Involvement of the Nrf2-proteasome pathway in the endoplasmic reticulum stress response in pancreatic β-cells

Toxicology and Applied Pharmacology 264 (2012) 431–438

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Involvement of the Nrf2-proteasome pathway in the endoplasmic reticulum stress response in pancreatic β-cells Sanghwan Lee a, Eu-gene Hur a, In-geun Ryoo b, Kyeong-Ah Jung b, Jiyeon Kwak c, Mi-Kyoung Kwak b,⁎ a b c

Yeungnam University, College of Pharmacy, Gyeongsan-si, Gyeongsangbuk-do 712‐749, Republic of Korea The Catholic University of Korea, College of Pharmacy, Wonmi-gu, Bucheon, Gyeonggi-do 420‐743, Republic of Korea Inha University, College of Medicine, 253 Yonghyun-dong, Nam-gu, Incheon 402‐751, Republic of Korea

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Article history: Received 23 April 2012 Revised 22 July 2012 Accepted 19 August 2012 Available online 30 August 2012 Keywords: Proteasome ER stress Nrf2 Pancreatic β-cells D3T Tunicamycin

a b s t r a c t The ubiquitin-proteasome system plays a central role in protein quality control through endoplasmic reticulum (ER)-associated degradation (ERAD) of unfolded and misfolded proteins. NF-E2‐related factor 2 (Nrf2) is a transcription factor that controls the expression of an array of phase II detoxification and antioxidant genes. Nrf2 signaling has additionally been shown to upregulate the expression of the proteasome catalytic subunits in several cell types. Here, we investigated the role of Nrf2 in tunicamycin-induced ER stress using a murine insulinoma β-cell line, βTC-6. shRNA-mediated silencing of Nrf2 expression in βTC-6 cells significantly increased tunicamycin-induced cytotoxicity, elevated the expression of the pro-apoptotic ER stress marker Chop10, and inhibited tunicamycin-inducible expression of the proteasomal catalytic subunits Psmb5 and Psmb6. The effects of 3H-1,2-dithiole-3-thione (D3T), a small molecule Nrf2 activator, on ER stress were also examined in βTC-6 cells. D3T pretreatment reduced tunicamycin cytotoxicity and attenuated the tunicamycin-inducible Chop10 and protein kinase RNA-activated‐like ER kinase (Perk). The protective effect of D3T was shown to be associated with increased ERAD. D3T increased the expression of Psmb5 and Psmb6 and elevated chymotrypsin-like peptidase activity; proteasome inhibitor treatment blocked D3T effects on tunicamycin cytotoxicity and ER stress marker changes. Similarly, silencing of Nrf2 abolished the protective effect of D3T against ER stress. These results indicate that the Nrf2 pathway contributes to the ER stress response in pancreatic β-cells by enhancing proteasome-mediated ERAD. © 2012 Elsevier Inc. All rights reserved.

Introduction The endoplasmic reticulum (ER) is the organelle that biosynthesizes and modifies secretory proteins. The fidelity of protein synthesis in the ER is ensured by multiple protein quality control mechanisms and the multi-step processes of protein folding and maturation are tightly controlled to maintain homeostasis. However, stress conditions such as glucose starvation, oxidative stress, hypoxia, and high fat or cholesterol perturb protein homeostasis and lead to accumulation of misfolded proteins in the ER lumen, which is referred to as ER stress (Schroder

Abbreviations: ER, endoplasmic reticulum; UPR, unfolded protein response; ERAD, ER-associated degradation; iNrf2, NRF2 knockdown βTC-6; iSc, nonspecific control βTC-6; ARE, antioxidant-response element; GSH, glutathione; Nqo1, NAP(D)H, quinone oxidoreductase-1; Gclc, catalytic subunit of γ-glutamate cysteine ligase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; D3T, 3H-1,2-dithiole3-thione. ⁎ Corresponding author at: The Catholic University of Korea, College of Pharmacy, 43 Jibong-ro, Wonmi-gu, Bucheon, Gyeonggi-do 420‐743, Republic of Korea. Fax: +82 2 2164 4059. E-mail address: [email protected] (M.-K. Kwak). 0041-008X/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.taap.2012.08.021

and Kaufman, 2005). In response to ER stress, cells initiate a specialized buffering system known as the unfolded protein response (UPR), which activates cellular signaling pathways that restore protein homeostasis and ER function (Hampton, 2000; Schroder and Kaufman, 2005; Walter and Ron, 2011). The UPR is activated by 3 stress sensors in the ER: protein kinase RNA-activated-like ER kinase (Perk), inositolrequiring enzyme 1α (Ire1α), and activating transcription factor 6 (Atf6). Signaling pathways from these sensors reduce the ER load by attenuating mRNA translation, increasing mRNA degradation, and increasing the expression of molecular chaperones and components of the ER-associated degradation (ERAD) pathway (Tsai and Weissman, 2010, 2011). Overall, the UPR alleviates ER stress by enhancing its protein folding capacity and facilitating the removal of unfolded proteins. Although the UPR promotes cell survival, prolonged and severe ER stress can induce apoptosis (Shore et al., 2011; Woehlbier and Hetz, 2011). Thus, UPR sensors can also elevate expression of pro-apoptotic factors such as Chop10 (also known as growth arrest and DNAdamage-inducible 153, GADD153) and suppress anti-apoptotic factors such as Bcl-2. The 26S proteasome, consisting of the 19S regulatory cap and the proteolytic 20S core complex, is responsible for the degradation of abnormal proteins in the cytosol, nucleus, and ER lumen (Poppek

