materials

17β-Estradiol (E2), ICI182,780 and LY294,002 were purchasedfrom TOCRICS Bioscience (Ballwin, MO, USA). LCT wasobtained from Sigma-Aldrich (St. Louis, MO, USA). ECL andloading buffer were purchased from Tanon Science & Tech-nology (Shanghai, China). Anti-phosphorylated Akt antibody,anti-Akt antibody, anti-phosphorylated 4E-BP1 antibody andanti-4E-BP1 antibody were purchased from Cell SignalingTechnology (Beverly, MA, USA). Anti-phosphorylated CREBantibody and anti-CREB antibody were purchased fromMillipore (Billerica, MA, USA). Anti-ERα antibody, anti-PSD95antibody and anti-glyceraldehyde-3-phosphate dehydroge-nase (GAPDH) antibody were purchased from Santa CruzBiotechnology (Santa Cruz, USA). The CCK-8 Cell CountingKit was obtained from Beyotime Biotechnology (Jiangsu,China).

1.2. Cell culture and treatment

The HT22 cell line is a subclone of HT4 (Morimoto & Koshland,1990), which is derived from themouse hippocampus (Li et al.,1997). The HT22 cell line (Cat No. 10122101, Jennio Biotech,Guangzhou, China) was obtained as a gift from Prof. Zhai(Department of Occupational and Environmental Health,Anhui Medical University, Anhui, China). Cells were culturedin Dulbecco's modified Eagle's medium (DMEM, Hyclone, USA)with 10% fetal bovine serum (FBS, Hyclone, USA) and 1%penicillin/streptomycin (Beyotime, China) in the presence of5% CO2 at 37°C to allow cells to be 80% confluent. To assay E2and LCT effects, cells were cultured in phenol red-freeNeurobasal media (GIBCO, USA) containing 2% B-27 supple-ment (GIBCO, USA), 1% L-Glutamine (Sigma-Aldrich, USA) and1% penicillin/streptomycin (Beyotime Biotechnology, China) for24 hr before adding E2 or/and LCT.When detecting the effect ofER inhibiters, ICI 182,780 was added 30 min before E2 or/andLCT incubation. After being incubated for 24 hr, the cells wereharvested for subsequent studies. LCT, E2, ICI182,780 andLY294,002 were dissolved in dimethyl sulfoxide (DMSO)(Sigma-Aldrich, USA).

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Fig. 1 – Viability assay of HT22 cells after LCT or/and E2treatment. The HT22 cells were treated with E2 (10 nmol/L),LCT (2, 10, 50 μmol/L), ICI182,780 (1 μmol/L), LY294,002(1 μmol/L), E2 (10 nmol/L) + ICI (1 μmol/L), E2 (10 nmol/L) +LY (1 μmol/L), LCT (50 μmol/L) + ICI (1 μmol/L), LCT(50 μmol/L) + LY (1 μmol/L) and LCT (50 μmol/L) + E2(10 nmol/L), respectively, for 24 hr. There was no significantdifference between the groups in cell viability, whichremained more than 80% in all treatments (n = 9). DMSO:dimethyl sulfoxide; E2: 17β-Estradiol; LCT:lambda-cyhalothrin; ICI: ICI182,780; LY: LY294002.

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1.3. Cell viability assay

Cells cultured in 96-well plates were treated with DMSO,10 nmol/L E2, 2–50 μmol/L LCT, or the combination of 50 μmol/LLCT and 10 nmol/L E2 (LCT + E2) for 24 hr before adding 10 μLCCK-8 detecting solution, and incubated for 1 hr at 37°C. Theoptical density was measured using a Biotek Synergy 4 platereader (Biotek, Vermont, USA) at the absorption wavelength of450 nm.

