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HERP depletion inhibits zearalenone-induced apoptosis through autophagy activation in mouse ovarian granulosa cells
Toxicology Letters 301 (2019) 1–10
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Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
HERP depletion inhibits zearalenone-induced apoptosis through autophagy activation in mouse ovarian granulosa cells Fenglei Chena,b,c,d, Xin Wena,b, Pengfei Lina,b, Huatao Chena,b, Aihua Wangb, Yaping Jina,b,
T
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a
Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Yangling, Shaanxi, 712100, China College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China c College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, Jiangsu, China d Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, Jiangsu, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: HERP Autophagy Zearalenone Apoptosis Granulosa cells
HERP is an endoplasmic reticulum (ER) membrane protein and is strongly induced by stress conditions. A recent study has indicated that HERP cooperates in apoptosis during zearalenone (ZEA) treatment. However, regulatory mechanisms and the role of HERP in ZEA-induced apoptosis remain elusive in ovarian granulosa cells. In this study, MTT and flow cytometry assays demonstrated that ZEA gradually decreased cell viability and increased apoptosis in granulosa cells in a dose-dependent manner. Western blot analysis showed that ZEA significantly activated autophagy by upregulating LC3-II. Chloroquine (CQ) significantly increased LC3-II and induced granulosa cell apoptosis. Moreover, Western blot analysis showed that ZEA inhibited the mTOR and ERK1/2 signaling pathways. Furthermore, we found that ZEA activated ER stress by upregulating the ER stress-related proteins GRP78, HERP and CHOP. 4-PBA significantly decreased GRP78, HERP, CHOP and LC3-II. In addition, knockdown of HERP (shHERP) significantly protected ovarian granulosa cells from apoptosis induced by ZEA. We found that HERP depletion activated autophagy and ERK1/2 signaling pathways, while it inhibited the mTOR and caspase-dependent mitochondrial signaling pathways. In summary, autophagy and ER stress cooperated in apoptosis induced by ZEA; HERP depletion inhibits ZEA-induced apoptosis of ovarian granulosa cells through autophagy activation and apoptotic pathway inhibition.
1. Introduction Zearalenone (ZEA), a nonsteroidal estrogenic mycotoxin from Fusarium fungi, causes reproductive toxicity in farm animals (Fushimi et al., 2015; Minervini and Dell’Aquila, 2008; Tiemann and Danicke, 2007; Yang et al., 2007). Since the structure of ZEA resembles that of 17β-estradiol, ZEA binds to estrogen receptors to exhibit estrogen functions (Adibnia et al., 2016; Gajecka et al., 2013; Minervini et al., 2001; Turcotte et al., 2005). In male animals, ZEA decreases the
testicular weight, the motility of spermatozoa and spontaneous acrosome reaction to affect the fertilization ability of sperm and reduces testosterone synthesis by Leydig cells by downregulating the expression of steroidogenic enzymes (Li et al., 2014; Long et al., 2016; Pang et al., 2017; Zheng et al., 2016). In female animals, ZEA impacts the maternal reproductive capability and embryonic implantation, delays fetal development, increases early death during early gestation and affects the levels of progesterone and estradiol in vivo and in vitro (Gao et al., 2017; Zhao et al., 2014, 2013). Ovarian granulosa cells play essential
Abbreviations: 4-PBA, 4-phenylbutyric acid; 7-AAD, 7-amino-actinomycin D 7; ANOVA, one-way analysis of variance; ATF, activating transcription factor; ATG, autophagy-related proteins; BAX, BCL-2 associated X protein; BCL-2, B-cell lymphoma-2; CHOP, CCAAT/enhancer binding protein homologous protein; CQ, chloroquine; DMEM/F12, Dulbecco’s modified Eagle’s medium: nutrient mixture F-12; DMSO, dimethyl sulfoxide; ECL, enhanced chemiluminescence; ELISA, enzyme linked immunosorbent assay; ER, endoplasmic reticulum; ERAD, ER-associated protein degradation; ERK1/2, extracellular signal-regulated kinase ½; FBS, fetal bovine serum; Fisher LSD, Fisher’s least significant difference test; GRP78, glucose-regulated protein 78; HERP, homocysteine-responsive ER-resident protein; IRE1α, inositol-requiring enzyme 1α; LC3, microtubule-associated protein light chain 3; MTT, 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide; mTOR, mammalian target of rapamycin; PERK, protein kinase (PKR)-like ER kinase; pERK1/2, phosphorylation of ERK1/2; pS6K1, phosphorylation of S6K1; PVDF, polyvinylidene difluoride; S6K1, p70 ribosomal protein S6 kinase; shHERP, HERP shRNA; shNC, non-silencing negative control; shRNAs, short hairpin interfering RNAs; SPSS, Statistical Package for the Social Science; UPR, unfolded protein response; ZEA, zearalenone ⁎ Corresponding author at: Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China. E-mail address: [email protected] (Y. Jin). https://doi.org/10.1016/j.toxlet.2018.10.026 Received 27 June 2018; Received in revised form 25 September 2018; Accepted 22 October 2018 Available online 28 October 2018 0378-4274/ © 2018 Elsevier B.V. All rights reserved.
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evidence of whether ZEA regulates upstream signaling pathways of autophagy, mTOR or ERK1/2 and whether HERP cooperates in apoptosis through regulating the mTOR or ERK1/2 signaling pathways in ovarian granulosa cells.
roles in follicular development, maturation and sex hormone secretion. Previous studies have demonstrated that ZEA exerts double functions on the proliferation and apoptosis of ovarian granulosa cells at different doses and various species in vitro (Caloni et al., 2014; Cortinovis et al., 2014; Hou et al., 2015; Kolesarova et al., 2017; Liu et al., 2018; Minervini et al., 2001, 2006; Zhu et al., 2012). Because of its structural similarity to 17β-estradiol, ZEA stimulates the proliferation of ovarian granulosa cells at low doses and induces apoptosis at high doses. A previous study has demonstrated that ZEA induces apoptosis and necrosis in porcine granulosa cells via Caspase-9 and Caspase-3 activation (Zhu et al., 2012). However, it remains elusive whether other mechanisms are involved in apoptosis induced by ZEA in ovarian granulosa cells. Our studies have confirmed that endoplasmic reticulum (ER) stress and autophagy cooperate in ZEA-induced apoptosis of RAW 264.7 macrophages and mouse Leydig cells (Chen et al., 2015; Lin et al., 2015; Yang et al., 2017). Hence, we speculated that ER stress and autophagy cooperate in ovarian granulosa cell apoptosis that is induced by ZEA. ER stress triggers the unfolded protein response (UPR) to restore cellular homeostasis (Hetz, 2012; Schroder and Kaufman, 2005). Inositol-requiring enzyme 1α (IRE1α), double-stranded RNA-dependent protein kinase (PKR)-like ER kinase (PERK), and activating transcription factor (ATF)-6 separate from glucose-regulated protein 78 (GRP78) to activate the UPR. GRP78 is known as a marker of ER stress activation (Rao et al., 2002). However, severe or long ER stress triggers apoptosis. CHOP (CCAAT/enhancer binding protein homologous protein) cooperates in ER-stress-induced apoptosis, and it is a popular marker to monitor ER stress-induced apoptosis (Oyadomari and Mori, 2004). The homocysteine-responsive ER-resident protein (HERP), which is localized in the ER membrane, plays an important role in ER-associated protein degradation (ERAD) that is induced by ER stress (Kim et al., 2008; Kny et al., 2011; Okuda-Shimizu and Hendershot, 2007; Siva et al., 2010). HERP also inhibits apoptosis through preventing the loss of ER Ca2+ and mitochondrial potential during ER stress (Chan et al., 2004; Hori et al., 2004a). However, a recent study has demonstrated that knockdown of HERP up-regulates autophagy-related protein 5 (ATG5) and BECLIN-1 to activate protective autophagy for cell survival during glucose starvation (Quiroga et al., 2013). Moreover, our results also showed that knockdown of HERP upregulates LC3-II and decreases CHOP to activate protective autophagy and inhibit ER stress for cell survival during ZEA treatment (Chen et al., 2016a). Other than autophagy and ER stress activation, little was known regarding whether HERP affects other signaling pathways to regulate apoptosis during ZEA treatment. Autophagy is an evolutionarily conserved self-degradative catabolic process to degrade and recycle damaged organelles and long-lived proteins. Although it cooperates in programmed cell death, autophagy also plays a protective role against apoptosis under numerous stressful conditions, such as glucose starvation, apoptosis induced by ZEA and elimination of microorganisms (Kirkegaard et al., 2004; Lu et al., 2018; Wang et al., 2014). Autophagy is a complex signaling pathway, and it involves many ATGs to regulate this catabolic process (Yin et al., 2016). Microtubule-associated protein light chain 3 (LC3), which is known as ATG8, plays a critical role in autophagosome formation by converting cytosolic form of LC3-I into the lapidated form of LC3-II, and is characterized as a marker of autophagy activation (Tanida et al., 2004). The molecular mechanism of autophagy is under the control of different signaling pathways. Mammalian target of rapamycin (mTOR)/p70 ribosomal protein S6 kinase (S6K1) and extracellular signal-regulated kinase 1/2 (ERK1/2) cooperate in regulating autophagy (Eom et al., 2010; Way et al., 2015; Zhang et al., 2017). It is well known that mTOR/S6K1 negatively regulates autophagy (Kim and Guan, 2015). ERK1/2 negatively adjusts mTOR phosphorylation (Cheng et al., 2013). ERK1/2 activation is mainly involved in cell proliferation to promote survival (Chen et al., 2010; Lu and Xu, 2006). Although ZEA induces apoptosis through regulating autophagy and ER stress, there is no
