Resveratrol induces antioxidant defence via transcription factor Yap1p

Yeast Yeast 2012; 29: 251–263. Published online 6 June 2012 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/yea.2903

Research Article

Resveratrol induces antioxidant defence via transcription factor Yap1p Xavier Escoté1*, Merce Miranda1, Sandra Menoyo2, Boris Rodríguez-Porrata3, Didac Carmona-Gutiérrez4, Helmut Jungwirth4, Frank Madeo4, Ricardo R. Cordero3, Albert Mas3, Francisco Tinahones5, Josep Clotet2 and Joan Vendrell1 1

Hospital Universitari de Tarragona Joan XXIII. IISPV, Universitat Rovira i Virgili, CIBERDEM, Spain Department of Molecular and Cellular Biology, Universitat Internacional de Catalunya, Sant Cugat del Vallès, Spain 3 Facultat d’Enologia, Universitat Rovira i Virgili, Tarragona, Spain 4 Institute of Molecular Biosciences, University of Graz, Graz, Austria 5 Servicio de Endocrinología y Nutrición, Hospital Clínico Virgen de la Victoria, Málaga. CIBER de Fisiopatología Obesidad y Nutrición (CIBEROBN) 2

*Correspondence to: X. Escoté, C/ Dr Mallafre Guasch 4, 43007 Tarragona, Spain. E-mail: [email protected]

Received: 28 June 2011 Accepted: 17 April 2012

Abstract Resveratrol is a polyphenol suggested to play a protective role against ageing and agerelated diseases. We demonstrate that administering low-doses of resveratrol causes ROS accumulation and transcriptional changes in yeast cells and human adipocytes. These changes in gene expression depend on the oxidative transcription factor Yap1p. In particular, resveratrol induces expression of Yap1p gene targets, such as TRX2, TRR1 or AHP1, in a Yap1p-dependent mode. Under resveratrol treatment, Yap1p is phosphorylated and accumulated in the nucleus. Yap1p knockout causes resveratrol sensitivity, which totally depends on the presence of the C-terminal region of Yap1p. Thus, resveratrol may enhance cellular lifespan by hormetic ROS accumulation, which leads to strengthening the cells’ antioxidant capacity. Copyright © 2012 John Wiley & Sons, Ltd. Keywords: oxidative stress; resveratrol; ROS; Yap1p; hormesis

Introduction Oxidative stress is involved in ageing as well as in many highly prevalent diseases, such as atherosclerosis and heart failure (Barnham et al., 2004). Mitochondrial respiration, fatty acid b-oxidation and exposure to radiation or to specific chemicals are among the different processes that may upset the redox balance (D’Autréaux and Toledano; 2007), eventually leading to an accumulation of radical oxygen species (ROS) and triggering oxidative stress (Storz and Imlay, 1999). It is well established that ROS can lead to cell injury at several levels, including DNA damage, protein oxidation and lipid peroxidation (Miller and Chang, 2007). Thus, the characterization of protective mechanisms against oxidative damage of exogenous Copyright © 2012 John Wiley & Sons, Ltd.

or metabolic origin, for instance via antioxidants (Willcox et al., 2004), has important implications as therapeutic targets for several diseases (Halliwell and Gutteridge, 2010). However, an antioxidant agent may in turn act as a pro-oxidant operating as a hormetic compound. In fact, several studies have demonstrated the pro-oxidant effects of antioxidant vitamins and other classes of plant-derived polyphenols in certain circumstances (Singh et al., 2001). Resveratrol is a polyphenol present in several plants, foods and in wines. Research on resveratrol increased as a result of the observation of a low incidence of cardiovascular diseases despite a highfat diet intake but moderate consumption of red wine, a phenomenon known as the ‘French paradox’ (Renaud and De Lorgeril, 1992). The exposure of human cells to low concentrations of

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resveratrol, comparable to those in red wine, inhibits hydrogen peroxide (H2O2) apoptosis, induced in the human leukaemia cell line HL60 (Ahmad et al., 2003). However, at these concentrations resveratrol elicited pro-oxidant properties, as evidenced by an increase in intracellular superoxide concentration (O–2), which created an intracellular environment that was not conducive to apoptotic execution (Ahmad et al., 2003). Saccharomyces cerevisiae is a valuable model for oxidative stress studies (Gasch et al., 2000; Gasch and Werner-Washburne, 2002), since it exhibits antioxidant responses analogous to those of mammals (Kuge et al., 1997). Several previous screens have identified the transcription factors that regulate antioxidant protection in yeast (Krems et al., 1995). Yap1p is a member of the AP-1-like transcription factors (Fernandes et al., 1997) as one of the central supervisors of the oxidative stress response (D’Autréaux and Toledano, 2007). Thereby, oxidative stress induces Yap1p relocalization into the nucleus, where it induces transcription of several antioxidant genes (Delaunay et al., 2000). The activity of Yap1p is primarily controlled at the nuclear level (Kuge et al., 1997). The C-terminal domain of Yap1p, which contains a nuclear export signal (NES), is responsible for the interaction with the export receptor Crm1p. During oxidative stress, nuclear export of Yap1p is inhibited due to dissociation of Yap1p from Crm1p, resulting in the retention of Yap1p in the nucleus, which induces enhanced transcriptional activity (Delaunay et al., 2000). Consequently, YAP1–depleted strains (yap1Δ) are sensitive to oxidative stress (Godon et al., 1998), whereas its overexpression prolongs the lifespan (Herker et al., 2004). The aim of this study was to evaluate the mechanism by which resveratrol acts on cell metabolism in S. cerevisiae. Our data reveal that resveratrol induces rapid changes in yeast and a rapid imbalance in the redox state, leading to an accumulation of ROS and the subsequent activation of Yap1p, finally inducing the expression of several antioxidant proteins. In yap1Δ ROS accumulation is increased, which translates into greater resveratrol sensitivity. Interestingly, resveratrol promotes ROS accumulation and the expression of detoxifying enzymes in human cells, indicating a possible evolutionarily conserved effect of resveratrol. Copyright © 2012 John Wiley & Sons, Ltd.