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and Grune, 2006; Tai and Schuman, 2008; Xie, 2010). The core 20S proteasome is composed of several structural subunits as well as the catalytic subunits Psmb5, Psmb6, and Psmb7, which possess chymotrypsin-like, caspase-like, and trypsin-like peptidase activities, respectively (Bedford et al., 2010). The 19S proteasome controls access of substrate proteins to the inner part of the 20S proteasome by recognizing and degrading polyubiquitinated proteins in an ATP-dependent manner. The proteasome is one component of the UPR that mediates ERAD (Rubinsztein, 2006; Tai and Schuman, 2008; Xie, 2010). However, little is known about the molecular control of proteasome expression in mammalian cells. Previously, we demonstrated that the transcription factor NF-E2‐related factor 2 (Nrf2) is involved in regulating the inducible expression of Psmb5 in murine cells (Kwak et al., 2003a). Nrf2 is an essential component of a transcriptional complex that activates expression of genes encoding detoxifying enzymes (e.g., NAD(P)H: quinone oxidoreductase 1 [Nqo1]) and antioxidant proteins (e.g., the GSH biosynthetic enzyme γ-glutamate cysteine ligase [Gcl]) (Li and Kong, 2009; Taguchi et al., 2011). Thus, the observation that Nrf2 upregulates Psmb5 has led to a novel concept that the proteasome can participate in the cellular defense system against divergent sources of stress. Chronic ER stress and dysregulation of UPR have been proposed to contribute to various human diseases, including neurodegenerative diseases and disorders of pancreatic β-cell function (Back and Kaufman, 2012; Rubinsztein, 2006). Alzheimer's and Parkinson's diseases show common pathological lesions of accumulated abnormal protein aggregates. Consistent with this, changes in proteasome activity have been identified in experimental cell systems as well as in pathologic samples from patients with Alzheimer's or Parkinson's diseases (Mittal and Ganesh, 2010; Rubinsztein, 2006; Zabel et al., 2010). In particular, multiple lines of evidence suggest that ER stress is involved in the pathology of type 2 diabetes (Back and Kaufman, 2012; Fonseca et al., 2011). For example, chronic ER stress is increased in pancreatic cells from patients with diabetes, which can lead to β-cell death. In the present study, we have investigated the role of Nrf2 in tunicamycin-induced ER stress using a murine pancreatic β-cell line. Tunicamycin is a mixture of homologous nucleotide antibiotics that inhibit the synthesis of N-linked glycoproteins and is commonly used to induce ER stress experimentally. Our results show that 3H-1,2-dithiole-3-thione (D3T), a small molecule Nrf2 activator, attenuates tunicamycin-induced cytotoxicity and inhibits the increase in ER stress markers such as Chop10. We show that D3T treatment protects cells from tunicamycin-induced stress by increasing expression of proteasome catalytic subunits in an Nrf2dependent manner. Consistent with this, Nrf2-silenced β-cells were more susceptible to tunicamycin cytotoxicity and this was not attenuated by D3T treatment. Materials and methods Materials. D3T was provided by Dr. Thomas Curphey (Dartmouth Medical School, NH). Tunicamycin was purchased from SigmaAldrich (Saint Louis, MO, USA). 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was purchased from Amersco Inc. (Solo, OH, USA). MG132 and substrates for measurement of proteasome activity were from EMD Chemicals (Billerica, MA, USA). Primary antibodies for Western blotting of Psmb5 and Psmb6 were purchased from Enzo life sciences (Farmingdale, NJ, USA). Antibodies recognizing Nrf2 and β-tubulin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and antibodies for phospho-eIF2α and phosphor-Perk were obtained from Cell Signaling Technology (Beverly, MA, USA). The lentiviral expression plasmid for mouse Nrf2 short hairpin RNA (shRNA), Mission™ Lentiviral Packaging Mix, hexadimethrine bromide, and puromycin were all from Sigma-Aldrich. Cell culture. The murine pancreatic β-insulinoma cell line βTC-6 was obtained from American Type Culture Collection (Manassas, VA,