1.4. Immunocytochemistry

For immunocytochemistry staining, cells were fixed inice-cold 4% paraformaldehyde in phosphate buffered saline(PBS) for 20 min, permeabilized with 0.1% Triton X-100 in PBSfor 10 min, blocked with 5% goat serum at 37°C for 30 min andthen incubated with primary antibody (1:200 in PBS buffer) at4°C overnight. The next day, the cells were rinsed with PBSthree times and incubated with the fluorophore-conjugatedsecondary antibodies Cy3 goat anti-rabbit IgG and FITC goatanti-mouse IgG (1:200 in PBS buffer, Molecular Probes, USA) for2 hr. After rinsing with PBS, 50 μg/mL DAPI solution was usedto stain the nucleus for 10 min. Finally, cells were observedusing an Olympus IX71 fluorescence microscope (IX71, Olym-pus, Japan) and the LSM570-metalaser scanning confocalmicroscope (LSM570, Carl Zeiss, Germany). All the imageswere analyzed with MacBIOphotonics ImageJ software (http://rsb.info.nih.gov/ij/).

1.5. Western blotting analysis

Cells were washed three times with ice-cold PBS and lysed inRIPA buffer (in mmol/L) (20 Tris-HCl (PH 7.5), 150 NaCl, 1Na2EDTA, 1 EGTA, 1% Triton X-100, 100 NaF, 1 Na3VO4) with1% PMSF for 5 min. The lysates were centrifuged at 15,000 g at4°C for 10 min. The supernatants were collected and proteinconcentrations were determined using the BCA protein assaykit (Thermo Fisher Scientific, USA). Cell lysates (30 μg/lane)were electrophoresed on 10% SDS-polyacrylamide gel electro-phoresis (PAGE), and transferred onto PVDFmembranes. Thenthe membrane was blocked with 5% skim milk at roomtemperature for 2 hr, incubated with specific primary anti-bodies (1:1000 in PBS buffer) at 4°C overnight; the membranewas then incubated with HRP-conjugated secondary antibody(1:50,000 in 5% skim milk, Santa Cruz, USA) at roomtemperature for 2 hr. The GAPDH was used as the internalcontrol. A digital gel image analysis system (Fine-do X6,Tanon, China) was used for protein quantification.

1.6. Quantitative polymerase chain reaction (Q-PCR)

The total RNA was extracted using an Eastep™ universal RNAextraction kit (Promega, USA), performed according to thetechnological manual. The ratio of OD 260/OD 280 was used todetermine the quality of total RNA. First-strand cDNAwas obtained by reverse transcription using oligo-dT primerfrom 2 μg of total RNA by a Reverse Transcription System kit(Promega, USA). For Q-PCR, the following primers were used.PSD95: Forward (-F) 5′-CGA CGA GAG TGG TCA AGG TT-3′ andReverse (-R) 3′-GTT GGC ACG GTC TTT GGT AG-5′. GAPDH:

Forward (-F) 5′-ACC CCA GCA AGG ACA CTG AGC AAG-3′ andReverse (-R) 3′-GGC CCC TCC TGT TAT TAT GGG GGT-5′. ThePCR was run on a ROCHE LightCycler 480 Real Time PCR(Roche Diagnostics) apparatus using GoTaq® qPCRMaster Mixkit (Promega, USA). The qPCR conditions were as follows:initiate denaturation at 95°C for 2 min, denaturation at 95°Cfor 30 sec, annealing at 59°C for 30 sec, and extension at 72°Cfor 30 sec, reaction with 40 cycles, and melting at 95°C for15 sec, 60°C for 1 min, and 95°C for 15 min. The comparativeCT-method was used to determine the amount of target,normalized to the endogenous reference (GAPDH) and relativeto a calibrator (2−△△Ct) using the ROCHE LightCycler 480 RealTime PCR software (Roche, version 1.5.0.)

1.7. Statistical analysis

The data were collected with Microsoft Office software andanalyzed using one way analysis of variance (ANOVA) withGraphPad prism 5 Software (USA), and Bonferroni's test wasused as a post hoc test. p < 0.05 was considered significant. Allvalues were expressed as Mean ± SD.

2. Results

2.1. LCT did not influence HT22 cell viability

Recent studies demonstrated that LCT could affect cell viability(Laffin et al., 2010; Saleem et al., 2014; Zhao et al., 2010). To verifywhether LCThas any cytotoxicity toHT22 cells, we exposedHT22cells to different concentrations of LCT (2–50 μmol/L) or E2(10 nmol/L) or DMSO for 24 hr before the cell viability assayswere performed. The results showed that the LCT or/and E2treatment groupshadno significant difference in the cell viabilitycompared to the DMSO group (Fig. 1). When ICI182,780 and

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LY294,002 were added 30 min before LCT or E2 treatment, theydid not show any significant effect on the cell viability (Fig. 1).

