Pyrethroid Insecticide Cypermethrin Modulates Gonadotropin Synthesis via Calcium Homeostasis and ERK1/2 Signaling in LβT2 Mouse Pituitary Cells

Pyrethroid Insecticide Cypermethrin Modulates Gonadotropin Synthesis via Calcium Homeostasis and... Abstract Pyrethroids are a class of widely used insecticides. Cypermethrin (CP) is one of most commonly used pyrethroid insecticides and its residue has been frequently detected in environmental media. Our recent animal study reported that early postnatal exposure to CP induced an increase in serum levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) as well as the expression of gonadotropin subunit genes (chorionic gonadotropin α [CGα], LHβ and FSHβ) in pituitary tissues. In this study, we further investigated the precise mechanism by which CP at concentrations of 1-100 nM affected the synthesis of gonadotropins using a murine pituitary gonadotropic cell line LβT2. We found that calcium (Ca2+)-dependent extracellular signal-regulated kinase 1/2 (ERK1/2) activity was required for CP-regulated transcription of CGαs, LHβ and FSHβ. We provided the novel evidence that CP caused both influx of extracellular Ca2+ through L-type voltage-gated calcium channels (VGCCs) and release of intracellular Ca2+ from endoplasmic reticulum (ER) via inhibition of Ca2+-ATPase. Our results showed that CP disrupted Ca2+ homeostasis via these two separate and independent pathways, thus resulting in the activation of protein kinase C /c-Raf/ERK1/2/immediate-early genes pathways and subsequent increase in the transcription of gonadotropin subunit genes. Our findings would have important implications for understanding the underlying mechanisms of the disrupting effects of some pyrethroids (such as CP) on the synthesis of pituitary gonadotropins. pyrethroids, cypermethrin, gonadotropin, pituitary, endocrine-disrupting chemicals Pyrethroids are a class of widely used insecticides that are synthetic esters derived from the naturally occurring toxin pyrethrins. Because of their large-scale use in residential and agricultural pest control, the residues of pyrethroids were frequently detected in residential homes and agricultural products (Liu et al., 2005). Therefore, the increased human exposure to pyrethroids through household environment and diets is expected. The urine metabolites of pyrethroids were prevalently found in both adults and children (Ye et al., 2017b,c; Meeker et al., 2009; Han et al., 2008). Cypermethrin (CP) is one of most widely used pyrethroid insecticides and has been extensively used for several decades (US EPA, 2016). The annual usage of CP has been more than 1 million pounds of active ingredient in Unite States, and its use continues to grow (US EPA, 2016). CP residue was frequently detected in environment, food and even in human breast-milk (Bouwman et al., 2006; Tulve et al., 2006; Yuan et al., 2014). Pyrethroids, including CP, have been considered as potential endocrine-disrupting chemicals (EDCs), since they were shown to have hormone-like activities and disrupt the function of endocrine and reproductive systems (Liu et al., 2011a,b; Wang et al., 2007; Zhang et al., 2016; Zhao et al., 2014; Ye et al., 2017a). The pituitary gonadotropins, ie, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), play critical roles in the control of mammalian reproduction and sexual maturity. The associations of pyrethroid exposure with altered levels of gonadotropins have been observed in human and animals. For example, two epidemiological studies showed that urinary metabolites of pyrethroids are positively associated with serum levels of FSH and/or LH in adult men (Han et al., 2008; Meeker et al., 2009). More recently, our human study reported on an association of increased pyrethroid exposure with elevated gonadotropins levels and earlier pubertal development in Chinese boys (Ye et al., 2017b). Our recent animal study showed that early postnatal exposure of male mice to CP induced a dose-dependent increase in serum levels of LH and FSH and significantly accelerated sexual maturity (Ye et al., 2017a). Moreover, our study demonstrated that CP could induce the expression of gonadotropin subunit genes in pituitary tissues (Ye et al., 2017a). Both LH and FSH are heterodimeric glycoproteins consisting of a common α-subunit (also known as chorionic gonadotropin α [CGα]) and unique β-subunits (LHβ and FSHβ) (Thackray et al., 2010). The transcription of these subunit genes is the rate-limiting step for production of LH and FSH (Thackray et al., 2010). Calcium (Ca2+) signaling plays an essential role in the signal transduction cascade necessary for the secretion of gonadotropins from anterior pituitary gonadotropic cells (Mulvaney et al., 1999; Reiss et al., 1997). We have previously indicated that the selective inhibitor for L-type voltage-gated calcium channels (VGCCs), nimodipine, was able to block CP-induced gonadotropin subunit gene transcription in pituitary tissues of male mice, suggesting Ca2+ signaling is involved in CP-induced gonadotropin synthesis (Ye et al., 2017a). However, the specific roles of spatial and temporal Ca2+ signals and the modifications of downstream signaling cascade in regulation of CP-stimulated gonadotropin production remains unclear. Previous studies have shown that activation of a mitogen-activated protein kinase (MAPK) family member, extracellular signal-regulated kinase 1/2 (ERK1/2), was absolutely required for the transcription of subunit genes of gonadotropins (Thackray et al., 2010). It has been reported that Ca2+ influx through L-type VGCCs was required for the gonadotropin-releasing hormone (GnRH) stimulation of ERK1/2 (Mulvaney et al., 1999). Thus, we proposed in this study that CP might activate ERK1/2 cascade and thereby stimulate the production of gonadotropins from pituitary gonadotropes, possibly through modification of Ca2+ signaling. In this study, we investigated the precise mechanism by which CP affected the synthesis of gonadotropins using a murine pituitary gonadotropic cell line LβT2, which expresses CGα, LHβ, and FSHβ subunit genes and secretes gonadotropins in response to GnRH and EDCs (Dooley et al., 2008, 2013; Zhou et al., 2014). MATERIALS AND METHODS Materials CP (CAS No. 52315-07-8,≥97% purity), GnRH, cetrorelix, PD184352, GF109203X, 1, 2-bis (2-aminophenoxy) ethane-N, N, N’, N’-tetraacetic acid acetoxymethyl ester (BAPTA-AM), ethylene glycol tetraacetic acid (EGTA), nimodipine and thapsgargin were purchased from Sigma-Aldrich Corp. (St Louis, Missouri). Tetrodotoxin (TTX) was purchased from Ruifang, Inc. (Dalian, China). W-7 and U73122 were purchased from Tocris Bioscience (Bristol, UK). All chemicals used in cell exposure experiments were dissolved in dimethylsulfoxide (DMSO). Antibodies for phospho-ERK1/2 (cat. no. 9101 s), ERK1/2 (cat. no. 9102), phospho-c-Raf (cat. no. 9427), c-Raf (cat. no. 9422 s), c-Jun (cat. no. 9165 s) and c-Fos (cat. no. 2250 s) were purchased from Cell Signaling Technology, Inc. (Beverly, Massachusetts). Antibody for Egr-1 (cat. no. sc-110) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, California). Antibody for GAPDH (10494-1-AP) was obtained from Proteintech Group, Inc. (Chicago, Illinois). Cell culture and exposure The murine pituitary gonadotrope cell line LβT2 was kindly provided by Dr Pamela Mellon at the University of California, San Diego. The LβT2 cells were cultured as described previously in Liu et al. (2006). Briefly, cells were grown in poly-L-lysine -coated flasks with DMEM medium (Hyclone, Logan, Uttah) supplement with 10% fetal bovine serum (Hyclone) and incubated at 37 °C in an atmosphere of 5% CO2. A 24-h serum starvation was performed before each chemical treatment for the cells. The starved cells were exposed to fresh serum-free media containing 1, 10, or 100 nM CP for 24 h. For inhibitors experiments, cells were pretreated with various inhibitors or vehicle (0.1% DMSO) for 30 min, and then treated with 100 nM CP. At defined time points, cells were collected for RNA or protein extraction. Measurement of LH and FSH LβT2 cells and conditioned media were collected after exposure to CP alone or in combination with inhibitors as described. To determine the concentrations of hormones in cytoplasm, cells were collected in 2.5 ml PBS buffer (105 cells/ml), repeatedly frozen and thawed, and then centrifuged and the supernatant was collected. LH and FSH levels were measured using the ELISA kits according to the manufacturer’s protocol (USCN Life Science Inc., Wuhan, China). Inter and intra-assay coefficient of variations for LH and FSH were <10%. The concentrations of hormones in cytoplasm were normalized by the total protein concentration. Quantification of mRNA Total RNA was isolated from cells or tissues using Trizol reagent (Life Technologies, Carlsbad, California) according to the manufacturer’s protocol. Synthesis of first-strand cDNA was performed by reverse transcription of 0.5 μg total RNA using ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan). Oligonucleotide primer sequences for mouse CGα, LHβ, FSHβ, and L32 were listed in Supplementary Table 1. The real-time PCR reactions were set up with SYBR Green PCR master mix (Toyobo) using a Mx3000P Real-time PCR system (Agilent Technologies, Palo Alto, California), as described previously (Liu et al., 2011a; Yang et al., 2014). The relative amount of each gene transcript was calculated using the 2−ΔΔCT method and normalized to the endogenous reference gene L32. Western blot analysis Western blotting was performed as described previously (Li et al., 2012; Liu et al., 2010). Total protein was isolated from cells. Fifty micrograms of protein was separated on a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane (Millipore, Billerica, Massachusetts). Membrane was incubated with the primary antibody overnight at 4 °C. Blots were analyzed using an enhanced chemiluminescence detection system (Pierce, Rockford, Illinois) and photographed using BioSpectrum 410 Imaging System (UVP, Upland, California). Each experiment was repeated at least three times. Determination of intracellular Ca2+ Intracellular Ca2+ levels were determined as previously described (Usmani et al., 2010). Briefly, LβT2 cells were incubated with HEPES-buffered saline containing 2 mM Fluo-4AM in the presences or absence of 10 μM nimodipine, 3 mM EGTA, or 50 μM BAPTA-AM. Cells were placed into an Infinite M200 PRO plate reader (Tecan Group, Männedorf, Switzerland) and kinetic cycle was measured with excitation at 490 nm and emission at 525 nm. Cells were stimulated after fluorescence reached a steady state. Then CP was added to the cells immediately prior to measurement for another 30 min. Relative fluorescence (ΔF/F) was calculated as fluorescence intensities normalized to fluorescence intensity measured at the last time point before CP addition. All the measurements were performed in triplicates. Ca2+-ATPase activity determination LβT2 cells were exposed to 1, 10, or 100 nM CP for 1 h. Cells were homogenized in 0.9% NaCl solution and then centrifuged. The supernatant was collected and employed to determine the total Ca2+-ATPase activity according to the manufacturer’s protocol of commercial kits (Jiancheng Bioengineering Institute, Nanjing, China). Intracellular inositol 1, 4, 5-trisphosphate measurements LβT2 cells were exposed to 100 nM GnRH or 1, 10, and 100 nM CP for 5 min. Cells were collected in 2.5 ml PBS buffer (105 cells/ml), repeatedly frozen and thawed, and then centrifuged. The supernatant was collected and employed to determine the inositol 1, 4, 5-trisphosphate (InsP3) concentrations according to the manufacturer’s protocol of commercial ELISA kits (Uscn Life Science, Wuhan, China). Statistical analysis Each experiment of cell treatment was performed in triplicate at least 3 times. All data were expressed as the mean ± SEM and tested by using 1-way ANOVA. If ANOVA revealed significant effects of treatments, the datasets from 2 groups were compared by 2-tailed unpaired Student’s t test, and p < .05 was considered significant. RESULTS Stimulation of Gonadotropin Synthesis by CP in LβT2 Cells LβT2 cells were exposed to CP at concentrations of 1, 10 or 100 nM for 24 h. Cell viability results indicated that CP at concentrations tested in this study had no cytotoxicity to LβT2 cells (Supplementary Figure 1). CP stimulated the mRNA