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ATF4-ATF3-CHOP Cascade Activation Play an Important Role in ER Stress-induced Sensitization of Tetrachlorobenzoquinone-Challenged PC12 Cells to ROS-mediated Apoptosis Through DR5 Signaling Zixuan Liu, Qiong Shi, Xiufang Song, Yuxin Wang, Yawen Wang, Erqun Song, and Yang Song Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.6b00181 • Publication Date (Web): 02 Aug 2016 Downloaded from http://pubs.acs.org on August 5, 2016

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Chemical Research in Toxicology

Activating transcription factor 4 (ATF4)-ATF3-C/EBP Homologous Protein (CHOP) Cascade Shows an Essential Role in the ER Stress-induced Sensitization of Tetrachlorobenzoquinone-Challenged PC12 Cells to ROSmediated Apoptosis via Death Receptor 5 (DR5) Signaling

Zixuan Liu, Qiong Shi, Xiufang Song, Yuxin Wang, Yawen Wang, 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, 2 Tiansheng

Rd., Beibei, Chongqing, 400715, P R China. Tel: +86-23-68251503. Fax: +86-23-68251225. Email addresses: [email protected] or [email protected]

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TABLE OF CONTENTS


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ABSTRACT Tetrachlorobenzoquinone (TCBQ) is a downstream metabolite of pentachlorophenol (PCP). Previously, we demonstrated that TCBQ caused cytotoxicity due to mitochondrial-related apoptosis. Here, we confirmed the upregulation of death receptor 5 (DR5) followed by the construction of the death-inducing signaling complex (DISC). We also detected the activation of the caspase cascade, which was correlated with TCBQ-induced apoptotic cell death in PC12 cells. The upregulation of DR5 included transcriptional activation and de novo protein synthesis in response to TCBQ. We also identified the endoplasmic reticulum (ER) as a new target for the TCBQ challenge in PC12 cells. The protein kinase R-like ER kinase/ eukaryotic translation initiation factor 2α (PERK/eIF2α)-mediated activating transcription factor 4 (ATF4)-ATF3C/EBP Homologous Protein (CHOP) signaling pathway contributed to the process of TCBQinduced ER stress. Blocking ATF4, ATF3 or CHOP signaling by gene silencing technology resulted in decreased cell apoptosis after exposure to TCBQ. Finally, NAC ameliorated TCBQinduced apoptosis and ER stress, which illustrated that TCBQ-induced apoptosis is somehow ROS-dependent. In summary, this study provided important mechanistic insight into how TCBQ utilizes ER stress-related signaling to exhibit pro-apoptotic activity in PC12 cells.

Keywords: TCBQ; apoptosis; UPR; ER stress; DR5



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INTRODUCTION The wildly distributed pollutant pentachlorophenol (PCP) has been classified by the International Agency for Research on Cancer (IARC) as a group 2B environmental carcinogen.1 Tetrachlorobenzoquinone (TCBQ) is a downstream metabolite of PCP. TCBQ exposure is associated with various toxicities, including the possible risk of bladder cancer.2, 3 The pathway for biodegradation of PCP was reported by occurred through soil bacteria, such as Mycobacterium chlorophenolicum comb. nov. and Sphingomonas chlorophenolica sp. nov.4, 5 The mode-of-action study demonstrated that TCBQ, similar to other quinones, can directly bind covalently with macromolecules.6, 7 TCBQ also participates in futile redox cycling to generate ROS, such as hydroxyl radicals.6 In addition to oxygen-centered radicals, TCBQ was found to contribute to the degradation of hydroperoxides to yield a carbon-centered quinone ketoxy radical.8 These mechanisms clearly suggested that TCBQ may be able to cause DNA damage. Furthermore, due to the electron withdrawing effect of halogen substitution, TCBQ may possess a higher reactivity than other quinones towards cellular components.9 Our previous studies demonstrated that TCBQ causes oxidative stress, cytotoxicity, inflammation and genotoxicity.10-14 In parallel, changes in DNA damage and DNA hydroxymethylation caused by halogenated quinones have been reported by another group.15 Recently, we reported that TCBQ induced neurotoxicity by activating apoptotic signaling, including the activation of caspases. More interestingly, we found that apoptotic signaling was amplified through a caspase 8/Bid-related positive feedback circle.16 Previous studies also provided evidence of the toxicity of PCP towards rat neurons through the p53-dependent apoptotic pathway, which was related to the mechanism of oxidative damage.17


