Thioredoxin-1 promotes survival in cells exposed to S-nitrosoglutathione: Correlation with reduction of intracellular levels of nitrosothiols and up-regulation of the ERK1/2 MAP Kinases

Toxicology and Applied Pharmacology 233 (2008) 227–237

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Thioredoxin-1 promotes survival in cells exposed to S-nitrosoglutathione:Correlation with reduction of intracellular levels of nitrosothiols and up-regulation ofthe ERK1/2 MAP Kinases

Roberto J. Arai a,⁎, Fernando T. Ogata a, Wagner L. Batista a, Hiroshi Masutani b, Junji Yodoi b, Victor Debbas a,Ohara Augusto c, Arnold Stern d, Hugo P. Monteiro a,⁎a Department of Biochemistry/Molecular Biology, CINTERGEN, Universidade Federal de São Paulo, Escola Paulista de Medicina, Rua 3 de Maio no. 100, 4° andar, CEP 04044-020,São Paulo, SP, Brazilb Department of Biological Responses, Institute for Virus Research, Kyoto University, Kyoto, Japanc Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazild Department of Pharmacology, New York University School of Medicine, NY, NY, USA

Abbreviations: ASK-1, apoptosis signal-regulating kminepentaacetic acid; GST-RBD, glutathione S-transferaGEF, Guanosine Nucleotide Exchange Factor; GSNO,horseradish peroxidase; MAP Kinase, mitogen activatedMaleimide; •NO, nitric oxide; PMSF, Phenylmethylsuliodide; RNS, reactive nitrogen species; ROS, reactive oxTRX-1, Thioredoxin-1.⁎ Corresponding authors. Fax: +55 11 5573 6407.

E-mail addresses: [email protected] (R.J. Arai), hp(H.P. Monteiro).

0041-008X/$ – see front matter © 2008 Elsevier Inc. Aldoi:10.1016/j.taap.2008.07.023

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Keywords:ThioredoxinNitrosothiolNitric oxideNitrotyrosinep21RasERK1/2 MAP kinases

cates that post-translational protein modifications by nitric oxide and its derivedspecies are critical effectors of redox signaling in cells. These protein modifications are most likely controlledby intracellular reductants. Among them, the importance of the 12 kDa dithiol protein thioredoxin-1 (TRX-1)has been increasingly recognized. However, the effects of TRX-1 in cells exposed to exogenous nitrosothiolsremain little understood. We investigated the levels of intracellular nitrosothiols and survival signaling inHeLa cells over-expressing TRX-1 and exposed to S-nitrosoglutahione (GSNO). A role for TRX-1 expression onGSNO catabolism and cell viability was demonstrated by the concentration-dependent effects of GSNO ondecreasing TRX-1 expression, activation of caspase-3, and increasing cell death. The over-expression of TRX-1in HeLa cells partially attenuated caspase-3 activation and enhanced cell viability upon GSNO treatment. Thiswas correlated with reduction of intracellular levels of nitrosothiols and increasing levels of nitrite andnitrotyrosine. The involvement of ERK, p38 and JNK pathways were investigated in parental cells treated withGSNO. Activation of ERK1/2 MAP kinases was shown to be critical for survival signaling. In cells over-expressing TRX-1, basal phosphorylation levels of ERK1/2 MAP kinases were higher and further increasedafter GSNO treatment. These results indicate that the enhanced cell viability promoted by TRX-1 correlateswith its capacity to regulate the levels of intracellular nitrosothiols and to up-regulate the survival signalingpathway mediated by the ERK1/2 MAP kinases.

© 2008 Elsevier Inc. All rights reserved.

Introduction

Nitric oxide (•NO) is a gaseous free radical which performs diverseactions in living organisms. At physiological (low) levels, •NO mayreact with protein cysteine thiols, converting them into S-nitro-sothiols. S-nitrosation of signaling proteins such as Ras, Jun-N-terminal Kinase (JNK), and caspase 3 is emerging as a redox-based

inase-1; DTPA, Diethylenetria-se-Raf-1-Ras Binding Domain;S-nitroso glutathione; HRP,protein kinase NEM, N-Ethyl

phonylfluoride; PI, propidiumygen species; •O2

- , superoxide;

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post-translational modification of proteins essential for NO-mediatedsignaling (Hess et al., 2005).

At toxic (high) levels such as those found in inflamed tissues, •NOproduced by inflammatory cells can damage DNA leading to increasedmutations and altered enzyme and protein function, essential to themultistage carcinogenesis process (Ohshima et al., 2004). On the otherhand, high levels of •NO and its related species are also implicated asantitumoral mediators by acting at distinct levels of cancer biology(Kim et al., 2001a,b; Mocellin et al., 2007). Several studies haveaddressed the potential use of •NO donors in the treatment of canceras agents capable of enhancing the tumor sensitivity for furtherchemotherapy or radiotherapy or even inducing apoptosis (Millet etal., 2002; Gao et al., 2005; Hirst and Robson, 2007; Lee et al., 2007).

There is a correspondence between physiological and toxicfunctions of NO and its production in low and high concentrations.It is well demonstrated that induction of cytostasis or apoptosisrequires relatively high concentrations of NO, a condition that may

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promote nitrosative stress (Eu et al., 2000; Jarry et al., 2004; Hirst andRobson, 2007).

