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Polychlorinated biphenyl quinone induces endoplasmic reticulum stress, unfolded protein response and calcium release Demei Xu, Chuanyang Su, Xiufang Song, Qiong Shi, Juanli Fu, Lihua Hu, Xiaomin Xia, Erqun Song, and Yang Song Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.5b00124 • Publication Date (Web): 07 May 2015 Downloaded from http://pubs.acs.org on May 9, 2015

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Polychlorinated biphenyl quinone induces endoplasmic reticulum stress, unfolded protein response and calcium release

Demei Xu, Chuanyang Su, Xiufang Song, Qiong Shi, Juanli Fu, Lihua Hu, Xiaomin Xia, Erqun Song, Yang Song*

Key Laboratory of Luminescence and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Pharmaceutical Sciences, Southwest University, Chongqing, People’s Republic of China, 400715

*

Corresponding author: College of Pharmaceutical Sciences, Southwest University, Beibei,

Chongqing, 400715, P R China. Tel: +86-23-68251503. Fax: +86-23-68251225. E-mail address: [email protected]

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Table of Contents

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ABSTRACT Organisms are able to respond to environmental insult to maintain cellular homeostasis, which include the activation of a wide range of cellular adaptive responses with tightly controlled mechanisms. The endoplasmic reticulum (ER) is an organelle responsible for protein folding and calcium storage. ER stress leads to the accumulation of unfolded proteins in the ER lumen. To against or respond to this effect, cells have a comprehensive signaling system, called unfolded protein response (UPR), to restore homeostasis and normal ER function, or activate cell death program. Therefore, it is critical to understand how environmental insult regulates the ingredients of ER stress and UPR signalings. Previously, we have demonstrated that polychlorinated biphenyl (PCB) quinone caused oxidative stress, cytotoxicity, genotoxicity and apoptosis in HepG2 cells. Here, we investigated the role of a PCB quinone, PCB29-pQ on the ER stress, UPR and calcium release. PCB29-pQ markedly increased the hallmark genes of ER stress, namely glucose-regulated protein 78 (GRP78), GRP94 and C/EBP homologous protein (CHOP) on both protein and mRNA levels in HepG2 cells. We also confirmed PCB29-pQ induced ER morphological defects by using transmission electron microscopy. Moreover, PCB29-pQ induced intracellular calcium accumulation and calpain activity, which were significantly inhibited by the pretreatment of BAPTA-AM (Ca2+ chelator). These results were correlated with the outcome that PCB29-pQ induces ER stress-related apoptosis through caspase family gene 12, whilst salubrinal and Z-ATAD-FMK (a specific inhibitor of caspase 12) partially ameliorated this effect, respectively. N-acetyl-L-cysteine (NAC) scavenged

ROS

formation,

and

consequently

alleviated

PCB29-pQ-induced

the

expression of ER stress-related genes. In conclusion, our result demonstrated for the first time that PCB quinone leads to ROS-dependent induction of ER stress, UPR and 3 ACS Paragon Plus Environment

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calcium release in HepG2 cells, and the evaluation of perturbations of ER stress, UPR and calcium signaling provide further information on the mechanisms of PCB-induced toxicity.

Keywords: PCB; ER stress; UPR; ROS; calcium release; apoptosis

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INTRODUCTION Polychlorinated biphenyl (PCB) congeners were manufactured for industrial usage, however, they became world-wide pollutants nowadays.1 Once they were introduced into the environment, they were likely to bio-accumulate in the environment and bioamplified through the food chain. PCB have been routinely detected in adipose tissues, liver and other organs, meanwhile, evidences supported they are associated with various adverse health effects.2 Although they are relative stable species, it has been widely validated that PCB metabolize into corresponding hydroxylated metabolites (OH-PCB) via a 1,2 shift (NIH shift) or direct oxygen insertion by the catalyzing of cytochrome P450 enzymatic system.3 Although hydrophilic OH-PCB are readily conjugated and excreted, several OH-PCB have been found to be retained in human plasma.4 For the last few years, attention has been paid to the metabolic pathway of PCB in humans and wildlife.3 PCB and their hydroxylated metabolites showed toxic effects in a number of in vitro and in vivo models.5-7 However, only few studies focused on the toxic effects of oxidized forms of OH-PCB, PCB quinone metabolites. Our previous studies indicated PCB quinones induce oxidative damage, cytotoxicity, genotoxicity and apoptosis in cultured human liver hepatocellular carcinoma HepG2 cells.8, 9 Mechanism study indicated the involvement of reactive oxygen species (ROS) in PCB quinone-induced cellular damage. Interestingly, strong evidence indicated the overexpression of ROS affects endoplasmic reticulum (ER) homeostasis and protein folding.10 ROS sensitize the calcium-release channels at the ER membrane and increase the leakage of calcium from the ER lumen to cytosol. However, the increase of cytosolic calcium in turn stimulates mitochondrial ROS production, which enhances challenges to

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proper protein folding. This implied that PCB quinone-induced toxicity may associate with ROS-mediated ER stress. ER is the multifunctional organelle which controls the processes of protein biosynthesis,

folding,

assembly,

posttranslational

modification

and

delivery,

and

regulates intracellular calcium homeostasis.11 Stimuli, such as glucose deprivation, hypoxia, calcium depletion, viral infection and oxidative stress impair ER function and lead to the accumulation of unfolded proteins in the ER lumen. To compromise these effects, cells activate an adaptive program called unfolded protein response (UPR), which consist of three canonical signal transduction pathways, i.e., inositol-requiring kinase/endonuclease 1α (IRE1α), protein kinase-like endoplasmic reticulum kinase (PERK) and activating transcription factor 6 (ATF6).12,

