The protective role of Nrf2-Gadd45b against antimony-induced oxidative stress and apoptosis in HEK293 cells

The protective role of Nrf2-Gadd45b against antimony-induced oxidative stress and apoptosis in HEK293 cells

Accepted Manuscript Title: The protective role of Nrf2-Gadd45b against antimony-induced oxidative stress and apoptosis in HEK293 cells Author: Xingkan...

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Accepted Manuscript Title: The protective role of Nrf2-Gadd45b against antimony-induced oxidative stress and apoptosis in HEK293 cells Author: Xingkang Jiang Zesheng An Chao Lu Yue Chen E Du Shiyong Qi Kuo Yang Zhihong Zhang Yong Xu PII: DOI: Reference:

S0378-4274(16)30113-8 http://dx.doi.org/doi:10.1016/j.toxlet.2016.05.016 TOXLET 9380

To appear in:

Toxicology Letters

Received date: Revised date: Accepted date:

10-4-2016 12-5-2016 17-5-2016

Please cite this article as: Jiang, Xingkang, An, Zesheng, Lu, Chao, Chen, Yue, Du, E, Qi, Shiyong, Yang, Kuo, Zhang, Zhihong, Xu, Yong, The protective role of Nrf2-Gadd45b against antimony-induced oxidative stress and apoptosis in HEK293 cells.Toxicology Letters http://dx.doi.org/10.1016/j.toxlet.2016.05.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

The protective role of Nrf2-Gadd45b against antimony-induced oxidative stress and apoptosis in HEK293 cells Running title: Nrf2-Gadd45b protects HEK293 cells from apoptosis Xingkang Jiang, Zesheng An, Chao Lu, Yue Chen, E Du, Shiyong Qi, Kuo Yang, Zhihong Zhang*, Yong Xu* Department of Urology, The Second Hospital of Tianjin Medical University, Tianjin Institute of Urology, Tianjin 300211, China

Corresponding Authors: (Xu, Y) E-mail: [email protected] , (Zhang, Z) E-mail: [email protected] Tel/Fax: 8622-88329692.

Address: Department of Urology, the Second Hospital of Tianjin Medical University, Tianjin Institute of Urology, Pingjiang Road 32, Hexi district, Tianjin 300211, China

Abbreviations: Antimony (Sb); N-acetylcysteine (NAC); NF-E2-related factor 2 (Nrf2); Reactive oxygen species (ROS); Antimony trioxide (Sb2O3); Tertiary butylhydroquinone (TBHQ).

Text word count: 3,615 (abstract 219) Number of figures: 5 Supplemental data: 1 1

Highlights 1. Sb2O3-induces apoptosis in a ROS-dependent manner in HEK293 cells 2. Nrf2 protects HEK293 cells against Sb2O3-induced apoptosis 3. Gadd45b drives activation of MAPKs upon Sb2O3 exposure. 4. Nrf2 transcriptionally actives Gadd45b expression against Sb2O3-induced apoptosis

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Abstract Antimony (Sb) is one of the most prevalent heavy metals and frequently causes biological toxicity. However, the specific mechanisms by which Sb elicits its toxic effects remains to be fully elucidated. In this study, we found antimony trioxide (Sb2O3) caused a dose-dependent cytotoxicity against HEK293 cells, and Sb2O3-induced excessive reactive oxygen species (ROS) was closely correlated with increased cell apoptosis. Mechanistic investigation manifested that nuclear factor NF-E2-related factor 2 (Nrf2) expression and nuclear translocation were significantly induced under Sb2O3 treatment in HEK293 cells, and Nrf2 knockdown aggregated Sb2O3-induced cell apoptosis. Moreover, elevated Gadd45b expression actives the phosphorylation of MAPKs upon Sb2O3 exposure, whereas Gadd45b knockdown diminished Sb2O3-induced activation of MAPKs and promoted cell apoptosis. In the meantime, however, the antioxidant N-acetylcysteine (NAC) was found to ameliorate Nrf2 expression and nuclear translocation as well as Gadd45b expression and MAPKs activation by repressing Sb2O3-induced ROS production. More importantly, we found Gadd45b was transcriptionally enhanced by Nrf2 through binding to three canonical antioxidant response elements (AREs) within its promoter region. Either Sb2O3 or TBHQ (a selective Nrf2 activator) treatment, Gadd45b expression was significantly increased by luciferase assay. Nrf2 inhibition greatly diminished Gadd45b expression due to reduced binding of Nrf2 in Gadd45b promoter under Sb2O3 treatment. To summarize, this study demonstrated the Nrf2-Gadd45b signaling axis exhibited a protective role in Sb-induced cell apoptosis.