Nitrosative stress conditions target critical intracellular compo-nents that control proliferation and survival. For instance, thenitrosation of cysteine residues of p21Ras is associated with itsactivation whereas the nitrosation of JNK leads to inhibition of its

Fig. 1. TRX-1 expression and cell viability. (A) HeLa cells were treated with increasing conceRelative mRNA levels of TRX-1 were determined by real-time PCR. Values represent as meconcentrations of GSNO (0.5–2 mM) for 8 h. Cells were harvested, lysed and the extracts wanti-β-Actin. (C) Caspase-3 activity was determined by Western blot analysis detectionrepresent means±S.D. from three independent experiments. (D — upper panel) In theAnnexin-V/PI labeling followed by FACS analysis. (D — lower panel) Percentages of viable cStatistical analysis were performed by one-way analysis of variance (ANOVA), followed b

kinase activity (Lander et al., 1997; Park et al., 2000). Severalenzymatic and non-enzymatic systems are involved in the catabolismof nitrosothiols, thereby altering cell-specific responses to nitrosativestress. Particularly, studies have shown that the thioredoxin system isinvolved in the catabolism of nitrosothiols (Nikitovic and Holmgren,1996; Nikitovic et al., 1998; Stoyanovsky et al., 2005).

ntrations of GSNO (0.5–2.0 mM) for 6 h. Cells were lysed and total RNA was extracted.an of three independent experiments±S.E. (B) HeLa cells were treated with increasingere submitted to double labeling Western blotting with anti-human TRX-1 mAB andof pro-caspase-3 (upper panel) or by a colorimetric assay (middle panel). Valuessame experimental conditions, cell viability assessments were performed by usingells shown in bars are expressed as mean±S.D. from three independent experiments.y Bonferroni multiple comparison test (⁎pb0.05).

Fig. 2. Overexpression of TRX-1 and cell viability. (A) HeLa cells transfected with Tet-off system encoding wild type TRX-1 (HeLa Tet-off-TRX-1) were induced to express TRX-1 byculturing cells in the absence of tetracycline (TC-) for 96 h. Cell extracts were submitted to double labeling Western blotting as indicated. (B) HeLa Tet-off-TRX-1 cells cultured in thepresence (TC+) or absence (TC-) of tetracycline were maintained in MEM containing 10% FBS and media were changed daily. Cell number was determined every-other day for up to5 days with use of a hematocytometer. Values represented aremean±S.D of three independent experiments. (C) Caspase-3 activity was determined byWestern blot detection of pro-caspase-3. (D— upper panel) HeLa Tet-off-TRX-1 cells cultured in the presence (TC+) or absence (TC-) of tetracycline were treated with 1 mMGSNO for 8 h and submitted to viabilityassessments by using AnnexinV/PI labeling followed by FACS analysis. (D — lower panel) Percentage of viable cells shown in bars are expressed as mean±S.D. from threeindependent experiments. A two-way ANOVA of the data indicates significant effects of GSNO (F=149.09; df=1; pb0.0001), TC- (F=15.06; df=1; p=0.005), and the interactionbetween GSNO and TC- (F=25. 84; df=1; p=0.001). ⁎ indicates that TC- values are significantly different from TC+ values (p=0.01; one-way ANOVA followed by Bonferroni's test).

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Thioredoxin (TRX-1) is a 12 kDa multifunctional dithiol reducingprotein that is found over-expressed in several tumors. Its over-expression in human cancers is correlated with poorer prognosis andresistance to several anticancer agents (Yokomizo et al., 1995; Raffel etal., 2003; Kim et al., 2005). TRX-1 is uniquely expressed in theextracellular, cytoplasmic and nuclear milieu, with distinct functionsin each compartment (Masutani et al.,1996; Hirota et al.,1999; Ueno etal., 1999; Nakamura et al., 2001; Watson et al., 2003; Monteiro et al.,2008). In vitro experiments showed that TRX-1 is capable of reducing

nitrosated substrates to thiols in a variety of proteins and peptidesincluding the transcription factor AP-1, Caspase-3, metallothionein andS-nitrosoglutathione (GSNO) (Nikitovic and Holmgren, 1996; Nikitovicet al., 1998; Stoyanovsky et al., 2005). The denitrosation reactionsmediated by TRX-1 may proceed by trans-S-nitrosation followed byhomolytic breakdown of S-N bonds to yield •NO, anion radical super-oxide (•O2

−) and the end product nitrite (NO2−), whereas heterolytic

breakdown yields nitroxyl (HNO) and hydroxylamines (NH2OH)(Nikitovic et al., 1996; Stoyanovsky et al., 2005). The combination of

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the secondary productsmay also generate other reactive species such asperoxynitrite (ONOO-) the product of a rapid diffusionally controlledreaction between •NOand •O2

− (Beckman et al.,1990; Kissner et al.,1997).ONOO-promotes nitration of tyrosine residues and is a very effectiveoxidant for thiols and lipids (Ischiropoulos, 1998).

Although the biochemical basis of the metabolism of nitrosothiolsby TRX-1 has been described earlier, only a few studies addressing themetabolism of nitrosothiols by TRX-1 and its implications on cellsignaling events have been performed (Haendeler et al., 2002; Kahloset al., 2003). In the present study we investigated the role of TRX-1expression in the cellular metabolism of nitrosothiols and itsconsequences on the Ras-ERK1/2 signaling pathway associated withcell survival in cells exposed to pro-apoptotic concentrations of GSNO.