13

Each of these signal inducer

contains an ER luminal domain that sequesters glucose-regulated protein 78 (GRP78, aka Bip) under normal conditions. However, under the status of ER stress, GRP78 dissociates from IRE1α, PERK or ATF6 to bind misfolded or unfolded protein, and enables their ATP-dependent protein folding.14 In turn, the release of these UPR transducers from GRP78 leads to their activation and initiate corresponding downstream signaling pathways, which are collectively combat ER stress. PERK is activated by dimerization and auto-phosphorylation, which allow PERK promotes the phosphorylation of eukaryotic translation initiation factor (eIF2α), and attenuates global mRNA translation to help cells against ER stress. Phosphorylated eIF2α enhances the translation of ATF4 transcription factor and leads to the transcription of many pro-survival genes.15 IRE1α is also activated by dimerization and autophosphorylation. Activated IRE1α initiates the splicing of X-box-binding protein-1 (XBP1) mRNA, with the resulting of open reading frame encoding full length XBP1 and up-regulated expression of genes involved in protein 6 ACS Paragon Plus Environment

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folding and ER-associated degradation (ERAD). After disassembling from GRP78, ATF6 translocate from ER to Golgi, where it is cleaved to cytosolic fragment ATF6 p50, then migrates to the nucleus to promote the transcription of genes encoding ER chaperones and enzymes that promote protein folding, secretion and ERAD.16 Although the idea of UPR activation is to reduce ER stress, the prolonged and irreversible ER stress showed implications for apoptosis.17 Once these three branches failed to recover cells, ER stress shifts the adaptive response of UPR to apoptotic cell death response. PERK induce ATF4-dependent up-regulation of the C/EBP homologous protein (CHOP, aka GADD153) transcription factor. IRE1α oligomerization leads to the activation of apoptotic signal regulating kinase 1 (ASK1) and its downstream target cJun NH2-terminal kinase (JNK).18 Both CHOP and JNK inhibit the expression of gene encoding anti-apoptotic Bcl-2 protein family members, which signal the mitochondrial apoptotic machinery. Thus, the extent of ER stress or UPR may determine the ultimate fate of cell by regulate pro-survival or anti-survival signals.ER lumen plays a central role in calcium storage and signaling, both protein folding reactions and protein chaperone need a high-calcium environment.19 Disturbances of calcium homeostasis in ER constitute a severe form of stress, which interfering with folding and processing of newly synthesized membrane and secretory proteins.20 Calpains are a family of calciumdependent intracellular cysteine proteases. Perturbations of calcium pools activate calpains in the cytosol, which activates Bax and Bid and caspases. Eventually, this event leads to the opening of permeability transition pore and the release of cytochrome c, which implicated the involvement of apoptosis.

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The assessment of ER and UPR signaling is likely to be informative in the analyzing of PCB quinone-induced toxicity. Here, we provided the first evidence to link ER stress and UPR to PCB quinone-induced toxicity and further elucidated the underlying mechanism.

MATERIALS AND METHODS Materials and reagents. 2,3,5-Trichloro-6-phenyl-[1,4]benzoquinone (PCB29-pQ, structure was shown in Fig 1A) was synthesized and characterized as previously described,21 and stock solution (50 mM) of PCB29-pQ was prepared in DMSO. 2’,7′Dichlorofluorescein diacetate (DCFH-DA), 1,2-bis(2-amino-phen-oxy)ethane-N,N,N,Ntetraacetic acid (BAPTA-AM) and salubrinal were purchased from Sigma-Aldrich Co. LLC (Shanghai, China). N-acetyl-L-cysteine (NAC) was purchased from Aladdin Reagent Database Inc. (Shanghai, China). S-LLVY-AMC was purchased from Boston Biochem Inc. (Cambridge, MA). Fluo-3 AM was purchased from Biotium Inc. (Hayward, CA). Z-ATADFMK was purchased from Chinese Peptide Co. (Hangzhou, China). Rabbit CHOP, GRP94, PERK, GRP78, ATF4, ATF6, caspase 12, eIF2α, JNK, c-Jun and β-actin polyclonal primary antibodies were obtained from Proteintech Group Co. Ltd. (Wuhan, China), Rabbit pPERK (Thr980), p-IRE1α (Ser726), p-JNK (Thr183) and IRE1α monoclonal primary antibodies were obtained from Biosynthesis Biotechnology Co. Ltd. (Beijing, China). Rabbit p-eIF2α (Ser51) monoclonal primary antibody was purchased from Bioworld Technology Inc. (Nanjing, China). Goat-anti-rabbit IgG-HRP conjugated secondary antibody was supplied by Dingguo Biotechnology Co. Ltd. (Beijing, China). All other chemicals used were of the highest commercial grade.