2. Materials and methods 2.1. Experimental animals and reagents Immature female Kunming White mice (21 days old) were purchased from the laboratory animal center of the Fourth Military Medical University (Xi’an, Shanxi, China), the case number of animal use certificate is SYXK (Shan) 2014-001. The mice were housed separately under the controlled conditions of relatively constant temperature (23 ± 2 °C), a 12-h light/dark cycle and were provided food and water ad libitum. This study was approved by the committee for the Ethics on Animal Care and Experiment of Northwest A&F University. ZEA, Chloroquine (CQ), 4-phenylbutyric acid (4-PBA) and anti-LC3 rabbit polyclonal antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). ZEA was dissolved in dimethyl sulfoxide (DMSO), and the concentration of the stock solution was 15 mM. 4-PBA and CQ were dissolved in PBS, and the concentrations of the stock solutions were 1 M and 5 mM, respectively. Rabbit polyclonal antibodies including antiCHOP (sc-575), anti-HERP (sc-98669) and anti-GRP78 (sc-1050) goat polyclonal antibody were purchased from Santa Cruz Biotechnology Inc. (Texas, USA). Rabbit polyclonal antibodies including anti-ERK1/2, anti-pERK1/2, anti-S6K1, anti-pS6K1 and anti-cleaved Caspase-3 were purchased from Cell Signaling Technology (Danvers, MA, USA). Rabbit polyclonal antibodies including anti-BCL-2 and anti-BAX were purchased from Wanlei Biotechnology Inc. (Shenyang, China). Anti-β-actin mouse monoclonal antibody was purchased from Tianjin Sungene Biotechnology Inc. (Tianjin, China). 2.2. Culture of mouse ovarian granulosa cells and treatment After superovulation, granulosa cells were isolated according to a previously described method (Yang et al., 2013). Briefly, the ovaries were excised, and the granulosa cells were released by puncturing with 26-gauge needles. The granulosa cells were collected and washed via brief filtration and centrifugation. Cell viability was determined using the trypan-blue-dye exclusion method. The cells were cultured in Dulbecco’s modified Eagle’s medium: nutrient mixture F-12 (DMEM/F12, HyClone, Beijing, China) that was supplemented with penicillin (100 IU/ml), streptomycin (100 μg/ml) and 10% fetal bovine serum (FBS, Invitrogen, USA) at 37 °C under a 5% CO2 atmosphere. After culturing for 72 h, the cells were treated with ZEA for further experiments. 2.3. Transducing short hairpin interfering RNAs (shRNAs) via lentiviral infection The U6 RNAi cassette fragment from pSilencer 2.1-U6 hygro (Cat. No. AM5760, Life Technologies, Carlsbad, CA, USA) was amplified and cloned into pCD513B-1 (SBI, Mountain View, CA, USA), which contains a GFP expression construct, to generate a pCD513B-U6 lentiviral vector. Lentivirus vectors encoding the HERP shRNA (shHERP) and nonsilencing negative control (shNC) were constructed by our group. The sequence of the shNC was 5′-GATCCGATGAAATGGGTAAGTACATTCAA GAGATGTACTTACCCATTTCATCTTTTTTG-3′. The sequence of the shHERP was 5′-GATCCGAGCAGCCGGACAACTCTAATCTCGAG ATTAG AGTTGTCCGGCTGCTCTTTTTG-3′. The recombinant lentivirus vector was packaged and transduced into HEK 293 T cells. The medium was harvested 48 h after transfection, purified via low-speed centrifugation, and filtered through a 0.45-μm PVDF filter. An appropriate number of lentiviral particles (MOI = 20) were transduced into primary granulosa cells using 8 μg/ml polybrene. After 12 h of incubation, the medium containing the virus was removed and replaced with fresh culture 2
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Fig. 1. ZEA induces apoptosis in mouse ovarian granulosa cells. (A) The granulosa cells were plated in 96-well culture plates and treated with ZEA (0–150 μM) for 24 h. Cell viability was measured via the MTT assay. (B and C) After exposure to 0, 15, 30 or 60 μM ZEA for 24 h, ovarian granulosa cells were collected for Annexin V-PE/7-AAD staining followed by flow cytometric analysis. The statistical analysis is shown in the bar graphs. Data are presented as the mean ± SEM of three independent experiments. Bars with different letters are significantly different (p < 0.05).
Jiangsu, China). Briefly, the cells were washed with cold PBS, trypsinized and harvested. The cells were resuspended in 500 μl of binding buffer containing 5 μl of 7-AAD and 1 μl of Annexin V-PE and incubated for 30 min. Measurement of the apoptotic rate was performed via flow cytometry (EPICS Altra, Beckman Coulter Cytomics Altra, Brea, CA). The normal cells, as the control, were used to compensate for spectral bleed-through. The experiments were repeated three times independently.
medium. The cells were harvested after an additional 48 h. 2.4. Measurement of cell viability Cell viability was tested according to a previously described method (Chen et al., 2015). Briefly, granulosa cells were seeded into a 96-well plate with 1 × 104 cells/well. After culturing for 72 h, the cells were exposed to 0–150 μM ZEA for 24 h. Next, the cells were treated with 0.5 mg/ml 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) for another 4 h. Finally, the medium was replaced with 150 μl DMSO, and colorimetric measurement was performed using an enzyme linked immunosorbent assay (ELISA) plate reader at 570 nm (Model 680, Bio-Rad, and Hercules, CA, USA). Experiments were repeated three times independently.
2.6. Western blot analysis The granulosa cells, which were treated with 30 μM ZEA for 0–24 h or 0–60 μM ZEA for 12 h, were lysed with the Total Protein Extraction Kit (Nanjing Keygen Biotech Co., Ltd., Nanjing, China). The protein concentration was measured using the BCA Protein Assay Kit (Nanjing Keygen Biotech Co., Ltd., Nanjing, China). Equal amount of total proteins (30 μg) were separated via a 12–15% SDS-PAGE gel and were electrotransferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). After blocking with 10% fatty acid-free milk in TBST for 1 h at room temperature, the membranes were
2.5. Flow cytometric analysis of granulosa cell apoptosis After 24 h of ZEA treatment, the apoptotic rate was quantified using the Annexin V-PE and 7-AAD Apoptosis Detection Kit according to the manufacturer’s instruction (Nanjing Keygen Biotech Co., Ltd, Nanjing, 3
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3.3. ZEA induced activation of ER stress in mouse granulosa cells
incubated overnight at 4 °C with primary antibodies. The next day, the membranes were incubated with secondary antibody conjugated to horseradish peroxidase (Zhongshan Golden Bridge Biotechnology, Nanjing, China; 1:5000 dilutions) at room temperature for 1 h. Finally, immunoreactive bands were visualized via enhanced chemiluminescence (ECL) substrate using the Gel Image System (Tannon, Biotech, Shanghai, China) and exposed to an X-ray film for visualization of the protein bands. The protein band intensities were digitized using Quantity One software (Bio-Rad Laboratories, Hercules, CA).
To investigate whether apoptosis induced by ZEA were related to ER stress in mouse ovarian granulosa cells, the levels of the ER stress-related proteins GRP78, HERP and CHOP were examined via western blot analysis. As shown in Fig. 3A and B, GRP78, HERP and CHOP were significantly increased upon treatment with ZEA at 30–60 μM for 12 h. Fig. 3C and D show that GRP78 and CHOP were gradually increased upon prolonging treatment for 0–24 h with 30 μM ZEA. HERP peaked at 12 h and then gradually decreased. To further confirm the functional relationship between ZEA and ER stress, we used 4-PBA, an inhibitor of ER stress, to block ER stress that was induced by ZEA. Compared with treatment with 30 μM ZEA alone for 12 h, granulosa cells that were pretreated with 1 mM 4-PBA for 1 h and then coincubated with 30 μM ZEA for another 12 h, showed a significant decrease in the level of GRP78, HERP and CHOP (Fig. 3E and F). We also detected that 4-PBA significantly inhibited LC3-II (Fig. 3E and F). ZEA not only actives autophagy but also induces ER stress.
2.7. Statistical analysis All results are presented as the mean ± SEM. Date analysis was performed using one-way analysis of variance (ANOVA), followed by Fisher’s least significant difference test (Fisher LSD) and the Independent-Samples t-test using Statistical Package for the Social Science (SPSS) software (Version 13.0; SPSS, Inc., Chicago, IL, USA). All the experiments were replicated at least three times for each group. Differences were considered significant when p < 0.05.
3.4. ZEA inhibited activation of the mTOR and ERK1/2 signaling pathways in granulosa cells
3. Results
The onset of autophagy is regulated through the mTOR and ERK1/2 signaling pathways. Thus, we examined the mTOR and ERK1/2 activation that was induced by ZEA in granulosa cells by detecting the levels of phosphorylated S6K1, a marker of mTORC1 activation, and phosphorylated ERK1/2. As shown in Fig. 4A and B, the ratios of pS6K1/S6K1 and pERK1/2/ERK1/2 were significantly decreased upon treatment with ZEA at 30–60 μM for 12 h. Fig. 4C and D show that pS6K1/S6K1 and pERK1/2/ERK1/2 were decreased upon prolonged treatment for 12–24 h with 30 μM ZEA, but pS6K1/S6K1 and pERK1/2/ ERK1/2 were increased upon treatment for on 6 h. These results demonstrated that the mTOR and ERK1/2 signaling pathways might cooperate in ZEA-induced autophagy or apoptosis of granulosa cells.