Materials and methods

Reagents. S-nitroso Glutathione (GSNO), farnesyltransferase inhibitorIII (FPT III), MEK inhibitor (PD98059), p38 inhibitor (SB220025),propidium iodide (PI), and a rabbit polyclonal anti-phospho JNK werepurchased from Calbiochem (San Diego, CA, USA). Anti-human TRX-1monoclonal antibody (11 mAB) was provided by Redox Bioscience(Kyoto, Japan). Anti-Pan Ras monoclonal antibody was purchased fromOncogene Science (Boston, MA, USA). Anti-phospho ERK1/2 MAPKinases, anti-ERK1/2 MAP Kinases rabbit polyclonal antibody, andanti-phospho p38 polyclonal antibody were purchased from CellSignaling Technologies (Beverly,MA,USA). Anti-nitrotyrosinepolyclonalantibody was purchased from Upstate Biotechnology (Lake Placid, NY,USA). Anti-β actin monoclonal antibody, nitrated serum bovinealbumine (BSANit), and N-ethyl-maleimide (NEM) were purchasedfrom Sigma-Aldrich (St. Louis, MO, USA). Anti-caspase 3 (CPP32) rabbitpolyclonal antibody was purchased from BD-Pharmingen (San Diego,CA, USA). Annexin-V-FITC, and Lipofectamine® reagent were purchasedfrom Invitrogen-Molecular Probes (Carlsbad, CA, USA).

Cell cultures. The cervical carcinoma cell line (HeLa) was cultured inMinimum Essential Medium (MEM) supplemented with 1 mMsodium pyruvate, 10% fetal bovine serum, penicillin (100 U/ml) andstreptomycin (100 μg/ml) at 37 °C, 5% CO2.

Plasmids and transfections. HeLa cells with tetracycline inducibleTRX-1 expressionwere generated by using the Tet-off system accordingto the manufacturer's protocol (Clontech, Mountain View, CA, USA).Briefly, HeLa cells were transfected with Tet-off system plasmids withLipofectamine® reagent. Transformed cells were selected, cloned andcultured with 0.5 mg/ml Geneticin® disulfate (ICN Biomedicals —

Aurora, OH, USA). Cloned cells were then co-transfected withresponsive plasmid encoding Wild Type TRX-1 and hygromicinresistant plasmid. HeLa-Tet-off-TRX-1 cells were selected and clonedin medium containing 2 μg/mL hygromycin, 0.5 mg/ml Geneticin® and1 μg/mL tetracycline. The control of gene expression was analyzed bythe luciferase assay. In all experiments, HeLa-Tet-off-TRX-1 cells werecultured in the absence or presence of tetracycline for 96 h. Then 1x106

cells were seeded in 100 mm cell culture dishes 16 h prior to the tests.

Quantitative real-time reverse transcription polymerase chain reaction(Real-time PCR). Two to 5 μg of total RNA were treated with DNase(Invitrogen) according to the manufacturer's instructions and an aliquotof 7.5 μl of the treated RNA was reverse-transcribed to cDNA using theSuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). Real-time PCR reactions were performed using SYBR Green PCR Master Mix(Applied Biosystems) in a GeneAmp 5700 Sequence Detection System(Applied Biosystems). The primers were obtained from IDT (Coralville,IA, USA), and the sequences optimized for real-time PCRwere as follows:

TRX-1: 5'-TGG TGA AGC AGA TCG AGA GCA AGA and 3'-ACC ACGTGG CTG AGA AGT CAA CTA, RPL13A: 5'-CCT GGA GGA GAA GAG GAAAGA GAand 3'-TTG AGG ACC TCT GTG TAT TTG TCA A.

Real-time PCR reactions were performed in triplicates andcontained 2 μl of a 1:10 dilution of the synthesized cDNA, primers toa final concentration of 600 nM each, 12.5 μl of the SYBR Green PCRMaster Mix and PCR-grade water to a total volume of 25 μl. Theparameters for the PCR reactionwere 50 °C for 2 min, 95 °C for 10min,40 cycles of 95 °C for 15 s and 60 °C for 1 min. The specificity of theamplified products was evaluated by the analysis of the dissociationcurves generated. The relative expression ratio (experimental/control)was determined based on the 2-[Delta][Delta]Ct method (Livak andSchmittgen, 2001). Basic procedures and calculations were performedas described previously (Batista et al., 2007).

Western blotting analysis for TRX-1, caspase-3, p38, JNK and ERK1/2. Westernblotting for TRX-1: At each time point, cells were lysed in lysis buffercontaining 20 mM Hepes pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 1.0 mMEGTA, 10% glycerol, 1% Triton, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and1.0 mM PMSF. Total cell lysates (60 μg/lane) were resolved in 15% SDS-PAGE gels, and blotted onto nitrocellulose sheets (GE Healthcare, UK).After incubation with a blocking solution containing 10% skim milk inPBS-T (PBS pH 7.6; Tween 0.1%), the membrane was incubated withantigen-specific monoclonal antibodies against human TRX-1 followedby incubationwith an appropriate secondary HRP-conjugated antibody.Western blots were developed using the Super Signal chemilumines-cence-based system (Pierce, Rockford, IL, USA). Western blotting tocaspase-3: Cell lysates (60 μg/lane) were resolved in 12% SDS-PAGE gels,and blotted onto nitrocellulose sheets (GE Healthcare, UK). Afterincubation with a blocking solution containing 10% skim milk in PBS-T(PBS pH 7.6; Tween 0.1%), the membrane was incubated with antigen-specific polyclonal antibodies against human pro-caspase-3 (BD-Pharmingen) followed by incubation with an appropriate secondaryHRP-conjugated antibody.Western Blotting for p38, JNK and ERK1/2: Ateach time point, cells were lysed in buffer containing 20 mM Hepes,150mMNaCl, 1.5 mMMgCl2, 1.0 mMEGTA,10% glycerol, 1% Triton,1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1.0 mM PMSF and phosphatasesinhibitors (2 mM sodium orthovanadate, 50 mM NaF, and 10 mMsodium pyrophosphate), pH 7.5. Total cell lysates (60 μg/lane) wereresolved in 12% SDS-PAGE gels, and blotted onto nitrocellulose sheets.Blots were probed using polyclonal antibodies against ERK1/2 MAPKinases and phospho-ERK1/2 MAP Kinases, polyclonal antibodiesagainst p38 MAP Kinase, or polyclonal antibodies against phospho-JNK MAP Kinase. After incubation with appropriate HRP-conjugatedsecondary antibodies, immunoblots were developed by using the SuperSignal chemiluminescence-based system (Pierce, Rockford, IL, USA).