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Cell culture. The human hepatoma cell line HepG2 was purchased from Nanjing KeyGEN Biotech. Co. Ltd. (Nanjing, China). Cells were cultured in DMEM medium with 10% newborn calf serum (Hangzhou Sijiqing Biological Engineering Materials Co. Ltd.) and antibiotics (100 U/ml penicillin and 100 U/ml streptomycin) with 5% CO2 atmosphere at 37°C. Transmission electron microscopy (TEM) and bright field microscopy. To observe the morphology and microstructure of HepG2 cells, TEM analysis was performed. After being treated with 10 µM PCB29-pQ for 48 h, HepG2 cells were harvested with 0.1% trypsin-EDTA solution, neutralized with 10% newborn calf serum containing fresh media and washed twice with PBS. Cells were fixed with ice-cold glutaraldehyde for 2 h. Then, cells were postfixed with 2% osmium tetroxide and embedded in Epon, counterstained with uranyl acetate and lead citrate, visualized in a Hitachi-7500 TEM (Hitachi Instrument, Tokyo, Japan). Alternatively, morphological signs of apoptosis were evaluated using bright field microscopy. Cells (5×105 cells per well) were incubated with 10 µM PCB29-pQ for 48 h. Cells were viewed under bright field and figures were captured by fluorescence microscopy (OLYMPUS IX71) at 200× magnification. Flow Cytometry. Frequency of apoptotic cells was detected by flow cytometry assay. After being exposed to PCB29-pQ for 48 h, cells were mixed with binding buffer and incubated with a fluorescein isothiocyanate (FITC)-conjugated annexin-V/propidium iodide (PI) double staining solution and incubated for 15 min in the dark. Fluorescence was determined on a BD FACS Vantage SE flow cytometer (BD Biosciences) and the percentage of apoptotic cells was calculated using BD Cell Quest software. Measurement of ROS. The generation of intracellular ROS was inspected by DCFHDA probe following a previously described procedure with minor modifications.9 After 9 ACS Paragon Plus Environment

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treatment with 10 µM PCB29-pQ for 48 h, cells were incubated with 10 µM solution of DCFH-DA at 37°C for 20 min, cells were washed twice with PBS and images were taken by fluorescent microscopy (OLYMPUS IX71). Protein preparation and quantification. HepG2 cells were washed twice with icecold PBS, then, total protein was extracted using RIPA lysis buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1% Triton X-100; 1% sodium deoxycholate; 0.1% sodium dodecyl sulfate; 5 mM EDTA; 1 mM EGTA) containing a protease inhibitor cocktail (0.5 mM DTT, 5 µg/µl leupeptin, 0.2 µM PMSF, 5 µg/µl aprotin, 1 µM Na2VO4 and 1 µM NaF). The cellular lysates were centrifuged at 12,000 g and 4°C for 15 min. The supernatant proteins were collected and quantified using the BCA Protein Assay Kit (Beyotime, China). Measurement of intracellular calcium level. The changes in intracellular calcium levels were determined by the fluorescent probe, Fluo-3AM. Cells were treated with the indicated concentrations of PCB29-pQ for 48 h. Then, cells were incubated 5 µM Fluo3AM for 1 h. Medium was replaced with fresh medium and the cells were placed at 37°C for another 30 min. Fluorescence was determined on BD FACS Vantage SE low cytometer (BD Biosciences). Data were analyzed using BD Cell Quest software. Measurement of calpain activity. Calpain activity was measured as previously described method with minor modification,22 using S-LLVY-AMC as the cell-permeable fluorogenic calpain substrate. Control and PCB29-pQ-treated cells were harvested with 0.25% trypsin-EDTA solution, neutralized with 10% newborn calf serum containing media and washed twice with PBS, re-suspended in PBS at 3.5×105 cells per ml, and pre-warmed at 37°C and 5% CO2 atmosphere for 10 min. S-LLVY-AMC (25 µM) was added and the AMC fluorescence intensity was measured on the spectrofluorometer 10 ACS Paragon Plus Environment

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(HITACHIF-7000) at an excitation wavelength of 345 nm and an emission wavelength of 445 nm. Western blotting. Proteins were electrophoresed on 10% or 12.5% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE), then, transferred onto a nitrocellulose membrane. The blots were blocked with 5% dried skim milk dissolved in TBST (10 mM TBS plus 1.0% Tween 20) for 1.5 h at room temperature and incubated with corresponding antibodies for 3 h. Blots were incubated with the secondary goat-antirabbit IgG–HRP-conjugated antibody for additional 1.5 h at room temperature. Target proteins were visualized and representative images were presented. Densitometric analysis of immunoblot was performed by ImageJ software. RNA extraction and real-time quantitative PCR (RT-qPCR). After treating HepG2 cells with PCB29-pQ for 48 h, total RNA was isolated using a total RNA kit I according to the manufacturer's instructions (Omega, Norcross, GA). RNA concentration and purity were assessed photometrically using A260/A280 ratio and RNA integrity was tested by agarose gel electrophoresis. The purified total RNA (2-3 µg) was employed for cDNA synthesis with the Transcriptor First Strand cDNA Synthesis Kit (Roche, Switzerland). cDNA was used to perform RT-qPCR analysis using LightCycler 96 instrument protocol with Faststart Essential DNA Green Master one-component hot start reaction mix (Roche, Switzerland). The sequences of the RT-qPCR primers were listed in Table 1. Three independent replicates were analyzed per sample and relative gene expression normalized to the internal housekeeping gene β-actin was obtained by the 2∆∆Ct

method.

XBP1 mRNA splicing assay. The cDNA was prepared as described above. XBP1 primers were used as shown in Table 1 and β-actin was used as an internal control. A 11 ACS Paragon Plus Environment

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331-bp PCR product was identified as the unspliced form of XBP1 mRNA and a 305-bp PCR product was identified as the spliced form of XBP1 mRNA. The amplified PCR products were separated by electrophoresis on a 1.5% (w/v) agarose gel and visualized under UV light. Statistical analysis. Data were presented as mean ± SD and statistical analysis was performed by SPSS 17.0 for Windows. Differences between the means of data were compared by one-way variance analysis (ANOVA) test and post hoc analysis of group differences was performed by least significant difference (LSD) test and a P value of < 0.05 was considered to be statistically significant.