Keywords: Antimony; Apoptosis; Nrf2; Gadd45b; ROS

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Introduction Antimony (Sb) is a silvery-white metal that is ubiquitous throughout the environment as a result of natural processes and human activities (Sundar et al., 2010). Due to mining and smelting processes, large quantities of Sb have been released resulting in serous Sb contamination of our living environments. Furthermore, coal combustion and Sb-associated products consumed in daily life are also important potential Sb contamination sources (He et al., 2012; Pierart et al., 2015). Environmental exposure of Sb may cause respiratory irritation, pneumoconiosis, spotting on the skin and gastrointestinal symptoms (Sundar et al., 2010). Although a large number of epidemiological studies of Sb exposure have been documented, its toxicity on health (e.g. nephrotoxicity) remains unknown and is of great concern. Reactive oxygen species (ROS) production is believed to be the major mechanism underlying toxic metal’s cytotoxicity to normal cells (Al-Gubory et al., 2014; Phaniendra et al., 2015). Even though excessive ROS production induced by Sb-containing compounds have been reported, a more complete characterization of its toxicity on normal cells is still limited (Lecureur et al., 2002; Lecureur et al., 2002; Losler et al., 2009; Mann et al., 2006). The transcription factor, NF-E2-related factor 2 (Nrf2), has been shown to regulate the expression of a network of cytoprotective enzymes for cellular defense against the environmental toxins and toxicants (Ma et al., 2013). Under normal conditions, the level of Nrf2 protein is present in the cytoplasm in association with its inhibitor Kelch-like ECH-asscociated protein 1 (Keap1), and binding of Keap1 to Nrf2 leads to biquitination of Nrf2 and proteosomal degradation. Upon exposure to endogenous activators (e.g. reactive oxygen species) or exogenous agents (e.g. heavy metals), Nrf2 dissociates from Keap1 and translocates into the nucleus. Through binding to antioxidant response elements (AREs), nucleus Nrf2 transcriptionally activates of its antioxidant target genes, such as glutathione S-tansferase, heme oxygenase-1, multidrug-resistance associated efflux pumps and NAD(P)H dehydrogenase quinone (NQO)-1 (Kobayashi and Yamamoto 2005; Li and Kong 2009). Until now, burgeoning evidence demonstrated the ability of Nrf2 to control cytoprotective adaptive response against environmental or industrial pollutions, such as arsenic, cadmium, chromium, copper, lead and mercury (Jiang et al., 2015; Lau et al., 2013; Simmons et al., 2011a; Son et al., 2014; Toyama et al., 2011; Ye et al., 2015). However, whether Nrf2 also conducts cytoprotection against Sb-induced cell toxicity still largely unknown thus far. 4

The members of Gadd45 gene family, Gadd45a, Gadd45b and Gadd45r, have been commonly implicated in stress signaling in response to physiological or environmental stressors, resulting in cell cycle arrest, DNA repair, cell survival and apoptosis (Salvador et al., 2013). Gadd45b has been implicated as an anti-apoptosis factor through activation MAPKs pathway in resistance to apoptosis initiated by a variety of stimuli (Ou et al., 2010; Salerno et al., 2012; Yang et al., 2009; Yoo et al., 2003). Nonetheless, we speculated whether Gadd45b could also provide protection from heavy metal induced cells apoptosis through MAPKs activation, and specifically, it is still unclear whether Gadd45b was regulated by Nrf2 under Sb-induced cell death. In the present study, we uncovered a critical protective role of Nrf2-Gadd45b pathways against oxidative damage induced by Sb in human embryonic kidney (HEK) 293 cells. Our combined data therefore highlighted a crucial role of Nrf2-Gadd45b in modulating Sb-induced apoptotic cell death.