Assay for caspase-3 (CPP32) activity. Caspase-3 activitywas assessedby using ApoAlert Caspase 3 (BD-Biosciences Clontech) according tomanufacturer's instructions. Spectrophotometric (405 nm) detectionof the cleavage product of the cromophore p-Nitroanilide (pNA) wasused to estimate CPP32 activity.

p21ras activity assay. The Raf-1 Ras binding domain (RBD) interactswith the GTP-bound state of the three isoforms of p21ras, H-Ras, K-Ras,andN-Ras. The activity of p21raswas assessed using the glutathione S-transferase-Raf-1-Ras Binding Domain (GST-RBD) fusion protein(Monteiro et al., 2005). Briefly, Glutathione-Sepharose® beads boundto the GST-RBD fusion protein were incubated with 300 μg of celllysates for 3 h. Bound protein was resolved by Western blot probedwith a mouse monoclonal anti-pan Ras antibody (Oncogene ScienceBoston, MA, USA). 50 μg of total cell lysateswere submitted toWesternblot analysis and probed with the same antibody to determineendogenous p21ras expression. For the GST-RBD loading control, thegel was stainedwith Coomassie BlueG250. Proteinswere visualized bythe Super Signal chemiluminescence-based detection system.

S-nitrosothiol, nitrite and nitrate determinations. Analyses of S-nitrosothiols, nitrite, and nitrate were performed following the

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procedure previously described by Feelisch et al., (2002) withminimalmodification. Cells were harvested and treated with lysis buffercontaining 10 mM NEM and 0.1 mM DTPA for 30 min. Cell extractswere treated with 10% (v/v) of 5% sulfanilamide in 1 N HCl for 30 minat room temperature to eliminate the nitrite background. To estimatemercury-stable complexes (nitrosamines and nitrosylhemes), cellextracts were treated with 10% (v/v) of 0.2% mercury chloride and 5%sulfanilamide in 1 N HCl (30 min). Treated extracts were injected intoa vessel containing potassium iodide (45 mM), iodine (10 mM),decanol (ca. 5%) in glacial acetic acid (HAc) maintained at 60 °C andactively purged with a helium stream in line with an NO analyzer(Sievers, Boulder, CO, USA). All procedures were performed in thedark. To measure nitrate, a saturated solution of vanadium (III)chloride in 1N hydrochloric acid (0.8 g/100 mL) was used to convertnitrate to nitric oxide. The reduction was performed at ∼90 °C in thepurge vessel of the NO Analyzer. The amount of nitrate present insamples was determined by the difference between the concentra-tions obtained using the reducting reagents VCl3/HCl and I2/I−/HAc.

Nitrotyrosine detection. At each time point, cells were lysed in buffercontaining 20 mM Hepes pH 7.5, 150 mM NaCl, 1.5 mMMgCl2, 1.0 mM

Fig. 3. Intracellular levels of GSNOmetabolites and breakdownproducts. (A) HeLa-Tet-off-TRX1 mM GSNO or vehicle (Control) for 1 h followed by cell lysis in NEM containing buffer. Cellschemiluminescence signals (mV)were obtained from 54 μg of extracted protein. (B) HeLa-Tet-harvested and lysed. The quantification of nitrosothiols (SNO) (upper panel), nitrite (NO2

−) (mbased assay as described inMaterials andmethods. Values are expressed asmean±S.D. of foutime and TC, as well as an interaction between time and TC, for SNO, NO2

− and NO3− (pb0.01 a

values (p=0.02, at least; two-way ANOVA followed by Bonferroni's test). (C) HeLa Tet-off-TRXcollected and 5 μg of extracted proteins were blotted onto a nitrocellulose membrane. Nitrabovine serum albumin (BSA) was used as a negative control, and nitrated-BSA was used as aloading control (lower panel). Relative densitometric values of dot plotting are shown in co

EGTA, 10% glycerol, 1% Triton, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and1.0 mM PMSF. Five micrograms of cell protein extracts were plottedonto a nitrocellulose membrane. Nitrotyrosine detection was per-formed by using a polyclonal anti-nitrotyrosine antibody. Bovineserum albumin was used as a negative control and nitrated bovineserum albumin as a positive control.

Assay for cell death. Cell viability was assessed by flow cytometry-based analysis (FACScalibur, BD Biosciences Immunocytometry Sys-tems, USA) using Annexin V-FITC and propidium iodide (PI). AnnexinV/PI labeling was performed according to the manufacturer's instruc-tions. Cell Quest software (BD Biosciences Immunocytometry Systems,USA) was used to analyze 10,000 gated events. Viable cells (Lower leftquadrant) were identified by their ability to exclude Annexin V and PI.

Densitometric analysis. Bands and plots optical densities wereevaluated by using Tollab TL100 software.