RESULTS PCB29-pQ induced an ER stress response in HepG2 cells. We first investigated whether ER stress was induced upon PCB29-pQ treatment. The choice of dosage and duration of PCB29-pQ exposure was based on our previous publication.23 As shown in Fig 1B, PCB29-pQ induced the expression of GRP78, CHOP and GRP94, three markers of ER stress in HepG2 cells in a concentration-dependent manner. Kinetic analysis further revealed a time-dependent up-regulation of these proteins after PCB29-pQ stimulation, Fig 1C. RT-qPCR analysis indicated that treatment of HepG2 cells with 1, 5 or 10 µM of PCB29-pQ for 48 h induced GRP78, CHOP and GRP94 mRNA expressions, respectively (Fig 1D). Next, we evaluated the effect of PCB29-pQ on ER morphology using TEM technology. As shown in Fig 1E, the chromatin in the nuclei of control group was well distributed and their ER showed normal flattened ultrastructure. However, PCB29-pQ induces serious ER dilatation in HepG2 cells. To sum up, these data indicated that the induction of ER stress is a general mode-of-action of PCB29-pQ in HepG2 cells. 12 ACS Paragon Plus Environment

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PCB29-pQ activated the PERK–eIF2α–ATF4 pathway in HepG2 cells. The perturbation of ER homeostasis may disrupt the protein folding process and activate the UPR. PERK–eIF2α–ATF4 represents the primary canonical arms of UPR signaling. Thus, to evaluate the effect of PCB29-pQ on PERK–eIF2α–ATF4 signaling, we treated HepG2 cells with the indicated concentration (or time) of PCB29-pQ, then, cells were subjected to Western blotting afterwards. Results showed that treatment of HepG2 cells with PCB29-pQ led to significant up-regulation in PERK and p-PERK expressions both concentration- (Fig 2A) and time-dependently (Fig 2B). Furthermore, although the expression of eIF2α has not significantly elevated with PCB29-pQ exposure, its phosphorylation form dramatically increased. Histograms indicated the ratio of peIF2α/eIF2α was elevated in a concentration- and time-dependently behavior. Similarly, ATF4 expression was stimulated on PCB29-pQ exposure. Salubrinal was identified as an inhibitor of eIF2α dephosphorylation, which promotes the survival of ER-stressed cells.24 As estimated, salubrinal (20 µM) significantly increased eIF2a phosphorylation level, Fig 2C. Surprisingly, salubrinal suppressed PCB29-pQ-induced ATF4 and CHOP expressions. PCB29-pQ activated the ATF6 and IRE1α–XBP1 pathways in HepG2 cells. We next examined the activation of two other UPR branches, ATF6 and IRE1–XBP1 pathways. As shown in Fig 3A & 3B, the increased expression of IRE1α and p-IRE1α were significantly observed in cells exposed to PCB29-pQ in both concentration- and timedependent manners. Although JNK phosphorylation is a known downstream event of IRE1 activation,17 however, our result indicated that JNK, p-JNK and c-Jun expressions have not been affected. According to the stimuli form of ER stress, a specific pathway among the ER stress responses can be selectively activated25 and our result suggested that

PCB29-pQ-induced

ER

stress

is

JNK-independent.

IRE1α

has

site-specific 13

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endoribonuclease activity, which removes a 26-nucleotide intron from XBP1.26 Previous study suggested the activation of XBP1 is a characteristic feature of ER stress,13 To further confirm the activation of IRE1α, we tested the effect of PCB29-pQ on splicing of XBP1 mRNA using RT-qPCR. As shown in Fig 3C, treatment with 1 to 10 µM PCB29-pQ for 48 h resulted in both the up-regulation of unspliced (XBP1u) and spliced (XBP1s). Consistently, RT-qPCR result indicated the total mRNA level of XBP was increased about 8-fold in 10 µM PCB29-pQ group, Fig 3D. ATF6 is an ER stress-regulated transmembrane protein, which is responsible for the activation of the transcription of ERrelated genes. Accumulation of misfolded proteins in the ER results in the proteolytic cleavage of ATF6.27 Treatment of HepG2 cells with PCB29-pQ caused concentration- and time-dependent decrease in the level of ATF6, respectively (Fig 3A & 3B). This result indicated the increased truncated ATF6, which is comparable with previous study.28 PCB29-pQ induces apoptosis via a caspase 12-dependent pathway. Caspase 12 is the first caspase reported to localize on the ER, which can be activated by UPR signaling in mammalian cells.29 Subsequently, activated caspase 12 may stimulate the activation of executor caspases. We found that there was a tendency of both concentration- and time-dependent increase of caspase 12 cleavages, Fig 4A & 4B. The morphology of HepG2 cells upon PCB29-pQ treatment was also investigated. As shown in Fig 4C, the control group cells showed normal cell morphology, whereas PCB29-pQtreated cells showed cytoplasmic vacuolization, cell shrinkage, irregular and rounded morphology, which are typical morphological changes of apoptosis. Moreover, the occurrence of apoptosis was supported by the direct observation of apoptotic body using TEM technique. The control cells were in normal morphology with intact nuclei, and the electron density of cytoplasm is homogeneous. However, early apoptotic cells, which 14 ACS Paragon Plus Environment

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were shown in PCB29-pQ group, as the feature of small cell volume, chromatin margination and the increased electron cloud density. Additionally, late apoptotic cells with visible karyopyknosis, nuclear fragmentation and the formation of apoptotic bodies were shown in PCB29-pQ group. Typical images were shown in Fig 4D. Our previous study illustrated that PCB29-pQ induces mitochondrial-driven apoptosis, accompanied with the increased cleavages of caspase 9 and 3.23 In this study, the specific caspase 12 inhibitor Z-ATAD-FMK suppressed the cleavage of caspase 12, as well as the cleavages of caspase 9 and 3 (Fig 4E), which confirm PCB29-pQ-induced apoptosis is caspase 12dependent. Caspases 12 and 9 are downstream molecules of ER and mitochondrial stress, respectively, and caspase 3 is the joint executor caspase for both initiator caspases. Thus, our result also implied the cross-talk of ER to mitochondrial signaling and caspase 12 acting as the upstream molecule of caspase 9. Moreover, apoptosis was quantitatively analyzed by Annexin V-FITC/PI double staining. In Fig 4F, HepG2 cells with 10 µM PCB29-pQ showed significant elevation of the early stage of apoptosis and late stage apoptosis, compared with the control group. However, salubrinal and Z-ATADFMK inhibited this effect partially, which implied PCB29-pQ-induced apoptosis in HepG2 cells via both PERK-eIF2α-ATF4 and caspase 12 signaling pathways. PCB29-pQ induces intracellular calcium level and calpain activity. A rise in cytosolic calcium (Ca2+) levels were a typical appearance of ER stress, some ER chaperones, such as GRP78 and GRP94 require Ca2+ for their activities.19 The effects of PCB29-pQ on intracellular calcium level were measured by a Fluo-3AM fluorescent probe in HepG2 cells treated with 1-10 µM PCB29-pQ for 48 h. In Fig 6A, PCB29-pQ increased intracellular calcium level in a dose-dependent manner, corresponding histogram of fluorescence intensity versus concentration of PCB29-pQ was presented in Fig 6B, and 15 ACS Paragon Plus Environment

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significant differences were found in 5 and 10 µM group, with P < 0.01 compared with the control group. To understand the effects of variations in cytoplasmic calcium on PCB29-pQ-induced ER stress, we measured the protein expressions of GRP78, GRP94 and CHOP of cells exposed to PCB29-pQ with or without BAPTA-AM. As shown in Fig 6C, the chelation of cytosolic Ca2+ by treatment with BAPTA-AM suppressed the upregulation of GRP78, GRP94 and CHOP expressions. In addition, PCB29-pQ significantly increased calpain activity as compared with the control group (Fig 6D, lane 3 vs. lane 1), and this increase was dramatically inhibited by BAPTA-AM (lane 4 vs. lane 3). Taken together,

these

results

demonstrated

that

PCB29-pQ-induced

an

elevation

of

intracellular Ca2+ that is associated with ER stress and UPR. PCB29-pQ-induced ER stress is mediated by ROS Production. Excess of ROS production has been linked with ER stress and UPR.30 To gain further insight into the mechanism of PCB29-pQ-induced ER stress, we measured intracellular ROS generation in PCB29-pQ treated HepG2 cells. Our result indicated a significant increase of fluorescence with PCB29-pQ exposure compared with the control group, Fig 7A. Surprisingly, the fluorescence in 10 µM PCB29-pQ group is comparable with 100 µM H2O2 group, suggested PCB29-pQ has great capability of inducing ROS. On the contrary, the

PCB29-pQ-induced

increase

of

fluorescence

intensity

was

inhibited

by

the

pretreatment of ROS scavenger NAC (5 mM). We next investigated whether PCB29-pQinduced excess of ROS play a role on the trigger of ER stress. As expected, NAC blocked PCB29-pQ-induced up-regulation of the ER stress markers GRP78, GRP94 and CHOP expressions on protein (Fig 7B) and mRNA levels (Fig 7C), respectively. These evidences indicated that PCB29-pQ-induced ER stress is mediated by ROS production.

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DISCUSSION PCB induces ER stress. Our previous studies identified a significant loss of cell viability and mitochondrial-driven apoptotic signal in PCB29-pQ-treated HepG2 cells.23 Furthermore, PCB29-pQ induces genotoxicity8 via free radical-mediated DNA damage.9 However, whether ER stress and UPR play certain roles in PCB29-pQ-induced apoptosis remains unclear.31 ER stress is associated with a wide range of diseases, such as neurodegeneration and inflammatory diseases,32 and even cancer.33 We found that PCB29-pQ significantly increased ER chaperon gene GRP78, GRP94 and CHOP protein and mRNA expressions. TEM analysis strongly indicated PCB29-pQ (10 µM) induced severe

ER

dilatation.

Moreover,

salubrinal,

a

selective

inhibitor

of

eIF2α

dephosphorylation, inhibited PCB29-PQ-induced apoptotic death, indicated PERK–eIF2α signaling pathway was critical for the regulation of CHOP. To the best of our knowledge, this is the first report to link ER stress-mediated cell death with the cytotoxic mechanism of PCB congeners (or metabolites). Our results showed that PCB29-pQ exposure led to ER stress and the activation of three branches UPR signaling pathways. To be clear, PCB29-pQ-treated cells showed an increase in eIF2α phosphorylation, ATF6 cleavage and XBP1 splicing. The activation of these UPR branches activates their downstream ER chaperon genes GRP78, GRP94 and CHOP on their transcriptional level. The up-regulated UPR response signals indicated the increased protein folding capacity of the ER and the attempt to prevent a buildup of irregular, toxic protein products, thus, the UPR signal is independent of cytotoxicity per se.34 Indeed, the activation of UPR responses even occurred under non-cytotoxic