USA). βTC-6 cells were maintained in Dulbecco's Modified Eagle's Medium (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone) and penicillin/streptomycin (Hyclone). Cells were maintained at 37 °C in a humidified 5% CO2 atmosphere. Production of shRNA lentiviral particles. Lentiviral particles containing the Nrf2-specific shRNA or control, scrambled (Sc) shRNA were produced by transfection of HEK293T cells with the relevant shRNA expression plasmid and Mission™ Lentiviral Packaging Mix (SigmaAldrich) as described previously (Kim et al., 2011). Briefly, HEK293T cells were seeded in 60 mm plates at a density of 7× 10 5 cells per well. The next day, the medium was replaced by OptiMEM (Invitrogen, Carlsbad, CA, USA) and cells were transfected with 1.5 μg pLKO.1-Nrf2 shRNA (mouse Nrf2-specific shRNA: 5′-CCGGCCAAAGCTAGTATAGCAA TAACTCGAGTTATTGCTATACTAGCTTTGGTTTTTG-3′) or pLKO.1-ScRNA and the Packaging Mix, using Lipofectamine™ 2000 (Invitrogen). On the second day, the medium was exchanged with fresh complete medium. The medium containing lentiviral particles was harvested after 4 days. Stable transduction of βTC-6 cells. βTC-6 cells in 6-well plates were transduced with lentiviral particles containing either pLKO.1-ScRNA or pLKO.1-Nrf2 shRNA in the presence of 8 μg/ml of hexadimethrine bromide (Sigma-Aldrich). Transduction was continued for 48 h followed by 24 h recovery in complete medium. Stable plasmid-expressing cells were selected by growth for 4 weeks in medium containing 1 μg/ml puromycin (Sigma-Aldrich). MTT assay. Cell viability was determined using the MTT assay. Briefly, βTC-6 cells were seeded at a density of 5×103 cells/well in 96-well plates and incubated with the relevant compounds (tunicamycin, D3T, MG132) for the indicated times. MTT solution (2 mg/ml) was added to the wells and plates were incubated for 4 h. The MTT solution was removed, 100 μl/well of DMSO was added, and the absorbance was measured at 540 nm using a Versamax microplate reader (Sunnyvale, CA, USA). Total RNA extraction and RT-PCR analysis. Total RNA was isolated from βTC-6 cells using TRIzol reagent (Invitrogen). For the synthesis of cDNAs, reverse transcriptase (RT) reactions were performed by incubating 200 ng of total RNA with a reaction mixture containing 0.5 μg/μl oligo dT12–18 and 200 U/μl Moloney murine leukemia virus RT (Invitrogen). Quantitative real-time RT-PCR was performed using a Roche LightCycler (Mannheim, Germany) with the Takara SYBR Premix ExTaq system (Otsu, Japan). Primers were synthesized by Bioneer (Daejeon, South Korea) and the primer sequences for the mouse genes are shown in the online material. All mRNA levels were normalized to the level of Hprt transcripts. Western blot analysis. βTC-6 cells were lysed with RIPA buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, and 1% NP40) containing a protease inhibitor cocktail (Sigma-Aldrich). Protein concentration was determined using a DC protein assay kit (Bio-Rad, Hercules, CA, USA). Protein samples were separated by electrophoresis on 6–12% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Whatman GmbH, Dassel, Germany) using a Trans-Blot® Semi-Dry Cell (Bio-Rad). The membrane was then blocked with 3% skim milk for 1 h, and antibody incubations were performed. The chemiluminescent image was captured using a Fujifilm LAS-4000 mini imager (Fujifilm, Tokyo, Japan). Measurement of luciferase activity. βTC-6 cells in 24-well plates were transfected with a mixture of 0.5 μg ARE-luciferase plasmid (Kim et al., 2011), 0.05 μg of pRLtk control plasmid (Promega, Madison, WI, USA), and WelFect transfection reagent (Welgene Inc., Daegu, South Korea). After 18 h, the transfection mixture was removed and cells

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were incubated in complete medium for 24 h. The cells were then lysed and Renilla and firefly luciferase activities were measured using the Dual Luciferase Assay System (Promega) with a luminometer (Turner Designs, Sunnyvale, CA, USA). Measurement of caspase activity. βTC-6 cells in 24-well plates were incubated with tunicamycin for the indicated times and total caspase activities were determined using the FLICA (Fluorochrome Inhibitor of Caspases) assay kit (ImmunoChemistry Technologies, Bloomington, MN, USA) according to the supplier's instructions (Cho et al., 2008). Briefly, 120 μl of 1× FLICA-containing medium was added to each well and the plates were incubated for 4 h at 37 °C in the dark. Then, the cells were mounted for fluorescence microscopy. Caspase activity (indicative of apoptosis) was detected as a green fluorescent signal (488 nm excitation/520 nm emission). Measurement of proteasome activity. The peptidase activities of the proteasomal subunits were measured by mixing cell lysates with 50 μM of fluorogenic substrate peptides Suc-LLVY-AMC (Succinyl-LeuLeu-Val-Tyr-7-amino-4-methylcoumarin), Z-LLE-AMC (Z-Leu-LeuGlu-7-amino-4-methylcoumarin), or Z-ARR-AMC (Z-Ala-Arg-Arg-7amino-4-methylcoumarin) (all EMD Chemicals) in a final volume of 100 μl of reaction buffer (50 mM Tris–HCl, pH 7.8, 20 mM KCl, 5 mM MgCl2, and 1 mM DTT). The mixture was incubated at 37 °C for 20 min and the reaction then stopped by addition of an equal volume of 125 mM sodium borate buffer (pH 9.0) containing 7.5% ethanol (Kwak et al., 2003a). The fluorescence of the cleaved AMC moiety was measured with excitation at 360 nm and emission at 460 nm. Fluorescence units were converted to AMC concentrations using a standard curve generated from free AMC. Statistical analyses. Statistical significance was analyzed by the Student t-test or one-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls test for multiple comparisons, using Prism software (GraphPad Prism, La Jolla, CA, USA).

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purpose, Nrf2-specific shRNA was stably introduced into βTC-6 cells and tunicamycin sensitivity was assessed. First, Nrf2 silencing in the stable cell lines (iNrf2 cells) was verified by measuring transcription of Nrf2 and its target genes. Nrf2, ARE-driven luciferase activity, and target gene Nqo1 mRNA levels were consistently reduced in iNrf2 cells compared to the control-transfected (iSc) cells (Figs. 2A, B, and C). Moreover, treatment of cells with D3T, a small molecule activator of Nrf2, elevated ARE-luciferase activity as well as Nqo1 expression in iSc cells but not in iNrf2 cells (Figs. 2B and C). Incubation of cells with tunicamycin (2–16 μg/ml) reduced the viability of iNrf2 cells to a greater extent than the control cells (Fig. 2D). Tunicamycin treatment had a differential effect on expression of UPR genes in the iNrf2 and iSc cells. Tunicamycin-induced increases in Chop10 and Atf3 mRNA levels were much greater in iNrf2 cells than in similarly treated iSc cells (Figs. 2E and F), whereas Perk and Atf4 levels were slightly lower in tunicamycin-treated iNrf2 cells than iSc cells (Figs. 2G and H). These results indicate that Nrf2 can influence the UPR upon tunicamycin treatment of pancreatic β-cells. Tunicamycin-mediated increase in proteasome expression is Nrf2dependent Because the Nrf2-silenced βTC-6 cells were more sensitive to the effects of tunicamycin, we hypothesized that Nrf2 might be involved in protection against tunicamycin-induced ER stress. To address this, we examined the effect of tunicamycin on Nrf2 transcriptional activity using an ARE-luciferase reporter assay and on Nrf2 target gene mRNA levels. Incubation of control βTC-6 cells (iSc) with 1 μg/ml tunicamycin for 24 h induced a small but significant increase in Nrf2 transcriptional activity (Fig. 3A), and in mRNA levels for Nqo1, Gclc, and Gsr (Fig. 3B). Of note, these effects were not observed in iNrf2 cells treated with tunicamycin. Similarly, expression of the proteasome subunits Psmb5 and Psmb6 was significantly elevated by tunicamycin treatment in iSc cells but not in iNrf2 cells (Figs. 3C and D). These data suggest that an Nrf2-dependent increase in proteasome activity may be involved in the tunicamycin-induced ER stress response.