3.1. Cytotoxic effect of ZEA on ovarian granulosa cells in vitro To examine the toxicity of ZEA on ovarian granulosa cells in vitro, the viability and apoptotic rate of granulosa cells was recorded via MTT and flow cytometry, after they were treated with different doses of ZEA for 24 h. As shown in Fig. 1A, the results of the MTT assay showed that there was very little effect on the growth of granulosa cells growth that were treated with 15 μM ZEA for 24 h compared with the control group. The cell viability was significantly inhibited by ZEA treatment at 30–150 μM in a dose dependent manner. To further analyze the apoptosis of granulosa cells that was induced by ZEA, the apoptotic rate was investigated via flow cytometry. As shown in Fig. 1B and C, treatment with ZEA at 15–60 μM resulted in a significant increase in the right lower quadrant and upright quadrant (19.8%, 50.15% and 73.15%, respectively). These results indicate that ZEA induces a cytotoxic effect in granulosa cells in a dose-dependent manner. Therefore, 30 μM of ZEA was employed for further experiments to evaluate its toxicity on granulosa cells.
3.5. HERP depletion inhibits ZEA-induced apoptosis in granulosa cells HERP is known to up-regulate protective autophagy that is induced by ZEA and glucose starvation. Here, we investigated whether HERP could protect granulosa cells from apoptosis that was induced by ZEA in an autophagy-dependent manner. To this end, we first constructed lentiviral vectors encoding shRNA of HERP (shHERP) (Chen et al., 2016b) and then transduced shHERP into granulosa cells to knockdown HERP before 30 μM ZEA treatment for 12 h, and the transduction efficiency was more than 80% (the results are not shown). As shown in Fig. 5A and B, the results of flow cytometry showed that the apoptotic rate of the shHERP group (42.2%) significantly decreased compared with that of the negative-control shRNA group (shNC) (58.2%). Western blot analysis showed that the shHERP group significantly inhibited the expression of HERP compared to the shNC; however, LC3-II was significantly increased after ZEA treatment (Fig. 5C and D). To further reveal how HERP regulated autophagy and apoptosis, we detected pS6K1, S6K1, pERK1/2, ERK1/2, BCL-2, BAX and cleaved Caspase-3 using western blot in the shHERP and shNC group after ZEA treatment. As shown in Fig. 5E and F, compared to the shNC, the ratios of pS6K1/ S6K1 and cleaved Caspase-3 were significantly decreased, and pERK1/ 2/ERK1/2 and BCL-2/BAX were significantly increased upon induction with by 30 μM ZEA for 12 h in the shHERP group. In summary, these results demonstrated that knockdown of HERP enhanced protective autophagy through regulating the mTOR and ERK1/2 signaling pathways and inhibited apoptosis through decreasing BAX and Caspase-3.
3.2. ZEA triggered autophagy responses in mouse granulosa cells We examined whether apoptosis of granulosa cells induced by ZEA is determined via activation of autophagy. Expression of LC3-II, a marker of autophagy activation signaling, was detected by western blot. As shown in Fig. 2A and B, LC3-II was significantly increased upon treatment with ZEA at 30–60 μM for 12 h compared with the control group. The level of LC3-II peaked at 30 μM and then gradually decreased. Fig. 2C and D show that LC3-II was gradually increased upon prolonged treatment for 0–24 h with 30 μM ZEA. To further confirm the functional relationship between ZEA and autophagy, we used CQ, a specific inhibitor of the late stage of autophagy, to block autophagy that was induced by ZEA. Compared with treatment with 30 μM ZEA for 12 h alone, granulosa cells that were pretreated with 5 μM CQ for 1 h and then coincubated with 30 μM ZEA for another 12 h, showed a significant increase in the level of LC3-II (Fig. 2E and F). We further detected the apoptotic rate after pretreatment with CQ to elucidate the relationship between apoptosis and autophagy in ZEA-induced toxicity. As shown in Fig. 2G and H, the results of flow cytometry showed that the apoptotic rate significantly increased after cotreatment with ZEA and CQ (63.0%) compared with ZEA alone (50.2%). In summary, ZEA induced autophagy and inhibited apoptosis in an autophagy-dependent manner.
4. Discussion Ovarian granulosa cells play an essential role in follicular development and maturation. Additional studies have revealed that ZEA 4
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Fig. 2. ZEA induces autophagy in mouse ovarian granulosa cells. (A and B) The granulosa cells were treated with the indicated concentration of ZEA and incubated for 12 h. Levels of LC3-I and LC3-II were measured via western blot assay. (C and D) The granulosa cells were treated with 30 μM ZEA for the indicated times. Levels of LC3-I and LC3-II were measured via western blot assay. (E and F) The granulosa cells were pretreated with 5 μM CQ for 1 h and coincubated with 30 μM ZEA for another 12 h. Levels of LC3-I and LC3-II were measured via western blot assay. (G and H) After pretreatment with 5 μM CQ for 1 h and coincubated with 30 μM ZEA for another 24 h, ovarian granulosa cells were collected for Annexin VePE/ 7-AAD staining followed by flow cytometric analysis. The statistical analysis is shown in the bar graphs. Analyses of band intensity on films are presented as the relative ratio of LC3-II to β-actin. Data are presented as the mean ± SEM. Bars with different letters are significantly different (p < 0.05).
granulosa cells. Consistent with the previous study, our results revealed that ZEA significantly inhibited proliferation and induced apoptosis in ovarian granulosa cells, which was dependent on the activation of autophagy and ER stress. The induction of autophagy and ER stress by
induces granulosa cell apoptosis (Hou et al., 2015; Liu et al., 2018; Minervini et al., 2006; Zhu et al., 2012). However, little was known regarding the detailed molecular mechanisms. In the present study, we detected that ZEA induced autophagy and apoptosis in ovarian 5
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Fig. 3. ZEA induces ER stress in mouse granulosa cells. (A and B) The granulosa cells were treated with the indicated concentration of ZEA and incubated for 12 h. Levels of ER stress-related proteins (GRP78, HERP, and CHOP) were measured via western blot assay. (C and D) The granulosa cells were treated with 30 μM ZEA for the indicated times. Levels of ER stress-related proteins (GRP78, HERP, and CHOP) were detected via western blot assay. (E and F) The granulosa cells were pretreated with 1 mM 4-PBA for 30 min and coincubated with 30 μM ZEA for another 12 h. Levels of ER stress-related proteins (GRP78, HERP and CHOP) were measured via western blot assay. The statistical analysis is shown in the bar graphs. Analyses of band intensity on films are presented as the relative ratio of GRP78, HERP or CHOP to β-actin. Data are presented as the mean ± SEM. Bars with different letters are significantly different (p < 0.05).
enhanced ZEA-induced apoptosis in ovarian granulosa cells. These results suggested that autophagy induced by ZEA played a protective role against apoptosis in ovarian granulosa cells. These results are in agreement with our previous studies that inhibition of autophagy by CQ significantly increased ZEA-induced apoptosis in RAW 264.7 cells (Chen et al., 2015). Collectively, these results suggest that autophagy could protect ovarian granulosa cells from apoptosis induced by ZEA. It is known that ZEA induces autophagy of various types of cells by a cascade of kinases (Ben Salem et al., 2017; Ben et al., 2015; Wang et al., 2014; Zheng et al., 2018). Additional evidence indicates that autophagy could be induced through the inhibition of the mTOR/S6K1 signaling pathway (Kim and Guan, 2015). mTOR is a major negative regulator of autophagy through inhibiting activation of ULK1 and ATG13 (Kim and Guan, 2015). Our results revealed that exposure of granulosa cells to ZEA caused a significant decrease in pS6K1, a marker of mTORC1 activation. Accordingly, we speculated that suppression of mTORC1
ZEA treatment was evidenced by the accumulation of LC3-II, GRP78, HERP and CHOP. Furthermore, knockdown of HERP protected granulosa cells from apoptosis induced by ZEA. HERP depletion activated autophagy and ERK1/2 signaling pathways by increasing LC3-II and pERK1/2 while inhibiting the mTOR and caspase-dependent mitochondrial signaling pathways by decreasing pS6K1, BAX and cleaved Caspase-3. Autophagy is a self-degradative process, and the amount of LC3-II is usually applied to monitor cellular autophagy (Tanida et al., 2004). In this study, our data revealed that ZEA raised the expression of LC3-II at different doses and times in ovarian granulosa cells. This finding is consistent with previous reports that ZEA induced the accumulation of LC3-II in primary Leydig cells and RAW 264.7 cells (Chen et al., 2016a; Wang et al., 2014). Furthermore, pretreatment with CQ, a special inhibitor of autophagy, which blocks the fusion of autophagosomes and lysosomes (Yoon et al., 2010), significantly inhibited autophagy and
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Fig. 4. ZEA inhibits the mTOR and ERK1/2 signaling pathways in mouse granulosa cells. (A and B) The granulosa cells were treated with the indicated concentration of ZEA and incubated for 12 h. Levels of pS6K1, S6K1, pERK1/2 and ERK1/2 were measured via western blot assay. (C and D) The granulosa cells were treated with 30 μM ZEA for the indicated times. Levels of pS6K1, S6K1, pERK1/2 and ERK1/2 were measured via western blot assay. The statistical analysis is shown in the bar graphs. Analyses of band intensity on films are presented as the relative ratio of pS6K1, S6K1, pERK1/2 or ERK1/2 to β-actin. Data are presented as the mean ± SEM. Bars with different letters are significantly different (p < 0.05).