Materials and methods Cell culture and reagents The HEK293 cells were obtained from the Shanghai Cell Bank of Type Culture Collection of Chinese Academy of Sciences. Cells were routinely maintained in 1640 medium (Hyclone) with 10% fetal bovine serum (Gibco) and 100 U/mL penicillin-streptomycin (Hyclone) at 37 C in humidified atmosphere with 5% CO2. Antimony trioxide (Sb2O3) and N-acetylcysteine (NAC) were purchased from Sigma-Aldrich. Tertiary butylhydroquinone (TBHQ) was obtained from Santa Cruz. Cytotoxicity assessment MTT assay was carried out to examine cytotoxicity upon chemical treatment following the instructions from manufacturer (Roche). In brief, cells were seeded in 96-well plates and then treated with Sb2O3 at different concentration. After treatment for 24 h, 20 μL of MTT (5 mg/mL) was added into complete culture media and cells were cultured for additional 4 h. Thereafter, the medium with MTT were aspirated, and 200 L of DMSO was added into each well. Finally, 96-well plates were read at 490nm on a microplate reader (Thermo). Intracellular ROS level Cells were cultured into 96-well pates and exposed to Sb2O3 in accordance with the manufacturer’s instruction (Sigma-Aldrich). Dichlorofluorescein diacetate (DCF-DA) was added at a final concentration of 10 M in the dark for 30 min before examination. Cells were then washed with PBS 5

three times, and DCF fluorescence was then monitored using a microplate reader. The excitation and emission wavelength were 488 and 525 nm, respectively, and 0.1% H2O2 was used as a positive control to induce ROS in cells. Apoptosis detection by flow Cytometry analysis Sb-induced apoptosis was determined by flow cytometry using the Annexin V-FITC apoptosis Detection Kit following the instructions from the manufacturer (BD Biosciences). In brief, cells after Sb2O3 treatment were collected and washed twice with PBS. Then, cells were stained with 5 L of Annexin V-FITC and 5 L of PI for 15 min. A total of 10,000 cells in each sample was analyzed by flow cytometry. The percentage distributions of early apoptosis and late apoptosis were calculated for comparison. Western blot analysis Whole cell proteins were extracted using ice-cold RIPA lysis buffer (Solarbio Biotechnology) supplemented with protease inhibitor cocktail (Roche). Nuclear or cytoplasmic protein were separated with the cytoplasmic and nuclear proteins extraction kit (Solarbio Biotechnology). Protein concentrations were determined with a Lowry protein concentration detection kit (Solarbio Biotechnology). About 50-150 g of protein from each sample was denatured, electrophoresed, and transferred onto nitrocellulose membranes (Millipore). Western blotting analysis was performed with specific antibodies against p-JNK, JNK, p-ERK, ERK, p-P38 and P38 (Santa Cruz), caspase3, caspase7, Bax and Bcl2 (Cell signaling). Antibodies against Nrf2, Gadd45b, Histone 3 and β-actin were purchased from Proteinteach. The intensities of the autoradiograms were quantitated with the software Image J, and the band intensity was normalized to those of loading control. qRT-PCR analysis Total RNAs were isolated from cells with Trizol reagent following the manufacturer’s instructions (Invitrogen). Approximately 2 g of total RNAs was reverse-transcribed into cDNA with M-MLV reverse transcriptase (Promega). Gene expression levels were evaluated using SYBR Green qPCR master mix (Promega) on Mx3005P qPCR machine (Stratagene). β-actin was used as an internal control to determine the relative expression of target genes. The primer sequences for real-time PCR are shown in Supplemental data. Lentiviral infection and luciferase reporter activity assay Human Nrf2 and Gadd45b shRNA constructs was cloned into lentiviral vector pLKO.1, respectively. 6