Results The Nrf2 activator D3T alleviates tunicamycin-mediated cytotoxicity Tunicamycin-mediated ER stress is enhanced by proteasome inhibition The murine insulinoma βTC-6 cell line was derived from a pancreatic tumor from an SV40 large T antigen transgenic mouse (Poitout et al., 1995). To test its cytotoxic effect on βTC-6 cells, tunicamycin (2, 4, 8, and 16 μg/ml) was added to cell cultures for 24 h and cell viability was measured using the MTT assay. Tunicamycin had a marginal affect on cell viability, with a reduction in viability (20%) following 16 μg/ml tunicamycin (Fig. 1A). ER stress was confirmed by measuring the expression of several UPR genes; mRNA levels for Perk and Chop10 were significantly increased by incubation of cells with 1 μg/ml tunicamycin for 24 h (Fig. 1B). Transcript levels of other ER stress markers including Atf3, Atf4, Atf6, and BIP were similarly elevated by tunicamycin treatment (data not shown). Of note, co-treatment of cells with the proteasome inhibitor MG132 (2.5 μM) substantially enhanced tunicamycin-mediated ER stress gene expression, implying that proteasome-mediated ERAD is important in the tunicamycin UPR (Fig. 1B). As a confirmation, tunicamycin-mediated ER stress in βTC-6 cells could be evidenced by immunoblot results showing the elevation of phospho-Perk and phospho-eIF2α in tunicamycin-treated cells at early time point (Fig. 1C). Nrf2 knockdown increases tunicamycin sensitivity We previously showed that the transcription factor Nrf2 is involved in regulating proteasome expression in murine cells (Kwak et al., 2003a). Based on this, we asked whether Nrf2 is involved in tunicamycininduced ER stress through modulation of the proteasome. For this

To confirm the role of Nrf2 in the ER stress response, we next examined the effect of a small molecule Nrf2 activator, D3T. Preincubation of βTC-6 cells with D3T for 18 h significantly reduced tunicamycin-induced cell death (Fig. 4A). Similarly, induction of apoptosis by tunicamycin was accompanied by an increase in caspase activity that was completely blocked by pretreating cells with D3T (Fig. 4B). Consistent with these results, tunicamycin-induced increase in expression of UPR-associated genes (Perk, Atf6, Atf4, and Chop10) was significantly suppressed by D3T treatment (Figs. 4C and D). These findings support the involvement of Nrf2 in cell survival following ER stress. D3T protection from ER stress is associated with increased proteasome expression D3T has been shown to increase the expression of proteasome subunits through Nrf2 signaling in mouse tissues and cell lines (Kwak et al., 2003a, 2007a, 2007b). Therefore, we hypothesized that D3T may mediate protection from tunicamycin cytotoxicity through its effects on proteasome expression. To test this, we used real-time PCR analysis to monitor expression of multiple subunits of the proteasome in βTC-6 cells following D3T treatment. D3T treatment increased Nrf2 target gene expression in βTC-6 cells, as shown by the levels of Nqo1 and Gclc mRNA (Fig. 5A). Expression of the catalytic subunits Psmb5, Psmb6, and Psmb7 was also significantly increased by incubation with 5 μM D3T for 24 h (Fig. 5B). In contrast, expression of the proteasomal structural subunits Psma1 and Psmc4, and

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Fig. 1. Proteasome involvement in tunicamycin-induced ER stress. (A) βTC-6 cells were incubated with tunicamycin (TUN, 2–16 μg/ml) or DMSO vehicle (Veh) for 24 h and viability was measured by the MTT assay. Values are means±SD of 8 wells. (B) Perk and Chop10 mRNA levels were measured after treatment of βTC-6 cells with vehicle, tunicamycin (1 μg/ml), or tunicamycin plus the proteasome inhibitor MG132 (2.5 μM) for 24 h. Values are means±SD of 4 experiments. aPb 0.05 compared with the vehicle control. (C) Protein levels for phospho-Perk and phospho-eIF2α were monitored in cells with tunicamycin incubation for 6 or 24 h.

(Fig. 5E). These results indicate that Nrf2 activation increased the expression of proteasome subunits and further implicate proteasome induction in cell survival following ER stress. In support of these findings, we observed that the ability of D3T to reduce tunicamycin-induced UPR gene expression, particularly the increase in Perk and Chop10, was blocked by the proteasome inhibitors MG132 and bortezomib (Figs. 6A and B). Similarly, alleviation effect of D3T on tunicamycin cytotoxicity was abolished by treatment with MG132 (Fig. 6C). Finally, we confirmed that the protective effect of D3T on tunicamycin cytotoxicity was not observed in Nrf2-silenced βTC-6 cells (Fig. 6D).