under ZEA treatment in RAW 264.7 cells (Chen et al., 2016a). Therefore, we investigated whether HERP deletion protects ovarian granulosa cells from the toxicity of ZEA through an autophagy-dependent pathway. Our results showed that knockdown of HERP significantly decreased apoptosis that was induced by ZEA in ovarian granulosa cells. The present study strengthened the idea that HERP cooperates in autophagy regulation. Autophagy, as an ERAD II pathway, can assist ERAD through degrading abnormally folded proteins accumulation (Fujita et al., 2007). HERP may be an important mediator of the crosstalk between ERAD and autophagy (Quiroga et al., 2013). Consistent with the previous study, our results revealed that knockdown of HERP enhanced autophagy via upregulating LC3-II in ovarian granulosa cells. Further study demonstrated that knockdown of HERP decreased the expression of pS6K1 and increased pERK1/2. Activation of ERK1/2 has been shown to inhibit apoptosis in response to a wide range of stimuli. ERK1/2 promotes cell survival by suppressing the functions of pro-apoptotic proteins and enhancing the activity of anti-apoptotic proteins of the BCL-2 family. ERK1/2 also regulates the caspase-dependent mitochondrial pathway by functioning downstream of Cytochrome c release and caspase protein activation. In this study, our results revealed that knockdown of HERP significantly down regulated BAX and cleaved Caspase-3. We speculated that knockdown of HERP might inhibit apoptosis by activating the ERK1/2 signaling pathway. Additional studies have revealed that the ERK1/2 signaling pathway negatively regulates mTORC1 and activates autophagy (Dai et al., 2014; Shinojima et al., 2007; Way et al., 2015; Zhang et al., 2017). Curcumin inhibits the apoptosis of chondrocytes through the activation of the ERK1/2 signaling pathways, which induces autophagy (Li et al., 2017). Cinnamtannin D1 induces autophagy via the inhibition of AKT/mTOR
activation might cooperate in ZEA-induced autophagy of granulosa cells. ERK1/2 regulates cellular growth and survival (Lu and Xu, 2006). We examined the ERK1/2 signaling pathway, which was downregulated upon exposure to ZEA. We speculated that the ERK1/2 signaling pathway may participate in ZEA-induced granulosa cell apoptosis through inhibiting its own expression. ER stress triggers transcriptional induction, translational attenuation and ERAD to restore cellular homeostasis. However, with severe or prolonged ER stress, apoptosis is activated. After exposure to ZEA, GRP78 and CHOP were significantly augmented in ovarian granulosa cells. GRP78 is an ER stress-response protein, and its expression is a typical marker of ER stress activation (Rao et al., 2002). CHOP cooperates in ER stress-induced apoptosis (Oyadomari and Mori, 2004). Our previous results showed that ZEA triggers ER stress and then induced apoptosis though CHOP activation in RAW 264.7 cells (Chen et al., 2015; Lin et al., 2015). The present study further demonstrated the effects of ZEA on stimulating ER stress in ovarian granulosa cells. In addition to GRP78 and CHOP activation, ZEA induced HERP, which is cooperated in ERAD induced by ER stress. ERAD degrades unfolded or misfolded proteins upon occurrence of ER stress. HERP is induced under classical ER stressors, proteasome inhibition and nutrient starvation conditions; it plays a protective role in ER stress-induced apoptosis (Diaz et al., 2013; George et al., 2018; Hori et al., 2004b; Mirabelli et al., 2016; Siva et al., 2010). However, during glucose starvation, knockdown of HERP rescues apoptosis by enhancing autophagy (Quiroga et al., 2013). Knockdown of HERP increased ATG5 and BECLIN-1, which leads to increased autophagic flux and clearing of poly-Ub protein aggregates. Our recent studies have suggested that knockdown of HERP inhibits apoptosis by upregulating autophagy 7
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Fig. 5. Depletion of HERP inhibited ZEA-induced cell apoptosis. (A and B) The granulosa cells were transduced with shHERP and shNC lentivirus for 48 h and then treated with 30 μM ZEA for another 24 h. Cells were stained with Annexin VePE/7-AAD, and analyzed via flow cytometry assay. (C and D) The granulosa cells were transduced with shHERP and shNC lentivirus for 48 h and treated with 30 μM ZEA for 12 h. Levels of HERP, LC3-I and LC3-IIwere measured via western blot assay. (E and F) The granulosa cells were transduced with shHERP and shNC lentivirus for 48 h, and then treated with 30 μM ZEA for 12 h. Levels of pS6K1, S6K1, pERK1/2, ERK1/2, BCL-2, BAX and cleaved Caspase-3 were measured via western blot assay. The statistical analysis is shown in the bar graphs. Analyses of band intensity on films are presented as the relative ratio of HERP, LC3-I, LC3-II, pS6K1, S6K1, pERK1/2, ERK1/2, BCL-2, BAX or cleaved Caspase-3 to β-actin. Data are presented as the mean ± SEM. Bars with different letters are significantly different (p < 0.05).
5. Conclusion
and activation of ERK1/2 in non-small-cell lung carcinoma cells (Way et al., 2015). Inhibition of the AKT/mTOR pathway significantly increases ERK1/2 signaling in the regulation of oligodendrocyte differentiation (Dai et al., 2014). We speculated that the ERK1/2 signaling pathway might also enhance autophagy to inhibit apoptosis in HERPknockdown granulosa cells that are induced by ZEA.
In summary, this study demonstrated that treatment with ZEA increased the apoptosis of ovarian granulosa cells in a dose-dependent manner. In parallel, ZEA induced autophagy by increasing the level of LC3-II in ovarian granulosa cells. Pretreatment with CQ attenuated ZEA-induced cellular autophagy, and enhanced apoptosis. Regarding 8
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the mechanisms, exposure to ZEA sequentially reduced the levels of phosphorylation of S6K1 and ERK1/2. In addition, ZEA augmented ER stress through upregulating the levels of GRP78, HERP and CHOP in ovarian granulosa cells. ZEA inhibited phosphorylation of S6K1 and ERK1/2. Interestingly, knockdown of HERP enhanced ZEA-induced cellular autophagy and inhibited apoptosis in ovarian granulosa cells. Knockdown of HERP decreased pS6K1, increased pERK1/2 and inhibited cleaved Caspase-3. Taken together, this study showed that HERP depletion induced autophagy and inhibited apoptosis induced by ZEA in ovarian granulosa cells through activating the mTOR- and ERK1/2-dependent signaling pathways and through inhibiting the caspase-dependent apoptotic pathway.
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Conflict of interest statement The authors declare that there are no conflicts of interest. Author contributions Yaping Jin conceived the experiments, interpreted the data and supervised the research project. Fenglei Chen and Xin Wen performed the experiments and wrote the manuscript. Pengfei Lin, Huatao Chen, and Aihua Wang participated in discussion and revised the manuscript. Funding Support for this research was provided by grants from the National Natural Science Foundation of China (Nos. 31702298, 31772817, 31771301 and 31602125), the Natural Science Foundation of Jiangsu Province, China (No. BK20170498), China Postdoctoral Science Foundation (Nos. 2017M621843, 2017M61065 and 2018T111112), the Natural Science Foundation of Jiangsu Higher Education Institutions of China (No. 17KJD230002), the Priority Academic Program Development of Jiangsu Higher Education Institution (PAPD). Transparency document The Transparency document associated with this article can be found in the online version. References Adibnia, E., Razi, M., Malekinejad, H., 2016. Zearalenone and 17 beta-estradiol induced damages in male rats reproduction potential; evidence for ER alpha and ER beta receptors expression and steroidogenesis. Toxicon 120, 133–146. Ben Salem, I., Boussabbeh, M., Da Silva, J.P., Guilbert, A., Bacha, H., Abid-Essefi, S., Lemaire, C., 2017. SIRT1 protects cardiac cells against apoptosis induced by zearalenone or its metabolites alpha- and beta-zearalenol through an autophagy-dependent pathway. Toxicol. Appl. Pharm. 314, 82–90. Ben, I., Boussabbeh, M., Da Silva, J.P., Lemaire, C., Bacha, H., Abid-Essefi, S., 2015. SIRT1/autophagy: a cardioprotective response to zearalenone-induced endoplasmic reticulum stress. FEBS J. 282 109-109. Caloni, F., Albonico, M., Schutz, L., Pizzo, F., Spicer, L.J., 2014. Differential effects of fumonisin B1 and zearalenone metabolites on estradiol and progesterone production in cultured bovine granulosa cells. Toxicol. Lett. 229 S57-S57. Chan, S.L., Fu, W.M., Zhang, P.S., Cheng, A.W., Lee, J.W., Kokame, K., Mattson, M.P., 2004. Herp stabilizes neuronal Ca2+ homeostasis and mitochondrial function during endoplasmic reticulum stress. J. Biol. Chem. 279, 28733–28743. Chen, F.L., Li, Q., Zhang, Z., Lin, P.F., Lei, L.J., Wang, A.H., Jin, Y.P., 2015. Endoplasmic reticulum stress cooperates in zearalenone-induced cell death of RAW 264.7 macrophages. Int. J. Mol. Sci. 16, 19780–19795. Chen, F.L., Lin, P.F., Wang, N., Yang, D.Q., Wen, X., Zhou, D., Wang, A.H., Jin, Y.P., 2016a. Herp depletion inhibits zearalenone-induced cell death in RAW 264.7 macrophages. Toxicol. In Vitro 32, 115–122. Chen, F.L., Wang, N., Yang, D.Q., Wen, X., Mahmoud, T.N., Zhou, D., Tang, K.Q., Lin, P.F., Wang, A.H., Jin, Y.P., 2016b. Herp depletion arrests the S phase of the cell cycle and increases estradiol synthesis in mouse granulosa cells. J. Reprod. Dev. 62, 159–166. Chen, Q.W., Edvinsson, L., Xu, C.B., 2010. Cigarette smoke extract promotes human vascular smooth muscle cell proliferation and survival through ERK1/2-and NFkappa B-dependent pathways. Sci. World J. 10, 2139–2156. Cheng, P., Ni, Z., Dai, X., Wang, B., Ding, W., Rae Smith, A., Xu, L., Wu, D., He, F., Lian, J., 2013. The novel BH-3 mimetic apogossypolone induces beclin-1- and ROS-mediated autophagy in human hepatocellular carcinoma [corrected] cells. Cell Death Dis. 4,
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HERP depletion inhibits zearalenone-induced apoptosis through autophagy activation in mouse ovarian granulosa cells Fenglei Chena,b,c,d, Xin Wena,b, Pengfei Lina,b, Huatao Chena,b, Aihua Wangb, Yaping Jina,b,
T
⁎
a
Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, Northwest A&F University, Yangling, Shaanxi, 712100, China College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China c College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, Jiangsu, China d Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, Jiangsu, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: HERP Autophagy Zearalenone Apoptosis Granulosa cells
HERP is an endoplasmic reticulum (ER) membrane protein and is strongly induced by stress conditions. A recent study has indicated that HERP cooperates in apoptosis during zearalenone (ZEA) treatment. However, regulatory mechanisms and the role of HERP in ZEA-induced apoptosis remain elusive in ovarian granulosa cells. In this study, MTT and flow cytometry assays demonstrated that ZEA gradually decreased cell viability and increased apoptosis in granulosa cells in a dose-dependent manner. Western blot analysis showed that ZEA significantly activated autophagy by upregulating LC3-II. Chloroquine (CQ) significantly increased LC3-II and induced granulosa cell apoptosis. Moreover, Western blot analysis showed that ZEA inhibited the mTOR and ERK1/2 signaling pathways. Furthermore, we found that ZEA activated ER stress by upregulating the ER stress-related proteins GRP78, HERP and CHOP. 4-PBA significantly decreased GRP78, HERP, CHOP and LC3-II. In addition, knockdown of HERP (shHERP) significantly protected ovarian granulosa cells from apoptosis induced by ZEA. We found that HERP depletion activated autophagy and ERK1/2 signaling pathways, while it inhibited the mTOR and caspase-dependent mitochondrial signaling pathways. In summary, autophagy and ER stress cooperated in apoptosis induced by ZEA; HERP depletion inhibits ZEA-induced apoptosis of ovarian granulosa cells through autophagy activation and apoptotic pathway inhibition.