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The endoplasmic reticulum (ER) is a critical regulator of protein synthesis. It also functions as one of most important hubs for signaling networks.18 Stimuli, such as ROS, may perturb the ER lumen, which dampens the efficiency of protein folding. This situation is usually termed as ER stress, and the corresponding feedback is the unfolded protein response (UPR).19, 20 The UPR acts to modulate the protein folding capacity and to restore cellular homeostasis to pro survival. ER stress, as well as UPR have implications in many human diseases and conditions. Three branches of UPR, protein kinase R-like ER kinase (PERK), activating transcriptional factor 6 (ATF6) and inositol-requiring enzyme 1 (IRE1), which were orchestrated by distinct ER stress transducers. In normal cells, all three ER stress receptors bind with chaperone glucoserelated protein 78 (GRP78) and maintain their inactive status. Upon the stimulation of stress, e.g., unfolded proteins, the receptors were activated by their dissociation from GRP78 and then trigger UPR. In principle, UPR signaling cascades maintain ER function. However, if ER stress cannot be removed by UPR cascades, corresponding signaling switch from pro-survival to proapoptotic through the very same receptors.21 For instance, the activation of PERK leads to the induction of transcription factor C/EBP homologous protein (CHOP), which mainly depends on ATF4.22 Consequently, CHOP activate the transcription of pro-apoptotic death receptor 5 (DR5), which leading to the drain of ER stress-induced apoptosis.20, 23 Here, we examined UPR signaling that results from the TCBQ challenge, as well as the mechanisms leading to ER-related induction of apoptosis. Our findings therefore suggested a novel mode of action of TCBQ-mediated toxic effect.

MATERIALS AND METHODS 5 ACS Paragon Plus Environment


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Materials and Reagents. Tetrachlorobenzoquinone (TCBQ) was purchased from Aladdin Reagent Database Inc. (Shanghai, China). 4-phenylbutyrate (4-PBA), N-acetyl-l-cysteine (NAC) and 2′, 7′-Dichlorofluorescein diacetate (DCFH-DA) were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). The cell counting kit-8 (CCK-8) was supplied by Gen-View Scientific Inc. (Shanghai, China). Rabbit PERK, eIF2α, GRP78, caspase 8, PARP-1, β-actin polyclonal primary antibodies, Cy3-conjugated Affinipure Donkey anti-goat IgG (H+L) and Fluorescein (FITC)-conjugated Affinipure Donkey anti-rabbit IgG (H+L) secondary antibody were obtained from Proteintech Group, Inc. (Wuhan, China). Rabbit p-eIF2α (Ser51) monoclonal primary antibody was obtained from Bioworld Technology Inc. (Nanjing, China). Rabbit p-PERK (Thr980), ATF4, ATF3, and FADD polyclonal primary antibodies were supplied by Biosynthesis Biotechnology Co. Ltd. (Beijing, China). The goat DR5 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Goat anti-rabbit IgG-HRP conjugated secondary antibody was supplied by Sangon Biotech Co., Ltd. (Shanghai, China). Rabbit CHOP and caspase 3 polyclonal primary antibodies were purchased from Ruiying Biological (Suzhou, China). The EasyBlot ECL kit, 4′, 6-Diamidino-2-phenylindole dihydrochloride (DAPI) and protein (A+G) agarose beads were obtained from Beyotime Institute of Biotechnology (Nanjing, China). ActinomycinD (ActD) and cycloheximide (CHX) were purchased from Yuanye Bio-Technology Co., Ltd. (Shanghai, China). RT-qPCR amplification primers of DR5, ATF3, β-actin and GAPDH were synthesized by Dingguo Biotechnology Co., Ltd. (Beijing, China). Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Keygen Biotech (Nanjing, China). ATF4, ATF3, CHOP and DR5 siRNA and siRNA-mate transfection reagent were supplied by GenePharma Co., Ltd. (Shanghai, China). All other chemicals used were of the highest commercial grade.


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Cell Culture and Treatment. The rat pheochromocytoma PC12 cell line (Nanjing Keygen Biotech. Co., Ltd, Nanjing, China) was maintained in DMEM supplemented with 10% newborn calf serum, 100 U/mL penicillin and 100 µg/mL streptomycin at 37°C in a humidified atmosphere of 5% CO2. For differentiation, nerve growth factor (NGF) was added to the culture medium at a final concentration of 50 ng/mL after plating cells for 24 h. Fresh media contained 50 ng/mL NGF was replaced every other day for 8 days. PC12 cells were exposed to different TCBQ concentrations for the indicated times. Control cells were treated with equal amount of DMSO. DMSO was always

Activating Transcription Factor 4 (ATF4) - ACS Publications - American

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