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the immunoproteasome subunits Lmp2, Lmp7, and Mecl1 were unaffected by D3T (Figs. 5C and D). While, expression of Rpn1, a non-ATPase subunit of the regulatory 19S proteasome, was elevated by D3T treatment (Fig. 5C). We confirmed that D3T treatment enhanced proteasome function by measuring levels of Psmb5 and Psmb6 protein (Fig. 5E) and subunit peptidase activities (Fig. 5F). The chymotrypsin-like activity of Psmb5 was significantly increased by D3T, whereas the trypsin-like activity of Psmb7 and caspase-like activity of Psmb6 were also increased but not to a statistically significant degree. In these cells, D3T-medaited Nrf2 activation was additionally confirmed by determining Nrf2 level in total cell lysates

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Fig. 2. Nrf2 involvement in tunicamycin-induced ER stress and cytotoxicity. (A) Nrf2 mRNA levels in βTC-6 cells expressing nonspecific scRNA (iSc) or Nrf2-specific shRNA (iNrf2). (B) ARE-driven luciferase activity in βTC-6 cells was measured following D3T (5 μM) incubation for 24 h. (C) Nqo1 mRNA in iSc and iNrf2 cells following treatment with DMSO vehicle (Veh) or D3T for 24 h. Values are means ± SD of 3 experiments. aP b 0.05 compared with the vehicle-treated group. (D) Cell viability of iSc and iNrf2 cells incubated for 24 h with DMSO vehicle (0) or tunicamycin (TUN, 2–16 μg/ml). Values are means ± SD of 8 wells. (E–H) Transcript levels of Chop10 (E), Atf3 (F), Perk (G), and Atf4 (H) in iSc and iNrf2 cells following incubation for 24 h with vehicle or tunicamycin (1 μg/ml). Values are means ± SD of 3–4 experiments. aP b 0.05 compared with each vehicle-treated group.

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Fig. 3. Tunicamycin-mediated proteasome expression is Nrf2 dependent. (A) ARE-luciferase reporter activity was measured in iSc and Nrf2i cells following incubation for 24 h with vehicle or tunicamycin (TUN, 1 μg/ml). (B–D) iSc and iNrf2 cells were incubated with vehicle or 1 μg/ml tunicamycin for 24 h and transcript levels for Nqo1, Gclm, Gsr (B), Psmb5 (C), and Psmb6 (D) were determined using real-time PCR analysis. Values are means ± SD of 3–4 experiments. aP b 0.05 compared with each vehicle-treated group.

Discussion Perturbation of the normal process of protein folding or modification within the ER initiates the UPR and ERAD adaptive responses. The UPR alleviates ER stress by modulating gene transcription and protein translation whereas the ERAD pathway enhances degradation of misfolded proteins (Tsai and Weissman, 2011; Walter and Ron, 2011). The 26S proteasome plays a major role in degradation of exported proteins from the ER (Tai and Schuman, 2008; Xie, 2010). Both UPR and ERAD contribute to normal homeostasis within the cell and their failure can activate signaling events that culminate in

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cell death. These observations have led to the recognition that ER stress plays an important role in various human diseases, including diabetes. Pancreatic β-cells often experience disturbances in ER homeostasis; for example, high glucose concentrations induce a rapid increase in insulin biosynthesis, which can generate a heavy burden of unfolded/misfolded proteins in the ER lumen (Fonseca et al., 2011; Oslowski and Urano, 2011). Short- and long-term exposure of β-cells to high glucose concentrations (> 16.7 mM) has been shown to increase expression of ER stress markers, leading to β-cell dysfunction and death (Lipson et al., 2006). In the Akita diabetes mouse model, the Cys96 mutation in insulin disrupts proinsulin folding and

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Fig. 4. D3T treatment protects βTC-6 cells from tunicamycin-mediated ER stress and cytotoxicity. (A) Cell viability was determined in βTC-6 cells treated with vehicle, tunicamycin (TUN) alone (8 and 16 μg/ml), or D3T (D, 5 μM) + tunicamycin for 48 h. Cells were preincubated with D3T for 18 h and incubated with tunicamycin for an additional 48 h. Values are means ± SD of 8 experiment wells. aP b 0.05 compared with each concentration of tunicamycin alone. (B) Caspase activity was measured in cells incubated with vehicle, tunicamycin alone (16 μg/ml), or D3T + tunicamycin for 48 h. Values are means ± SD of 3 experiments. aP b 0.05 compared with the vehicle group, bP b 0.05 compared with the tunicamycin group. (C–D) Perk, Atf6, Atf4, Chop10, and Bip mRNA levels following treatment with vehicle, tunicamycin (1 μg/ml) alone, or D3T + tunicamycin. Cells were preincubated with D3T (5 μM) for 18 h and incubated with tunicamycin for an additional 24 h. Values are means ± SD of 3–4 experiments. aP b 0.05 compared with the vehicle group. bP b 0.05 compared with the tunicamycin group.

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Fig. 5. D3T induces proteasome expression in βTC-6 cells. (A–D) Transcript levels for Nrf2 target genes Nqo1 and Gclc (A), catalytic subunits Psmb5, Psmb6, and Psmb7 (B), structural subunits Psma1, Psmc4, and Rpn1 (C), and immunoproteasome subunits Lmp2, Lmp7, and Mecl1 (D). Expression was determined in cells treated with vehicle (DMSO, veh) or D3T (2.5 and 5 μM) for 24 h. Values are means±SD of 3–4 experiments. aPb 0.05 compared with the vehicle group. (E) Protein levels for Psmb5, Psmb6, and Nrf2 in total cell lysates were determined using immunoblot analysis. Similar results were obtained in 3 individual experiments. (F) βTC-6 cells were incubated with 5 μM D3T for 24 h and proteasome catalytic activities were monitored by measuring chymotrypsin-like (Chymo), trypsin-like (Tryp), and caspase-like (Casp) peptidase activities. Values are means±SD of 3 experiments. a Pb 0.05 compared with the vehicle group.

