The Mechanisms Of Tetrachlorobenzoquinone-induced Cytotoxicity Via Endoplasmic Reticulum Stress And Cells Resistance In PC12 Cells

Back GroundTetrachlorobenzoquinone（TCBQ）,an example of halogenated quinoid compounds,has been widely used as an electron acceptor and is observed as a major reactive metabolite in oxidation processes of degrading the widely used fungicide HCB and PCP.Due to their stable nature and long-range transport ability,HCB and PCP chronic exposure still threat to human.Some studies have shown that the biological toxicity of environmental pollutant HCB or PCP may be caused by their oxidative metabolite TCBQ.In addition,TCBQ was widely used as a fungicide,a basic agent for pigments production,and an electron acceptor in chemical industry.TCBQ analogues have also been identified as byproducts of drinking water disinfection.A study shows that the most toxic of 14 p-benzoquinone congeners is TCBQ.Thus,understanding the mechanisms by which cells respond to TCBQ is key to determine ways of improving the therapy or reducing cell damage.Neurodegenerative diseases are a large class of neurological diseases,and also known as protein misfolding diseases associated with the misfolded protein accumulation of plaques.The pathogenesis of neurodegenerative diseases are complex,including oxidative stress and endoplasmic reticulum stress.The latest data shows that ferroptosis also occurs in neurodegenerative diseases.In vitro and in vivo studies have demonstrated that TCBQ cause oxidative stress,endoplasmic reticulum stress,inflammation and genotoxicity.Recently,we reported that TCBQ induced neurotoxicity by activating the apoptotic signaling.However,there is limited evidence of the molecular mechanism of TCBQ neurotoxicity.ObjectivesA better understanding of the mechanisms whereby cells respond to TCBQ is essential to improve therapy or reduce cell damage.The aim of this work is to investigate the molecular mechanisms of TCBQ-induced neurotoxicity in PC12 cells.We further detecte the mechanisms of cell resistanced to TCBQ neurotoxicity,providing a theoretical basis for the prevention and treatment of TCBQ-induced neurotoxicityMethods and Results（1）Effect of TCBQ on endoplasmic reticulum（ER）stress in PC12 cells.We first investigated whether GRP78 and the ER lumen were changed under the treatment of TCBQ.Western blot results showed that TCBQ markedly increased the expression of GRP78,a hallmark gene of ER stress.Then we tested the effect of TCBQ on ER morphology using TEM technology.TCBQ treatment resulted in a obvious ER dilatation in PC12 cells.In this regard,we hypothesized that TCBQ could potentially induce ER stress in PC12 cells.We then examined the activation status of three branches in unfolded protein response（UPR）signaling pathways with TCBQ challenge.Western blot analysis have shown that TCBQ activated PERK-eIF2α-ATF4-CHOP signaling pathway.4-PBA,a specific inhibitor of ER stress,significantly suppressed TCBQ-induced GRP78,p-PERK and p-eIF2αexpressions.Subsequently,we examined the impact of TCBQ on IRE1αsignaling pathway.Western blot analysis showed that the phosphorylated form of IRE1αwas significantly increased in PC12 cells exposed to TCBQ,but TCBQ treatment showed no effects in JNK activation and the downstream expression of c-Jun.Given the important roles of XBP-1 in signaling pathways induced by ER stress,we next examined the effect of TCBQ on the level of XBP-1s mRNA using RT-PCR and RT-qPCR.TCBQ stimuli resulted in the up-regulation of spliced XBP-1s.Next,we investigated the impact of TCBQ on ATF6 signaling pathway.However,neither precursor nor the cleavage of ATF6 was significantly altered.Finally,we also studied the effect of TCBQ on intracellular Ca²⁺homeostasis.The results showed that TCBQ breaked Ca²⁺balance by upregulating intracellular free calcium concentration[Ca²⁺]_i.Consistent with the increase of[Ca²⁺]_i,we also found that TCBQ treatment activated Caspase 12.Therefore,we elucidated the molecular mechanism of TCBQ-induced ER stress in PC12 cells.