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concentration (< 2.5 µM), indicating that PCB29-pQ could stimulate adaptive responses without triggering cell death. PCB29-pQ induces UPR signaling. Although the primary aim of UPR is to prevent (or relief) cell damage, previous study suggested the cross-talk of UPR and apoptosis.35 When the response UPR fails to restore folding capacity, three UPR branches contribute to the inducing of cell apoptosis. Indeed, a high concentration (10 µM) of PCB29-pQ exposure induces caspase 12-driven apoptosis via the PERK-eIF2α pathway. CHOP is a downstream target of the PERK-eIF2α signaling,36 although PERK-eIF2α pathway is essential in prolonged ER stress, CHOP transcription is often being induced. CHOP is unlikely to induce apoptosis directly rather down-regulates the expression of the antiapoptotic protein Bcl-2 and up-regulates the expressions of pro-apoptotic Bcl-2 family members, then activates caspase cascades,37 or through the increase of cellular ROS.15 Alternatively, JNK pathway may activated in response to ER stress through the autophosphorylation of IRE1α.38 Phosphorylated IRE1α activates the apoptosis signalregulating kinase (ASK1) through the recruitment of TNF receptor-associated factor 2 (TRAF2) to the ER membrane, which in turn activates the JNK signal and leading to cell death. Although the action of JNK signaling was identified in various stimulus conditions, including TNFα and H2O2, the exact role of JNK in ER stress is not clear.39 In the current study, our result demonstrated PCB29-pQ has no effect on JNK, as well as c-Jun expressions.

Rather,

PCB29-pQ-induced

apoptotic

process

accompanied

with

mitochondrial dysfunction,23 suggested a cross-talk of apoptotic signaling between ER and mitochondria, which is consistent with other studies.40-42 The study of Verma et al, but not our study suggested the involvement of JNK in the ER-mitochondrial cross-talk during apoptotic cell death,43 indicated the role of JNK is controversial. Unlike PERK18 ACS Paragon Plus Environment

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eIF2α pathway which was shared with other stresses, IRE1α and AFT6 signaling were specifically mediated by ER stress.44 The activation of XBP1 is controlled by both ATF6 and IRE1α, which is responsible for the up-regulation of the amount of XBP1 mRNA and removing

of

26-nucleotide

intron

within

XBP1

mRNA

(called

XBP1

splicing),

respectively.45, 46 In the current study, we showed an elevation of the XBP mRNA level upon PCB29-pQ treatment and the amount of both sXBP1 and uXBP1 were increased proportionally, which indicating the effect of ATF6 and IRE1α. This result implied that the level of XBP1 mRNA was increased prior to the IRE1α-mediated splicing reaction. Yoshida et al suggested the active form of ATF6 is produced faster than that of XBP1 because ATF6 is derived from precursor protein whilst XBP1 need to be translated from mRNA.45 However, uXBP1 is able to autoregulates its own transcription, which keeps its function in a sustained fashion although ATF6 mRNA is not changed during ER stress.27 PCB29-pQ induces calcium turbulence. We also examined the effect of PCB29-pQ on calcium release from ER to cytosol, and how this links with PCB29-pQ-induced ER stress. As shown, PCB29-pQ increased cytosolic calcium concentration in a dosedependent manner suggested the disruption of intracellular Ca2+ homeostasis, which leads to the promotion of cell dysfunction and apoptosis. Pre-incubation with BAPTA-AM significantly suppressed PCB29-pQ-induced apoptosis. Calpain is a calcium-dependent intracellular cysteine protease, the elevation of cytosolic calcium caused by PCb29-pQ in HepG2 cells leads to the accumulation and activation of calpain at the ER membrane, which in turn activates caspase 12.39,

47

Taking together, these results suggested that

PCB29-pQ induces the increase of intracellular calcium level via the release of calcium from the ER. Considering that GRP78, GRP94 and CHOP need high calcium concentration for their activities, these results strongly suggested that PCB29-pQ play an important 19 ACS Paragon Plus Environment

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role in calcium turbulence and ultimately leading to the failure of protein folding, cell dysfunction and apoptosis. PCB29-pQ-induced ER and UPR is ROS-dependent. Previous study suggested PCBs-induced toxicity is highly associated with oxidative stress.48 The mode-of-action study described PCB congeners, especially co-planar PCBs interact with aryl hydrocarbon receptor (AhR) and induce various iso-enzymes of P450 cytochrome families, eventually promote the release of ROS and oxidative stress.49,

50

Interestingly, PCB29-pQ, which

possesses a highly chlorinated quinone moiety, induces high concentration of hydroxyl radical in cell free system.21,

51

That is, PCB quinone-induced oxidative stress can be

independent with AhR binding and P450 cytochrome enzymes activation. ROS production induced by exogenous chemicals has the capability of induce ER stress and the UPR.52 We found that PCB29-pQ induces cellular ROS and glutathione depletion in a concentration- and time-dependent manner in HepG2 cells.9 Here, NAC treatment not only reduced cellular ROS level, it also inhibited GRP78, GRP94 and CHOP expressions on the protein and mRNA levels, suggesting that ROS are the keys to controlling intracellular signaling and biological outcomes of ER stress and UPR. The cross-talk of UPR with other PCB29-pQ-induced stress responses. There are many signal pathways may related to UPR signaling. For instance, IRE1α can activate p53, which resulting in commitment of the cell to mitochondrial-initiated apoptosis.54 Pro-apoptotic Bcl-2 family proteins have been found to interact with IRE1α directly on ER.53 However, the UPR is a defensive response, which is important for cell survival. Thus, we are wondering whether other protective responses were also activated. Indeed, our previous study indicated PCB29-pQ acts as a potential activator of NF-E2-related factor2 (Nrf2) and induces Phase II detoxification enzymes, such as heme 20 ACS Paragon Plus Environment