2.2. LCT disrupted the up-regulation effect of E2 on PSD 95protein levels

To investigate the effects of LCT and E2 on the PSD95 expression,HT22 cellswere treatedwith LCT (50 μmol/L) or E2 (10 nmol/L) for24 hr. The western blotting and immunocytochemistry analyseswere performed. First, we confirmed that PSD95 existed in theHT22 cells (Fig. 2a). Treatment with LCT or E2 could significantlyup-regulate PSD95 expression (Fig. 2b, c, d). Both ICI182,780 and

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Fig. 2 – Changes of PSD95 protein expression after LCT or/and E2Red: PSD95. Green: Tuj. Blue: DAPI. Bar = 50 μm, (b) E2, LCT, ICI1immunocytochemistry method. Red: PSD95. Blue: DAPI. Bar = 50Akt signalingwas involved in PSD95 expression. *p < 0.05 compacompared to LCT group (n = 5). PSD95: postsynaptic density 95; G

LY294,002 blocked this process (Fig. 2b, c, d). Interestingly, whenHT22 cells were treated with LCT + E2, the PSD95 protein levelshad no significant difference compared to the DMSO group(Fig. 2b, c, d). Moreover, the PSD95 expression decreased com-pared to E2 or LCT treatment groups, suggesting that LCT coulddisrupt the promoting effect of E2 on the PSD95 protein level.

2.3. LCT disrupted the up-regulation effect of E2 on ERαexpression levels

ERα is localized with PSD95 at the PSD, and the fact that E2increases ERα level has been well established (Z. Chen et al.,

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red to DMSO group, #p < 0.05 compared to E2 group, & p < 0.05APDH: glyceraldehyde-3-phosphate dehydrogenase.

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Fig. 3 – Changes of ERα expression after LCT or/and E2 treatment. (a) Effect of ICI182,780 on ERα expression and (b) effect ofLY294,002 on ERα expression. *p < 0.05 compared to DMSO group, #p < 0.05 compared to E2 group, &p < 0.05 compared to LCTgroup (n = 5). ERα: estrogen receptor α.

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1999; McEwen, et al., 2001). Recent study has also demon-strated that LCT can competitively bind to ERα (Saito et al.,2000). Next we sought to examine whether ERα is involved inthe process of LCT-induced PSD95 expression. In the study,both LCT and E2 were found to be able to enhance ERαexpression, respectively, and could both be blocked byICI182,780 and LY294,002 (Fig. 3). Meanwhile, when HT22 cellswere treated with LCT + E2, ERα levels were not up-regulatedcompared to the DMSO or E2 groups (Fig. 3). These resultssuggested that interactions between LCT and E2 may affect ERαexpression in HT22 cells.

2.4. LCT had no significant effects on PSD95 mRNA level

Protein expression can be regulated at transcription andtranslational levels. To investigate whether LCT or E2 affectPSD95 at the transcriptional level, cells were exposed to E2 or/and LCT for 24 hr, then PSD95 expression levels were assessedby Q-PCR. Fig. 4 shows that there was no significant differenceamong the treatment groups, suggesting that LCT may notinfluence ERα-dependent nuclear signaling.

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Fig. 4 – Changes of PSD95 mRNA expression after LCT or/andE2 treatment.

2.5. LCT disrupts E2-mediated and ERα-dependent Aktpathway activation

E2 activates ERα-mediated membrane rapid signaling (Campbellet al., 2001), which affects the phosphorylation of Akt and 4E-BP1(Akama&McEwen, 2003). To test this, cellswere treatedwith LCTor E2, and the results showed that LCT or E2 indeed increasedAktand 4E-BP1 phosphorylation, and all these effects could beblocked by ICI182,780 and LY294,002 (Fig. 5). However, whencells were treated with LCT + E2, the phosphorylation of Akt and4E-BP1 decreased compared to the E2 or LCT group, but remainedunchanged compared to the DMSO group (Fig. 5). These resultssuggested that LCT could activate the Akt signaling pathwayto increase PSD95 translation, and LCT could disrupt theERα-dependent Akt pathway activation mediated by E2.