（2）TCBQ-induced apoptosis was mediated by ER stress-mediated death receptor（DR5）signaling.The findings of this study illustrated that TCBQ-induced ER stress resulted the activation of pro-apoptotic signaling in PC12 cells by upregulating the expression of DR5.Immunofluorescence double staining and immunoprecipitation assay showed TCBQ influenced DR5 distribution and transport in PC12 cells.TCBQ activated the apoptosis of PC12 cells by upregulating the expression of DR5 and enhancing the formation of DISC.TCBQ-induced cleavage of caspase 8,caspase 3 and PARP-1 were partly reversed in DR5 siRNA-transfected cells compared with control siRNA group.RT-qPCR analysis showed that actinomycin D（ActD）completely blocked the TCBQ-induced DR5 expression and the addition of cycloheximide（CHX）also reduce the increase in DR5 expression induced by TCBQ,indicating that increase in DR5 mRNA expression required transcription activation and de novo protein synthesis.As CHOP is one of the most powerful inducers of DR5 transcription,to elucidate the relationship amongst ATF4,ATF3 and CHOP in DR5-dependent cell apoptosis,cells were transfected with ATF4,ATF3 or CHOP siRNA,respectively.We found ATF4-ATF3-CHOP pathway was required for the fully activation of the DR5gene in response to TCBQ.These results suggested that TCBQ triggered ER stress in PC12 cells,and induced ATF4-ATF3-CHOP pathway that leading to DR5-dependent apoptosis.Finally,N-acetylcysteine（NAC）reversed TCBQ-induced ROS,ER stress and the expression of DR5 in PC12 cells.These evidence have strongly supported that TCBQ-induced apoptosis and ER stress were mediated by ROS production.（3）TCBQ induced apoptosis by altering the subcellular distribution of protein disulfide isomerase（PDI）through ER stress in PC12 cells.PDIs regulate the progress of various neurodegenerative disorders,including Parkinson’s disease and Alzheimer’s disease.We tested the hypothesis that PDIs subcellular translocation implicates survival/death signaling switch.PC12 cells were exposed to TCBQ.Interestingly,pharmaceutical（or siRNA）abrogation of PDIA1/PDIA3 aggravated cell viability loss induced by relative low concentration（10μM）of TCBQ.However,PDIA1/PDIA3 inhibition rescued high concentration（20μM）of TCBQ-caused cell death.We further quest the mechanism of PDIs as a pleiotropic apoptotic regulator under TCBQ treatment.Western blot and biotin-switch assay showed TCBQ did not influence the protein expression and S-nitrosylation modification of PDIs in PC12 cells.This indicated that TCBQ may not affect the activity of PDIs.However,subcellular fractionation and immunofluorescent double staining assay revealed the release of PDIs from ER lumen to cytosol upon TCBQ treatment.In order to study the mechanism of apoptosis by the changes of PDIs subcellular localization,we examined the effect of PDIs on MOMP.Cytochrome c release and MMP were greatly stimulated by TCBQ in a PDIs-dependent manner,indicating that PDIs play an important role in TCBQ-mediated MOMP.Next,we investigated how PDIs manipulate TCBQ-induced MOMP.Cross-linking assay was performed to detecting Bak and Bax oligomerization on mitochondria.These results showed Bak oligomerization occurred simultaneously with the increase of TCBQ-mediated MOMP,suggesting that PDIA1 and PDIA3 may be involved in TCBQ-mediated MOMP by triggering Bak,but not Bax,oligomerization in mitochondria and subsequently mitochondrial dysfunction in PC12 cells.Finally,NAC or 4-PBA was employed in this study as an inhibitor of ROS or ER stress,respectively.Western blot analysis revealed that NAC and 4-PBA both prevented TCBQ-induced increase of PDIs in cytosol fractions.TUNEL,CCK-8 and LDH assays revealed the blockage of ROS with NAC inhibited TCBQ-induced cell death.These results suggested that ROS formation was an initial step of TCBQ-induced PDIs release from ER stress and consequent apoptosis.（4）The mechanism of Nrf2 as a defensive system to alleviate the ER stress induced by TCBQ.Although the mechanisms of neurotoxicity induced by TCBQ have been studied,it remains unclear whether TCBQ causes the opening of cellular defense responses.Here we found that activation of nuclear factor erythroid-derived 2-like 2（Nrf2）triggered an adaptive response against the neurotoxicity induced by TCBQ through the upregulation of intracellular glutathione（GSH）levels in PC12 cells.TCBQ upregulated the levels of GSH mainly by the following two ways:（i）Nrf2 activation induced the expression of cystine/glutamate antiporter solute carrier family 7 member11（SLC7A11,also called xCT）.SLC7A11 regulates GSH levels by promoting cystine import,which is quickly converted to cysteine（a precursor of GSH synthesis）;（ii）Nrf2activation resulted in increased expression of GSH rate-limiting enzyme GCL,which is composed of GCLM and GCLC.GSH is involved in cell antioxidant ability and protein thiol homeostasis,especially in the ER.Therefore,GSH has the ability to inhibite ER stress and promote cell survival.Our data showed that decreasing GSH levels through Nrf2 knockdown or GSH inhibition with BSO exacerbated TCBQ-induced depletion of protein-SH,particularly in the ER.Conversely,increasing GSH levels with NAC attenuated TCBQ-induced protein damage,degree of ER stress,and cell death.This findings demonstrated that GSH-inhibited cells were vulnerable to TCBQ-induced ER stress and apoptosis.Overall,our results analysed the relationships between Nrf2 and ER stress in response to TCBQ and showed that activation of Nrf2-GSH played a protective role against TCBQ-induced ER stress-associated neurotoxicity in PC12 cells via regulating GSH synthesis and protein thiol homeostasis.