levels of CGα, LHβ, and FSHβ in a concentration-dependent manner (Figure 1A). The protein levels of LH and FSH in cytoplasm and conditioned culture media were significantly up-regulated by CP (Figs. 1B and 1C). Figure 1. View largeDownload slide Stimulation of gonadotropin synthesis by CP. LβT2 cells were exposed to CP at concentrations of 1, 10, or 100 nM for 24 h. A, The mRNA levels for CGα, LHβ, and FSHβ in LβT2 cells. B, The levels of LH and FSH in cytoplasm. C, The levels of LH and FSH in cultured media. Values shown represent mean ±SEM for three independent experiments performed in triplicate. * p < .05 treatment versus control. Figure 1. View largeDownload slide Stimulation of gonadotropin synthesis by CP. LβT2 cells were exposed to CP at concentrations of 1, 10, or 100 nM for 24 h. A, The mRNA levels for CGα, LHβ, and FSHβ in LβT2 cells. B, The levels of LH and FSH in cytoplasm. C, The levels of LH and FSH in cultured media. Values shown represent mean ±SEM for three independent experiments performed in triplicate. * p < .05 treatment versus control. Requirement of ERK1/2 Activity in CP-Induced Gonadotropin Subunit Gene Expression As shown in Figure 2A, ERK1/2 inhibitor PD184352 prevented the induction of CGα, LHβ, and FSHβ gene expression by 100 nM CP. Addition of 100 nM CP stimulated a rapid and very strong increase in ERK1/2 phosphorylation that reached its peak at 5 min (Figure 2B). A concentration-dependent increase in ERK1/2 activity was observed in LβT2 cells exposed to 1, 10, or 100 nM CP for 5 min (Figure 2C) and PD184352 completely attenuated ERK1/2 activity induced by CP (Figure 2D). These data suggest that activation of ERK1/2 is required for CP-induced gonadotropin subunit gene expression. Figure 2. View largeDownload slide Role of ERK1/2 activity in CP-induced gonadotropin subunit gene expression. A, The mRNA levels for CGα, LHβ and FSHβ in LβT2 cells exposed to 100 nM CP alone or in combination with 1 μM PD184352 (PD). Values shown represent mean ± SEM for 3 independent experiments performed in triplicate. * p < .05 treatment versus control. B, Phosphorylation of ERK1/2 (p-ERK1/2) in LβT2 cells exposed to 100 nM CP for 5, 10, 30, or 60 min, respectively. C, p-ERK1/2 in LβT2 cells treated with CP at concentrations of 1, 10 or 100 nM for 5 min. D, p-ERK1/2 in LβT2 cells exposed to 100 nM CP alone or in combination with 1 μM PD. t-ERK1/2: total ERK1/2. Figure 2. View largeDownload slide Role of ERK1/2 activity in CP-induced gonadotropin subunit gene expression. A, The mRNA levels for CGα, LHβ and FSHβ in LβT2 cells exposed to 100 nM CP alone or in combination with 1 μM PD184352 (PD). Values shown represent mean ± SEM for 3 independent experiments performed in triplicate. * p < .05 treatment versus control. B, Phosphorylation of ERK1/2 (p-ERK1/2) in LβT2 cells exposed to 100 nM CP for 5, 10, 30, or 60 min, respectively. C, p-ERK1/2 in LβT2 cells treated with CP at concentrations of 1, 10 or 100 nM for 5 min. D, p-ERK1/2 in LβT2 cells exposed to 100 nM CP alone or in combination with 1 μM PD. t-ERK1/2: total ERK1/2. Noninvolvement of GnRH Receptor/Phospholipase C/InsP3 Pathway in CP-Induced Gonadotropin Subunit Gene Expression and ERK1/2 Activation It is known that, in pituitary gonadotropic cells, binding of ligand (GnRH or its agonists) to GnRH receptor (GnRHR) initiates phospholipase C (PLC) activity to generate InsP3, which leads to elevation of intracellular Ca2+ levels, activation of protein kinase C (PKC) and MAPK cascades to regulate the synthesis of gonadotropins (Thackray et al., 2010). To investigate whether GnRHR mediates the effects of CP on gonadotropin gene expression and ERK1/2 activity, LβT2 cells were treated with CP in combination with the GnRH antagonist cetrorelix. Cetrorelix significantly blocked GnRH-induced expression of CGα, LHβ, and FSHβ genes, whereas it was unable to prevent the stimulation of gonadotropin subunit gene expression and ERK1/2 activation by CP (Supplementary Figs. 2A and 2B). Furthermore, the PLC inhibitor U73122 could not abolish the activity of ERK1/2 by CP (Supplementary Figure 2C). The InsP3 concentrations did not change in cells exposed to CP (Supplementary Figure 2D). These results indicate that GnRHR/PLC/InsP3 pathway is not involved in CP-induced gonadotropin gene expression and ERK1/2 activation. Noninvolvement of Voltage-Gated Sodium Channels in CP-Induced Gonadotropin Subunit Gene Expression and ERK1/2 Activation It has been well-documented that voltage-gated sodium channels (VGSCs) are primary target sites for pyrethroids (Shafer et al., 2005). We examined whether VGSCs were involved in CP-induced gonadotropin subunit gene expression and ERK1/2 activation in LβT2 cells. The VGSCs specific inhibitor TTX at concentration of 10 μM was without effects on the induction of gonadotropin subunit gene expression and ERK1/2 phosphorylation by CP (Supplementary Figure 3). Requirement of Ca2+ Signaling in CP-Induced ERK1/2 Activation To examine the VGCCs and Ca2+ requirement, LβT2 cells were pretreated with 10 μM nimodipine to block L-type VGCCs, 3 mM EGTA to chelate extracellular Ca2+, or 50 μM BAPTA-AM to chelate intracellular Ca2+. Nimodipine and EGTA caused a partial reduction in ERK1/2 activity by CP stimulation, while BAPTA-AM almost completely attenuated CP-induced ERK1/2 activation (Figure 3A). The effects of CP on changes in intracellular Ca2+ levels ([Ca2+]i) in LβT2 cells were further investigated. As shown in Figure 3B, CP treatment caused an increase in the amount of [Ca2+]i compared with untreated cells, and pretreatment of cells with nimodipine, EGTA or BAPTA-AM significantly attenuated the increase in [Ca2+]i when the cells were treated with CP. These data suggest that CP increased Ca2+ influx through L-type VGCCs and Ca2+ signal is required for CP-induced ERK1/2 activation. Figure 3. View largeDownload slide Role of Ca2+ signaling in CP-induced ERK1/2 activation. A, Phosphorylation of ERK1/2 (p-ERK1/2) in LβT2 cells exposed to 100 nM CP for 5 min in the presence or absence of 10 μM nimodipine (Nimo), 3 mM EGTA, or 50 μM BAPTA-AM (BAPTA). B, Time course of [Ca2+]i changes induced by 100 nM CP in the presence or absence of Nimo, EGTA or BAPTA. C, p-ERK1/2 in LβT2 cells treated with 100 nM CP for 5 min or 2 mM thapsigargin (Thap) for 5, 10, or 30 min, or they were treated with Thap 30 min prior to and during CP application. D, Ca2+-ATPase activity in LβT2 cells exposed to CP at concentrations of 1, 10 or 100 nM. E, p-ERK1/2 in LβT2 cells exposed to 100 nM CP for 5 min in the presence or absence of 15 μM W-7. Values shown represent mean ± SEM for 3 independent experiments performed in triplicate. * p < .05 treatment versus control. t-ERK1/2: total ERK1/2. Figure 3. View largeDownload slide Role of Ca2+ signaling in CP-induced ERK1/2 activation. A, Phosphorylation of ERK1/2 (p-ERK1/2) in LβT2 cells exposed to 100 nM CP for 5 min in the presence or absence of 10 μM nimodipine (Nimo), 3 mM EGTA, or 50 μM BAPTA-AM (BAPTA). B, Time course of [Ca2+]i changes induced by 100 nM CP in the presence or absence of Nimo, EGTA or BAPTA. C, p-ERK1/2 in LβT2 cells treated with 100 nM CP for 5 min or 2 mM thapsigargin (Thap) for 5, 10, or 30 min, or they were treated with Thap 30 min prior to and during CP application. D, Ca2+-ATPase activity in LβT2 cells exposed to CP at concentrations of 1, 10 or 100 nM. E, p-ERK1/2 in LβT2 cells exposed to 100 nM CP for 5 min in the presence or absence of 15 μM W-7. Values shown represent mean ± SEM for 3 independent experiments performed in triplicate. * p < .05 treatment versus control. t-ERK1/2: total ERK1/2. To rule out a potential contribution of intracellular Ca2+ stores in this signaling pathway, cells were treated with thapsigargin, an inhibitor of the Ca2+-ATPase that pumps Ca2+ into intracellular stores. Thapsigargin is known to increase cytoplasmic Ca2+ levels and eventually cause depletion of intracellular stores (Mulvaney et al., 1999). Thapsigargin treatment alone stimulated ERK1/2 phosphorylation and CP-induced ERK1/2 activation was markedly reduced in thapsigargin-pretreated cells (Figure 3C), indicating that the thapsigargin-stimulated Ca2+ signal activates ERK1/2 and depletion of Ca2+ intracellular stores by thapsigargin results in an attenuation of CP-induced ERK1/2 activation. We further showed that CP exposure caused a dose-dependent decrease in Ca2+-ATPase activity in LβT2 cells (Figure 3D). These results suggest that CP could also inhibit Ca2+-ATPase activity, which leads to a release of Ca2+ from intracellular stores and consequently activate ERK1/2. Calmodulin (CaM) is the prototypical Ca2+-binding protein and serves important roles as a Ca2+ sensor in a number of different intracellular signaling scenarios (Takemoto-Kimura et al., 2017). In this study, pretreatment with the CaM inhibitor W-7 abolished CP-induced ERK1/2 activation (Figure 3E), indicating that CaM is also involved in CP-dependent ERK1/2 pathway. Involvement of Ca2+ Signaling in CP-Induced c-Raf Activation Next studies focused on examining the effects of Ca2+ pathway on upstream catalytic activity of c-Raf kinase that has been known as a key signaling intermediate within the ERK1/2 cascade in gonadotropic cells (Roberson et al., 2005). Addition of 100 nM CP caused a significant increase in the phosphorylation of c-Raf (p-c-Raf) kinase at serine 338 (Figure 4A). The p-c-Raf with CP was blocked by nimodipine, EGTA and BAPTA-AM (Figure4A). Treatment with thapsigargin alone also stimulated c-Raf phosphorylation and CP-induced c-Raf activation was reduced in thapsigargin-treated cells (Figure 4B). Pretreatment of LβT2 cells with W-7 resulted in an inhibition of c-Raf kinase phosphorylation (Figure 4C). These data suggest that VGCCs and Ca2+-ATPase/Ca2+/CaM signaling pathways mediate CP-induced c-Raf activation. Figure 4. View largeDownload slide Involvement of Ca2+ signaling in CP-induced c-Raf activation. A, Phosphorylation of c-Raf (p-c-Raf) in LβT2 cells exposed to 100 nM CP for 5 min in the presence or absence of 10 μM nimodipine (Nimo), 3 mM EGTA or 50 μM BAPTA-AM (BAPTA). B, p-c-Raf in LβT2 cells treated with 100 nM CP for 5 min or 2 mM thapsigargin (Thap) for 5, 10, or 30 min, or they were treated with Thap 30 min prior to and during CP application. C, p-c-Raf in LβT2 cells exposed to 100 nM CP for 5 min in the presence or absence of 15 μM W-7. t-c-Raf: total c-Raf. Figure 4. View largeDownload slide Involvement of Ca2+ signaling in CP-induced c-Raf activation. A, Phosphorylation of c-Raf (p-c-Raf) in LβT2 cells exposed to 100 nM CP for 5 min in the presence or absence of 10 μM nimodipine (Nimo), 3 mM EGTA or 50 μM BAPTA-AM (BAPTA). B, p-c-Raf in LβT2 cells treated with 100 nM CP for 5 min or 2 mM thapsigargin (Thap) for 5, 10, or 30 min, or they were treated with Thap 30 min prior to and during CP application. C, p-c-Raf in LβT2 cells exposed to 100 nM CP for 5 min in the presence or absence of 15 μM W-7. t-c-Raf: total c-Raf. Requirement of PKC in CP-Induced c-Raf/ERK1/2 Signaling and Gonadotropin Gene Expression It is known that Ca2+-dependent PKC controls the expression of three gonadotropin subunit genes in gonadotropic cells (Thackray et al., 2010). We further tested whether PKC mediates the up-regulation of gonadotropin gene expression by CP. The results showed that GF109203X, a specific inhibitor for PKC, significantly diminished CP-enhanced expression of CGα, LHβ, and FSHβ genes in LβT2 cells (Figure 5A). GF109203X abolished the p-c-Raf and ERK1/2 in CP-treated cells (Figure 5B). These data indicate that PKC is the upstream activator of c-Raf/ERK1/2 upon CP stimulation. Figure 5. View largeDownload slide Requirement of PKC in CP-induced c-Raf/ERK1/2 signaling and gonadotropin gene expression. A, The mRNA levels for CGα, LHβ and FSHβ in LβT2 cells exposed to 100 nM CP for 24 h in the presence or absence of 5 μM GF109203X (GF). Values shown represent mean ± SEM for 3 independent experiments performed in triplicate. * p < .05 treatment versus control. B, Phosphorylation of c-Raf (p-c-Raf) or ERK1/2 (p-ERK1/2) in LβT2 cells exposed to 100 nM CP for 5 min in the presence or absence of 5 μM GF. t-c-Raf: total c-Raf; t-ERK1/2: total ERK1/2. Figure 5. View largeDownload slide Requirement of PKC in CP-induced c-Raf/ERK1/2 signaling and gonadotropin gene expression. A, The mRNA levels for CGα, LHβ and FSHβ in LβT2 cells exposed to 100 nM CP for 24 h in the presence or absence of 5 μM GF109203X (GF). Values shown represent mean ± SEM for 3 independent experiments performed in triplicate. * p < .05 treatment versus control. B, Phosphorylation of c-Raf (p-c-Raf) or ERK1/2 (p-ERK1/2) in LβT2 cells exposed to 100 nM CP for 5 min in the presence or absence of 5 μM GF. t-c-Raf: total c-Raf; t-ERK1/2: total ERK1/2. Induction of the Immediate-Early Gene Expression by CP via Ca2+/PKC/ERK1/2 Signaling Pathway It has been shown that stimulation with GnRH or agonists results in increased mRNA and protein for the immediate-early genes (IEGs), ie, Egr-1, c-Fos, and c-Jun, in pituitary gonadotropic cells (Cheng et al., 2000; Liu et al., 2002; Wolfe and Call, 1999). In response to extracellular signals, Egr-1 and the c-Fos/c-Jun heterodimers (activator protein-1, AP-1) serve as transcription factors to promote gonadotropin subunit gene transcription (Cheng et al., 2000; Liu et al., 2002; Wolfe and Call, 1999). Therefore, we examined the effects of CP on the mRNA and protein expression of Egr-1, c-Fos and c-Jun. As shown in Figures 6A and 6B, the mRNA and protein levels for these IEGs were concentration-dependently increased in cells exposed to 10 or 100 nM CP. Chelation of intracellular Ca2+ with BAPTA-AM, or extracellular Ca2+ with EGTA blocked CP-induced expression of Egr-1, c-Fos, and c-Jun proteins (Figure 6C). Inhibition of PKC with GF109203X prevented the induction of Egr-1, c-Fos and c-Jun by CP (Figure 6D). Inhibition of ERK1/2 with PD184352, or CaM with W-7 caused significant reduction in expression levels of Egr-1, c-Fos, and c-Jun (Figure 6E). These observations suggest that the CP-induced expression of IEGs is dependent on Ca2+/PKC/ERK1/2 signaling. Figure 6. View largeDownload slide Induction of the IEGs expression by CP. A, The mRNA levels for Egr-1, c-Fos and c-Jun in LβT2 cells exposed to CP at concentrations of 1, 10, or 100 nM for 2 h. Values shown represent mean ± SEM for 3 independent experiments performed in triplicate. * p < 0.05 treatment versus control. B, The protein levels for Egr-1, c-Fos and c-Jun in LβT2 cells exposed to 1-100 nM CP for 2 h. C–E, The protein levels for Egr-1, c-Fos and c-Jun in LβT2 cells exposed to 100 nM CP for 2 h in the presence or absence of 50 μM BAPTA-AM (BAPTA), 3 mM EGTA, 5 μM GF109203X (GF), 1 μM PD184352 (PD) or 15 μM W-7, respectively. Figure 6. View largeDownload slide Induction of the IEGs expression by CP. A, The mRNA levels for Egr-1, c-Fos and c-Jun in LβT2 cells exposed to CP at concentrations of 1, 10, or 100 nM for 2 h. Values shown represent mean ± SEM for 3 independent experiments performed in triplicate. * p < 0.05 treatment versus control. B, The protein levels for Egr-1, c-Fos and c-Jun in LβT2 cells exposed to 1-100 nM CP for 2 h. C–E, The protein levels for Egr-1, c-Fos and c-Jun in LβT2 cells exposed to 100 nM CP for 2 h in the presence or absence of 50 μM BAPTA-AM (BAPTA), 3 mM EGTA, 5 μM GF109203X (GF), 1 μM PD184352 (PD) or 15 μM W-7, respectively. DISCUSSION We have previously demonstrated in mice that exposure to CP stimulated the transcription of gonadotropin subunit genes in pituitary through interference with VGCCs (Ye et al., 2017a). In the present study, we found that Ca2+-dependent ERK1/2 activity was required for CP-regulated gonadotropin synthesis. We provided the evidence that CP caused both influx of extracellular Ca2+ through interference with VGCCs and release of intracellular Ca2+ via inhibition of Ca2+-ATPase in LβT2 cells. Our results showed that CP disrupted Ca2+ homeostasis via these two separate and independent pathways, thus resulting in the activation of PKC/c-Raf/ERK1/2/IEGs cascade and subsequent increase in the transcription of gonadotropin subunit genes (summary in Figure 7). Figure 7. View largeDownload slide Schematic model of CP-induced production of gonadotropins through Ca2+/PKC/c-Raf/ERK1/2/IEGs signaling in pituitary gonadotropic cells. Figure 7. View largeDownload slide Schematic model of CP-induced production of gonadotropins through Ca2+/PKC/c-Raf/ERK1/2/IEGs signaling in pituitary gonadotropic cells. Neuronal VGSCs are identified as primary targets for the insecticidal and neurotoxic effects of pyrethroids (Soderlund, 2012). The expression of VGSCs has been previously identified in isolated rat gonadotropic cells and mouse gonadotropic cell line (Bosma and Hille, 1992; Tse and Hille, 1993). Veratridine, a neurotoxin that abolishes inactivation of VGSCs in a similar manner as CP, was found to induce LH secretion in primary rat pituitary cell cultures and veratridine-stimulated LH release was attenuated by blockade of VGSCs with TTX (Conn and Rogers, 1980). However, the current findings in LβT2 cells showed that TTX failed to block CP-induced gonadotropin subunit gene expression and ERK1/2 activation. Although perturbation of VGSCs is the primary mode of pyrethroid action in both insects and mammals, mammalian VGSCs sensitivity to pyrethroids is 1000-fold less sensitive than the sodium channel of insect (Vais et al., 2001). The treatment concentrations of veratridine (10−6 M to 10−4 M) used in the previous study were 1000 times higher than the environmentally relevant concentrations of CP (10−9 M to 10−7 M) used in our study. Our previous study also demonstrated that the VGSCs-mediated effects of CP on mouse hypothalamic explants were only observed at higher concentrations of 10−6 M and 10−5 M (Ye et al., 2017a). Therefore, VGSCs are nonresponsible for CP’s action in gonadotropic cells may be due to the insensitivity of mammalian VGSCs to CP at low concentrations. In addition to VGSCs, other ion channels, particularly VGCCs, have been implicated as alternative sites of action for a subset of pyrethroids (Soderlund, 2012). A previous study in mouse neocortical neurons reported that exposure to CP and other pyrethroids produced concentration-dependent elevations in intracellular Ca2+ concentration (Cao et al., 2011). Another study found that CP triggered the release of Ca2+ in primary culture of rat astrocytes (Maurya et al., 2014). The activation of VGCCs by pyrethroids also has been observed in non-neural cells. For instance, allethrin induced apoptosis was associated with VGCCs mediated intracellular Ca2+ release in rat testicular carcinoma cells (Madhubabu and Yenugu, 2014). Deltamethrin induced [Ca2+]i rise that involved Ca2+ entry through L-type VGCCs in human prostate cancer cells (Lee et al., 2016). In agreement with these previous observations, we found that CP exposure at non-cytotoxic concentrations caused an increase in [Ca2+]i in pituitary gonadotropic cells. [Ca2+]i can be elevated by extracellular Ca2+ entry through VGCCs or Ca2+ release from intracellular Ca2+ stores such as the endoplasmic reticulum (ER) (Verkhratsky, 2005). In this study, the L-type VGCCs selective blocker nimodipine was significantly but not completely blocked CP-stimulated c-Raf/ERK1/2 activity, while CP-stimulated c-Raf/ERK activation required refilling of thapsigargin-sensitive Ca2+ stores. These data suggest that the release of Ca2+ from ER was also sufficient for activation of c-Raf/ERK cascade by CP. The ER acts as a dynamic Ca2+ store and possesses independent pathways for intracellular Ca2+ influx and efflux (Verkhratsky, 2005). Release of Ca2+ from the ER usually occurs through an InsP3-stimulated calcium channel, commonly known as InsP3 receptors (InsP3Rs) (Verkhratsky, 2005). GnRH-induced release of Ca2+ from intracellular stores in gonadotropic cells has been shown to respond to elevation of InsP3, which is dependent on the activation of GnRHR/PLC pathway (Thackray et al., 2010). However, our data showed that exposure to CP did not alter cellular InsP3 concentrations in LβT2 cells. GnRHR/PLC/InsP3/InsP3Rs pathway seems not to be responsible for CP-induced gonadotropin subunit gene expression and ERK1/2 activation. Consistent with our observations, another pyrethroid deltamethrin evoked [Ca2+]i rise was also required for PLC-independent Ca2+ release from the ER (Lee et al., 2016). On the other hand, Ca2+ influx into the ER lumen results from the activity of Ca2+ pumps of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) (Verkhratsky, 2005). In this study, we found that CP could inhibit the activity of Ca2+-ATPase in a concentration-dependent manner. The results of this study support speculation that the inhibition of the SERCA pumps by CP, which resulted in a relatively slow emptying of releasable Ca2+ from the ER, may at least partially contribute to CP-induced [Ca2+]i in gonadotropic cells. The ERK1/2 branch of the MAPK pathways is a critical signaling molecule to mediate a variety of actions of pyrethroids on cells. For example, the requirement of ERK1/2 pathway has been reported in studies examining the neurotrophic effects of deltamethrin on cultured neurons (Ihara et al., 2012). CP-induced macrophages apoptosis through oxidative stress-mediated ERK1/2 signaling (Huang et al., 2016). We showed here that Ca2+-dependent ERK1/2 activation was required for CGα, LHβ or FSHβ induction by CP in gonadotropic cells. Stimulation of [Ca2+]i with CP was also a sufficient stimulus for activation of c-Raf protein, which is part of the ERK1/2 pathway as a MAPK kinase (MAP3K) (Roskoski, 2010). Consistent with our observations, many previous studies demonstrated that the expression of gonadotropic subunit genes was Ca2+ dependent and augmented by c-Raf/ERK1/2 signaling (Thackray et al., 2010). We further demonstrated that induction of gonadotropic subunit gene expression by CP was dependent upon PKC signaling, since the PKC inhibitor GF109203X prevented gonadotropic subunit gene transcription. Activation of c-Raf and ERK1/2 kinases by CP was also inhibited by GF109203X. These data suggest that PKC is acting downstream of Ca2+ but upstream of c-Raf and ERK1/2, since a role for Ca2+ in the activation of PKC has been well documented (Huang, 1989). Regulation of the LHβ and FSHβ genes occurs through the induction of intermediate IEGs, which constitute Egr-1 and AP-1 consisting of c-Fos and c-Jun (Thackray et al., 2010). In our present study, CP-increased [Ca2+]i appeared to be sufficient to induce increased protein for the ERK1/2 substrates Egr-1, c-Fos, and c-Jun, since chelation of Ca2+ and inhibition of PKC/ERK1/2 significantly attenuated CP-induced increases in the expression of these IEGs in LβT2 cells. Previous study showed that CaM had a critical role as a Ca2+ sensor in specifically linking Ca2+ flux with ERK1/2 activation and c-Raf kinase was a CaM-binding protein in a Ca2+-dependent manner (Roberson et al., 2005). In this study, CaM inhibition by W-7 effectively blocked c-Raf kinase phosphorylation at S338, subsequently leading to inhibition of ERK1/2 phosphorylation and IEGs induction by CP. These data suggest that CaM serving as Ca2+ binding protein effector is required for sensing local Ca2+ influx and activation of c-Raf/ERK by CP. Further studies will be of interest to determine the roles of two downstream pathways of CaM, the CaM-dependent kinases and the phosphatase calcineurin, in transducing CP-activated signals to gonadotropin gene transcription. In conclusion, this study provided novel evidence in support that a commonly used pyrethroid insecticide, CP, regulated Ca2+ homeostasis by two different pathways, subsequently activated PKC/c-Raf/ERK1/2/IEGs signaling to promote gonadotropin subunit gene transcription in LβT2 cells. We also found the crosstalk between Ca2+/CaM pathway and PKC-dependent ERK1/2 pathway in coordinate regulation of gonadotropin subunit gene expression. This would have important implications for understanding the precise mechanisms of the disrupting effects of pyrethroids on the synthesis of pituitary gonadotropins. Our findings suggest that like endocrine cell types such as gonadotropic cells may make use of elementary Ca2+ signals in the regulation of specific hormone synthesis by EDCs, such as pyrethroids. SUPPLEMENTARY DATA Supplementary data are available at Toxicological Sciences online. ACKNOWLEDGMENTS We are grateful to Dr Zhengxing Wu at Huazhong University of Science & Technology for kindly technical assistance for the cultures of LβT2 cells. FUNDING This work was supported by the National Natural Science Foundation of China (21377113 and 81670971) and Zhejiang Provincial Natural Science Foundation of China (LR15B070001 and LY15C120001). REFERENCES Bosma M. M., Hille B. ( 1992). Electrophysiological properties of a cell-line of the gonadotrope lineage. Endocrinology  130, 3411– 3420. http://dx.doi.org/10.1210/endo.130.6.1317783 Google Scholar CrossRef Search ADS PubMed  Bouwman H., Sereda B., Meinhardt H. M. ( 2006). Simultaneous presence of DDT and pyrethroid residues in human breast milk from a malaria endemic area in South Africa. Environ. Pollut.  