2.6. LCT had no significant effects on CREB phosphorylation

Another Akt downstream signaling factor cAMP responseelement binding protein (CREB) also plays an important role inneurodevelopment (Pugazhenthi et al., 2000). However, in ourstudy, there were no changes on CREB phosphorylation afterLCT or E2 exposure (Fig. 6).

3. Discussion

In this study, we used mouse hippocampal cell line HT22cells to investigate the mechanisms of LCT contributing toneurodevelopment. Our results demonstrated a potentialpathway of LCT affecting PSD95 expression that could disruptE2 function. LCT may affect ERα expression and activateERα-dependent rapid signaling to up-regulate PSD95 expres-sion, which shows a similar effect to that of E2 (Fig. 7). Inagreement with a previous study, E2 mediation of PSD95expression ismainly via ER rapid signaling (Akama &McEwen,2003). However, LCT disrupted the promoting effect of E2 onPSD95 expression as an anti-estrogen agent, although LCT orE2 could up-regulate PSD95 protein level respectively.

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Fig. 5 – Rapid signaling activated after LCT or/and E2 treatment in HT22 cells. (a) ERα signaling was involved in Aktphosphorylation, (b) PI3K signaling was involved in Akt phosphorylation, (c) ERα signaling was involved in 4E-BP1phosphorylation, (d) PI3K signaling was involved in 4E-BP1 phosphorylation. *p < 0.05 compared to DMSO group, #p < 0.05compared to E2 group, &p < 0.05 compared to LCT group (n = 5). Akt: protein kinase B; pAkt: phosphorylated protein kinase B;4E-BP1: eukaryotic initiation factor 4E-binding protein 1; p4E-BP1: phosphorylated eukaryotic initiation factor 4E-bindingprotein 1.

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Fig. 6 – Changes of CREB phosphorylation after LCT or/and E2 treatment in HT22 cells. There was no significant difference inCREB phosphorylation levels between the groups (n = 5). (a) ERα signaling has no effects in CREB phosphorylation, (b) PI3Ksignaling has no effects in CREB phosphorylation. CREB: cAMP-response element binding protein; pCREB: phosphorylatedcAMP-response element binding protein.

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Fig. 7 – LCT increases PSD95 translation via ERα-dependent PI3K/Akt signaling. LCT or E2 can bind to ERα and activate PI3K/Aktsignaling, and activated Akt will phosphorylate the downstream factor 4E-BP1. The phosphorylated 4E-BP1 plays a vital role inPSD95 mRNA translation. Both ICI182,780 and LY294,002 can block this effect. However, LCT can disrupt the ERα-dependentAkt signaling pathway, which is activated by E2.

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Our initial experiment was to detect the cell viability ofHT22 cells using a CCK-8 kit. There was no difference in cellviability for the treatments of E2 or/and LCT compared to thecontrol.

Next, we investigated the effects of LCT on neuro-development. The neural scaffolding protein PSD95, whichwas the postsynaptic marker, was detected in our study.Treatment with LCT or/and E2 resulted in increased PSD95protein expression. Our results are in line with the recentstudies (Akama & McEwen, 2003; Chen, et al., 2012). Theseresults suggested that there may be a common effect onneurodevelopment between LCT and E2. Recent studiesdemonstrated that pyrethroids could inhibit the binding ofE2 to ERs (Tange et al., 2014) and affected E2 levels in plasma(Forsgren et al., 2013). We observed a similar phenomenon,that the PSD95 level did not increase significantly whenco-treated with LCT + E2. These results suggested there maybe a potential competitive antagonism between E2 and LCT,and that LCT could disrupt the promoting effect of E2 onneurodevelopment to some extent. Further studies areneeded to investigate the mechanism for this phenomenon.