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oxygenase-1 and NAD(P)H: quinone oxidoreductase 1, which is considered as a protective and pro-survival response to cells.54 Mechanism study suggested the formation of dimer of sulfhydryl-containing protein Kelch-like ECH-associated protein 1 (the negative regulator of Nrf2), resulting the release and activation of Nrf2.55 Actually, PCB29-pQ showed its nucleophilic characteristics and readily reacts with glutathione yielding a conjugated quinone.51 Based on this result, we further speculate PCB29-pQ may induce the ER stress through the inhibition of disulfide bonds formation in newly synthesized proteins. Previous studies indicated UPR inhibits cell cycle at G1 phase through inhibition of cyclin D1 translation via the PERK-eIF2α signaling.56, 57 In addition, the PERK-ATF4 branch up-regulates DNA damage repair signaling. Similarly, we also demonstrated PCB29-pQ induces DNA damage response58 to coordinate stress damages. However, further investigation is needed to decipher the cross-talk of UPR with these stress responses. In conclusion, numbers of evidences indicated ER stress is highly related with a variety of diseases,59 which encourage us to use this knowledge to decipher environmental

pollutants-related

diseases

and

to

translate

it

into

therapeutic

opportunities. In this regard, the current study improved the understanding of PCB quinone-induced toxicity by revealed the interconnection between oxidative stress and ER stress, UPR and calcium release. Thus, the obligatory signaling molecules on ER stress, UPR and calcium release might be considered as the novel targets for drug discovery.

AUTHOR INFORMATION *Corresponding author 21 ACS Paragon Plus Environment

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Phone: +86-23-68251503. Fax: +86-23-68251225. E-mail address: [email protected]

Author Contributions The manuscript was written through the contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding This work is supported by The National Natural Science Foundation of China (21477098),

Science

and

Technology

Talent

Cultivation

Project

of

Chongqing

(cstc2014kjrc-qnrc00001), Scientific Research Foundation for the Returned Overseas Chinese Scholars from State Education Ministry (2011[508]) and Fundamental Research Funds for the Central Universities (XDJK2014A020, XDJK2013B009).

Notes The authors declare no competing financial interest.

ABBREVIATIONS ATF6, activating transcription factor 6; CHOP, C/EBP homologous protein (aka GADD153); eIF2α, eukaryotic translation initiation factor; ER, endoplasmic reticulum; FITC, fluorescein isothiocyanate; GRP78, glucose-regulated protein 78 (aka Bip); IRE1α, inositol-requiring kinase/endonuclease 1α; JNK, c-Jun NH2-terminal kinase; NAC, N22 ACS Paragon Plus Environment

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acetyl-L-cysteine; Nrf2, NF-E2-related factor2; PCB, polychlorinated biphenyl; PERK, protein kinase-like endoplasmic reticulum kinase; PI, propidium iodide; RT-qPCR, realtime quantitative PCR; ROS, reactive oxygen species; UPR, unfolded protein response; XBP1, X-box-binding protein 1

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Dong, H., Shi, Q., Song, X., Fu, J., Hu, L., Xu, D., Su, C., Xia, X., Song, E., and Song, Y. (2015) Polychlorinated biphenyl quinone induces oxidative DNA damage and repair responses: The activations of NHEJ, BER and NER via ATM-p53 signaling axis. Toxicol. Appl. Pharmacol. In press. DOI: 10.1016/j.taap.2015.03.017.

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Table 1. Primer pairs used in RT-qPCR. Gene

Primer sequences (5’–3′)

GRP78

(F)GTTTGCTGAGGAAGACAAAAAGCTC (R)CACTTCCATAGAGTTTGCTGATAATTG

GRP94

(F)CTCACCATTTGGATCCTGTGTG (R)ACATGACAAGATTTTACATCAAGA

CHOP

(F)CCTCCTGGAAATGAAGAGGAAGAA (R)CTCTGGGAGGTGCTTGTGAC

XBP1

(F)CAGCGCTTGGGGATGGATGC (R)CCATGGGGAGATGTTCTGGA

β-actin

(F)TCCTCCCTGGAGAAGAGCTAC (R)TCCTGCTTGCTGATCCACAT

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Figure 1. (A) Chemical structure of PCB29-pQ. Concentration and time-dependent effects of PCB29-pQ on ER stress chaperone genes GRP78, GRP94 and CHOP expressions. (B) HepG2 cells were treated with 0, 1, 5 or 10 µM PCB29-pQ for 48 h. (C) HepG2 cells were treated with 10 µM PCB29-pQ for 0, 12, 24 or 48 h. Cell lysates prepared from cells treated with PCB29-pQ were subjected to Western blotting analysis. (D) PCB29-pQ treatment up-regulates GRP78, GRP94 and CHOP mRNA expressions. HepG2 cells were treated with 0, 1, 5 or 10 µM PCB29-pQ for 48 h. Total RNA was isolated and analyzed by RT-qPCR as described in the method section. Relative mRNA 32 ACS Paragon Plus Environment

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expression levels after normalization to β-actin from three separate experiments were presented as mean ± SD. **P < 0.01 and ***P < 0.001 as compared with the control group. (E) Morphological observation of ER changes. HepG2 cells were treated 10 µM PCB29-pQ for 48 h, then visualized by TEM and representative images from three independent experiments are shown. ER, endoplasmic reticulum; Mi, mitochondria; Nu, Nucleus; CM, cell membrane. Bar= 0.5 µM.