144, 902– 917. http://dx.doi.org/10.1016/j.envpol.2006.02.002 Google Scholar CrossRef Search ADS PubMed  Cao Z., Shafer T. J., Murray T. F. ( 2011). Mechanisms of pyrethroid insecticide-induced stimulation of calcium influx in neocortical neurons. J. Pharmacol. Exp. Ther.  336, 197– 205. http://dx.doi.org/10.1124/jpet.110.171850 Google Scholar CrossRef Search ADS PubMed  Cheng K. W., Ngan E. S., Kang S. K., Chow B. K., Leung P. C. ( 2000). Transcriptional down-regulation of human gonadotropin-releasing hormone (GnRH) receptor gene by GnRH: Role of protein kinase C and activating protein 1. Endocrinology  141, 3611– 3622. Google Scholar CrossRef Search ADS PubMed  Conn P. M., Rogers D. C. ( 1980). Gonadotropin-release from pituitary cultures following activation of endogenous ion channels. Endocrinology  107, 2133– 2134. http://dx.doi.org/10.1210/endo-107-6-2133 Google Scholar CrossRef Search ADS PubMed  Dooley G. P., Reardon K. F., Prenni J. E., Tjalkens R. B., Legare M. E., Foradori C. D., Tessari J. E., Hanneman W. H. ( 2008). Proteomic analysis of diaminochlorotriazine adducts in wister rat pituitary glands and L beta T2 rat pituitary cells. Chem. Res. Toxicol.  21, 844– 851. Google Scholar CrossRef Search ADS PubMed  Dooley G. P., Tjalkens R. B., Hanneman W. H. ( 2013). The atrazine metabolite diaminochlorotriazine suppresses LH release from murine L beta T2 cells by suppressing GnRH-induced intracellular calcium transients. Toxicol. Res.  2, 180– 186. http://dx.doi.org/10.1039/c3tx20088d Google Scholar CrossRef Search ADS   Han Y., Xia Y., Han J., Zhou J., Wang S., Zhu P., Zhao R., Jin N., Song L., Wang X. ( 2008). The relationship of 3-PBA pyrethroids metabolite and male reproductive hormones among non-occupational exposure males. Chemosphere  72, 785– 790. Google Scholar CrossRef Search ADS PubMed  Huang F., Liu Q. Y., Xie S. J., Xu J., Huang B., Wu Y. H., Xia D. J. ( 2016). Cypermethrin Induces Macrophages Death through Cell Cycle Arrest and Oxidative Stress-Mediated JNK/ERK Signaling Regulated Apoptosis. Int. J. Mol. Sci.  17, 885. Google Scholar CrossRef Search ADS   Huang K. P. ( 1989). The Mechanism of Protein Kinase-C Activation. Trends Neurosci.  12, 425– 432. http://dx.doi.org/10.1016/0166-2236(89)90091-X Google Scholar CrossRef Search ADS PubMed  Ihara D., Fukuchi M., Honma D., Takasaki I., Ishikawa M., Tabuchi A., Tsuda M. ( 2012). Deltamethrin, a type II pyrethroid insecticide, has neurotrophic effects on neurons with continuous activation of the Bdnf promoter. Neuropharmacology  62, 1091– 1098. Google Scholar CrossRef Search ADS PubMed  Lee H. H., Chou C. T., Liang W. Z., Chen W. C., Wang J. L., Yeh J. H., Kuo C. C., Shieh P., Kuo D. H., Chen F. A., et al.   ( 2016). Ca2+ Movement Induced by Deltamethrin in PC3 Human Prostate Cancer Cells. Chin. J. Physiol.  59, 148– 155. Google Scholar CrossRef Search ADS PubMed  Li F., Jo M., Curry T. E.Jr, Liu J. ( 2012). Hormonal induction of polo-like kinases (Plks) and impact of Plk2 on cell cycle progression in the rat ovary. PLoS One  7, e41844. Google Scholar CrossRef Search ADS PubMed  Liu F., Austin D. A., Mellon P. L., Olefsky J. M., Webster N. J. ( 2002). GnRH activates ERK1/2 leading to the induction of c-fos and LHbeta protein expression in LbetaT2 cells. Mol. Endocrinol.  16, 419– 434. Google Scholar PubMed  Liu H. S., Hu Z. T., Zhou K. M., Jiu Y. M., Yang H., Wu Z. X., Xu T. ( 2006). Heterogeneity of the Ca2+ sensitivity of secretion in a pituitary gonadotrope cell line and its modulation by protein kinase C and Ca2+. J. Cell Physiol.  207, 668– 674. Google Scholar CrossRef Search ADS PubMed  Liu J., Park E. S., Curry T. E.Jr, Jo M. ( 2010). Periovulatory expression of hyaluronan and proteoglycan link protein 1 (Hapln1) in the rat ovary: Hormonal regulation and potential function. Mol. Endocrinol.  24, 1203– 1217. http://dx.doi.org/10.1210/me.2009-0325 Google Scholar CrossRef Search ADS PubMed  Liu J., Yang Y., Yang Y., Zhang Y., Liu W. ( 2011a). Disrupting effects of bifenthrin on ovulatory gene expression and prostaglandin synthesis in rat ovarian granulosa cells. Toxicology  282, 47– 55. Google Scholar CrossRef Search ADS   Liu J., Yang Y., Zhuang S., Yang Y., Li F., Liu W. ( 2011b). Enantioselective endocrine-disrupting effects of bifenthrin on hormone synthesis in rat ovarian cells. Toxicology  290, 42– 49. Google Scholar CrossRef Search ADS   Liu W. P., Gan J. Y., Schlenk D., Jury W. A. ( 2005). Enantioselectivity in environmental safety of current chiral insecticides. Proc. Natl. Acad. Sci. U.S.A . 102, 701– 706. http://dx.doi.org/10.1073/pnas.0408847102 Google Scholar CrossRef Search ADS PubMed  Madhubabu G., Yenugu S. ( 2014). Allethrin induces oxidative stress, apoptosis and calcium release in rat testicular carcinoma cells (LC540). Toxicol. In Vitro  28, 1386– 1395. http://dx.doi.org/10.1016/j.tiv.2014.07.008 Google Scholar CrossRef Search ADS PubMed  Maurya S. K., Mishra J., Tripathi V. K., Sharma R., Siddiqui M. H. ( 2014). Cypermethrin induces astrocyte damage: Role of aberrant Ca2+, ROS, JNK, P38, matrix metalloproteinase 2 and migration related reelin protein. Pestic. Biochem. Physiol.  111, 51– 59. Google Scholar CrossRef Search ADS PubMed  Meeker J. D., Barr D. B., Hauser R. ( 2009). Pyrethroid insecticide metabolites are associated with serum hormone levels in adult men. Reprod. Toxicol.  27, 155– 160. http://dx.doi.org/10.1016/j.reprotox.2008.12.012 Google Scholar CrossRef Search ADS PubMed  Mulvaney J. M., Zhang T., Fewtrell C., Roberson M. S. ( 1999). Calcium influx through L-type channels is required for selective activation of extracellular signal-regulated kinase by gonadotropin-releasing hormone. J. Biol. Chem.  274, 29796– 29804. Google Scholar CrossRef Search ADS PubMed  Reiss N., Llevi L. N., Shacham S., Harris D., Seger R., Naor Z. ( 1997). Mechanism of mitogen-activated protein kinase activation by gonadotropin-releasing hormone in the pituitary alpha T3-1 cell line: Differential roles of calcium and protein kinase C. Endocrinology  138, 1673– 1682. http://dx.doi.org/10.1210/endo.138.4.5057 Google Scholar CrossRef Search ADS PubMed  Roberson M. S., Bliss S. P., Xie J., Navratil A. M., Farmerie T. A., Wolfe M. W., Clay C. M. ( 2005). Gonadotropin-releasing hormone induction of extracellular-signal regulated kinase is blocked by inhibition of calmodulin. Mol. Endocrinol.  19, 2412– 2423. Google Scholar CrossRef Search ADS PubMed  Roskoski R. ( 2010). RAF protein-serine/threonine kinases: Structure and regulation. Biochem. Biophys. Res. Commun.  399, 313– 317. http://dx.doi.org/10.1016/j.bbrc.2010.07.092 Google Scholar CrossRef Search ADS PubMed  Shafer T. J., Meyer D. A., Crofton K. M. ( 2005). Developmental neurotoxicity of pyrethroid insecticides: Critical review and future research needs. Environ. Health Perspect.  113, 123– 136. Google Scholar CrossRef Search ADS PubMed  Soderlund D. M. ( 2012). Molecular mechanisms of pyrethroid insecticide neurotoxicity: Recent advances. Arch. Toxicol.  86, 165– 181. http://dx.doi.org/10.1007/s00204-011-0726-x Google Scholar CrossRef Search ADS PubMed  Takemoto-Kimura S., Suzuki K., Horigane S. I., Kamijo S., Inoue M., Sakamoto M., Fujii H., Bito H. ( 2017). Calmodulin kinases: Essential regulators in health and disease. J. Neurochem.  141, 808– 818. Google Scholar CrossRef Search ADS PubMed  Thackray V. G., Mellon P. L., Coss D. ( 2010). Hormones in synergy: Regulation of the pituitary gonadotropin genes. Mol Cell Endocrinol  314, 192– 203. http://dx.doi.org/10.1016/j.mce.2009.09.003 Google Scholar CrossRef Search ADS PubMed  Tse A., Hille B. ( 1993). Role of voltage-gated Na+ and Ca2+ channels in gonadotropin-releasing hormone-induced membrane-potential changes in identified rat gonadotropes. Endocrinology  132, 1475– 1481. Google Scholar CrossRef Search ADS PubMed  Tulve N. S., Jones P. A., Nishioka M. G., Fortmann R. C., Croghan C. W., Zhou J. Y., Fraser A., Cave C., Friedman W. ( 2006). Pesticide measurements from The First National Environmental Health Survey of Child Care Centers using a multi-residue GC/MS analysis method. Environ. Sci. Technol.  40, 6269– 6274. Google Scholar CrossRef Search ADS PubMed  US EPA. ( 2016) Ecological Risk Management Rationale for Pyrethroids in Registration Review. Washington, DC, USA https://www.regulations.gov/document? D=EPA-HQ-OPP-2012-0167-0047: Usmani S. M., Fois G., Albrecht S., von A. S., Dietl P., Wittekindt O. H. ( 2010). 2-APB and capsazepine-induced Ca2+ influx stimulates clathrin-dependent endocytosis in alveolar epithelial cells. Cell Physiol. Biochem.  25, 91– 102. Google Scholar CrossRef Search ADS PubMed  Vais H., Williamson M. S., Devonshire A. L., Usherwood P. N. R. ( 2001). The molecular interactions of pyrethroid insecticides with insect and mammalian sodium channels. Pest Manage. Sci.  57, 877– 888. Google Scholar CrossRef Search ADS   Verkhratsky A. ( 2005). Physiology and pathophysiology of the calcium store in the endoplasmic reticulum of neurons. Physiol. Rev.  85, 201– 279. http://dx.doi.org/10.1152/physrev.00004.2004 Google Scholar CrossRef Search ADS PubMed  Wang L. M., Liu W., Yang C. X., Pan Z. Y., Gan J. Y., Xu C., Zhao M. R., Schlenk D. ( 2007). Enantioselectivity in estrogenic potential and uptake of bifenthrin. Environ. Sci. Technol.  41, 6124– 6128. Google Scholar CrossRef Search ADS PubMed  Wolfe M. W., Call G. B. ( 1999). Early growth response protein 1 binds to the luteinizing hormone-beta promoter and mediates gonadotropin-releasing hormone-stimulated gene expression. Mol. Endocrinol.  13, 752– 763. Google Scholar PubMed  Yang Y., Ma H. H., Zhou J. H., Liu J., Liu W. P. ( 2014). Joint toxicity of permethrin and cypermethrin at sublethal concentrations to the embryo-larval zebrafish. Chemosphere  96, 146– 154. http://dx.doi.org/10.1016/j.chemosphere.2013.10.014 Google Scholar CrossRef Search ADS PubMed  Ye X., Li F., Zhang J., Ma H., Ji D., Huang X., Curry T. E.Jr, Liu W., Liu J. ( 2017a) Pyrethroid insecticide cypermethrin accelerates pubertal onset in male mice via disrupting hypothalamic-pituitary-gonadal axis. Environ. Sci. Technol. 51, 10212– 10221. Ye X., Pan W., Zhao S., Zhao Y., Zhu Y., Liu J., Liu W. ( 2017b). Relationships of pyrethroid exposure with gonadotropins levels and pubertal development in Chinese boys. Environ. Sci. Technol.  51, 6379– 6386. Google Scholar CrossRef Search ADS   Ye X., Pan W., Zhao Y., Zhao S., Zhu Y., Liu W., Liu J. ( 2017c). Association of pyrethroids exposure with onset of puberty in Chinese girls. Environ. Pollut.  277, 606– 612. Google Scholar CrossRef Search ADS   Yuan Y. W., Chen C., Zheng C. M., Wang X. L., Yang G. L., Wang Q., Zhang Z. H. ( 2014). Residue of chlorpyrifos and cypermethrin in vegetables and probabilistic exposure assessment for consumers in Zhejiang Province, China. Food Control  36, 63– 68. Google Scholar CrossRef Search ADS   Zhang J. Y., Zhang J., Liu R., Gan J., Liu J., Liu W. P. ( 2016). Endocrine-Disrupting Effects of Pesticides through Interference with Human Glucocorticoid Receptor. Environmental Science & Technology  50, 435– 443. Google Scholar CrossRef Search ADS PubMed  Zhao M. R., Zhang Y., Zhuang S. L., Zhang Q., Lu C. S., Liu W. P. ( 2014). Disruption of the hormonal network and the enantioselectivity of bifenthrin in trophoblast: Maternal-fetal health risk of chiral pesticides. Environ. Sci. Technol.  48, 8109– 8116. Google Scholar CrossRef Search ADS PubMed  Zhou J., Yang Y., Xiong K., Liu J. ( 2014). Endocrine disrupting effects of dichlorodiphenyltrichloroethane analogues on gonadotropin hormones in pituitary gonadotrope cells. Environ. Toxicol. Pharmacol.  37, 1194– 1201. http://dx.doi.org/10.1016/j.etap.2014.04.018 Google Scholar CrossRef Search ADS PubMed  © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Toxicological Sciences Oxford University Press