1. Introduction Zearalenone (ZEA), a nonsteroidal estrogenic mycotoxin from Fusarium fungi, causes reproductive toxicity in farm animals (Fushimi et al., 2015; Minervini and Dell’Aquila, 2008; Tiemann and Danicke, 2007; Yang et al., 2007). Since the structure of ZEA resembles that of 17β-estradiol, ZEA binds to estrogen receptors to exhibit estrogen functions (Adibnia et al., 2016; Gajecka et al., 2013; Minervini et al., 2001; Turcotte et al., 2005). In male animals, ZEA decreases the
testicular weight, the motility of spermatozoa and spontaneous acrosome reaction to affect the fertilization ability of sperm and reduces testosterone synthesis by Leydig cells by downregulating the expression of steroidogenic enzymes (Li et al., 2014; Long et al., 2016; Pang et al., 2017; Zheng et al., 2016). In female animals, ZEA impacts the maternal reproductive capability and embryonic implantation, delays fetal development, increases early death during early gestation and affects the levels of progesterone and estradiol in vivo and in vitro (Gao et al., 2017; Zhao et al., 2014, 2013). Ovarian granulosa cells play essential
Abbreviations: 4-PBA, 4-phenylbutyric acid; 7-AAD, 7-amino-actinomycin D 7; ANOVA, one-way analysis of variance; ATF, activating transcription factor; ATG, autophagy-related proteins; BAX, BCL-2 associated X protein; BCL-2, B-cell lymphoma-2; CHOP, CCAAT/enhancer binding protein homologous protein; CQ, chloroquine; DMEM/F12, Dulbecco’s modified Eagle’s medium: nutrient mixture F-12; DMSO, dimethyl sulfoxide; ECL, enhanced chemiluminescence; ELISA, enzyme linked immunosorbent assay; ER, endoplasmic reticulum; ERAD, ER-associated protein degradation; ERK1/2, extracellular signal-regulated kinase ½; FBS, fetal bovine serum; Fisher LSD, Fisher’s least significant difference test; GRP78, glucose-regulated protein 78; HERP, homocysteine-responsive ER-resident protein; IRE1α, inositol-requiring enzyme 1α; LC3, microtubule-associated protein light chain 3; MTT, 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide; mTOR, mammalian target of rapamycin; PERK, protein kinase (PKR)-like ER kinase; pERK1/2, phosphorylation of ERK1/2; pS6K1, phosphorylation of S6K1; PVDF, polyvinylidene difluoride; S6K1, p70 ribosomal protein S6 kinase; shHERP, HERP shRNA; shNC, non-silencing negative control; shRNAs, short hairpin interfering RNAs; SPSS, Statistical Package for the Social Science; UPR, unfolded protein response; ZEA, zearalenone ⁎ Corresponding author at: Key Laboratory of Animal Biotechnology of the Ministry of Agriculture, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China. E-mail address: [email protected] (Y. Jin). https://doi.org/10.1016/j.toxlet.2018.10.026 Received 27 June 2018; Received in revised form 25 September 2018; Accepted 22 October 2018 Available online 28 October 2018 0378-4274/ © 2018 Elsevier B.V. All rights reserved.
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evidence of whether ZEA regulates upstream signaling pathways of autophagy, mTOR or ERK1/2 and whether HERP cooperates in apoptosis through regulating the mTOR or ERK1/2 signaling pathways in ovarian granulosa cells.
roles in follicular development, maturation and sex hormone secretion. Previous studies have demonstrated that ZEA exerts double functions on the proliferation and apoptosis of ovarian granulosa cells at different doses and various species in vitro (Caloni et al., 2014; Cortinovis et al., 2014; Hou et al., 2015; Kolesarova et al., 2017; Liu et al., 2018; Minervini et al., 2001, 2006; Zhu et al., 2012). Because of its structural similarity to 17β-estradiol, ZEA stimulates the proliferation of ovarian granulosa cells at low doses and induces apoptosis at high doses. A previous study has demonstrated that ZEA induces apoptosis and necrosis in porcine granulosa cells via Caspase-9 and Caspase-3 activation (Zhu et al., 2012). However, it remains elusive whether other mechanisms are involved in apoptosis induced by ZEA in ovarian granulosa cells. Our studies have confirmed that endoplasmic reticulum (ER) stress and autophagy cooperate in ZEA-induced apoptosis of RAW 264.7 macrophages and mouse Leydig cells (Chen et al., 2015; Lin et al., 2015; Yang et al., 2017). Hence, we speculated that ER stress and autophagy cooperate in ovarian granulosa cell apoptosis that is induced by ZEA. ER stress triggers the unfolded protein response (UPR) to restore cellular homeostasis (Hetz, 2012; Schroder and Kaufman, 2005). Inositol-requiring enzyme 1α (IRE1α), double-stranded RNA-dependent protein kinase (PKR)-like ER kinase (PERK), and activating transcription factor (ATF)-6 separate from glucose-regulated protein 78 (GRP78) to activate the UPR. GRP78 is known as a marker of ER stress activation (Rao et al., 2002). However, severe or long ER stress triggers apoptosis. CHOP (CCAAT/enhancer binding protein homologous protein) cooperates in ER-stress-induced apoptosis, and it is a popular marker to monitor ER stress-induced apoptosis (Oyadomari and Mori, 2004). The homocysteine-responsive ER-resident protein (HERP), which is localized in the ER membrane, plays an important role in ER-associated protein degradation (ERAD) that is induced by ER stress (Kim et al., 2008; Kny et al., 2011; Okuda-Shimizu and Hendershot, 2007; Siva et al., 2010). HERP also inhibits apoptosis through preventing the loss of ER Ca2+ and mitochondrial potential during ER stress (Chan et al., 2004; Hori et al., 2004a). However, a recent study has demonstrated that knockdown of HERP up-regulates autophagy-related protein 5 (ATG5) and BECLIN-1 to activate protective autophagy for cell survival during glucose starvation (Quiroga et al., 2013). Moreover, our results also showed that knockdown of HERP upregulates LC3-II and decreases CHOP to activate protective autophagy and inhibit ER stress for cell survival during ZEA treatment (Chen et al., 2016a). Other than autophagy and ER stress activation, little was known regarding whether HERP affects other signaling pathways to regulate apoptosis during ZEA treatment. Autophagy is an evolutionarily conserved self-degradative catabolic process to degrade and recycle damaged organelles and long-lived proteins. Although it cooperates in programmed cell death, autophagy also plays a protective role against apoptosis under numerous stressful conditions, such as glucose starvation, apoptosis induced by ZEA and elimination of microorganisms (Kirkegaard et al., 2004; Lu et al., 2018; Wang et al., 2014). Autophagy is a complex signaling pathway, and it involves many ATGs to regulate this catabolic process (Yin et al., 2016). Microtubule-associated protein light chain 3 (LC3), which is known as ATG8, plays a critical role in autophagosome formation by converting cytosolic form of LC3-I into the lapidated form of LC3-II, and is characterized as a marker of autophagy activation (Tanida et al., 2004). The molecular mechanism of autophagy is under the control of different signaling pathways. Mammalian target of rapamycin (mTOR)/p70 ribosomal protein S6 kinase (S6K1) and extracellular signal-regulated kinase 1/2 (ERK1/2) cooperate in regulating autophagy (Eom et al., 2010; Way et al., 2015; Zhang et al., 2017). It is well known that mTOR/S6K1 negatively regulates autophagy (Kim and Guan, 2015). ERK1/2 negatively adjusts mTOR phosphorylation (Cheng et al., 2013). ERK1/2 activation is mainly involved in cell proliferation to promote survival (Chen et al., 2010; Lu and Xu, 2006). Although ZEA induces apoptosis through regulating autophagy and ER stress, there is no