Lentiviral particle package and infection were performed accordingly. We also constructed the Gadd45b-Luc reporter with human Gadd45b promoter region. In detail, a 3k bp fragment of the gene for Gadd45b was inserted into the pGL3 vector. To analyze the significance of the ARE sites (TGA-nnnn-GC) in the Gadd45b promoter, the sequence was mutated to TAG-nnnn-CG. The luciferase activity was measured by Luciferase Reporter Assay System (Promega) according to the manufacturer’s protocol. The luciferase activity was normalized to cell viability. The primer sequences are presented in Supplemental data. Chromatin Immunoprecipitation (ChIP) assay ChIP assay was performed using a ChIP assay Kit (Millipore) according to the manufacturer’s instruments. Briefly, cells were fixed with 1% formaldehyde for 10 min at room temperature, and washed three times with cold PBS. Fixed cells were re-suspended in lysis buffer, and then subjected to sonication to shear DNA into fragments with length between 200 to 1000 bp. After centrifugation, a small aliquot of chromatin was saved as input controls, and the rest was diluted in dilution buffer. The diluted chromatin was incubated with 1 g corresponding antibody or normal immunoglobulin G. Immunoprecipitated DNA was purified, and then analyzed by RT-PCR. The primer sequences are listed in Supplemental data. Statistical analysis Statistical analysis was undertaken using the SPSS statistic 17.0 package. The difference in data between two experimental groups were assessed using the two-tailed Student’s t-test or One-way ANOVA test. Data are presented as mean ± SD. P value <0.05 was considered statistically significant.

Results Sb2O3 induces apoptosis of HEK293 cells in a ROS-dependent manner To determine the biological toxicity of Sb exposure on cells, we evaluated the effect of Sb2O3 treatment on the cytotoxicity of HEK293 cells using the MTT-based assay. As depicted in Fig 1A, Sb2O3 exhibited a dose-dependent cytotoxicity against HEK293 cells for 24 h (P < 0.05). Moreover, we also assessed Sb-induced apoptosis of HEK293 cells using Annexin V-FITC and PI staining. Flow cytometry analysis showed there were almost 20% and 35% cell death when cells were treated with Sb2O3 at 8 and 16 M respectively (P < 0.05) (Fig 1B-C). When cells undergo apoptosis, 7

anti-apoptotic Bcl2 level was reduced and pro-apoptotic Bax expression was elevated in 8 and 16 M Sb2O3 treated HEK293 cells (Fig 1D-E). Under different stress condition, Bax undergoes a conformational change and translocates to the mitochondrial membrane, thereby activating apoptosis-related proteins caspase3 and caspase7 (Finucane et al., 1999). Therefore, we found that caspase3 and caspase7 cleavage product levels increased 1.5 fold and 2-fold in HEK293 cells treated with Sb2O3 at 8 and 16 M compared with those of control (P < 0.05) (Fig 1D). These results together indicated cell apoptosis in Sb2O3-treated HEK293 cells, especially for those treated at 8 and 16 M. Besides, to determine whether the apoptosis was associated with oxidase stress, we measured ROS content after HEK293 cells were exposure to Sb2O3. As shown in Fig 1F, intracellular ROS content increased in a dose dependent manner with the peak reaching at 6 h, suggesting that Sb2O3 induced ROS accumulation in HEK293 cells. Moreover, the production of Sb2O3–induced ROS production could be inhibited by NAC (a ROS scavenger) pretreatment, compared to cells without pretreatment of NAC (P < 0.05) (Fig 1F). In the meantime, Sb2O3–induced cell death was also greatly ameliorated when ROS was scavenged by NAC, as the cell viability and apoptosis was improved in Sb2O3 and NAC co-treated HEK293 cells (P < 0.05) (Fig 1G-H). In short, these data demonstrated that Sb2O3 induces apoptosis of HEK293 cells in a ROS-dependent manner. Nrf2 protects HEK293 cells against Sb2O3-induced apoptosis Since Nrf2 is known to be induced by oxidative stress, we hypothesized Nrf2 was also involved in Sb-induced apoptosis. Consequently, we assessed the expression of Nrf2 upon exposure of HEK293 cells to Sb2O3. As illustrated in Fig 2A, the mRNA expression level of Nrf2 was significantly elevated and peaked on 12 h (1.8-fold and 2.2-fold) after Sb2O3 treatment at 8 M and 16 M, and then gradually decreased but was still higher than baseline at 48 h (P < 0.05). To further investigate the activation of Nrf2, we assessed nuclear translocalization of Nrf2 protein by separating cellular fractions into cytoplasmic and nuclear fractions. The Nrf2 proteins in the nucleus was rapidly accumulated almost 2 fold at 12 h, whereas cytoplasmic Nrf2 protein level was decreased gradually upon Sb2O3 exposure at 8 M (Fig 2B). These data suggested that Sb2O3 can induce Nrf2 expression and activation in HEK293 cells. To determine whether Nrf2 involved Sb-induced apoptosis in HEK293 cells, we used Nrf2 shRNA to knockdown Nrf2 expression. Fig 2C showed that the whole Nrf2 protein level was decreased almost 8