Statistical analysis. All values are expressed as mean± S.D. Eachvalue is the mean of at least three separate experiments in eachgroup. Statistical significance was assessed by one- or two-way

-1 cells cultured in the absence (TC-) or presence (TC+) of tetracyclinewere treated withextracts were treated with sulfanilamide (A) or sulfanilamide + HgCl2 (B). The detectedoff-TRX-1 cells were treatedwith 1mMof GSNO for 1–8 h. At each time point, cells wereiddle panel) and nitrate (NO3

- ) (lower panel) was determined by a chemiluminescence-r independent experiments. A two-way ANOVA of the data indicates significant effects oft least, in all such cases). ⁎ indicates that TC- values are significantly different from TC+-1 cells were culture with 1 mM GSNO at indicated times. At each time point cells wereted tyrosine residues were detected by using an anti-nitrotyrosine polyclonal antibodypositive control. The visualization of proteins on membrane by Ponceau-S was used aslumns. Image is representative of three independent experiments.

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ANOVA as appropriate with post-hoc Bonferroni test for comparisonbetween groups. Pb0.05 was considered statistically significant.

Results

Down-regulation of TRX-1 expression by GSNO correlates with cell death

A critical role for TRX-1 in regulating the viability of cells submittedto several stressors has been previously reported (for review:Nakamura et al., 1997; Hirota et al., 2002; Watson et al., 2003; Benharand Stamler, 2005). In addition, stress conditions may also regulatethe expression levels of TRX-1 (Hirota et al., 2002; Hansen et al., 2004).We then evaluate the mRNA levels of TRX-1 in HeLa cell culturesexposed to increasing concentrations of the low molecular weightnitrosothiol GSNO. TRX-1 mRNA expression did not change afterincubation of HeLa cells with increasing concentrations of GSNO(Fig. 1A). However, at the protein level, the addition of increasingconcentrations of GSNO to HeLa cell cultures resulted in down-regulation of TRX-1 expression (Fig. 1B) while inducing activation ofcaspase 3 and cell death (Figs. 1C and D).

Overexpression of TRX-1 correlates with increased cell viability in HeLacultures treated with GSNO

To investigate a role for TRX-1 expression in cells exposed toGSNO, we transfected HeLa cells with the tetracycline induciblesystem (Tet-off System) harboring wild type TRX-1 (see Materialsand methods for details) (Fig. 2A). HeLa-Tet-off cells induced toexpress TRX-1 (TC-) showed no toxicity, but rather enhanced cellproliferation as observed previously (Powis et al., 1994) (Fig. 2B). Wethen examined the effects of 1 mM GSNO on HeLa cells over-expressing TRX-1. TRX-1 over-expression did not alter caspaseexpression and activation in cells without GSNO treatment, butattenuated the caspase 3 cleavage and activity (Fig. 2C). Accordingly,the resulting action of TRX-1 over-expression (TC-) conferred asignificant increase in cell viability when compared to cellsexpressing basal levels of TRX-1 (TC+). Indeed, upon 1 mM GSNOtreatment of TC- and TC+ cells, they lost their viability by 23% and54%, respectively (Fig 2D). Similar results were obtained when weuse the MTT cell viability assay (data not shown).

Intracellular levels of GSNO metabolites and breakdown products

Studies of GSNO uptake andmetabolism bywhole animals (Bryan etal., 2004), cell cultures (Zenget al., 2001; Zhang andHogg, 2004) and cellfractions (Beer et al., 2004; Dahmet al., 2006) have indicated that GSNOcatabolism yields low levels of •NO and high levels of intracellularnitrosothiols. By using an ozone-based chemiluminescence assay, weassessed the intracellular levels of nitrosothiols in HeLa Tet-off cellsexposed to 1 mM GSNO for 1 to 8 h. Although in different proportions,HeLa cells over-expressingTRX-1or not (TC−or TC+) showed an increasein the intracellular levels of nitrosothiols, but not nitrosylhemes andnitrosoamines (as indicate in samples treated with sulfanilamide+HgCl2(see Materials and methods for details — Fig. 3A). The signal obtainedfrom the extracts of cells over-expressing TRX-1 showed a lower peak(mV) than that observed from cells expressing basal levels of TRX-1 (Fig3A). Intracellular nitrosothiols were significantly lower in cells over-expressing TRX-1 (in 1 and 2h)when compared to cells expressing basallevels of TRX-1 (Fig 3B— upper panel). However, the intracellular levelsof nitrite found in cells over-expressing TRX-1 treatedwithGSNO for 1 hand 2 h were found to be significantly higher than in cells expressingbasal levels of TRX-1 (Fig. 3B—middle panel), suggesting that the excessof TRX-1 may further promote the catabolism of nitrosothiols.Conversely, the levels of nitrate in cells expressing basal levels of TRX-1 were found significantly higher than in cells over-expressing TRX-1(Fig 3B — lower panel).

Previous observations indicated that the secondary products ofnitrosothiols catabolismmay include •NO and •O2

− and possibly ONOO-,a powerful nitrating agent (Nikitovic and Holgrem, 1996; Ischiropou-los, 2003). We then tried to detect nitrotyrosines (protein 3-nitrotyrosine), the “footprint” for nitrating agents. In HeLa cellsover-expressing TRX-1 the levels of nitrotyrosines were maximal after1–2 h of incubationwith 1 mM GSNO followed by a decrease in 4–8 h.In cells expressing basal levels of TRX-1, nitrotyrosine levels increasedonly after 4–8 h of treatment with 1 mM GSNO and were markedlylower than in cells over-expressing TRX-1 (Fig. 3C).