Pyrethroid Insecticide Cypermethrin Modulates Gonadotropin Synthesis via Calcium Homeostasis and ERK1/2 Signaling in LβT2 Mouse Pituitary Cells

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Abstract

Abstract Pyrethroids are a class of widely used insecticides. Cypermethrin (CP) is one of most commonly used pyrethroid insecticides and its residue has been frequently detected in environmental media. Our recent animal study reported that early postnatal exposure to CP induced an increase in serum levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) as well as the expression of gonadotropin subunit genes (chorionic gonadotropin α [CGα], LHβ and FSHβ) in pituitary tissues. In this study, we further investigated the precise mechanism by which CP at concentrations of 1-100 nM affected the synthesis of gonadotropins using a murine pituitary gonadotropic cell line LβT2. We found that calcium (Ca2+)-dependent extracellular signal-regulated kinase 1/2 (ERK1/2) activity was required for CP-regulated transcription of CGαs, LHβ and FSHβ. We provided the novel evidence that CP caused both influx of extracellular Ca2+ through L-type voltage-gated calcium channels (VGCCs) and release of intracellular Ca2+ from endoplasmic reticulum (ER) via inhibition of Ca2+-ATPase. Our results showed that CP disrupted Ca2+ homeostasis via these two separate and independent pathways, thus resulting in the activation of protein kinase C /c-Raf/ERK1/2/immediate-early genes pathways and subsequent increase in the transcription of gonadotropin subunit genes. Our findings would have important implications for understanding the underlying mechanisms of the disrupting effects of some pyrethroids (such as CP) on the synthesis of pituitary gonadotropins. pyrethroids, cypermethrin, gonadotropin, pituitary, endocrine-disrupting chemicals Pyrethroids are a class of widely used insecticides that are synthetic esters derived from the naturally occurring toxin pyrethrins. Because of their large-scale use in residential and agricultural pest control, the residues of pyrethroids were frequently detected in residential homes and agricultural products (Liu et al., 2005). Therefore, the increased human exposure to pyrethroids through household environment and diets is expected. The urine metabolites of pyrethroids were prevalently found in both adults and children (Ye et al., 2017b,c; Meeker et al., 2009; Han et al., 2008). Cypermethrin (CP) is one of most widely used pyrethroid insecticides and has been extensively used for several decades (US EPA, 2016). The annual usage of CP has been more than 1 million pounds of active ingredient in Unite States, and its use continues to grow (US EPA, 2016). CP residue was frequently detected in environment, food and even in human breast-milk (Bouwman et al., 2006; Tulve et al., 2006; Yuan et al., 2014). Pyrethroids, including CP, have been considered as potential endocrine-disrupting chemicals (EDCs), since they were shown to have hormone-like activities and disrupt the function of endocrine and reproductive systems (Liu et al., 2011a,b; Wang et al., 2007; Zhang et al., 2016; Zhao et al., 2014; Ye et al., 2017a). The pituitary gonadotropins, ie, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), play critical roles in the control of mammalian reproduction and sexual maturity. The associations of pyrethroid exposure with altered levels of gonadotropins have been observed in human and animals. For example, two epidemiological studies showed that urinary metabolites of pyrethroids are positively associated with serum levels of FSH and/or LH in adult men (Han et al., 2008; Meeker et al., 2009). More recently, our human study reported on an association of increased pyrethroid exposure with elevated gonadotropins levels and earlier pubertal development in Chinese boys (Ye et al., 2017b). Our recent animal study showed that early postnatal exposure of male mice to CP induced a dose-dependent increase in serum levels of LH and FSH and significantly accelerated sexual maturity (Ye et al., 2017a). Moreover, our study demonstrated that CP could induce the expression of gonadotropin subunit genes in pituitary tissues (Ye et al., 2017a). Both LH and FSH are heterodimeric glycoproteins consisting of a common α-subunit (also known as chorionic gonadotropin α [CGα]) and unique β-subunits (LHβ and FSHβ) (Thackray et al., 2010). The transcription of these subunit genes is the rate-limiting step for production of LH and FSH (Thackray et al., 2010). Calcium (Ca2+) signaling plays an essential role in the signal transduction cascade necessary for the secretion of gonadotropins from anterior pituitary gonadotropic cells (Mulvaney et al., 1999; Reiss et al., 1997). We have previously indicated that the selective inhibitor for L-type voltage-gated calcium channels (VGCCs), nimodipine, was able to block CP-induced gonadotropin subunit gene transcription in pituitary tissues of male mice, suggesting Ca2+ signaling is involved in CP-induced gonadotropin synthesis (Ye et al., 2017a). However, the specific roles of spatial and temporal Ca2+ signals and the modifications of downstream signaling cascade in regulation of CP-stimulated gonadotropin production remains unclear. Previous studies have shown that activation of a mitogen-activated protein kinase (MAPK) family member, extracellular signal-regulated kinase 1/2 (ERK1/2), was absolutely required for the transcription of subunit genes of gonadotropins (Thackray et al., 2010). It has been reported that Ca2+ influx through L-type VGCCs was required for the gonadotropin-releasing hormone (GnRH) stimulation of ERK1/2 (Mulvaney et al., 1999). Thus, we proposed in this study that CP might activate ERK1/2 cascade and thereby stimulate the production of gonadotropins from pituitary gonadotropes, possibly through modification of Ca2+ signaling. In this study, we investigated the precise mechanism by which CP affected the synthesis of gonadotropins using a murine pituitary gonadotropic cell line LβT2, which expresses CGα, LHβ, and FSHβ subunit genes and secretes gonadotropins in response to GnRH and EDCs (Dooley et al., 2008, 2013; Zhou et al., 2014). MATERIALS AND METHODS Materials CP (CAS No. 52315-07-8,≥97% purity), GnRH, cetrorelix, PD184352, GF109203X, 1, 2-bis (2-aminophenoxy) ethane-N, N, N’, N’-tetraacetic acid acetoxymethyl ester (BAPTA-AM), ethylene glycol tetraacetic acid (EGTA), nimodipine and thapsgargin were purchased from Sigma-Aldrich Corp. (St Louis, Missouri). Tetrodotoxin (TTX) was purchased from Ruifang, Inc. (Dalian, China). W-7 and U73122 were purchased from Tocris Bioscience (Bristol, UK). All chemicals used in cell exposure experiments were dissolved in dimethylsulfoxide (DMSO). Antibodies for phospho-ERK1/2 (cat. no. 9101 s), ERK1/2 (cat. no. 9102), phospho-c-Raf (cat. no. 9427), c-Raf (cat. no. 9422 s), c-Jun (cat. no. 9165 s) and c-Fos (cat. no. 2250 s) were purchased from Cell Signaling Technology, Inc. (Beverly, Massachusetts). Antibody for Egr-1 (cat. no. sc-110) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, California). Antibody for GAPDH (10494-1-AP) was obtained from Proteintech Group, Inc. (Chicago, Illinois). Cell culture and exposure The murine pituitary gonadotrope cell line LβT2 was kindly provided by Dr Pamela Mellon at the University of California, San Diego. The LβT2 cells were cultured as described previously in Liu et al. (2006). Briefly, cells were grown in poly-L-lysine -coated flasks with DMEM medium (Hyclone, Logan, Uttah) supplement with 10% fetal bovine serum (Hyclone) and incubated at 37 °C in an atmosphere of 5% CO2. A 24-h serum starvation was performed before each chemical treatment for the cells. The starved cells were exposed to fresh serum-free media containing 1, 10, or 100 nM CP for 24 h. For inhibitors experiments, cells were pretreated with various inhibitors or vehicle (0.1% DMSO) for 30 min, and then treated with 100 nM CP. At defined time points, cells were collected for RNA or protein extraction. Measurement of LH and FSH LβT2 cells and conditioned media were collected after exposure to CP alone or in combination with inhibitors as described. To determine the concentrations of hormones in cytoplasm, cells were collected in 2.5 ml PBS buffer (105 cells/ml), repeatedly frozen and thawed, and then centrifuged and the supernatant was collected. LH and FSH levels were measured using the ELISA kits according to the manufacturer’s protocol (USCN Life Science Inc., Wuhan, China). Inter and intra-assay coefficient of variations for LH and FSH were <10%. The concentrations of hormones in cytoplasm were normalized by the total protein concentration. Quantification of mRNA Total RNA was isolated from cells or tissues using Trizol reagent (Life Technologies, Carlsbad, California) according to the manufacturer’s protocol. Synthesis of first-strand cDNA was performed by reverse transcription of 0.5 μg total RNA using ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan). Oligonucleotide primer sequences for mouse CGα, LHβ, FSHβ, and L32 were listed in Supplementary Table 1. The real-time PCR reactions were set up with SYBR Green PCR master mix (Toyobo) using a Mx3000P Real-time PCR system (Agilent Technologies, Palo Alto, California), as described previously (Liu et al., 2011a; Yang et al., 2014). The relative amount of each gene transcript was calculated using the 2−ΔΔCT method and normalized to the endogenous reference gene L32. Western blot analysis Western blotting was performed as described previously (Li et al., 2012; Liu et al., 2010). Total protein was isolated from cells. Fifty micrograms of protein was separated on a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane (Millipore, Billerica, Massachusetts). Membrane was incubated with the primary antibody overnight at 4 °C. Blots were analyzed using an enhanced chemiluminescence detection system (Pierce, Rockford, Illinois) and photographed using BioSpectrum 410 Imaging System (UVP, Upland, California). Each experiment was repeated at least three times. Determination of intracellular Ca2+ Intracellular Ca2+ levels were determined as previously described (Usmani et al., 2010). Briefly, LβT2 cells were incubated with HEPES-buffered saline containing 2 mM Fluo-4AM in the presences or absence of 10 μM nimodipine, 3 mM EGTA, or 50 μM BAPTA-AM. Cells were placed into an Infinite M200 PRO plate reader (Tecan Group, Männedorf, Switzerland) and kinetic cycle was measured with excitation at 490 nm and emission at 525 nm. Cells were stimulated after fluorescence reached a steady state. Then CP was added to the cells immediately prior to measurement for another 30 min. Relative fluorescence (ΔF/F) was calculated as fluorescence intensities normalized to fluorescence intensity measured at the last time point before CP addition. All the measurements were performed in triplicates. Ca2+-ATPase activity determination LβT2 cells were exposed to 1, 10, or 100 nM CP for 1 h. Cells were homogenized in 0.9% NaCl solution and then centrifuged. The supernatant was collected and employed to determine the total Ca2+-ATPase activity according to the manufacturer’s protocol of commercial kits (Jiancheng Bioengineering Institute, Nanjing, China). Intracellular inositol 1, 4, 5-trisphosphate measurements LβT2 cells were exposed to 100 nM GnRH or 1, 10, and 100 nM CP for 5 min. Cells were collected in 2.5 ml PBS buffer (105 cells/ml), repeatedly frozen and thawed, and then centrifuged. The supernatant was collected and employed to determine the inositol 1, 4, 5-trisphosphate (InsP3) concentrations according to the manufacturer’s protocol of commercial ELISA kits (Uscn Life Science, Wuhan, China). Statistical analysis Each experiment of cell treatment was performed in triplicate at least 3 times. All data were expressed as the mean ± SEM and tested by using 1-way ANOVA. If ANOVA revealed significant effects of treatments, the datasets from 2 groups were compared by 2-tailed unpaired Student’s t test, and p < .05 was considered significant. RESULTS Stimulation of Gonadotropin Synthesis by CP in LβT2 Cells LβT2 cells were exposed to CP at concentrations of 1, 10 or 100 nM for 24 h. Cell viability results indicated that CP at concentrations tested in this study had no cytotoxicity to LβT2 cells (Supplementary Figure 1). CP stimulated the mRNA levels of CGα, LHβ, and FSHβ in a concentration-dependent manner (Figure 1A). The