80% in Nrf2-knockdown cells (P < 0.05). Nrf2 knockdown increased cells susceptibility to Sb2O3-induced cytotoxicity at 8 and 16 M, compared with those of controls (P < 0.05) (Fig 2D). Besides, to determine the role of ROS in modulating Sb2O3-induced Nrf2 activation, we pretreated HEK293 cells with NAC upon Sb2O3 exposure. As shown in Fig 2E-F, NAC pretreatment greatly ameliorated elevated Nrf2 expression and nuclear accumulation under Sb2O3 treatment, indicating ROS production was required for Sb2O3-induced Nrf2 activation. These combined data suggested a protective role of Nrf2 against Sb2O3-induced cytotoxicity. Gadd45b drives activation of MAPKs upon Sb2O3 exposure The mRNA expression level of Gadd45b was increased almost 1.5-fold at 12 h and persistent at high level upon exposure of HEK293 cells to Sb2O3 (P < 0.05) (Fig 3A). Since Gadd45b has been reported to active MAPKs pathway, we monitored the level of phosphorylation of JNK, p38 and Erk under oxidative stress. As illustrated in Fig 3B, Sb2O3 exposure increased the kinase activity of JNK and p38 at 8 and 16 M, whereas phosphorylation of Erk level was unchanged. As expected, phosphorylation of JNK and p38 could also be induced by H2O2 treatment (Fig 3B). These findings demonstrated that Sb-induced ROS accumulation promotes Gadd45b expression and activation of MAPKs. To further confirm whether Gadd45b was capable of inhibiting Sb-induced apoptotic cell death, HEK293 cells were transfected with shRNA-mediated knockdown of Gadd45b, and then incubated with a dose of 8 M Sb2O3. Fig 3C showed that the protein level of Gadd45b and activation of MAPKs were inhibited upon Gadd45b knockdown. After the treatment, the cell viability was significantly inhibited in Gadd45b-knockdown cells under Sb2O3 treatment (P < 0.05) (Fig 3D). In addition, NAC pretreatment greatly reversed the elevated expression of Gadd45b and phosphorylation of JNK and p38 upon Sb2O3 exposure, demonstrating a critical of ROS in Sb2O3-induced Gadd45b expression and MAPKs activation (P < 0.05) (Fig 3E-F). These data together showed a protective effect of Gadd45b on Sb-induced HEK293 cells apoptosis. Nrf2 transcriptionally actives Gadd45b expression against Sb2O3-induced apoptosis We next attempted to identify a possible targeted transcription factors that up-regulates Gadd45b expression. Sequence analysis showed the existence of putative three antioxidant responsive elements (ARE1, -2221 to -2213; ARE2, -1985 to -1977; ARE3, -1670 to -1662) in the Gadd45b promoter (Fig 4A). These canonical AREs were typically matched to the general consensus sequence 9

of 5’-TGA-nnnn-GC-3’, which is the binding site of Nrf2. To interrogate the binding of Nrf2 to the AREs within Gadd45b promoter region, we performed the quantitative ChIP assay in HEK293 cells. As shown in Fig 4B, significant enrichment were verified within ARE1, ARE2 and ARE3 upon Nrf2 Ab precipitation, demonstrating the binding of Nrf2 to the promoter of Gadd45b (P < 0.05). Moreover, luciferase assay was also used to determine whether Nrf2 transcriptionally activates Gadd45b expression under Sb2O3 exposure, with TBHQ as a positive control. As a result, the wild type (WT) promoter activity treated induced by Sb2O3 and TBHQ was increased approximately 2.5-fold and 3.2-fold, respectively. (P < 0.05) (Fig 4C). To determine whether lack of ARE sites for Nrf2 binding decrease the promoter activity, we constructed four Gadd45b reporter constructs within mutation of the ARE sites. As showed in Fig 4C, promoter activity of each mutant construct (pGL-3-ARE1(Mut), pGL-3-ARE2(Mut), pGL-3-ARE3(Mut)) was remarkably decreased, compared with those of WT construct (P < 0.05), with the highest level in ARE1 mutation construct. By mutating all ARE sites (ARE1-3), the promoter activity was completely diminished (Fig 4C). Moreover, we further found that Sb2O3-induced Gadd45b expression and MAPKs activation were largely ameliorated in Nrf2-knockdown HEK293 cells, in comparison to scrambled control (Fig 4D). ChIP assays also revealed more than 70%, 55% and 60% decrease binding of Nrf2 in ARE1, ARE2 and ARE3 of Gadd45b promoter region in Nrf2-knockdown HEK293 cells under Sb2O3 treatment (P < 0.05) (Fig 4E). Nrf2-knockdown in HEK293 cells diminished the luciferase activity of pGL3-WT, pGL3-ARE1, pGL3-ARE2 and pGL3-ARE3, when cells were underwent Sb2O3 treatment (P < 0.05) (Fig 4F). Collectively, these data demonstrated the transcriptional modulation of Gadd45b expression by Nrf2, and also indicated the crucial role of Nrf2-Gadd45b signaling in protecting cells against Sb-induced apoptosis.