2. Materials and methods 2.1. Experimental animals and reagents Immature female Kunming White mice (21 days old) were purchased from the laboratory animal center of the Fourth Military Medical University (Xi’an, Shanxi, China), the case number of animal use certificate is SYXK (Shan) 2014-001. The mice were housed separately under the controlled conditions of relatively constant temperature (23 ± 2 °C), a 12-h light/dark cycle and were provided food and water ad libitum. This study was approved by the committee for the Ethics on Animal Care and Experiment of Northwest A&F University. ZEA, Chloroquine (CQ), 4-phenylbutyric acid (4-PBA) and anti-LC3 rabbit polyclonal antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). ZEA was dissolved in dimethyl sulfoxide (DMSO), and the concentration of the stock solution was 15 mM. 4-PBA and CQ were dissolved in PBS, and the concentrations of the stock solutions were 1 M and 5 mM, respectively. Rabbit polyclonal antibodies including antiCHOP (sc-575), anti-HERP (sc-98669) and anti-GRP78 (sc-1050) goat polyclonal antibody were purchased from Santa Cruz Biotechnology Inc. (Texas, USA). Rabbit polyclonal antibodies including anti-ERK1/2, anti-pERK1/2, anti-S6K1, anti-pS6K1 and anti-cleaved Caspase-3 were purchased from Cell Signaling Technology (Danvers, MA, USA). Rabbit polyclonal antibodies including anti-BCL-2 and anti-BAX were purchased from Wanlei Biotechnology Inc. (Shenyang, China). Anti-β-actin mouse monoclonal antibody was purchased from Tianjin Sungene Biotechnology Inc. (Tianjin, China). 2.2. Culture of mouse ovarian granulosa cells and treatment After superovulation, granulosa cells were isolated according to a previously described method (Yang et al., 2013). Briefly, the ovaries were excised, and the granulosa cells were released by puncturing with 26-gauge needles. The granulosa cells were collected and washed via brief filtration and centrifugation. Cell viability was determined using the trypan-blue-dye exclusion method. The cells were cultured in Dulbecco’s modified Eagle’s medium: nutrient mixture F-12 (DMEM/F12, HyClone, Beijing, China) that was supplemented with penicillin (100 IU/ml), streptomycin (100 μg/ml) and 10% fetal bovine serum (FBS, Invitrogen, USA) at 37 °C under a 5% CO2 atmosphere. After culturing for 72 h, the cells were treated with ZEA for further experiments. 2.3. Transducing short hairpin interfering RNAs (shRNAs) via lentiviral infection The U6 RNAi cassette fragment from pSilencer 2.1-U6 hygro (Cat. No. AM5760, Life Technologies, Carlsbad, CA, USA) was amplified and cloned into pCD513B-1 (SBI, Mountain View, CA, USA), which contains a GFP expression construct, to generate a pCD513B-U6 lentiviral vector. Lentivirus vectors encoding the HERP shRNA (shHERP) and nonsilencing negative control (shNC) were constructed by our group. The sequence of the shNC was 5′-GATCCGATGAAATGGGTAAGTACATTCAA GAGATGTACTTACCCATTTCATCTTTTTTG-3′. The sequence of the shHERP was 5′-GATCCGAGCAGCCGGACAACTCTAATCTCGAG ATTAG AGTTGTCCGGCTGCTCTTTTTG-3′. The recombinant lentivirus vector was packaged and transduced into HEK 293 T cells. The medium was harvested 48 h after transfection, purified via low-speed centrifugation, and filtered through a 0.45-μm PVDF filter. An appropriate number of lentiviral particles (MOI = 20) were transduced into primary granulosa cells using 8 μg/ml polybrene. After 12 h of incubation, the medium containing the virus was removed and replaced with fresh culture 2
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Fig. 1. ZEA induces apoptosis in mouse ovarian granulosa cells. (A) The granulosa cells were plated in 96-well culture plates and treated with ZEA (0–150 μM) for 24 h. Cell viability was measured via the MTT assay. (B and C) After exposure to 0, 15, 30 or 60 μM ZEA for 24 h, ovarian granulosa cells were collected for Annexin V-PE/7-AAD staining followed by flow cytometric analysis. The statistical analysis is shown in the bar graphs. Data are presented as the mean ± SEM of three independent experiments. Bars with different letters are significantly different (p < 0.05).
Jiangsu, China). Briefly, the cells were washed with cold PBS, trypsinized and harvested. The cells were resuspended in 500 μl of binding buffer containing 5 μl of 7-AAD and 1 μl of Annexin V-PE and incubated for 30 min. Measurement of the apoptotic rate was performed via flow cytometry (EPICS Altra, Beckman Coulter Cytomics Altra, Brea, CA). The normal cells, as the control, were used to compensate for spectral bleed-through. The experiments were repeated three times independently.
medium. The cells were harvested after an additional 48 h. 2.4. Measurement of cell viability Cell viability was tested according to a previously described method (Chen et al., 2015). Briefly, granulosa cells were seeded into a 96-well plate with 1 × 104 cells/well. After culturing for 72 h, the cells were exposed to 0–150 μM ZEA for 24 h. Next, the cells were treated with 0.5 mg/ml 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) for another 4 h. Finally, the medium was replaced with 150 μl DMSO, and colorimetric measurement was performed using an enzyme linked immunosorbent assay (ELISA) plate reader at 570 nm (Model 680, Bio-Rad, and Hercules, CA, USA). Experiments were repeated three times independently.
2.6. Western blot analysis The granulosa cells, which were treated with 30 μM ZEA for 0–24 h or 0–60 μM ZEA for 12 h, were lysed with the Total Protein Extraction Kit (Nanjing Keygen Biotech Co., Ltd., Nanjing, China). The protein concentration was measured using the BCA Protein Assay Kit (Nanjing Keygen Biotech Co., Ltd., Nanjing, China). Equal amount of total proteins (30 μg) were separated via a 12–15% SDS-PAGE gel and were electrotransferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). After blocking with 10% fatty acid-free milk in TBST for 1 h at room temperature, the membranes were
2.5. Flow cytometric analysis of granulosa cell apoptosis After 24 h of ZEA treatment, the apoptotic rate was quantified using the Annexin V-PE and 7-AAD Apoptosis Detection Kit according to the manufacturer’s instruction (Nanjing Keygen Biotech Co., Ltd, Nanjing, 3
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3.3. ZEA induced activation of ER stress in mouse granulosa cells
incubated overnight at 4 °C with primary antibodies. The next day, the membranes were incubated with secondary antibody conjugated to horseradish peroxidase (Zhongshan Golden Bridge Biotechnology, Nanjing, China; 1:5000 dilutions) at room temperature for 1 h. Finally, immunoreactive bands were visualized via enhanced chemiluminescence (ECL) substrate using the Gel Image System (Tannon, Biotech, Shanghai, China) and exposed to an X-ray film for visualization of the protein bands. The protein band intensities were digitized using Quantity One software (Bio-Rad Laboratories, Hercules, CA).
To investigate whether apoptosis induced by ZEA were related to ER stress in mouse ovarian granulosa cells, the levels of the ER stress-related proteins GRP78, HERP and CHOP were examined via western blot analysis. As shown in Fig. 3A and B, GRP78, HERP and CHOP were significantly increased upon treatment with ZEA at 30–60 μM for 12 h. Fig. 3C and D show that GRP78 and CHOP were gradually increased upon prolonging treatment for 0–24 h with 30 μM ZEA. HERP peaked at 12 h and then gradually decreased. To further confirm the functional relationship between ZEA and ER stress, we used 4-PBA, an inhibitor of ER stress, to block ER stress that was induced by ZEA. Compared with treatment with 30 μM ZEA alone for 12 h, granulosa cells that were pretreated with 1 mM 4-PBA for 1 h and then coincubated with 30 μM ZEA for another 12 h, showed a significant decrease in the level of GRP78, HERP and CHOP (Fig. 3E and F). We also detected that 4-PBA significantly inhibited LC3-II (Fig. 3E and F). ZEA not only actives autophagy but also induces ER stress.
2.7. Statistical analysis All results are presented as the mean ± SEM. Date analysis was performed using one-way analysis of variance (ANOVA), followed by Fisher’s least significant difference test (Fisher LSD) and the Independent-Samples t-test using Statistical Package for the Social Science (SPSS) software (Version 13.0; SPSS, Inc., Chicago, IL, USA). All the experiments were replicated at least three times for each group. Differences were considered significant when p < 0.05.
3.4. ZEA inhibited activation of the mTOR and ERK1/2 signaling pathways in granulosa cells
3. Results
The onset of autophagy is regulated through the mTOR and ERK1/2 signaling pathways. Thus, we examined the mTOR and ERK1/2 activation that was induced by ZEA in granulosa cells by detecting the levels of phosphorylated S6K1, a marker of mTORC1 activation, and phosphorylated ERK1/2. As shown in Fig. 4A and B, the ratios of pS6K1/S6K1 and pERK1/2/ERK1/2 were significantly decreased upon treatment with ZEA at 30–60 μM for 12 h. Fig. 4C and D show that pS6K1/S6K1 and pERK1/2/ERK1/2 were decreased upon prolonged treatment for 12–24 h with 30 μM ZEA, but pS6K1/S6K1 and pERK1/2/ ERK1/2 were increased upon treatment for on 6 h. These results demonstrated that the mTOR and ERK1/2 signaling pathways might cooperate in ZEA-induced autophagy or apoptosis of granulosa cells.