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Figure 2. PCB29-pQ stimulated the UPR and activated the PERK–eIF2α–ATF4 pathway in HepG2 cells. Concentration and time-dependent effects of PCB29-pQ on PERK, p-PERK, eIF2α, p-eIF2α and ATF4 expressions. (A) HepG2 cells were treated with 0, 1, 5 or 10 µM PCB29-pQ for 48 h. (B) HepG2 cells were treated with 10 µM PCB29-pQ for 0, 12, 24 or 48 h. Western blotting analysis was used to detect these protein expressions. Densitometric analysis of immunoblot was performed with ImageJ software and relative density of p-eIF2α/eIF2α were shown in the histogram. The data represent the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 as compared with the control group.

(C) The effect of salubrinal, a selective inhibitor of

eIF2α dephosphorylation on PCB29-pQ-induced p-eIF2α, ATF4 and CHOP expressions.

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Figure 3. PCB29-pQ activated the ATF6 and IRE1α–XBP1 pathways. Concentration and time-dependent effect of PCB29-pQ on IRE1α, p-RE1α, JNK, p-JNK, c-Jun and ATF6 expressions. (A) HepG2 cells were treated with 0, 1, 5 or 10 µM PCB29-pQ for 48 h. (B) HepG2 cells were treated with 10 µM PCB29-pQ for 0, 12, 24 or 48 h. Western blotting analysis was used to detect these protein expressions. Cell lysates were subjected to Western blotting analysis. RNA was prepared from cells treated with indicated 35 ACS Paragon Plus Environment

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concentrations of PCB29-pQ and XBP1 mRNA was determined using RT-qPCR. (C) HepG2 cells were treated with 0, 1, 5 or 10 µM PCB29-pQ for 48 h. A representative gel for XBP1u and XBP1s was shown. (D) HepG2 cells were treated with 0, 1, 5 or 10 µM PCB29-pQ for 48 h. XBP1 mRNA expression was measured by RT-qPCR and normalized to β-actin. The data represent the mean ± SD of three independent experiments. ***p < 0.001 represent significant differences compared with the control group.

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Figure

Chemical Research in Toxicology

4.

PCB29-pQ

activated

caspase

12

and

induced

ER-related

apoptosis.

Concentration and time-dependent effect of PCB29-pQ on caspase 12 activation. (A) HepG2 cells were treated with 0, 1, 5 or 10 µM PCB29-pQ for 48 h. (B) HepG2 cells were treated with 10 µM PCB29-pQ for 0, 12, 24 or 48 h. Western blotting analysis was used to detect these protein expressions. Cell lysates were subjected to Western blotting analysis. Morphological changes of HepG2 cells were analyzed after the 37 ACS Paragon Plus Environment

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treatment of 10 µM PCB29-pQ for 48 h. (C) The bright field image. 200x magnification. (D) TEM image. 8000x magnification. (E) The effect of caspase 12 inhibitor Z-ATADFMK on caspase family genes activation. Cells were pretreated with 25 µM Z-ATAD-FMK for 1 h and then exposed to 10 µM PCB29-pQ for 48 h, the activation of caspase 12, 9 and 3 were determined by Western blotting analysis. (F) The effect of caspase 12 inhibitor Z-ATAD-FMK or salubrinal on PCB29-pQ-induced apoptosis. Cells were pretreated with 25 µM Z-ATAD-FMK or 20 µM salubrinal for 1 h and then exposed to 10 µM PCB29-pQ for 48 h. Flow cytometry analysis was performed by Annexin V-FITC and PI double-staining. Cells in lower right quadrant correspond to early apoptotic cells (Annexin V+/PI-). Cells in upper right quadrant correspond to late apoptotic/dead cells (Annexin V+/PI+). Data are presented as the mean ± SD of three independent experiments.

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Figure 5. PCB29-pQ induced intracellular Ca2+ imbalance and calpain activity in HepG2 cells. (A) Cells were treated with the indicated concentration of PCB29-pQ for 48 h, intracellular calcium level of HepG2 cells was analyzed by flow cytometry. (B) Cells were incubated in the presence or absence of 10 µM BAPTA-AM for 1 h then treated with PCB29-pQ for 48 h. Western blot analysis of GRP78, GRP94 and CHOP expressions. (C) PCB29-pQ induced calpain activation was blocked by BAPTA. Calpain activity was assessed by using S-LLVY-AMC as the cell-permeable calpain substrate. The results are expressed as the mean ± SD of the fluorescence intensity. **P < 0.01 and ***P<0.001, as compared with the control group,

###

P<0.001, as compared with 10 µM PCB29-pQ

group.

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Figure 6. PCB29-pQ-induced ER stress is mediated by ROS Production, which is inhibited by NAC. HepG2 cells were pre-incubated with 5 mM NAC for 1 h, then incubated with 10 µM PCB29-pQ for 48 h. (A) Cells were stained with DCFH-DA probe and representative images were taken by fluorescence microscopy. The cells were treated with 100 µM H2O2 as positive control. (B) Western blot analysis of GRP78, GRP94 and CHOP expressions. (C) GRP78, GRP94 and CHOP mRNA expressions. Relative mRNA expression levels after normalization to β-actin from three separate experiments are presented as mean ± SD. ***P < 0.001 as compared with the control group,

###

P<

0.001, as compared with 10 µM PCB29-pQ group.

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Scheme 1. The proposed signaling pathway of ER-stress induced by PCB29-pQ in HepG2 cells

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Polychlorinated biphenyl quinone induces endoplasmic reticulum stress, unfolded protein response, and calcium release.

Organisms are able to respond to environmental insult to maintain cellular homeostasis, which include the activation of a wide range of cellular adapt...
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