causes ER stress and β-cell death (Wang et al., 1999). Similarly, specific mutations in human insulin lead to neonatal diabetes due to proinsulin misfolding (Stoy et al., 2007). These findings suggest that ER stress can be a cause of β-cell damage in diabetes. Aberrant levels of free fatty acids, inflammatory cytokines, and islet amyloid polypeptide are additional stimulators of β-cell ER stress (Oslowski and Urano, 2011). Thus, identification of factors that regulate UPR and ERAD and determine ER stress-induced survival or death is essential to our understanding of the pathophysiology of β-cell dysfunction. The proteasome system controls the degradation of cellular proteins that regulate the cell cycle, transcription, apoptosis, and many other cellular processes. In addition, the proteasome is largely responsible for the degradation of abnormal proteins such as unfolded/misfolded proteins, damaged proteins, and mutated proteins located in the cytosol and nucleus (Bedford et al., 2010). Under ER stress conditions, misfolded proteins, which are exported from the ER lumen to the cytosol through specific protein channels, are subsequently marked by polyubiquitination for degradation by the proteasome (Tsai and Weissman, 2010, 2011; Xie, 2010). Accumulating evidence suggests that proteasome malfunction plays a pathogenic role in various human diseases, including neurodegenerative diseases, and in aging (Rubinsztein, 2006; Tai and Schuman, 2008). Therefore, the identification of proteasome subunit assembly and gene regulation will contribute to our understanding of protein degradation in human disease. In Saccharomyces cerevisiae, the transcription factor Rpn4 coordinately controls the expression of proteasome genes under stress conditions through binding to the proteasome-associated control element (Mannhaupt et al., 1999; Xie and Varshavsky, 2001). Consistent with this, Rpn4 mutant cells have significantly reduced constitutive and inducible proteasome expression and peptidase activity compared with

wild-type cells. Proteasome genes in animal cells are also coordinately upregulated under various conditions, suggesting transcriptional regulation of proteasome expression. For example, siRNA-mediated silencing of a single proteasome subunit induces a compensatory elevation in expression of the remaining proteasome subunits (Lundgren et al., 2003). Similarly, treatment of animal cells with proteasome inhibitors enhances the expression of multiple proteasome subunits (Meiners et al., 2003). Several studies have demonstrated the role of NF-E2 transcription factors in proteasome gene regulation. The small molecule Nrf2 activator D3T was shown to elevate expression of multiple proteasome subunits in liver of wild-type mice, but not nrf2 knockout animals (Kwak et al., 2003b). Nrf2 mediated the inducible expression of the mouse catalytic subunit Psmb5 through the proximal transcriptional control element ARE, whereas basal Psmb5 expression appeared not to be affected by Nrf2 (Kwak et al., 2003a). Consequently, proteasome induction by Nrf2 signaling contributed to the protection of murine neuroblastoma cells against hydrogen peroxide-induced cytotoxicity by reducing the accumulation of oxidized proteins (Kwak et al., 2007a). Other groups have demonstrated that pharmacological induction of the proteasome delayed senescence of human fibroblasts through Nrf2 signaling (Kapeta et al., 2010) and proteasome induction by Nrf2 activators was blocked by Nrf2 siRNA introduction (Pickering et al., 2012). In addition to the involvement of Nrf2, another NF-E2‐related factor, Nrf1, was shown to mediate upregulation of proteasome genes. Treatment of murine embryonic fibroblasts (MEF) with proteasome inhibitors induced proteasome subunit expression through Nrf1 signaling (Radhakrishnan et al., 2010). Consequently, a genetic ablation of nrf1 in mouse brain led to dysregulated proteasome expression and caused neurodegeneration (Lee et al., 2011). Similar results have been reported in human cells,

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Fig. 6. Protective effect of D3T on tunicamycin-induced ER stress is Nrf2-dependent. (A–B) βTC-6 cells were preincubated with D3T (5 μM) for 18 h and tunicamycin (1 μg/ml) was added for an additional 24 h. MG132 (2.5 μM) and bortezomib (0.1 μM) were co-incubated with tunicamycin and Perk (A) and Chop10 (B) mRNA levels were determined using real-time PCR analysis. Values are means ± SD of 3–4 experiments. aP b 0.05 compared with the D3T + tunicamycin group. (C) Cell viability was monitored in βTC-6 cells following treatment with tunicamycin (TUN, 16 μg/ml) and D3T (D, 5 μM) for 24 h. MG132 was co-incubated with tunicamycin. (D) Effects of D3T on tunicamycin-mediated cytotoxicity were assessed in iSc and iNrf2 cells. Cells were incubated with tunicamycin or D3T + tunicamycin for 24 h. Values are means ± SD of 8 experimental wells. aP b 0.05 compared with the vehicle group. bP b 0.05 compared with the tunicamycin-treated group.