Critical role of the ERK1/2 MAP kinases in cell viability

Earlier studies have demonstrated that protein modificationsmediated by •NO are key regulators of signaling pathways associatedwith cell viability (Marshall and Stamler, 2002, Sumbayev andYasinska,2005). Among them, the Mitogen Activated-Protein Kinases (MAPKs);c-Jun-N-Terminal Kinase (JNK), p38 MAP Kinase and the ExtracellularSignal-Regulating Kinases 1 and 2 (ERK1/2), have been implicated:(Callsen and Brune.,1999; Benhar and Stamler, 2005). •NOmay regulateJNK and p38 activity through nitrosation reactions targeting ApoptosisSignal-Regulated Kinase-1 (Ask-1) (Sumbayev and Yasinska, 2005)whereas ERK1/2 may be regulated by nitrosation of p21Ras (Lander etal., 1997; Rocha Oliveira et al., 2003). In this study, we assessed theactivity of ERK1/2, JNK1/2 and p38 through the immunodetection oftheir phosphorylated forms (active forms) by using specific antibodies(see Materials and methods). ERK1/2 was strongly phosphorylated inHeLa parental cells treatedwith 1mMGSNO for 4 h followed by partialdephosphorylation after 8 h incubation. These findings are similar toearlier work that showed that ERK1/2 phosphorylation followed by itsdephosphorylation is an essential step for triggering •NO-inducedapoptosis (Pervin et al., 2003). P38 was increasingly phosphorylatedover the period of incubation whereas phosphorylation of JNK was notobserved (Fig. 4A).

To address the importance of ERK1/2 and p38 activation on cellviability, parental HeLa cells were pretreated with a selective in-hibitor of p38 (SB220025). Alternatively, cells were pretreated withPD98059, a MEK selective inhibitor. After treatment with 1 mMGSNO for 8 h, cultures pretreated with PD98059 showed a markeddecreased in cell viability as compared to cells treated with GSNOalone whereas in cells pretreated with SB220025 no significantalteration in cell viability was observed (Fig. 4B). This indicated thatalthough ERK1/2 is not activated in 8 h GSNO treatment, theinhibition of its initial activation (within 4 h) by PD98059 decreasedcell resistance to GSNO.

Up-regulation of the ERK1/2 MAP kinases activity in cellsover-expressing TRX-1

To investigate whether or not the enhanced cell survival mediatedby TRX-1 may involve survival signaling through ERK1/2 MAP kinasesactivities were measured in basal and over-expressing TRX-1 cellsexposed to increasing concentrations of GSNO. HeLa cells over-expressing TRX-1 showed elevation on the ERK1/2 phosphorylationlevels over the entire range of GSNO concentrations, while in cellsexpressing basal levels of TRX-1, phosphorylation levels of ERK1/2 at 1and 2 mM were lower (Fig. 5A). In order to investigate the •NO-mediated upstream regulation of ERK1/2, we evaluated the activity ofthe small G protein p21Ras. Although activation was observed eitherin cells expressing basal or high levels of TRX-1, the basal activity ofp21Ras and its activation by GSNO were lower in comparison to cellsexpressing basal levels of TRX-1 (Fig. 5B). To confirm the participationof p21Ras in ERK1/2 activation, HeLa cells were treated with aselective inhibitor of p21Ras farnesylation (farnesyltransferase inhi-bitor type III - FPT III) prior to addition of GSNO. In cells expressingbasal levels of TRX-1, FPTIII promoted a partial decrease on the

Fig. 4. Phosphorylation levels of JNK, p38, and ERK1/2 MAP Kinases, and cell viability. (A) HeLa parental cells were treated with 1 mM GSNO at indicated times. At each time point,cells were harvested and lysed. Cell extracts were submitted to Western blotting analysis and the membrane was probed with anti-phospho JNK (upper panel), anti-phosphop38 (middle panel), and anti-phospho ERK1/2 (lower panel). Immunodetection of β-actin was used as a protein loading control. (B-upper panel) HeLa parental cells were pretreatedwith 2 μMof p38 inhibitor, SB220025 (SB) for 2 h or 50 μMofMEK inhibitor, PD98059 (PD) for 30min prior to GSNO treatment or vehicle (control) for an additional period of 8 h. Cellswere harvested and viability was determined by labeling with AnnexinV/PI, followed by FACS analysis. (B-lower panel) Percentages of viable cells shown in bars are expressed asmeans±S.D. from three independent experiments. Statistical analysis were performed by one-way analysis of variance (ANOVA), followed by Bonferroni multiple comparison test(⁎pb0.05).

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phosphorylation levels of ERK1/2. In cells over-expressing TRX-1,however, the inhibitory effects of FPTIII on the phosphorylation levelsof ERK1/2 were no longer observed (Fig. 5C) suggesting an alternativemechanism for the activation of ERK1/2.

Discussion

The adaptive induction of TRX-1 expression in activatedmonocyte/macrophage THP-1 cells and in PC12 phaeochromocytoma cell line,

234 R.J. Arai et al. / Toxicology and Applied Pharmacology 233 (2008) 227–237

has been consistently shown as a key event in cell protection from thetoxic effects of •NO (Ferret et al., 2000; Bai et al., 2003). The inductionof TRX-1 gene expression by •NO may be dependent on the redoxsensitive transcription factors Nrf2 and AP-1 (Kim et al., 2001a,b;Jaiswal, 2004). The regulation of both Nrf-2/DNA and AP-1/DNAbinding is in turn, dependent on the nuclear migration of TRX-1,comprising a unique redox control of transcription factors activityunder oxidative stress (Hirota et al., 1997; Hansen et al., 2004). •NO has