3.1. Cytotoxic effect of ZEA on ovarian granulosa cells in vitro To examine the toxicity of ZEA on ovarian granulosa cells in vitro, the viability and apoptotic rate of granulosa cells was recorded via MTT and flow cytometry, after they were treated with different doses of ZEA for 24 h. As shown in Fig. 1A, the results of the MTT assay showed that there was very little effect on the growth of granulosa cells growth that were treated with 15 μM ZEA for 24 h compared with the control group. The cell viability was significantly inhibited by ZEA treatment at 30–150 μM in a dose dependent manner. To further analyze the apoptosis of granulosa cells that was induced by ZEA, the apoptotic rate was investigated via flow cytometry. As shown in Fig. 1B and C, treatment with ZEA at 15–60 μM resulted in a significant increase in the right lower quadrant and upright quadrant (19.8%, 50.15% and 73.15%, respectively). These results indicate that ZEA induces a cytotoxic effect in granulosa cells in a dose-dependent manner. Therefore, 30 μM of ZEA was employed for further experiments to evaluate its toxicity on granulosa cells.
3.5. HERP depletion inhibits ZEA-induced apoptosis in granulosa cells HERP is known to up-regulate protective autophagy that is induced by ZEA and glucose starvation. Here, we investigated whether HERP could protect granulosa cells from apoptosis that was induced by ZEA in an autophagy-dependent manner. To this end, we first constructed lentiviral vectors encoding shRNA of HERP (shHERP) (Chen et al., 2016b) and then transduced shHERP into granulosa cells to knockdown HERP before 30 μM ZEA treatment for 12 h, and the transduction efficiency was more than 80% (the results are not shown). As shown in Fig. 5A and B, the results of flow cytometry showed that the apoptotic rate of the shHERP group (42.2%) significantly decreased compared with that of the negative-control shRNA group (shNC) (58.2%). Western blot analysis showed that the shHERP group significantly inhibited the expression of HERP compared to the shNC; however, LC3-II was significantly increased after ZEA treatment (Fig. 5C and D). To further reveal how HERP regulated autophagy and apoptosis, we detected pS6K1, S6K1, pERK1/2, ERK1/2, BCL-2, BAX and cleaved Caspase-3 using western blot in the shHERP and shNC group after ZEA treatment. As shown in Fig. 5E and F, compared to the shNC, the ratios of pS6K1/ S6K1 and cleaved Caspase-3 were significantly decreased, and pERK1/ 2/ERK1/2 and BCL-2/BAX were significantly increased upon induction with by 30 μM ZEA for 12 h in the shHERP group. In summary, these results demonstrated that knockdown of HERP enhanced protective autophagy through regulating the mTOR and ERK1/2 signaling pathways and inhibited apoptosis through decreasing BAX and Caspase-3.
3.2. ZEA triggered autophagy responses in mouse granulosa cells We examined whether apoptosis of granulosa cells induced by ZEA is determined via activation of autophagy. Expression of LC3-II, a marker of autophagy activation signaling, was detected by western blot. As shown in Fig. 2A and B, LC3-II was significantly increased upon treatment with ZEA at 30–60 μM for 12 h compared with the control group. The level of LC3-II peaked at 30 μM and then gradually decreased. Fig. 2C and D show that LC3-II was gradually increased upon prolonged treatment for 0–24 h with 30 μM ZEA. To further confirm the functional relationship between ZEA and autophagy, we used CQ, a specific inhibitor of the late stage of autophagy, to block autophagy that was induced by ZEA. Compared with treatment with 30 μM ZEA for 12 h alone, granulosa cells that were pretreated with 5 μM CQ for 1 h and then coincubated with 30 μM ZEA for another 12 h, showed a significant increase in the level of LC3-II (Fig. 2E and F). We further detected the apoptotic rate after pretreatment with CQ to elucidate the relationship between apoptosis and autophagy in ZEA-induced toxicity. As shown in Fig. 2G and H, the results of flow cytometry showed that the apoptotic rate significantly increased after cotreatment with ZEA and CQ (63.0%) compared with ZEA alone (50.2%). In summary, ZEA induced autophagy and inhibited apoptosis in an autophagy-dependent manner.
4. Discussion Ovarian granulosa cells play an essential role in follicular development and maturation. Additional studies have revealed that ZEA 4
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Fig. 2. ZEA induces autophagy in mouse ovarian granulosa cells. (A and B) The granulosa cells were treated with the indicated concentration of ZEA and incubated for 12 h. Levels of LC3-I and LC3-II were measured via western blot assay. (C and D) The granulosa cells were treated with 30 μM ZEA for the indicated times. Levels of LC3-I and LC3-II were measured via western blot assay. (E and F) The granulosa cells were pretreated with 5 μM CQ for 1 h and coincubated with 30 μM ZEA for another 12 h. Levels of LC3-I and LC3-II were measured via western blot assay. (G and H) After pretreatment with 5 μM CQ for 1 h and coincubated with 30 μM ZEA for another 24 h, ovarian granulosa cells were collected for Annexin VePE/ 7-AAD staining followed by flow cytometric analysis. The statistical analysis is shown in the bar graphs. Analyses of band intensity on films are presented as the relative ratio of LC3-II to β-actin. Data are presented as the mean ± SEM. Bars with different letters are significantly different (p < 0.05).
granulosa cells. Consistent with the previous study, our results revealed that ZEA significantly inhibited proliferation and induced apoptosis in ovarian granulosa cells, which was dependent on the activation of autophagy and ER stress. The induction of autophagy and ER stress by
induces granulosa cell apoptosis (Hou et al., 2015; Liu et al., 2018; Minervini et al., 2006; Zhu et al., 2012). However, little was known regarding the detailed molecular mechanisms. In the present study, we detected that ZEA induced autophagy and apoptosis in ovarian 5
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Fig. 3. ZEA induces ER stress in mouse granulosa cells. (A and B) The granulosa cells were treated with the indicated concentration of ZEA and incubated for 12 h. Levels of ER stress-related proteins (GRP78, HERP, and CHOP) were measured via western blot assay. (C and D) The granulosa cells were treated with 30 μM ZEA for the indicated times. Levels of ER stress-related proteins (GRP78, HERP, and CHOP) were detected via western blot assay. (E and F) The granulosa cells were pretreated with 1 mM 4-PBA for 30 min and coincubated with 30 μM ZEA for another 12 h. Levels of ER stress-related proteins (GRP78, HERP and CHOP) were measured via western blot assay. The statistical analysis is shown in the bar graphs. Analyses of band intensity on films are presented as the relative ratio of GRP78, HERP or CHOP to β-actin. Data are presented as the mean ± SEM. Bars with different letters are significantly different (p < 0.05).
enhanced ZEA-induced apoptosis in ovarian granulosa cells. These results suggested that autophagy induced by ZEA played a protective role against apoptosis in ovarian granulosa cells. These results are in agreement with our previous studies that inhibition of autophagy by CQ significantly increased ZEA-induced apoptosis in RAW 264.7 cells (Chen et al., 2015). Collectively, these results suggest that autophagy could protect ovarian granulosa cells from apoptosis induced by ZEA. It is known that ZEA induces autophagy of various types of cells by a cascade of kinases (Ben Salem et al., 2017; Ben et al., 2015; Wang et al., 2014; Zheng et al., 2018). Additional evidence indicates that autophagy could be induced through the inhibition of the mTOR/S6K1 signaling pathway (Kim and Guan, 2015). mTOR is a major negative regulator of autophagy through inhibiting activation of ULK1 and ATG13 (Kim and Guan, 2015). Our results revealed that exposure of granulosa cells to ZEA caused a significant decrease in pS6K1, a marker of mTORC1 activation. Accordingly, we speculated that suppression of mTORC1
ZEA treatment was evidenced by the accumulation of LC3-II, GRP78, HERP and CHOP. Furthermore, knockdown of HERP protected granulosa cells from apoptosis induced by ZEA. HERP depletion activated autophagy and ERK1/2 signaling pathways by increasing LC3-II and pERK1/2 while inhibiting the mTOR and caspase-dependent mitochondrial signaling pathways by decreasing pS6K1, BAX and cleaved Caspase-3. Autophagy is a self-degradative process, and the amount of LC3-II is usually applied to monitor cellular autophagy (Tanida et al., 2004). In this study, our data revealed that ZEA raised the expression of LC3-II at different doses and times in ovarian granulosa cells. This finding is consistent with previous reports that ZEA induced the accumulation of LC3-II in primary Leydig cells and RAW 264.7 cells (Chen et al., 2016a; Wang et al., 2014). Furthermore, pretreatment with CQ, a special inhibitor of autophagy, which blocks the fusion of autophagosomes and lysosomes (Yoon et al., 2010), significantly inhibited autophagy and
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Fig. 4. ZEA inhibits the mTOR and ERK1/2 signaling pathways in mouse granulosa cells. (A and B) The granulosa cells were treated with the indicated concentration of ZEA and incubated for 12 h. Levels of pS6K1, S6K1, pERK1/2 and ERK1/2 were measured via western blot assay. (C and D) The granulosa cells were treated with 30 μM ZEA for the indicated times. Levels of pS6K1, S6K1, pERK1/2 and ERK1/2 were measured via western blot assay. The statistical analysis is shown in the bar graphs. Analyses of band intensity on films are presented as the relative ratio of pS6K1, S6K1, pERK1/2 or ERK1/2 to β-actin. Data are presented as the mean ± SEM. Bars with different letters are significantly different (p < 0.05).