where transcription of the PSMB6 gene after proteasome inhibitor treatment was regulated by Nrf1 through the proximal ARE (Steffen et al., 2010). The involvement of NF-E2‐related factors in proteasome induction led to the hypothesis that the proteasome participates in the ER stress response in pancreatic β-cells. Our results confirm this hypothesis: i) Nrf2-silenced βTC-cells are more sensitive to tunicamycin cytotoxicity than are wild-type cells and show higher increases in the expression of pro-apoptotic ER stress markers. Tunicamycin-mediated activation of Nrf2 and induction of the proteasome are also attenuated by Nrf2 knockdown. ii) Treatment with the Nrf2 activator D3T protects βTC-6 cells from tunicamycin cytotoxicity and attenuated ER stress marker increases, which appear to be associated with proteasome induction. iii) D3T elevates expression and peptidase activity of the catalytic proteasome subunits in an Nrf2-dependent manner; this effect was diminished by proteasome inhibition or Nrf2 silencing. Our results linking Nrf2 to ER stress can be also supported by reports that activated Perk, a direct sensor of ER stress, can stimulate Nrf2 signaling. Perk phosphorylates eIF2α and leads to a global attenuation of protein translation to reduce ER burden (Hetz, 2012; Schroder and Kaufman, 2005). Several proteins including Atf4 and Chop10 are selectively increased by Perk, but whether this change results in cell survival or death depends on the intensity and duration of stress (Hetz, 2012; Woehlbier and Hetz, 2011). On the other hand, Perk directly phosphorylates Nrf2 to facilitate its nuclear translocation and subsequent target gene induction, which has been suggested to promote survival following ER stress (Cullinan and Diehl, 2004; Cullinan et al., 2003). In our βTC-6 cell system, tunicamycin treatment increased Nrf2 transcription activity with a concomitant elevation of Nqo1 in an Nrf2-dependent manner, indicating that the ER stress can activate Nrf2 signaling through Perk. This indicates that Perk-mediated Nrf2 activation together with enhanced ERAD can account for the protective effect of Nrf2-proteasome signaling on tunicamycin-induced ER stress. Collectively, our current results provide evidence that Nrf2 signaling participates in the ER stress defense system of pancreatic β-cells by enhancing proteasome-mediated ERAD. This information expands

our knowledge of how cells integrate and initiate the cellular response networks under various stress conditions, such as ER stress. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgments This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010–0016808, 2011–0017977). This work was also supported by the Research Fund, 2011 of The Catholic University of Korea. Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.taap.2012.08.021. References Back, S.H., Kaufman, R.J., 2012. Endoplasmic reticulum stress and type 2 diabetes. Annu. Rev. Biochem. 81, 767–793. Bedford, L., Paine, S., Sheppard, P.W., Mayer, R.J., Roelofs, J., 2010. Assembly, structure, and function of the 26S proteasome. Trends Cell Biol. 20, 391–401. Cho, J.M., Manandhar, S., Lee, H.R., Park, H.M., Kwak, M.K., 2008. Role of the Nrf2antioxidant system in cytotoxicity mediated by anticancer cisplatin: implication to cancer cell resistance. Cancer Lett. 260, 96–108. Cullinan, S.B., Diehl, J.A., 2004. PERK-dependent activation of Nrf2 contributes to redox homeostasis and cell survival following endoplasmic reticulum stress. J. Biol. Chem. 279, 20108–20117. Cullinan, S.B., Zhang, D., Hannink, M., Arvisais, E., Kaufman, R.J., Diehl, J.A., 2003. Nrf2 is a direct PERK substrate and effector of PERK-dependent cell survival. Mol. Cell. Biol. 23, 7198–7209. Fonseca, S.G., Gromada, J., Urano, F., 2011. Endoplasmic reticulum stress and pancreatic beta-cell death. Trends Endocrinol. Metab. 22, 266–274. Hampton, R.Y., 2000. ER stress response: getting the UPR hand on misfolded proteins. Curr. Biol. 10, R518–R521.

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Hetz, C., 2012. The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nat. Rev. Mol. Cell Biol. 13, 89–102. Kapeta, S., Chondrogianni, N., Gonos, E.S., 2010. Nuclear erythroid factor 2-mediated proteasome activation delays senescence in human fibroblasts. J. Biol. Chem. 285, 8171–8184. Kim, T.H., Hur, E.G., Kang, S.J., Kim, J.A., Thapa, D., Lee, Y.M., Ku, S.K., Jung, Y., Kwak, M.K., 2011. NRF2 blockade suppresses colon tumor angiogenesis by inhibiting hypoxia-induced activation of HIF-1alpha. Cancer Res. 71, 2260–2275. Kwak, M.K., Wakabayashi, N., Greenlaw, J.L., Yamamoto, M., Kensler, T.W., 2003a. Antioxidants enhance mammalian proteasome expression through the Keap1-Nrf2 signaling pathway. Mol. Cell. Biol. 23, 8786–8794. Kwak, M.K., Wakabayashi, N., Itoh, K., Motohashi, H., Yamamoto, M., Kensler, T.W., 2003b. Modulation of gene expression by cancer chemopreventive dithiolethiones through the Keap1-Nrf2 pathway. Identification of novel gene clusters for cell survival. J. Biol. Chem. 278, 8135–8145. Kwak, M.K., Cho, J.M., Huang, B., Shin, S., Kensler, T.W., 2007a. Role of increased expression of the proteasome in the protective effects of sulforaphane against hydrogen peroxide-mediated cytotoxicity in murine neuroblastoma cells. Free Radic. Biol. Med. 43, 809–817. Kwak, M.K., Huang, B., Chang, H., Kim, J.A., Kensler, T.W., 2007b. Tissue specific increase of the catalytic subunits of the 26S proteasome by indirect antioxidant dithiolethione in mice: enhanced activity for degradation of abnormal protein. Life Sci. 80, 2411–2420. Lee, C.S., Lee, C., Hu, T., Nguyen, J.M., Zhang, J., Martin, M.V., Vawter, M.P., Huang, E.J., Chan, J.Y., 2011. Loss of nuclear factor E2-related factor 1 in the brain leads to dysregulation of proteasome gene expression and neurodegeneration. Proc. Natl. Acad. Sci. U. S. A. 108, 8408–8413. Li, W., Kong, A.N., 2009. Molecular mechanisms of Nrf2-mediated antioxidant response. Mol. Carcinog. 48, 91–104. Lipson, K.L., Fonseca, S.G., Ishigaki, S., Nguyen, L.X., Foss, E., Bortell, R., Rossini, A.A., Urano, F., 2006. Regulation of insulin biosynthesis in pancreatic beta cells by an endoplasmic reticulum-resident protein kinase IRE1. Cell Metab. 4, 245–254. Lundgren, J., Masson, P., Realini, C.A., Young, P., 2003. Use of RNA interference and complementation to study the function of the Drosophila and human 26S proteasome subunit S13. Mol. Cell. Biol. 23, 5320–5330. Mannhaupt, G., Schnall, R., Karpov, V., Vetter, I., Feldmann, H., 1999. Rpn4p acts as a transcription factor by binding to PACE, a nonamer box found upstream of 26S proteasomal and other genes in yeast. FEBS Lett. 450, 27–34. Meiners, S., Heyken, D., Weller, A., Ludwig, A., Stangl, K., Kloetzel, P.M., Kruger, E., 2003. Inhibition of proteasome activity induces concerted expression of proteasome genes and de novo formation of Mammalian proteasomes. J. Biol. Chem. 278, 21517–21525. Mittal, S., Ganesh, S., 2010. Protein quality control mechanisms and neurodegenerative disorders: checks, balances and deadlocks. Neurosci. Res. 68, 159–166. Oslowski, C.M., Urano, F., 2011. The binary switch that controls the life and death decisions of ER stressed beta cells. Curr. Opin. Cell Biol. 23, 207–215. Pickering, A.M., Linder, R.A., Zhang, H., Forman, H.J., Davies, K.J., 2012. Nrf2-dependent