Discussion It is well-documented that exposure to heavy metals could induce oxidative stress, disruption of cell metabolism and epigenetic changes (Jomova et al., 2011; Leonard et al., 2004). Sb is a toxic trace element widely distributed in the lithosphere and mainly associated with arsenic. Trivalent antimony are considered to be pollutants of high interest, however, the biogeochemical behavior of Sb is still largely unknown, especially compared to other well-known toxic elements. To date, Losler et al found Sb2O3-induced apoptosis are enhanced by modulators of cellular glutathione redox system in 10

myelogenic and lymphatic cells (Losler et al., 2009). Besides, Mann and colleagues further indicated that SEK1/JNK signaling plays an important role on Sb2O3-induced ROS related-apoptosis in acute promyelocytic leukemia cell lines (Mann et al., 2006). In the present study, we found that Sb2O3 exhibited a dose-dependent cytotoxicity against HEK293 cells, and excessive ROS production was closely correlated with increased cell apoptosis. Sb2O3 exposure also led to the imbalance of Bax/Bcl2, thereby activating caspase3 and caspase7. Mechanistically, Nrf2 expression and nuclear translocation were induced under Sb2O3 treatment in HEK293 cells, and elevated Gadd45b expression actives the phosphorylation of MAPKs under Sb2O3 exposure. In contrast, NAC was found to ameliorate Nrf2 and Gadd45b expression by repressing Sb2O3-induced ROS production. More importantly, we found Gadd45b was transcriptionally enhanced by Nrf2 through ARE binding sites within its promoter region. Metals represent the ultimate form of persistent environmental pollutants since they are chemically and biologically indestructible. The understanding of possible biological changes caused by heavy metals exposure will probably be of great significance in drug development. Nrf2 is an antioxidant transcriptional factor that protects cells from ROS-induced damage through modulating the expression of cytoprotective and antioxidant genes (Li et al., 2009; Ma 2013). The important role of Nrf2 in anti-stress has been documented in heavy metals (Simmons et al., 2011b). Ye et al reported the protective role of Nrf2 against Pb-induced oxidative stress and apoptosis in human neuroblastoma SH-SY5Y cells (Ye et al., 2015). Yue et al found Nrf2 is responsible for the resistance of human osteosarcoma cells to arsenic trioxide treatment (Yue et al., 2015). Moreover, Jiang et al also indicated that copper exposure caused oxidative damage to fish muscle by decreasing the antioxidant enzyme activities via the destruction of Nrf2/ARE signaling (Jiang et al., 2015). In current study, we identified a ROS-dependent manner of Nrf2 expression upon oxidative stress from Sb2O3 exposure. Moreover, elevated Nrf2 protein was translocated into the nucleus under Sb2O3 treatment, leading to promote cytoprotective activity against oxidant stress-induced apoptosis. In contrast, removal of intracellular ROS through pretreatment with NAC could significantly prevent Sb2O3-induced apoptosis in HEK293 cells. Regarding the role of Gadd45b in cell stress response, the current literature provides a lot of information. However, it has been noted that there are still inconsistent and even conflicting results among the different reports, possibly due to different simulators, concentrations, and cell and animal 11