under ZEA treatment in RAW 264.7 cells (Chen et al., 2016a). Therefore, we investigated whether HERP deletion protects ovarian granulosa cells from the toxicity of ZEA through an autophagy-dependent pathway. Our results showed that knockdown of HERP significantly decreased apoptosis that was induced by ZEA in ovarian granulosa cells. The present study strengthened the idea that HERP cooperates in autophagy regulation. Autophagy, as an ERAD II pathway, can assist ERAD through degrading abnormally folded proteins accumulation (Fujita et al., 2007). HERP may be an important mediator of the crosstalk between ERAD and autophagy (Quiroga et al., 2013). Consistent with the previous study, our results revealed that knockdown of HERP enhanced autophagy via upregulating LC3-II in ovarian granulosa cells. Further study demonstrated that knockdown of HERP decreased the expression of pS6K1 and increased pERK1/2. Activation of ERK1/2 has been shown to inhibit apoptosis in response to a wide range of stimuli. ERK1/2 promotes cell survival by suppressing the functions of pro-apoptotic proteins and enhancing the activity of anti-apoptotic proteins of the BCL-2 family. ERK1/2 also regulates the caspase-dependent mitochondrial pathway by functioning downstream of Cytochrome c release and caspase protein activation. In this study, our results revealed that knockdown of HERP significantly down regulated BAX and cleaved Caspase-3. We speculated that knockdown of HERP might inhibit apoptosis by activating the ERK1/2 signaling pathway. Additional studies have revealed that the ERK1/2 signaling pathway negatively regulates mTORC1 and activates autophagy (Dai et al., 2014; Shinojima et al., 2007; Way et al., 2015; Zhang et al., 2017). Curcumin inhibits the apoptosis of chondrocytes through the activation of the ERK1/2 signaling pathways, which induces autophagy (Li et al., 2017). Cinnamtannin D1 induces autophagy via the inhibition of AKT/mTOR
activation might cooperate in ZEA-induced autophagy of granulosa cells. ERK1/2 regulates cellular growth and survival (Lu and Xu, 2006). We examined the ERK1/2 signaling pathway, which was downregulated upon exposure to ZEA. We speculated that the ERK1/2 signaling pathway may participate in ZEA-induced granulosa cell apoptosis through inhibiting its own expression. ER stress triggers transcriptional induction, translational attenuation and ERAD to restore cellular homeostasis. However, with severe or prolonged ER stress, apoptosis is activated. After exposure to ZEA, GRP78 and CHOP were significantly augmented in ovarian granulosa cells. GRP78 is an ER stress-response protein, and its expression is a typical marker of ER stress activation (Rao et al., 2002). CHOP cooperates in ER stress-induced apoptosis (Oyadomari and Mori, 2004). Our previous results showed that ZEA triggers ER stress and then induced apoptosis though CHOP activation in RAW 264.7 cells (Chen et al., 2015; Lin et al., 2015). The present study further demonstrated the effects of ZEA on stimulating ER stress in ovarian granulosa cells. In addition to GRP78 and CHOP activation, ZEA induced HERP, which is cooperated in ERAD induced by ER stress. ERAD degrades unfolded or misfolded proteins upon occurrence of ER stress. HERP is induced under classical ER stressors, proteasome inhibition and nutrient starvation conditions; it plays a protective role in ER stress-induced apoptosis (Diaz et al., 2013; George et al., 2018; Hori et al., 2004b; Mirabelli et al., 2016; Siva et al., 2010). However, during glucose starvation, knockdown of HERP rescues apoptosis by enhancing autophagy (Quiroga et al., 2013). Knockdown of HERP increased ATG5 and BECLIN-1, which leads to increased autophagic flux and clearing of poly-Ub protein aggregates. Our recent studies have suggested that knockdown of HERP inhibits apoptosis by upregulating autophagy 7
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Fig. 5. Depletion of HERP inhibited ZEA-induced cell apoptosis. (A and B) The granulosa cells were transduced with shHERP and shNC lentivirus for 48 h and then treated with 30 μM ZEA for another 24 h. Cells were stained with Annexin VePE/7-AAD, and analyzed via flow cytometry assay. (C and D) The granulosa cells were transduced with shHERP and shNC lentivirus for 48 h and treated with 30 μM ZEA for 12 h. Levels of HERP, LC3-I and LC3-IIwere measured via western blot assay. (E and F) The granulosa cells were transduced with shHERP and shNC lentivirus for 48 h, and then treated with 30 μM ZEA for 12 h. Levels of pS6K1, S6K1, pERK1/2, ERK1/2, BCL-2, BAX and cleaved Caspase-3 were measured via western blot assay. The statistical analysis is shown in the bar graphs. Analyses of band intensity on films are presented as the relative ratio of HERP, LC3-I, LC3-II, pS6K1, S6K1, pERK1/2, ERK1/2, BCL-2, BAX or cleaved Caspase-3 to β-actin. Data are presented as the mean ± SEM. Bars with different letters are significantly different (p < 0.05).
5. Conclusion
and activation of ERK1/2 in non-small-cell lung carcinoma cells (Way et al., 2015). Inhibition of the AKT/mTOR pathway significantly increases ERK1/2 signaling in the regulation of oligodendrocyte differentiation (Dai et al., 2014). We speculated that the ERK1/2 signaling pathway might also enhance autophagy to inhibit apoptosis in HERPknockdown granulosa cells that are induced by ZEA.
In summary, this study demonstrated that treatment with ZEA increased the apoptosis of ovarian granulosa cells in a dose-dependent manner. In parallel, ZEA induced autophagy by increasing the level of LC3-II in ovarian granulosa cells. Pretreatment with CQ attenuated ZEA-induced cellular autophagy, and enhanced apoptosis. Regarding 8
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the mechanisms, exposure to ZEA sequentially reduced the levels of phosphorylation of S6K1 and ERK1/2. In addition, ZEA augmented ER stress through upregulating the levels of GRP78, HERP and CHOP in ovarian granulosa cells. ZEA inhibited phosphorylation of S6K1 and ERK1/2. Interestingly, knockdown of HERP enhanced ZEA-induced cellular autophagy and inhibited apoptosis in ovarian granulosa cells. Knockdown of HERP decreased pS6K1, increased pERK1/2 and inhibited cleaved Caspase-3. Taken together, this study showed that HERP depletion induced autophagy and inhibited apoptosis induced by ZEA in ovarian granulosa cells through activating the mTOR- and ERK1/2-dependent signaling pathways and through inhibiting the caspase-dependent apoptotic pathway.
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Conflict of interest statement The authors declare that there are no conflicts of interest. Author contributions Yaping Jin conceived the experiments, interpreted the data and supervised the research project. Fenglei Chen and Xin Wen performed the experiments and wrote the manuscript. Pengfei Lin, Huatao Chen, and Aihua Wang participated in discussion and revised the manuscript. Funding Support for this research was provided by grants from the National Natural Science Foundation of China (Nos. 31702298, 31772817, 31771301 and 31602125), the Natural Science Foundation of Jiangsu Province, China (No. BK20170498), China Postdoctoral Science Foundation (Nos. 2017M621843, 2017M61065 and 2018T111112), the Natural Science Foundation of Jiangsu Higher Education Institutions of China (No. 17KJD230002), the Priority Academic Program Development of Jiangsu Higher Education Institution (PAPD). Transparency document The Transparency document associated with this article can be found in the online version. References Adibnia, E., Razi, M., Malekinejad, H., 2016. Zearalenone and 17 beta-estradiol induced damages in male rats reproduction potential; evidence for ER alpha and ER beta receptors expression and steroidogenesis. Toxicon 120, 133–146. Ben Salem, I., Boussabbeh, M., Da Silva, J.P., Guilbert, A., Bacha, H., Abid-Essefi, S., Lemaire, C., 2017. SIRT1 protects cardiac cells against apoptosis induced by zearalenone or its metabolites alpha- and beta-zearalenol through an autophagy-dependent pathway. Toxicol. Appl. Pharm. 314, 82–90. Ben, I., Boussabbeh, M., Da Silva, J.P., Lemaire, C., Bacha, H., Abid-Essefi, S., 2015. SIRT1/autophagy: a cardioprotective response to zearalenone-induced endoplasmic reticulum stress. FEBS J. 282 109-109. Caloni, F., Albonico, M., Schutz, L., Pizzo, F., Spicer, L.J., 2014. Differential effects of fumonisin B1 and zearalenone metabolites on estradiol and progesterone production in cultured bovine granulosa cells. Toxicol. Lett. 229 S57-S57. Chan, S.L., Fu, W.M., Zhang, P.S., Cheng, A.W., Lee, J.W., Kokame, K., Mattson, M.P., 2004. Herp stabilizes neuronal Ca2+ homeostasis and mitochondrial function during endoplasmic reticulum stress. J. Biol. Chem. 279, 28733–28743. Chen, F.L., Li, Q., Zhang, Z., Lin, P.F., Lei, L.J., Wang, A.H., Jin, Y.P., 2015. Endoplasmic reticulum stress cooperates in zearalenone-induced cell death of RAW 264.7 macrophages. Int. J. Mol. Sci. 16, 19780–19795. Chen, F.L., Lin, P.F., Wang, N., Yang, D.Q., Wen, X., Zhou, D., Wang, A.H., Jin, Y.P., 2016a. Herp depletion inhibits zearalenone-induced cell death in RAW 264.7 macrophages. Toxicol. In Vitro 32, 115–122. Chen, F.L., Wang, N., Yang, D.Q., Wen, X., Mahmoud, T.N., Zhou, D., Tang, K.Q., Lin, P.F., Wang, A.H., Jin, Y.P., 2016b. Herp depletion arrests the S phase of the cell cycle and increases estradiol synthesis in mouse granulosa cells. J. Reprod. Dev. 62, 159–166. Chen, Q.W., Edvinsson, L., Xu, C.B., 2010. Cigarette smoke extract promotes human vascular smooth muscle cell proliferation and survival through ERK1/2-and NFkappa B-dependent pathways. Sci. World J. 10, 2139–2156. Cheng, P., Ni, Z., Dai, X., Wang, B., Ding, W., Rae Smith, A., Xu, L., Wu, D., He, F., Lian, J., 2013. The novel BH-3 mimetic apogossypolone induces beclin-1- and ROS-mediated autophagy in human hepatocellular carcinoma [corrected] cells. Cell Death Dis. 4,
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