protein levels of LH and FSH in cytoplasm and conditioned culture media were significantly up-regulated by CP (Figs. 1B and 1C). Figure 1. View largeDownload slide Stimulation of gonadotropin synthesis by CP. LβT2 cells were exposed to CP at concentrations of 1, 10, or 100 nM for 24 h. A, The mRNA levels for CGα, LHβ, and FSHβ in LβT2 cells. B, The levels of LH and FSH in cytoplasm. C, The levels of LH and FSH in cultured media. Values shown represent mean ±SEM for three independent experiments performed in triplicate. * p < .05 treatment versus control. Figure 1. View largeDownload slide Stimulation of gonadotropin synthesis by CP. LβT2 cells were exposed to CP at concentrations of 1, 10, or 100 nM for 24 h. A, The mRNA levels for CGα, LHβ, and FSHβ in LβT2 cells. B, The levels of LH and FSH in cytoplasm. C, The levels of LH and FSH in cultured media. Values shown represent mean ±SEM for three independent experiments performed in triplicate. * p < .05 treatment versus control. Requirement of ERK1/2 Activity in CP-Induced Gonadotropin Subunit Gene Expression As shown in Figure 2A, ERK1/2 inhibitor PD184352 prevented the induction of CGα, LHβ, and FSHβ gene expression by 100 nM CP. Addition of 100 nM CP stimulated a rapid and very strong increase in ERK1/2 phosphorylation that reached its peak at 5 min (Figure 2B). A concentration-dependent increase in ERK1/2 activity was observed in LβT2 cells exposed to 1, 10, or 100 nM CP for 5 min (Figure 2C) and PD184352 completely attenuated ERK1/2 activity induced by CP (Figure 2D). These data suggest that activation of ERK1/2 is required for CP-induced gonadotropin subunit gene expression. Figure 2. View largeDownload slide Role of ERK1/2 activity in CP-induced gonadotropin subunit gene expression. A, The mRNA levels for CGα, LHβ and FSHβ in LβT2 cells exposed to 100 nM CP alone or in combination with 1 μM PD184352 (PD). Values shown represent mean ± SEM for 3 independent experiments performed in triplicate. * p < .05 treatment versus control. B, Phosphorylation of ERK1/2 (p-ERK1/2) in LβT2 cells exposed to 100 nM CP for 5, 10, 30, or 60 min, respectively. C, p-ERK1/2 in LβT2 cells treated with CP at concentrations of 1, 10 or 100 nM for 5 min. D, p-ERK1/2 in LβT2 cells exposed to 100 nM CP alone or in combination with 1 μM PD. t-ERK1/2: total ERK1/2. Figure 2. View largeDownload slide Role of ERK1/2 activity in CP-induced gonadotropin subunit gene expression. A, The mRNA levels for CGα, LHβ and FSHβ in LβT2 cells exposed to 100 nM CP alone or in combination with 1 μM PD184352 (PD). Values shown represent mean ± SEM for 3 independent experiments performed in triplicate. * p < .05 treatment versus control. B, Phosphorylation of ERK1/2 (p-ERK1/2) in LβT2 cells exposed to 100 nM CP for 5, 10, 30, or 60 min, respectively. C, p-ERK1/2 in LβT2 cells treated with CP at concentrations of 1, 10 or 100 nM for 5 min. D, p-ERK1/2 in LβT2 cells exposed to 100 nM CP alone or in combination with 1 μM PD. t-ERK1/2: total ERK1/2. Noninvolvement of GnRH Receptor/Phospholipase C/InsP3 Pathway in CP-Induced Gonadotropin Subunit Gene Expression and ERK1/2 Activation It is known that, in pituitary gonadotropic cells, binding of ligand (GnRH or its agonists) to GnRH receptor (GnRHR) initiates phospholipase C (PLC) activity to generate InsP3, which leads to elevation of intracellular Ca2+ levels, activation of protein kinase C (PKC) and MAPK cascades to regulate the synthesis of gonadotropins (Thackray et al., 2010). To investigate whether GnRHR mediates the effects of CP on gonadotropin gene expression and ERK1/2 activity, LβT2 cells were treated with CP in combination with the GnRH antagonist cetrorelix. Cetrorelix significantly blocked GnRH-induced expression of CGα, LHβ, and FSHβ genes, whereas it was unable to prevent the stimulation of gonadotropin subunit gene expression and ERK1/2 activation by CP (Supplementary Figs. 2A and 2B). Furthermore, the PLC inhibitor U73122 could not abolish the activity of ERK1/2 by CP (Supplementary Figure 2C). The InsP3 concentrations did not change in cells exposed to CP (Supplementary Figure 2D). These results indicate that GnRHR/PLC/InsP3 pathway is not involved in CP-induced gonadotropin gene expression and ERK1/2 activation. Noninvolvement of Voltage-Gated Sodium Channels in CP-Induced Gonadotropin Subunit Gene Expression and ERK1/2 Activation It has been well-documented that voltage-gated sodium channels (VGSCs) are primary target sites for pyrethroids (Shafer et al., 2005). We examined whether VGSCs were involved in CP-induced gonadotropin subunit gene expression and ERK1/2 activation in LβT2 cells. The VGSCs specific inhibitor TTX at concentration of 10 μM was without effects on the induction of gonadotropin subunit gene expression and ERK1/2 phosphorylation by CP (Supplementary Figure 3). Requirement of Ca2+ Signaling in CP-Induced ERK1/2 Activation To examine the VGCCs and Ca2+ requirement, LβT2 cells were pretreated with 10 μM nimodipine to block L-type VGCCs, 3 mM EGTA to chelate extracellular Ca2+, or 50 μM BAPTA-AM to chelate intracellular Ca2+. Nimodipine and EGTA caused a partial reduction in ERK1/2 activity by CP stimulation, while BAPTA-AM almost completely attenuated CP-induced ERK1/2 activation (Figure 3A). The effects of CP on changes in intracellular Ca2+ levels ([Ca2+]i) in LβT2 cells were further investigated. As shown in Figure 3B, CP treatment caused an increase in the amount of [Ca2+]i compared with untreated cells, and pretreatment of cells with nimodipine, EGTA or BAPTA-AM significantly attenuated the increase in [Ca2+]i when the cells were treated with CP. These data suggest that CP increased Ca2+ influx through L-type VGCCs and Ca2+ signal is required for CP-induced ERK1/2 activation. Figure 3. View largeDownload slide Role of Ca2+ signaling in CP-induced ERK1/2 activation. A, Phosphorylation of ERK1/2 (p-ERK1/2) in LβT2 cells exposed to 100 nM CP for 5 min in the presence or absence of 10 μM nimodipine (Nimo), 3 mM EGTA, or 50 μM BAPTA-AM (BAPTA). B, Time course of [Ca2+]i changes induced by 100 nM CP in the presence or absence of Nimo, EGTA or BAPTA. C, p-ERK1/2 in LβT2 cells treated with 100 nM CP for 5 min or 2 mM thapsigargin (Thap) for 5, 10, or 30 min, or they were treated with Thap 30 min prior to and during CP application. D, Ca2+-ATPase activity in LβT2 cells exposed to CP at concentrations of 1, 10 or 100 nM. E, p-ERK1/2 in LβT2 cells exposed to 100 nM CP for 5 min in the presence or absence of 15 μM W-7. Values shown represent mean ± SEM for 3 independent experiments performed in triplicate. * p < .05 treatment versus control. t-ERK1/2: total ERK1/2. Figure 3. View largeDownload slide Role of Ca2+ signaling in CP-induced ERK1/2 activation. A, Phosphorylation of ERK1/2 (p-ERK1/2) in LβT2 cells exposed to 100 nM CP for 5 min in the presence or absence of 10 μM nimodipine (Nimo), 3 mM EGTA, or 50 μM BAPTA-AM (BAPTA). B, Time course of [Ca2+]i changes induced by 100 nM CP in the presence or absence of Nimo, EGTA or BAPTA. C, p-ERK1/2 in LβT2 cells treated with 100 nM CP for 5 min or 2 mM thapsigargin (Thap) for 5, 10, or 30 min, or they were treated with Thap 30 min prior to and during CP application. D, Ca2+-ATPase activity in LβT2 cells exposed to CP at concentrations of 1, 10 or 100 nM. E, p-ERK1/2 in LβT2 cells exposed to 100 nM CP for 5 min in the presence or absence of 15 μM W-7. Values shown represent mean ± SEM for 3 independent experiments performed in triplicate. * p < .05 treatment versus control. t-ERK1/2: total ERK1/2. To rule out a potential contribution of intracellular Ca2+ stores in this signaling pathway, cells were treated with thapsigargin, an inhibitor of the Ca2+-ATPase that pumps Ca2+ into intracellular stores. Thapsigargin is known to increase cytoplasmic Ca2+ levels and eventually cause depletion of intracellular stores (Mulvaney et al., 1999). Thapsigargin treatment alone stimulated ERK1/2 phosphorylation and CP-induced ERK1/2 activation was markedly reduced in thapsigargin-pretreated cells (Figure 3C), indicating that the thapsigargin-stimulated Ca2+ signal activates ERK1/2 and depletion of Ca2+ intracellular stores by thapsigargin results in an attenuation of CP-induced ERK1/2 activation. We further showed that CP exposure caused a dose-dependent decrease in Ca2+-ATPase activity in LβT2 cells (Figure 3D). These results suggest that CP could also inhibit Ca2+-ATPase activity, which leads to a release of Ca2+ from intracellular stores and consequently activate ERK1/2. Calmodulin (CaM) is the prototypical Ca2+-binding protein and serves important roles as a Ca2+ sensor in a number of different intracellular signaling scenarios (Takemoto-Kimura et al., 2017). In this study, pretreatment with the CaM inhibitor W-7 abolished CP-induced ERK1/2 activation (Figure 3E), indicating that CaM is also involved in CP-dependent ERK1/2 pathway. Involvement of Ca2+ Signaling in CP-Induced c-Raf Activation Next studies focused on examining the effects of Ca2+ pathway on upstream catalytic activity of c-Raf kinase that has been known as a key signaling intermediate within the ERK1/2 cascade in gonadotropic cells (Roberson et al., 2005). Addition of 100 nM CP caused a significant increase in the phosphorylation of c-Raf (p-c-Raf) kinase at serine 338 (Figure 4A). The p-c-Raf with CP was blocked by nimodipine, EGTA and BAPTA-AM (Figure4A). Treatment with thapsigargin alone also stimulated c-Raf phosphorylation and CP-induced c-Raf activation was reduced in thapsigargin-treated cells (Figure 4B). Pretreatment of LβT2 cells with W-7 resulted in an inhibition of c-Raf kinase phosphorylation (Figure 4C). These data suggest that VGCCs and Ca2+-ATPase/Ca2+/CaM signaling pathways mediate CP-induced c-Raf activation. Figure 4. View largeDownload slide Involvement of Ca2+ signaling in CP-induced c-Raf activation. A, Phosphorylation of c-Raf (p-c-Raf) in LβT2 cells exposed to 100 nM CP for 5 min in the presence or absence of 10 μM nimodipine (Nimo), 3 mM EGTA or 50 μM BAPTA-AM (BAPTA). B, p-c-Raf in LβT2 cells treated with 100 nM CP for 5 min or 2 mM thapsigargin (Thap) for 5, 10, or 30 min, or they were treated with Thap 30 min prior to and during CP application. C, p-c-Raf in LβT2 cells exposed to 100 nM CP for 5 min in the presence or absence of 15 μM W-7. t-c-Raf: total c-Raf. Figure 4. View largeDownload slide Involvement of Ca2+ signaling in CP-induced c-Raf activation. A, Phosphorylation of c-Raf (p-c-Raf) in LβT2 cells exposed to 100 nM CP for 5 min in the presence or absence of 10 μM nimodipine (Nimo), 3 mM EGTA or 50 μM BAPTA-AM (BAPTA). B, p-c-Raf in LβT2 cells treated with 100 nM CP for 5 min or 2 mM thapsigargin (Thap) for 5, 10, or 30 min, or they were treated with Thap 30 min prior to and during CP application. C, p-c-Raf in LβT2 cells exposed to 100 nM CP for 5 min in the presence or absence of 15 μM W-7. t-c-Raf: total c-Raf. Requirement of PKC in CP-Induced c-Raf/ERK1/2 Signaling and Gonadotropin Gene Expression It is known that Ca2+-dependent PKC controls the expression of three gonadotropin subunit genes in gonadotropic cells (Thackray et al., 2010). We further tested whether PKC mediates the up-regulation of gonadotropin gene expression by CP. The results showed that GF109203X, a specific inhibitor for PKC, significantly diminished CP-enhanced expression of CGα, LHβ, and FSHβ genes in LβT2 cells (Figure 5A). GF109203X abolished the p-c-Raf and ERK1/2 in CP-treated cells (Figure 5B). These data indicate that PKC is the upstream activator of c-Raf/ERK1/2 upon CP stimulation. Figure 5. View largeDownload slide Requirement of PKC in CP-induced c-Raf/ERK1/2 signaling and gonadotropin gene expression. A, The mRNA levels for CGα, LHβ and FSHβ in LβT2 cells exposed to 100 nM CP for 24 h in the presence or absence of 5 μM GF109203X (GF). Values shown represent mean ± SEM for 3 independent experiments performed in triplicate. * p < .05 treatment versus control. B, Phosphorylation of c-Raf (p-c-Raf) or ERK1/2 (p-ERK1/2) in LβT2 cells exposed to 100 nM CP for 5 min in the presence or absence of 5 μM GF. t-c-Raf: total c-Raf; t-ERK1/2: total ERK1/2. Figure 5. View largeDownload slide Requirement of PKC in CP-induced c-Raf/ERK1/2 signaling and gonadotropin gene expression. A, The mRNA levels for CGα, LHβ and FSHβ in LβT2 cells exposed to 100 nM CP for 24 h in the presence or absence of 5 μM