model used. For example, Gupta et al showed that Gadd45b plays a survival function to protect hematopoietic cells from DNA-damaging agents under ultra violet-induced apoptosis (Gupta et al., 2006). Similarly, Yu et al identified that Gadd45b-mediated p53 protein degradation was also crucial for anti-apoptosis effect due to arsenate exposure (Yu et al., 2013). On the contrary, Kim et al reported that Gadd45b could induce cardiomyocyte apoptosis under ischaemia/hypoxia both in vitro and in vivo (Kim et al., 2010). Moreover, Chen et al further demonstrated that Gadd45b aggravated puromycin aminonucleoside-induced podocyte injury and proteinuria (Chen et al., 2016). In the current study, we found that Gadd45b was significantly upregulated and subsequent activation of MAPKs under Sb2O3 exposure, whereas shRNA-mediated Gadd45b knockdown diminished MAPK activation and aggravated cell apoptosis. In addition, pretreated with NAC ameliorated Sb2O3–induced Gadd45b expression and MAPK phosphorylation, supporting that Sb2O3-induced ROS accumulation was closely correlated with up-regulation of Gadd45b and MAPKs activation. Although accumulating studies suggested elevated Gadd45b expression under oxidative stress, the specific transcriptional regulation of Gadd45b are still poorly understand. In 2014, Kim et al found that PPARα indirectly induces the Gadd45b gene in liver through promoting degradation of the repressor STAT3 as a result of elevated oxidative stress. In their study, STAT3 was found to be a repressor of Gadd45b through binding to upstream regulatory elements (Kim et al., 2014). In addition,

p53

induces

transcriptionally

activation

of

Gadd45b

through

p38α-mediated

phosphorylation, leading to apoptotic death during ischemia/anoxia (Kim et al., 2013). In our study, we found Nrf2 functions as a novel transcriptional regulator of the Gadd45b via activation of the ARE sequence in its promoter region. Elevated expression and nuclear translocation of Nrf2 under Sb2O3 exposure promoted the expression of Gadd45b through Nrf2-ARE binding activity. Nrf2 knockdown diminished Gadd45b expression through inhibiting the binding ability of Nrf2 to AREs in Sb2O3 treated of HEK293 cells. To summarize, this study demonstrated that a protective role of Nrf2-Gadd45b against antimony-induced oxidative stress and apoptosis in HEK293 cells (Fig 5). This findings not only signified a novel toxicity feature under Sb2O3 exposure but also implied a significant protection measure against Sb exposure.

Conflict of interest 12

No potential conflicts of interest was disclosed from the authors. ACKNOWLEDGMENTS This work was supported by grants under the Strategic Priority Research Program of the Chinese Academy of Science (no. XDB14000000) and the National Nature Science Foundation of China (no. 81472416, 21577097, 81402092).

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Figures Legend

Fig 1. Sb2O3 induces apoptosis of HEK293 cells in a ROS-dependent manner. (A) Cytotoxicity was determined through the MTT assay in HEK293 cells upon exposure to Sb2O3 at various concentrations for 24 h. Data are expressed as means ± SD for five independent experiments. (B) Cell apoptosis was examined by FACS analysis when cells were treated with Sb2O3 at 8 and 16 M. (C) Representative means ± SD of cell apoptosis from three independent samples. (D) Cleaved capase-3, cleaved caspase-7, Bcl2 and Bax were assessed by Western blot in HEK293 cells treated with Sb2O3 at 8 and 16 M. β-actin levels were used as a loading control. Protein bands were quantified by Image J software. (E) Representative mean ± SD of Bax/Bal2 ratio for three independent experiments. (F) The time course of intracellular ROS content was monitored by DCF fluorescent intensities in HEK293 cells treated with Sb2O3 at 8 and 16 M with or without NAC pretreatment. Data are means ± SD of three samples. (G) Cytotoxicity was assessed by MTT assay and (H) apoptosis was assessed by flow cytometry in HEK293 cells treated with Sb2O3 at 8 and 16 M with or without NAC pretreatment. Results are expressed as means ± SD of three experiments. Asterisk (*) indicates P<0.05.