（5）To explore the mechanisms of ferroptosis in TCBQ-induced neurotoxicity and reveal the role of Nrf2 in ferroptosis.In this study,we first demonstrated that ferroptosis could occur in PC12 cells.Then we tested whether ferroptosis was involved in the neurotoxicity of TCBQ.Ferrostatin-1（Fer-1）,a specific inhibitor of ferroptosis,DFP,an iron specific chelator,rescued cell death in TCBQ-treated cells.In contrast,additional iron accelerated TCBQ-induced cell death.By transmission electron microscopy of TCBQ-treated cells,we observed shrunken mitochondria with increased membrane density.All of these results indicated that TCBQ induced ferroptosis in PC12cells.We further investigated potential triggers of ferroptosis and a possible crosstalk with other pathways.However,well-established regulators of ferroptosis,i.e.,p53,ferritinophagy,NOX2,and JNK were not required for TCBQ-mediated ferroptosis,whereas TCBQ-induced ferroptosis might be dependent on the activation of PKCαand VDAC2/3 signaling pathways.Iron metabolism and lipid peroxidation are increasingly recognized as central mediators of ferroptosis.Therefore,we tested whether TCBQ-induced ferroptosis promoted cellular ROS accumulation by increasing free iron levels in cells.TCBQ treatment have increased the expression of protein transferrin receptor 1（TFRC）and transferrin（TF）,then promoted iron uptake.Surprisingly,we observed an increase of endogenous ferritin light chain 1（FTH1,an iron storage protein）during ferroptosis,whereas knockdown of Nrf2 inhibited the expression of FTH1.Although TCBQ initially caused the increase of free iron levels,then the levels of iron gradually recovered,which coincided with the activation of Nrf2 and the expression of FTH1.From our results,it was found that TCBQ caused a low degree of ferroptosis in PC12 cells,probably because TCBQ activated Nrf2,a negative regulator of ferroptosis.Moreover,inactivation of GPX4 or GSH depletion trigger ferroptosis by accumulation of lipid peroxidation.Although we observed a decrease in the amount of GPX4 protein in the present of TCBQ,the activity of GPX4 gradually recovered along with the up-regulation of GSH.In addition,depletion of GSH by BSO aggravated lipid peroxidation and the loss of cell viability caused by TCBQ.Knockdown of Nrf2enhanced the ability of TCBQ to produce ROS and increased the concentration of free iron.These findings suggested that TCBQ induced intracellular iron overload and lipid peroxide production,whereas the activation of Nrf2 protected against TCBQ-induced ferroptosis by regulating iron metabolism and GPX4 activity.Conclusions（1）TCBQ induced ER stress and activated the major downstream pathways.Unlike the effects of ATF6 and JNK,PERK/eIF2α,XBP-1 and Caspase 12 signaling pathway contributed to the process of TCBQ-induced ER stress.（2）The ATF4-ATF3-CHOP pathway leaded to increase the expression of DR5.The upregulating the expression of DR5 and enhancing the formation of DISC,followed by activation of caspase cascade,correlated with TCBQ-induced apoptosis in PC12 cells.NAC reversed these signaling,indicating a ROS-dependent mechanism.（3）TCBQ-induced constant ER stress promoted signaling switch to pro-apoptotic by the release of PDIA1/PDIA3 from ER lumen to induce Bak-dependent MOMP.ROS was an initial step of TCBQ-induced PDIs release from ER.PDIs subcellular translocation contributed to the“live or death”fate of PC12 cells in the present of TCBQ.（4）Nrf2 was activated by TCBQ as a adaptive cell defense response against xenobiotics, which resulted in enhancing GSH biosynthesis by inducing GCL subunits and SLC7A11.We demonstrated that GSH enhanced the ability of ER proteins to resist TCBQ damage,followed by reducing ER stress.These findings offered a better understanding of TCBQ-induced cell response and suggested that Nrf2 could be used as a target for the treatment and prevention of TCBQ toxicity.（5）TCBQ could induce ferroptosis in PC12 cells.p53,ferritinophagy,NOX2,and JNK were not involved in TCBQ-induced PC12 cells.PKCαand VDAC2/3 played important roles in TCBQ-induced ferroptosis.Remarkably,Nrf2 activation increased iron storage capacity and reduced the production of lipid peroxides, prompting PC12 cells to resist ferroptosis.