induction of proteasome and Pa28alphabeta regulator are required for adaptation to oxidative stress. J. Biol. Chem. 287, 10021–10031. Poitout, V., Stout, L.E., Armstrong, M.B., Walseth, T.F., Sorenson, R.L., Robertson, R.P., 1995. Morphological and functional characterization of beta TC-6 cells—an insulin-secreting cell line derived from transgenic mice. Diabetes 44, 306–313. Poppek, D., Grune, T., 2006. Proteasomal defense of oxidative protein modifications. Antioxid. Redox Signal. 8, 173–184. Radhakrishnan, S.K., Lee, C.S., Young, P., Beskow, A., Chan, J.Y., Deshaies, R.J., 2010. Transcription factor Nrf1 mediates the proteasome recovery pathway after proteasome inhibition in mammalian cells. Mol. Cell 38, 17–28. Rubinsztein, D.C., 2006. The roles of intracellular protein-degradation pathways in neurodegeneration. Nature 443, 780–786. Schroder, M., Kaufman, R.J., 2005. The mammalian unfolded protein response. Annu. Rev. Biochem. 74, 739–789. Shore, G.C., Papa, F.R., Oakes, S.A., 2011. Signaling cell death from the endoplasmic reticulum stress response. Curr. Opin. Cell Biol. 23, 143–149. Steffen, J., Seeger, M., Koch, A., Kruger, E., 2010. Proteasomal degradation is transcriptionally controlled by TCF11 via an ERAD-dependent feedback loop. Mol. Cell 40, 147–158. Stoy, J., Edghill, E.L., Flanagan, S.E., Ye, H., Paz, V.P., Pluzhnikov, A., Below, J.E., Hayes, M.G., Cox, N.J., Lipkind, G.M., Lipton, R.B., Greeley, S.A., Patch, A.M., Ellard, S., Steiner, D.F., Hattersley, A.T., Philipson, L.H., Bell, G.I., 2007. Insulin gene mutations as a cause of permanent neonatal diabetes. Proc. Natl. Acad. Sci. U. S. A. 104, 15040–15044. Taguchi, K., Motohashi, H., Yamamoto, M., 2011. Molecular mechanisms of the Keap1Nrf2 pathway in stress response and cancer evolution. Genes Cells 16, 123–140. Tai, H.C., Schuman, E.M., 2008. Ubiquitin, the proteasome and protein degradation in neuronal function and dysfunction. Nat. Rev. Neurosci. 9, 826–838. Tsai, Y.C., Weissman, A.M., 2010. The unfolded protein response, degradation from endoplasmic reticulum and cancer. Genes Cancer 1, 764–778. Tsai, Y.C., Weissman, A.M., 2011. Ubiquitylation in ERAD: reversing to go forward? PLoS Biol. 9, e1001038. Walter, P., Ron, D., 2011. The unfolded protein response: from stress pathway to homeostatic regulation. Science 334, 1081–1086. Wang, J., Takeuchi, T., Tanaka, S., Kubo, S.K., Kayo, T., Lu, D., Takata, K., Koizumi, A., Izumi, T., 1999. A mutation in the insulin 2 gene induces diabetes with severe pancreatic beta-cell dysfunction in the Mody mouse. J. Clin. Invest. 103, 27–37. Woehlbier, U., Hetz, C., 2011. Modulating stress responses by the UPRosome: a matter of life and death. Trends Biochem. Sci. 36, 329–337. Xie, Y., 2010. Structure, assembly and homeostatic regulation of the 26S proteasome. J. Mol. Cell Biol. 2, 308–317. Xie, Y., Varshavsky, A., 2001. RPN4 is a ligand, substrate, and transcriptional regulator of the 26S proteasome: a negative feedback circuit. Proc. Natl. Acad. Sci. U. S. A. 98, 3056–3061. Zabel, C., Nguyen, H.P., Hin, S.C., Hartl, D., Mao, L., Klose, J., 2010. Proteasome and oxidative phoshorylation changes may explain why aging is a risk factor for neurodegenerative disorders. J. Proteomics 73, 2230–2238.