Fig 2. Nrf2 protects HEK293 cells against Sb2O3-induced apoptosis. (A) Nrf2 mRNA expression levels were determined by qRT-PCR in HEK293 cells treated with Sb2O3 at 8 and 16 M during different time course. β-actin levels were used as an endogenous housekeeping gene. Results are expressed as means ± SD of three independent samples. (B) Nrf2 protein levels in nuclear and cytosolic were assessed by Western blot in HEK293 cells treated with Sb2O3 at 8 M during different time course. Histone H3 was used as control for nuclear proteins, whereas β-actin for cytosolic marker. Protein bands were quantified by Image J software. (C) Nrf2 protein level was detected under Sb2O3 exposure when cells were transfected with shRNA Nrf2 and shRNA control construct. β-actin levels were used as a loading control. (D) Cytotoxicity was assessed by MTT assay under Sb2O3 exposure when cells were transfected with shRNA Nrf2 and shRNA control construct. Data 16

are means ± SD of five independent samples. (E) Nrf2 mRNA expression levels were assessed by qRT-PCR and (F) Nrf2 protein level in nuclear and cytosolic were examined by Western blot in HEK293 cells treated with Sb2O3 with or without NAC pretreatment. Asterisk (*) indicates P<0.05.

Fig 3. Gadd45b drives activation of MAPKs upon Sb2O3 exposure. (A) The expression levels of Gadd45b were determined by qRT-PCR in HEK293 cells treated with Sb2O3 at 8 and 16 M during different time course. β-actin levels were used as an endogenous housekeeping gene. Results are expressed as means ± SD of three samples. (B) P-p38, p38, P-JNK, JNK, p-Erk and Erk protein level were detected by Western blot HEK293 cells treated with Sb2O3 at 8 16 M during different time course. H2O2 was used as a positive control to induce ROS. β-actin levels were used as a loading control. (C) The protein level of Gadd45b, P-p38, p38, P-JNK and JNK were detected under Sb2O3 exposure when cells were transfected with shRNA Gadd45b and shRNA control. β-actin levels were used as a loading control. Protein bands were quantified by Image J software. (D) Cytotoxicity was assessed by MTT assay under Sb2O3 exposure when cells were transfected with shRNA Gadd45b and shRNA control. Data are expressed as means ± SD for five independent experiments. (E) Gadd45b mRNA expression levels were assessed by qRT-PCR, and (F) the protein level of Gadd45b, P-p38, p38, P-JNK and JNK were examined by Western blot in HEK293 cells treated with Sb2O3 with or without NAC pretreatment. β-actin levels were used as a loading control. Asterisk (*) indicates P<0.05.

Fig 4. Nrf2 transcriptionally actives Gadd45b expression against Sb2O3-induced apoptosis. (A) The Gadd45b promoter of 3k bp contains the three Nrf2 binding sites (ARE1-3). (B) ChIP assay was performed to detect the three AREs in Gadd45b promoter region using an anti-Nrf2 antibody or normal IgG. Data are means ± SD of three independent samples. (C) Transcriptional activity of Gadd45b upon elevated endogenous Nrf2 expression was determined by luciferase assay under Sb2O3 treatment, with TBHQ act as a positive control. HEK293 cells were transfected with pGL3-Gadd45b promoter (WT, ARE1 Mutation, ARE2 Mutation, ARE3 Mutation and ARE1-3 Mutation) when cells were underwent Sb2O3 exposure. Results are expressed as means ± SD of three samples. (D) The protein level of Gadd45b and P-p38, p38, P-JNK, and JNK were detected by Western blot under Sb2O3 exposure when cells were transfected with shRNA Nrf2 and shRNA 17

control construct. β-actin levels were used as a loading control. Protein bands were quantified by Image J software. (E) Nrf2 binding ability was determined by ChIP assay under Sb2O3 treatment when cells were transfected with shRNA Nrf2 and shRNA control. Data are means ± SD of three independent samples. (F) Transcriptional activity of Gadd45b upon increased endogenous Nrf2 expression was determined under Sb2O3 treatment when cells were transfected with shRNA Nrf2 and shRNA control construct. Results are expressed as means ± SD of three samples. Asterisk (*) indicates P<0.05.

Fig 5. A scheme diagram deciphering the mechanism underlying the Nrf2-Gadd45b signaling axis exhibited a protective role in Sb2O3-induced cell apoptosis

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