Recent reports have demonstrated that E2 can enhancePSD95 expression through binding to ERs, and ERα plays animportant role in this process (Lu et al., 2012; Sato et al., 2007).Our next question was to identify whether the effect of LCT onPSD95 level is through the ERα-dependent signaling pathway.ICI182,780 has been commonly used to block ERα effects(Akama & McEwen, 2003). Moreover, LCT presented a similareffect on PSD95 expression to that of E2. ICI82,780 andLY294,002 blocked the promoting effect of LCT or E2 onPSD95 expression (Fig. 2). Concerning the inhibiter effects,the decrease of PSD95 after E2 or LCT treatment indicated that

ERα-related signaling was essential in the promotion of LCT.However, our results also showed that there was an antago-nism effect between LCT and E2 on up-regulating PSD95expression, and these results demonstrated that LCT mightdisrupt the promoting effect of E2 on neurodevelopment.Moreover, the changes of ERα protein expression wereconsistent with PSD95. These results supported the hypoth-esis that LCT could influence PSD95 protein expressionthrough ERα-dependent signaling.

However, E2 is not the only chemical known that canincrease PSD95 in neurons. If LCT can truly influence thePSD95 level through ERα, it could also act on the downstreamgrowth effectors. It is well known that E2 treatment inducesneuroplasticity in hippocampal neurons via ERα-dependentnucleus and membrane signaling (Flores et al., 2003; Guo etal., 2006; Kao et al., 2013; Sato, et al., 2007; Varea et al., 2013).However, our results showed that there were no changes inPSD95 mRNA among the treatment groups, which wasconsistent with the results from Akama's group (Akama &McEwen, 2003). These results indicated that LCT or/and E2treatment for 24 hr may not influence PSD95 at the transcrip-tional level, and nucleus-initiated signaling of ERα may havehad no effect in our study. To further elucidate the membranesignaling effects in this study, we investigated the phosphor-ylation level of Akt, which was the downstream effector ofactivated PI3K. The PI3K-AKT-mTOR pathway is an importantintracellular signaling pathway that drives cellular growthand survival. Recently, some studies have demonstratedthat E2 is involved in the phosphorylation of Akt in themembrane-initiated signaling pathway through interactingdirectly with the ERα and G-protein coupled estrogen receptor(GPER) (Ciruelos Gil, 2014; Ruiz-Palmero et al., 2013; Simoncini

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et al., 2000). Activated PI3K triggers cascades of signalingfactor activity such as Akt and mTOR (Varea, et al., 2013). Ourresults showed an increase in phosphorylated Akt after LCT orE2 exposure. Moreover, activated Akt induced the phosphor-ylation of the downstream factor 4E-BP1, which plays animportant role in the translational process (Showkat et al.,2014). However, the interference between LCT and E2 occurredin the Akt rapid signaling pathway as well. The result of theco-treatment of LCT and E2 is quite the contrary to LCT orE2 treatment. These results supported the supposition thatLCT could truly interrupt E2 activation effects in the Aktsignaling pathway. Moreover, recent studies have demon-strated that Akt activation induced Bcl2 expression occurs viathe CREB-dependent mechanism in PC12 cells (Pugazhenthi etal, 2000). However, our results showed no significant differ-ence in CREB activation after exposure to LCT or/and E2.Considering that several signaling pathways (PKA, PKC, MAPK)could also regulate CREB activation, our results are more likelyto be explainedby the above signaling pathways rather than thesingle ERα-dependent PI3K-Akt signaling (Purves-Tyson &Keast, 2004). In addition, we observed an interesting result,that co-treatment with E2 and LCT had no effects on theERα-dependent signaling activation. However, the mechanismfor interaction between E2 and LCT effects has not beendetermined, and a further study is needed to investigate it.

E2 is an important hormone in the brain, which could belocally synthesized and transported from systemic circula-tion. E2 is indispensable in the normal neurodevelopmentprocess, so a combination of E2 and LCT, rather than LCTalone, is more typical than exposure to LCT. Although LCTcould promote PSD95 expression alone, the disruption to E2 ismore dangerous.

4. Conclusions

We provided evidence that LCT promotes PSD95 expression atthe protein translation level via ERα-dependent signaling, andthat LCT could disrupt the promoting effect of E2 on PSD95expression via the same pathway. Our study provides a newinsight into pyrethroid toxicity in neurodevelopment.However, the mechanisms of the LCT-mediated effects arenot very clear and further studies are needed.

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (No. H2607-30571585). Useful linguisticmodifications given by Prof. Chen (Department of Toxicology& Cancer Biology, University of Kentucky, USA) are alsoacknowledged.

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