GF109203X (GF). Values shown represent mean ± SEM for 3 independent experiments performed in triplicate. * p < .05 treatment versus control. B, Phosphorylation of c-Raf (p-c-Raf) or ERK1/2 (p-ERK1/2) in LβT2 cells exposed to 100 nM CP for 5 min in the presence or absence of 5 μM GF. t-c-Raf: total c-Raf; t-ERK1/2: total ERK1/2. Induction of the Immediate-Early Gene Expression by CP via Ca2+/PKC/ERK1/2 Signaling Pathway It has been shown that stimulation with GnRH or agonists results in increased mRNA and protein for the immediate-early genes (IEGs), ie, Egr-1, c-Fos, and c-Jun, in pituitary gonadotropic cells (Cheng et al., 2000; Liu et al., 2002; Wolfe and Call, 1999). In response to extracellular signals, Egr-1 and the c-Fos/c-Jun heterodimers (activator protein-1, AP-1) serve as transcription factors to promote gonadotropin subunit gene transcription (Cheng et al., 2000; Liu et al., 2002; Wolfe and Call, 1999). Therefore, we examined the effects of CP on the mRNA and protein expression of Egr-1, c-Fos and c-Jun. As shown in Figures 6A and 6B, the mRNA and protein levels for these IEGs were concentration-dependently increased in cells exposed to 10 or 100 nM CP. Chelation of intracellular Ca2+ with BAPTA-AM, or extracellular Ca2+ with EGTA blocked CP-induced expression of Egr-1, c-Fos, and c-Jun proteins (Figure 6C). Inhibition of PKC with GF109203X prevented the induction of Egr-1, c-Fos and c-Jun by CP (Figure 6D). Inhibition of ERK1/2 with PD184352, or CaM with W-7 caused significant reduction in expression levels of Egr-1, c-Fos, and c-Jun (Figure 6E). These observations suggest that the CP-induced expression of IEGs is dependent on Ca2+/PKC/ERK1/2 signaling. Figure 6. View largeDownload slide Induction of the IEGs expression by CP. A, The mRNA levels for Egr-1, c-Fos and c-Jun in LβT2 cells exposed to CP at concentrations of 1, 10, or 100 nM for 2 h. Values shown represent mean ± SEM for 3 independent experiments performed in triplicate. * p < 0.05 treatment versus control. B, The protein levels for Egr-1, c-Fos and c-Jun in LβT2 cells exposed to 1-100 nM CP for 2 h. C–E, The protein levels for Egr-1, c-Fos and c-Jun in LβT2 cells exposed to 100 nM CP for 2 h in the presence or absence of 50 μM BAPTA-AM (BAPTA), 3 mM EGTA, 5 μM GF109203X (GF), 1 μM PD184352 (PD) or 15 μM W-7, respectively. Figure 6. View largeDownload slide Induction of the IEGs expression by CP. A, The mRNA levels for Egr-1, c-Fos and c-Jun in LβT2 cells exposed to CP at concentrations of 1, 10, or 100 nM for 2 h. Values shown represent mean ± SEM for 3 independent experiments performed in triplicate. * p < 0.05 treatment versus control. B, The protein levels for Egr-1, c-Fos and c-Jun in LβT2 cells exposed to 1-100 nM CP for 2 h. C–E, The protein levels for Egr-1, c-Fos and c-Jun in LβT2 cells exposed to 100 nM CP for 2 h in the presence or absence of 50 μM BAPTA-AM (BAPTA), 3 mM EGTA, 5 μM GF109203X (GF), 1 μM PD184352 (PD) or 15 μM W-7, respectively. DISCUSSION We have previously demonstrated in mice that exposure to CP stimulated the transcription of gonadotropin subunit genes in pituitary through interference with VGCCs (Ye et al., 2017a). In the present study, we found that Ca2+-dependent ERK1/2 activity was required for CP-regulated gonadotropin synthesis. We provided the evidence that CP caused both influx of extracellular Ca2+ through interference with VGCCs and release of intracellular Ca2+ via inhibition of Ca2+-ATPase in LβT2 cells. Our results showed that CP disrupted Ca2+ homeostasis via these two separate and independent pathways, thus resulting in the activation of PKC/c-Raf/ERK1/2/IEGs cascade and subsequent increase in the transcription of gonadotropin subunit genes (summary in Figure 7). Figure 7. View largeDownload slide Schematic model of CP-induced production of gonadotropins through Ca2+/PKC/c-Raf/ERK1/2/IEGs signaling in pituitary gonadotropic cells. Figure 7. View largeDownload slide Schematic model of CP-induced production of gonadotropins through Ca2+/PKC/c-Raf/ERK1/2/IEGs signaling in pituitary gonadotropic cells. Neuronal VGSCs are identified as primary targets for the insecticidal and neurotoxic effects of pyrethroids (Soderlund, 2012). The expression of VGSCs has been previously identified in isolated rat gonadotropic cells and mouse gonadotropic cell line (Bosma and Hille, 1992; Tse and Hille, 1993). Veratridine, a neurotoxin that abolishes inactivation of VGSCs in a similar manner as CP, was found to induce LH secretion in primary rat pituitary cell cultures and veratridine-stimulated LH release was attenuated by blockade of VGSCs with TTX (Conn and Rogers, 1980). However, the current findings in LβT2 cells showed that TTX failed to block CP-induced gonadotropin subunit gene expression and ERK1/2 activation. Although perturbation of VGSCs is the primary mode of pyrethroid action in both insects and mammals, mammalian VGSCs sensitivity to pyrethroids is 1000-fold less sensitive than the sodium channel of insect (Vais et al., 2001). The treatment concentrations of veratridine (10−6 M to 10−4 M) used in the previous study were 1000 times higher than the environmentally relevant concentrations of CP (10−9 M to 10−7 M) used in our study. Our previous study also demonstrated that the VGSCs-mediated effects of CP on mouse hypothalamic explants were only observed at higher concentrations of 10−6 M and 10−5 M (Ye et al., 2017a). Therefore, VGSCs are nonresponsible for CP’s action in gonadotropic cells may be due to the insensitivity of mammalian VGSCs to CP at low concentrations. In addition to VGSCs, other ion channels, particularly VGCCs, have been implicated as alternative sites of action for a subset of pyrethroids (Soderlund, 2012). A previous study in mouse neocortical neurons reported that exposure to CP and other pyrethroids produced concentration-dependent elevations in intracellular Ca2+ concentration (Cao et al., 2011). Another study found that CP triggered the release of Ca2+ in primary culture of rat astrocytes (Maurya et al., 2014). The activation of VGCCs by pyrethroids also has been observed in non-neural cells. For instance, allethrin induced apoptosis was associated with VGCCs mediated intracellular Ca2+ release in rat testicular carcinoma cells (Madhubabu and Yenugu, 2014). Deltamethrin induced [Ca2+]i rise that involved Ca2+ entry through L-type VGCCs in human prostate cancer cells (Lee et al., 2016). In agreement with these previous observations, we found that CP exposure at non-cytotoxic concentrations caused an increase in [Ca2+]i in pituitary gonadotropic cells. [Ca2+]i can be elevated by extracellular Ca2+ entry through VGCCs or Ca2+ release from intracellular Ca2+ stores such as the endoplasmic reticulum (ER) (Verkhratsky, 2005). In this study, the L-type VGCCs selective blocker nimodipine was significantly but not completely blocked CP-stimulated c-Raf/ERK1/2 activity, while CP-stimulated c-Raf/ERK activation required refilling of thapsigargin-sensitive Ca2+ stores. These data suggest that the release of Ca2+ from ER was also sufficient for activation of c-Raf/ERK cascade by CP. The ER acts as a dynamic Ca2+ store and possesses independent pathways for intracellular Ca2+ influx and efflux (Verkhratsky, 2005). Release of Ca2+ from the ER usually occurs through an InsP3-stimulated calcium channel, commonly known as InsP3 receptors (InsP3Rs) (Verkhratsky, 2005). GnRH-induced release of Ca2+ from intracellular stores in gonadotropic cells has been shown to respond to elevation of InsP3, which is dependent on the activation of GnRHR/PLC pathway (Thackray et al., 2010). However, our data showed that exposure to CP did not alter cellular InsP3 concentrations in LβT2 cells. GnRHR/PLC/InsP3/InsP3Rs pathway seems not to be responsible for CP-induced gonadotropin subunit gene expression and ERK1/2 activation. Consistent with our observations, another pyrethroid deltamethrin evoked [Ca2+]i rise was also required for PLC-independent Ca2+ release from the ER (Lee et al., 2016). On the other hand, Ca2+ influx into the ER lumen results from the activity of Ca2+ pumps of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) (Verkhratsky, 2005). In this study, we found that CP could inhibit the activity of Ca2+-ATPase in a concentration-dependent manner. The results of this study support speculation that the inhibition of the SERCA pumps by CP, which resulted in a relatively slow emptying of releasable Ca2+ from the ER, may at least partially contribute to CP-induced [Ca2+]i in gonadotropic cells. The ERK1/2 branch of the MAPK pathways is a critical signaling molecule to mediate a variety of actions of pyrethroids on cells. For example, the requirement of ERK1/2 pathway has been reported in studies examining the neurotrophic effects of deltamethrin on cultured neurons (Ihara et al., 2012). CP-induced macrophages apoptosis through oxidative stress-mediated ERK1/2 signaling (Huang et al., 2016). We showed here that Ca2+-dependent ERK1/2 activation was required for CGα, LHβ or FSHβ induction by CP in gonadotropic cells. Stimulation of [Ca2+]i with CP was also a sufficient stimulus for activation of c-Raf protein, which is part of the ERK1/2 pathway as a MAPK kinase (MAP3K) (Roskoski, 2010). Consistent with our observations, many previous studies demonstrated that the expression of gonadotropic subunit genes was Ca2+ dependent and augmented by c-Raf/ERK1/2 signaling (Thackray et al., 2010). We further demonstrated that induction of gonadotropic subunit gene expression by CP was dependent upon PKC signaling, since the PKC inhibitor GF109203X prevented gonadotropic subunit gene transcription. Activation of c-Raf and ERK1/2 kinases by CP was also inhibited by GF109203X. These data suggest that PKC is acting downstream of Ca2+ but upstream of c-Raf and ERK1/2, since a role for Ca2+ in the activation of PKC has been well documented (Huang, 1989). Regulation of the LHβ and FSHβ genes occurs through the induction of intermediate IEGs, which constitute Egr-1 and AP-1 consisting of c-Fos and c-Jun (Thackray et al., 2010). In our present study, CP-increased [Ca2+]i appeared to be sufficient to induce increased protein for the ERK1/2 substrates Egr-1, c-Fos, and c-Jun, since chelation of Ca2+ and inhibition of PKC/ERK1/2 significantly attenuated CP-induced increases in the expression of these IEGs in LβT2 cells. Previous study showed that CaM had a critical role as a Ca2+ sensor in specifically linking Ca2+ flux with ERK1/2 activation and c-Raf kinase was a CaM-binding protein in a Ca2+-dependent manner (Roberson et al., 2005). In this study, CaM inhibition by W-7 effectively blocked c-Raf kinase phosphorylation at S338, subsequently leading to inhibition of ERK1/2 phosphorylation and IEGs induction by CP. These data suggest that CaM serving as Ca2+ binding protein effector is required for sensing local Ca2+ influx and activation of c-Raf/ERK by CP. Further studies will be of interest to determine the roles of two downstream pathways of CaM, the CaM-dependent kinases and the phosphatase calcineurin, in transducing CP-activated signals to gonadotropin gene transcription. In conclusion, this study provided novel evidence in support that a commonly used pyrethroid insecticide, CP, regulated Ca2+ homeostasis by two different pathways, subsequently activated PKC/c-Raf/ERK1/2/IEGs signaling to promote gonadotropin subunit gene transcription in LβT2 cells. We also found the crosstalk between Ca2+/CaM pathway and PKC-dependent ERK1/2 pathway in coordinate regulation of gonadotropin subunit gene expression. This would have important implications for understanding the precise mechanisms of the disrupting effects of pyrethroids on the synthesis of pituitary gonadotropins. Our findings suggest that like endocrine cell types such as gonadotropic cells may make use of elementary Ca2+ signals in the regulation of specific hormone synthesis by EDCs, such as pyrethroids. SUPPLEMENTARY DATA Supplementary data are available at Toxicological Sciences online. ACKNOWLEDGMENTS We are grateful to Dr Zhengxing Wu at Huazhong University of Science & Technology for kindly technical assistance for the cultures of LβT2 cells. FUNDING This work was supported by the National Natural Science Foundation of China (21377113 and 81670971) and Zhejiang Provincial Natural Science Foundation of China (LR15B070001 and LY15C120001). REFERENCES Bosma M. M., Hille B. ( 1992). Electrophysiological properties of a cell-line of the gonadotrope lineage. Endocrinology  130, 3411– 3420. http://dx.doi.org/10.1210/endo.130.6.1317783 Google Scholar CrossRef Search ADS PubMed  Bouwman H., Sereda B., Meinhardt H. M. ( 2006). Simultaneous presence of DDT and pyrethroid residues in human breast milk from a malaria endemic area in South Africa. Environ. Pollut.  