Fig. 5. Phosphorylation levels of ERK 1/2 MAP Kinases and p21Ras dependent ERK1/2 activa(TC-) or presence (TC+) of tetracycline were treated with GSNO at indicated concentrationsERK1/2 (upper panel) and anti-ERK1/2 (lower panel). Image represents three independent eRBD) as described in Materials andmethods. HeLa-Tet-off-TRX-1 cells were treated with 1 mMby GST-RBD (upper panel). Western blotting of total cell extract identified total expression ooff-TRX-1 cells were pretreated with 30 μM of the Farnesyltransferase inhibitor III (FPTII) foadditional 4 h. Cell extracts were then submitted to Western blotting by using anti-phosphobands are shown in columns. Blots presented are representative of three independent expe

also been shown to down-regulate TRX-1 expression by interferingwith NF-kB expression and signaling (Zhang et al., 1999). Thisparadoxical effect of •NO in controlling TRX-1 expression might beexplained in part due to the prevalence of its derived species. •NOmayreact with other reactive species such as •O2

− to produce peroxynitrite(ONOO-) a strong oxidant (Beckman et al., 1990; Ischiropoulos, 2003).The excess of •NOwould react with ONOO-producing N2O3, a powerfulnitrosating agent (Wink et al., 1997). This competing reaction

tion in cells over-expressing TRX-1. (A) HeLa-Tet-off-TRX-1 cells cultured in the absencefor 4 h. Then cell extracts were submitted to Western blotting by using anti-phospho

xperiments. (B) Assessment of p21Ras activity by using Raf-1 Ras Binding Domain (GST-GSNO or vehicle for 30min followed by protein extraction. Active p21Ras was detected

f p21Ras (lower panel). Image represents three independent experiments (C) HeLa-Tet-r 30 min followed by additional treatment by 1 mM GSNO or vehicle (Control) for anERK1/2 (upper panel) and anti-ERK1/2 (lower panel). Relative densitometric values ofriments.

235R.J. Arai et al. / Toxicology and Applied Pharmacology 233 (2008) 227–237

determines whether or not nitrosative stress conditions might beachieved in vivo (Wink et al., 1997; Ullrich and Kissner, 2006). •NOpromotes either oxidative stress or nitrosative stress thereby alteringgene expressions that are specifically responsive to each stress. Theprevalence of nitrosative stress may inhibit transcription factorsincluding AP-1 and NF-kB (Nikitovic et al., 1998; Marshall and Stamler,2002) whereas oxidative stress may activate Nrf2 (Hansen et al.,2004). The observation that GSNO-treated HeLa cells caused anincrease in intracellular nitrosothiols levels indicates that the balanceof •NO-derived species or its availability favors nitrosation reactions.This may be the cause of down-regulation of the TRX-1 geneexpression. However, under our experimental conditions increasingconcentrations of GSNO did not affect the mRNA levels of TRX-1suggesting that down-regulation may occur after post-translationalmodifications of TRX-1. Since TRX-1 can be nitrated (Tao et al., 2006),and this post-translational modification has been associated withprotein degradation (Souza et al., 2000), TRX-1 down-regulation couldinvolve the proteasome pathway.

The observed down-regulation of TRX-1 expression in HeLa cellsupon treatment with GSNO may be considered an important eventthat renders cells susceptible to death stimuli. The down-regulation ofTRX-1 may result in the impairment of the cellular redox homeostasisand the activation of death mechanisms (Nakamura et al., 1997; Powisand Montfort, 2001).

Caspase-3 activity was attenuated by the over-expression ofTRX-1 which may point to the question of whether or not TRX-1could protect cells by acting on the execution program ofapoptosis. TRX-1 may act on caspase-3 by nitrosating (andinactivating) this protease (Tao et al 2004; Mitchell et al., 2007).Conversely, TRX-1 may also denitrosate caspase-3 (Stoyanovsky etal 2005). However, the mechanism of protection provided by TRX-1 regarding the modulation of caspase-3 activity is controversial.Alternatively, we can refer to studies conducted by Marshall andStamler (2002) that showed that NF-kB is nitrosated duringnitrosative stress and this may decrease cell viability or resistanceto stressors. Once TRX-1 denitrosate specific proteins that interactwith it, the regulation of the NF-kB pathway by TRX-1 may occurvia denitrosation.

HeLa cells over-expressing TRX-1 reduced the intracellular levels ofnitrosothiols with greater efficiency than cells expressing basal TRX-1.Studies have demonstrated that the concentrations of exogenoussources of •NO or nitrosothiols are not directly related to the inductionof death response, but rather related to the accumulation ofintracellular nitrosothiols that ultimately reach apoptotic levels (Eu etal., 2000; Marshall and Stamler, 2002). There are limitations for thetransport of S-nitrosothiols from the extracellular space to theintracellular compartment. Therefore, concentrations of GSNO rangingfrom 500 μM to 1 mM has to be used in the extracellular compartmentin order to get picomols-nanomols/mg protein levels of S-nitrosothiolsin the intracellular compartment and induction of apoptosis (Eu et al.,2000; Zhang and Hogg, 2004). On the other hand, the elevatedexpression of TRX-1 (this work) associated with its capacity inpromoting denitrosation reactions (Nikitovic and Holmgren, 1996;Stoyanovsky et al., 2005; Benhar et al., 2008) may contribute to cellsurvival.

The products generated by denitrosation reactions will performdifferent functions in biological systems; hence the idea that thereduction of nitrosothiols levels would rescue cells from death isobviously simplistic. The denitrosation reaction may proceed viahomolytic or heterolytic cleavage of S-N bonds, however, thepredominance of each reaction in biological systems is still unknown(Nikitovic and Holmgren, 1996; Stoyanovsky et al., 2005).