144, 902– 917. http://dx.doi.org/10.1016/j.envpol.2006.02.002 Google Scholar CrossRef Search ADS PubMed  Cao Z., Shafer T. J., Murray T. F. ( 2011). Mechanisms of pyrethroid insecticide-induced stimulation of calcium influx in neocortical neurons. J. Pharmacol. Exp. Ther.  336, 197– 205. http://dx.doi.org/10.1124/jpet.110.171850 Google Scholar CrossRef Search ADS PubMed  Cheng K. W., Ngan E. S., Kang S. K., Chow B. K., Leung P. C. ( 2000). Transcriptional down-regulation of human gonadotropin-releasing hormone (GnRH) receptor gene by GnRH: Role of protein kinase C and activating protein 1. Endocrinology  141, 3611– 3622. Google Scholar CrossRef Search ADS PubMed  Conn P. M., Rogers D. C. ( 1980). Gonadotropin-release from pituitary cultures following activation of endogenous ion channels. Endocrinology  107, 2133– 2134. http://dx.doi.org/10.1210/endo-107-6-2133 Google Scholar CrossRef Search ADS PubMed  Dooley G. P., Reardon K. F., Prenni J. E., Tjalkens R. B., Legare M. E., Foradori C. D., Tessari J. E., Hanneman W. H. ( 2008). Proteomic analysis of diaminochlorotriazine adducts in wister rat pituitary glands and L beta T2 rat pituitary cells. Chem. Res. Toxicol.  21, 844– 851. Google Scholar CrossRef Search ADS PubMed  Dooley G. P., Tjalkens R. B., Hanneman W. H. ( 2013). The atrazine metabolite diaminochlorotriazine suppresses LH release from murine L beta T2 cells by suppressing GnRH-induced intracellular calcium transients. Toxicol. Res.  2, 180– 186. http://dx.doi.org/10.1039/c3tx20088d Google Scholar CrossRef Search ADS   Han Y., Xia Y., Han J., Zhou J., Wang S., Zhu P., Zhao R., Jin N., Song L., Wang X. ( 2008). The relationship of 3-PBA pyrethroids metabolite and male reproductive hormones among non-occupational exposure males. Chemosphere  72, 785– 790. Google Scholar CrossRef Search ADS PubMed  Huang F., Liu Q. Y., Xie S. J., Xu J., Huang B., Wu Y. H., Xia D. J. ( 2016). Cypermethrin Induces Macrophages Death through Cell Cycle Arrest and Oxidative Stress-Mediated JNK/ERK Signaling Regulated Apoptosis. Int. J. Mol. Sci.  17, 885. Google Scholar CrossRef Search ADS   Huang K. P. ( 1989). The Mechanism of Protein Kinase-C Activation. Trends Neurosci.  12, 425– 432. http://dx.doi.org/10.1016/0166-2236(89)90091-X Google Scholar CrossRef Search ADS PubMed  Ihara D., Fukuchi M., Honma D., Takasaki I., Ishikawa M., Tabuchi A., Tsuda M. ( 2012). Deltamethrin, a type II pyrethroid insecticide, has neurotrophic effects on neurons with continuous activation of the Bdnf promoter. Neuropharmacology  62, 1091– 1098. Google Scholar CrossRef Search ADS PubMed  Lee H. H., Chou C. T., Liang W. Z., Chen W. C., Wang J. L., Yeh J. H., Kuo C. C., Shieh P., Kuo D. H., Chen F. A., et al.   ( 2016). Ca2+ Movement Induced by Deltamethrin in PC3 Human Prostate Cancer Cells. Chin. J. Physiol.  59, 148– 155. Google Scholar CrossRef Search ADS PubMed  Li F., Jo M., Curry T. E.Jr, Liu J. ( 2012). Hormonal induction of polo-like kinases (Plks) and impact of Plk2 on cell cycle progression in the rat ovary. PLoS One  7, e41844. Google Scholar CrossRef Search ADS PubMed  Liu F., Austin D. A., Mellon P. L., Olefsky J. M., Webster N. J. ( 2002). GnRH activates ERK1/2 leading to the induction of c-fos and LHbeta protein expression in LbetaT2 cells. Mol. Endocrinol.  16, 419– 434. Google Scholar PubMed  Liu H. S., Hu Z. T., Zhou K. M., Jiu Y. M., Yang H., Wu Z. X., Xu T. ( 2006). Heterogeneity of the Ca2+ sensitivity of secretion in a pituitary gonadotrope cell line and its modulation by protein kinase C and Ca2+. J. Cell Physiol.  207, 668– 674. Google Scholar CrossRef Search ADS PubMed  Liu J., Park E. S., Curry T. E.Jr, Jo M. ( 2010). Periovulatory expression of hyaluronan and proteoglycan link protein 1 (Hapln1) in the rat ovary: Hormonal regulation and potential function. Mol. Endocrinol.  24, 1203– 1217. http://dx.doi.org/10.1210/me.2009-0325 Google Scholar CrossRef Search ADS PubMed  Liu J., Yang Y., Yang Y., Zhang Y., Liu W. ( 2011a). Disrupting effects of bifenthrin on ovulatory gene expression and prostaglandin synthesis in rat ovarian granulosa cells. Toxicology  282, 47– 55. Google Scholar CrossRef Search ADS   Liu J., Yang Y., Zhuang S., Yang Y., Li F., Liu W. ( 2011b). Enantioselective endocrine-disrupting effects of bifenthrin on hormone synthesis in rat ovarian cells. Toxicology  290, 42– 49. Google Scholar CrossRef Search ADS   Liu W. P., Gan J. Y., Schlenk D., Jury W. A. ( 2005). Enantioselectivity in environmental safety of current chiral insecticides. Proc. Natl. Acad. Sci. U.S.A . 102, 701– 706. http://dx.doi.org/10.1073/pnas.0408847102 Google Scholar CrossRef Search ADS PubMed  Madhubabu G., Yenugu S. ( 2014). Allethrin induces oxidative stress, apoptosis and calcium release in rat testicular carcinoma cells (LC540). Toxicol. In Vitro  28, 1386– 1395. http://dx.doi.org/10.1016/j.tiv.2014.07.008 Google Scholar CrossRef Search ADS PubMed  Maurya S. K., Mishra J., Tripathi V. K., Sharma R., Siddiqui M. H. ( 2014). Cypermethrin induces astrocyte damage: Role of aberrant Ca2+, ROS, JNK, P38, matrix metalloproteinase 2 and migration related reelin protein. Pestic. Biochem. Physiol.  111, 51– 59. Google Scholar CrossRef Search ADS PubMed  Meeker J. D., Barr D. B., Hauser R. ( 2009). Pyrethroid insecticide metabolites are associated with serum hormone levels in adult men. Reprod. Toxicol.  27, 155– 160. http://dx.doi.org/10.1016/j.reprotox.2008.12.012 Google Scholar CrossRef Search ADS PubMed  Mulvaney J. M., Zhang T., Fewtrell C., Roberson M. S. ( 1999). Calcium influx through L-type channels is required for selective activation of extracellular signal-regulated kinase by gonadotropin-releasing hormone. J. Biol. Chem.  274, 29796– 29804. Google Scholar CrossRef Search ADS PubMed  Reiss N., Llevi L. N., Shacham S., Harris D., Seger R., Naor Z. ( 1997). Mechanism of mitogen-activated protein kinase activation by gonadotropin-releasing hormone in the pituitary alpha T3-1 cell line: Differential roles of calcium and protein kinase C. Endocrinology  138, 1673– 1682. http://dx.doi.org/10.1210/endo.138.4.5057 Google Scholar CrossRef Search ADS PubMed  Roberson M. S., Bliss S. P., Xie J., Navratil A. M., Farmerie T. A., Wolfe M. W., Clay C. M. ( 2005). Gonadotropin-releasing hormone induction of extracellular-signal regulated kinase is blocked by inhibition of calmodulin. Mol. Endocrinol.  19, 2412– 2423. Google Scholar CrossRef Search ADS PubMed  Roskoski R. ( 2010). RAF protein-serine/threonine kinases: Structure and regulation. Biochem. Biophys. Res. Commun.  399, 313– 317. http://dx.doi.org/10.1016/j.bbrc.2010.07.092 Google Scholar CrossRef Search ADS PubMed  Shafer T. J., Meyer D. A., Crofton K. M. ( 2005). Developmental neurotoxicity of pyrethroid insecticides: Critical review and future research needs. Environ. Health Perspect.  113, 123– 136. Google Scholar CrossRef Search ADS PubMed  Soderlund D. M. ( 2012). Molecular mechanisms of pyrethroid insecticide neurotoxicity: Recent advances. Arch. Toxicol.  86, 165– 181. http://dx.doi.org/10.1007/s00204-011-0726-x Google Scholar CrossRef Search ADS PubMed  Takemoto-Kimura S., Suzuki K., Horigane S. I., Kamijo S., Inoue M., Sakamoto M., Fujii H., Bito H. ( 2017). Calmodulin kinases: Essential regulators in health and disease. J. Neurochem.  141, 808– 818. Google Scholar CrossRef Search ADS PubMed  Thackray V. G., Mellon P. L., Coss D. ( 2010). Hormones in synergy: Regulation of the pituitary gonadotropin genes. Mol Cell Endocrinol  314, 192– 203. http://dx.doi.org/10.1016/j.mce.2009.09.003 Google Scholar CrossRef Search ADS PubMed  Tse A., Hille B. ( 1993). Role of voltage-gated Na+ and Ca2+ channels in gonadotropin-releasing hormone-induced membrane-potential changes in identified rat gonadotropes. Endocrinology  132, 1475– 1481. Google Scholar CrossRef Search ADS PubMed  Tulve N. S., Jones P. A., Nishioka M. G., Fortmann R. C., Croghan C. W., Zhou J. Y., Fraser A., Cave C., Friedman W. ( 2006). Pesticide measurements from The First National Environmental Health Survey of Child Care Centers using a multi-residue GC/MS analysis method. Environ. Sci. Technol.  40, 6269– 6274. Google Scholar CrossRef Search ADS PubMed  US EPA. ( 2016) Ecological Risk Management Rationale for Pyrethroids in Registration Review. Washington, DC, USA https://www.regulations.gov/document? D=EPA-HQ-OPP-2012-0167-0047: Usmani S. M., Fois G., Albrecht S., von A. S., Dietl P., Wittekindt O. H. ( 2010). 2-APB and capsazepine-induced Ca2+ influx stimulates clathrin-dependent endocytosis in alveolar epithelial cells. Cell Physiol. Biochem.  25, 91– 102. Google Scholar CrossRef Search ADS PubMed  Vais H., Williamson M. S., Devonshire A. L., Usherwood P. N. R. ( 2001). The molecular interactions of pyrethroid insecticides with insect and mammalian sodium channels. Pest Manage. Sci.  57, 877– 888. Google Scholar CrossRef Search ADS   Verkhratsky A. ( 2005). Physiology and pathophysiology of the calcium store in the endoplasmic reticulum of neurons. Physiol. Rev.  85, 201– 279. http://dx.doi.org/10.1152/physrev.00004.2004 Google Scholar CrossRef Search ADS PubMed  Wang L. M., Liu W., Yang C. X., Pan Z. Y., Gan J. Y., Xu C., Zhao M. R., Schlenk D. ( 2007). Enantioselectivity in estrogenic potential and uptake of bifenthrin. Environ. Sci. Technol.  41, 6124– 6128. Google Scholar CrossRef Search ADS PubMed  Wolfe M. W., Call G. B. ( 1999). Early growth response protein 1 binds to the luteinizing hormone-beta promoter and mediates gonadotropin-releasing hormone-stimulated gene expression. Mol. Endocrinol.  13, 752– 763. Google Scholar PubMed  Yang Y., Ma H. H., Zhou J. H., Liu J., Liu W. P. ( 2014). Joint toxicity of permethrin and cypermethrin at sublethal concentrations to the embryo-larval zebrafish. Chemosphere  96, 146– 154. http://dx.doi.org/10.1016/j.chemosphere.2013.10.014 Google Scholar CrossRef Search ADS PubMed  Ye X., Li F., Zhang J., Ma H., Ji D., Huang X., Curry T. E.Jr, Liu W., Liu J. ( 2017a) Pyrethroid insecticide cypermethrin accelerates pubertal onset in male mice via disrupting hypothalamic-pituitary-gonadal axis. Environ. Sci. Technol. 51, 10212– 10221. Ye X., Pan W., Zhao S., Zhao Y., Zhu Y., Liu J., Liu W. ( 2017b). Relationships of pyrethroid exposure with gonadotropins levels and pubertal development in Chinese boys. Environ. Sci. Technol.  51, 6379– 6386. Google Scholar CrossRef Search ADS   Ye X., Pan W., Zhao Y., Zhao S., Zhu Y., Liu W., Liu J. ( 2017c). Association of pyrethroids exposure with onset of puberty in Chinese girls. Environ. Pollut.  277, 606– 612. Google Scholar CrossRef Search ADS   Yuan Y. W., Chen C., Zheng C. M., Wang X. L., Yang G. L., Wang Q., Zhang Z. H. ( 2014). Residue of chlorpyrifos and cypermethrin in vegetables and probabilistic exposure assessment for consumers in Zhejiang Province, China. Food Control  36, 63– 68. Google Scholar CrossRef Search ADS   Zhang J. Y., Zhang J., Liu R., Gan J., Liu J., Liu W. P. ( 2016). Endocrine-Disrupting Effects of Pesticides through Interference with Human Glucocorticoid Receptor. Environmental Science & Technology  50, 435– 443. Google Scholar CrossRef Search ADS PubMed  Zhao M. R., Zhang Y., Zhuang S. L., Zhang Q., Lu C. S., Liu W. P. ( 2014). Disruption of the hormonal network and the enantioselectivity of bifenthrin in trophoblast: Maternal-fetal health risk of chiral pesticides. Environ. Sci. Technol.  48, 8109– 8116. Google Scholar CrossRef Search ADS PubMed  Zhou J., Yang Y., Xiong K., Liu J. ( 2014). Endocrine disrupting effects of dichlorodiphenyltrichloroethane analogues on gonadotropin hormones in pituitary gonadotrope cells. Environ. Toxicol. Pharmacol.  37, 1194– 1201. http://dx.doi.org/10.1016/j.etap.2014.04.018 Google Scholar CrossRef Search ADS PubMed  © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

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Toxicological SciencesOxford University Press

Published: Mar 1, 2018

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