The one-electron mediated denitrosation (homolytic cleavage)would produce •NO, •O2

− and eventually ONOO-. The oxidationproducts of •NO and ONOO- are primarily nitrite and nitrate,respectively (Nikitovic and Holmgren 1996; Radi et al., 2001). The

two-electron mediated denitrosation mechanism (heterolytic clea-vage) would produce hydrogen oxynitrate, nitrous oxide and hydro-xylamine (HNO, N2O and NH2OH) (Stoyanovsky et al., 2005). HeLacells over-expressing TRX-1 showed lower levels of nitrosothiolsaccompanied by higher levels of nitrite in comparison with those incells expressing basal levels of TRX-1. These cells also showed a fasterand higher accumulation of nitrotyrosines than cells expressing basallevels of TRX-1, suggesting that nitrating species are being produced.That nitrite levels were higher than nitrate levels in cells over-expressing TRX-1, suggests that excess of TRX-1 may further scavengeany accumulating ONOO-. Alternatively, TRX-1 in concert withthioredoxin peroxidase may reduce ONOO- to nitrite, because it is arapid and effective reaction (7×107 M−1 s−1) (Masumoto et al., 1996).The sum of nitrosothiol, nitrite and nitrate however, did not increasein cells over-expressing TRX-1, indicating an unbalanced reaction inwhich the heterolytic pathway may be accounted. The products of thetwo-electron denitrosation cannot be ruled out in our experimentalmodel, since HNO, N2O and NH2OH are not detectable by themethodology employed.

The observed catabolism of nitrosothiols mediated by TRX-1 withelevation of intracellular nitrotyrosine levels may impact on severalredox-regulated signaling pathways. In different cell types, theactivation of the JNK and the p38 pathways by •NO leads to apoptosis(Callsen and Brune, 1999; Kim et al., 2001; Tsujita et al., 2008). •NOregulates both MAP kinases by preventing the inhibitory effects ofTRX-1 on ASK-1 (Sumbayev and Yasinska, 2005). The activation of p38showed a marginal effect on cell viability in the presence of itsinhibitor SB220025. In contrast, the activation of ERK1/2 played a keyrole in cell viability.

A major signaling cascade that leads to ERK1/2 activation isinitiated by p21Ras (Vojtek and Der, 1998; Rocha Oliveira et al.,2003; Arai et al., 2006). p21Ras is redox regulated via its criticalCysteine residue 118. The nitrosation of Cysteine 118 displays thesame effect as the binding of the Guanine Nucleotide ExchangeFactor to p21Ras (Lander et al., 1997). In HeLa cells expressingbasal levels of TRX-1, GSNO stimulated p21Ras and ERK1/2 MAPkinases activities, while in cells over-expressing TRX-1, where thenitrosothiols levels were down-regulated, the activation of p21Raswas barely observed. Phosphorylation levels of ERK1/2 werehigher in cells over-expressing TRX-1. This suggests that ERK path-way activation occur through a mechanism not involving eitherthe p21Ras pathway or nitrosation levels in cells over-expressingTRX-1.

In cells over-expressing TRX-1, total nitrotyrosine levels weremaximal after 1–2 h incubation of cells with GSNO and decreased after4–8 h incubation. The decrease on nitrotyrosine levels found in cellsresistant to GSNOdue to the over-expression of TRX-1might be relatedto their degradation by the proteasome pathway or perhaps denitra-tion, suggesting a role for this oxidative-basedmodification of proteinson cell signaling (Grune et al., 1998; Monteiro, 2002; Irie et al., 2003;Aulak et al., 2004, Ullrich and Kissner, 2006). The nitration of tyrosineresidues has been implicated in the up-regulation or down-regulationof various signaling pathways relying on protein tyrosine phosphor-ylation (MacMillan-Crow et al., 2000; Monteiro, 2002).

As observed by others, the redox-based up-regulation of the ERK1/2 MAP kinases independent of p21Ras may be dependent on nitrationof ERK (Pesse et al., 2005). Although the phosphorylation of ERK1/2MAP kinases requires MEK activation, tyrosine nitration of ERK1/2MAP kinases is capable of contributing to their activation (Pinzar et al.,2005). Our findings suggest that the secondary products of denitrosa-tion derived from the metabolic actions of TRX-1 may be involved inthe ERK1/2 MAP kinases up-regulation via nitrative and/or oxidative-based modifications on these kinases or in related phosphatases. TheERK pathway has been implicated as a critical mechanism ofcytoprotection capable of delaying or offsetting cell death (Callsenand Brune, 1999; Kim et al., 2001). Thus TRX-1-mediated up-

236 R.J. Arai et al. / Toxicology and Applied Pharmacology 233 (2008) 227–237

regulation of ERK1/2 MAP kinases activities may be considered animportant signaling event that contributes to increased cell resistanceto nitrosative stress.

The over-expression of TRX-1 conferred increased cell viability bymeans of distinct mechanisms. TRX-1 is capable of attenuatingnitrosative stress while up-regulating critical anti-apoptotic MAPkinases. The crosstalk mechanism between the denitrosation reac-tions and MAP kinases activation might be dependent on products ofdenitrosation. The precise mechanism by which the ERK pathway isactivated independent of p21Ras is still unclear, but it is apparentlydependent on oxidative second messengers.

Acknowledgments

This work was supported by grants from Fundação de Amparo aPesquisa do Estado de S. Paulo (FAPESP) grant no. 00/12154-2 and 02/10192-0, and from Conselho Nacional de Desenvolvimento Científico eTecnológico (CNPq) Instituto do Milênio — Redoxoma (Projeto no.420011/2005).

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