Materials and methods Yeast strains and plasmids The strains used were derivatives of BY4741 (MATa; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0) and BY4742 (MATa; his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0) and null mutants were obtained from EUROSCARF. The strains obtained for this study were XY44 (MATa; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0 yap1:: LEU2) and XY72 (MATa; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0 YAP1–3xHA::KanMX) and the oligonucleotides used are shown in Table S1 (see Supporting information). The plasmid pCen– YAP1–11, containing a mutant allele of gene YAP1 (YAP1–C620F) carrying a single mutation in the YAP1 gene resulting in a C620F exchange in the Yap1 protein, and the plasmid pHJ100, containing a C-terminal truncated version of the YAP1 gene encoding for the first 438 amino acids, have been previously described (Wendler et al., 1997). The plasmid pCYC59 (3HA–ADH1t–KanMX), used as a template for chromosomal tagging. The Yap1 ORF was cloned into pGREG576, as essentially described previously (Jansen et al., 2005), to obtain an N-terminal GFP-tagged version.

Growth conditions, cell synchrony, cell morphology and flow-cytometry analyses Rich medium (YPD) and synthetic medium with amino acids were supplemented with 2% dextrose. Cells were analysed in a FACScan flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA) and the captured files were processed using WinMDI 2.9 flow-cytometry software (Joseph Trotter, Salk Institute for Biological Studies, La Jolla, CA, USA). Resveratrol was resuspended with DMSO, following the manufacturers’ instructions (SigmaAldrich, UK). Free intracellular radicals were detected using dihydrorhodamine 123 (DHR; CAS 109244-58-8) or dihydroethidium (DHET; CAS 104821-25-2) (Sigma-Aldrich, UK), as described essentially by Madeo et al. (1999). The cells were incubated with fluorescent probes (25 mg/ml DHR, 50 mg/ml DHET) and subsequently were washed twice with water and then resuspended in PBS. Fluorescence intensity was quantified by flow cytometry and the results are given as the mean fluorescence value. Yeast 2012; 29: 251–263. DOI: 10.1002/yea

Yap1p activation by resveratrol

mRNA analysis by real time RT–PCR and protein analysis by Western blotting Total RNA was extracted from approximately 1.8  107 cells, using the RNeasy Mini Kit (Qiagen Science, Germantown, MD, USA) following the manufacturer’s instructions. Total RNA was quantified by absorbance measurement and its purity assessed by the OD260:OD280 ratio. For real-time PCR, 1 mg RNA was retrotranscribed to cDNA using the Reverse Transcription System (Promega, Madison, WI, USA) in a final volume of 20 ml, following the manufacturer’s instructions. CDC28 was used as a reference gene for standardization in yeast studies and cyclophilin A in human cell line studies. All primers were synthesized by Roche (Indianapolis, IN, USA). Gene expression analysis was performed on a LightCycler Instrument (Roche), using the SYBR Green fluorescence method. Quantification of 2 ml cDNA was carried out in 20 ml of a mixture containing 0.3 mM oligonucleotides and 2 ml LC-FastStart DNA Master SYBR Green I (Roche). The final concentration of MgCl2 was adjusted for each gene. The purity of each amplified product was confirmed by melting curve analysis, and detection of the fluorescent signal was adjusted to avoid primer– dimer detection. For each experiment, quantification was calculated relative to the reference gene and to the control, using LightCycler Software v 3.5 (Roche). The oligonucleotides used for RT–PCR are shown in Table S1 (see Supporting information). Western blotting to detect Yap1p change mobility was resolved by electrophoresis in 10% SDS–PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA) by electroblotting, essentially as described by Escoté et al. (2004); HA was detected with anti-HA monoclonal antibody (12CA5, Roche) and Hog1 was used as a loading and non-phosphorylated control (Santa Cruz, CA, USA) and developed with Supersignal West-Femto (Thermo Scientific, Waltham, MA, USA). Images were captured using VersaDoc (BioRad, Hercules, CA, USA). Cell extracts were treated with phosphatase alkaline (Roche) with a final concentration of 2 U/ml for 30 min at 37  C.

Microscopy ROS was stained with DHR 123 (Sigma-Aldrich) (Madeo et al., 1999). Stained and DIC images were visualized using a Leica DM 4000B fluorescence Copyright © 2012 John Wiley & Sons, Ltd.

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microscope (Allendale, NJ, USA) with a 100 oilimmersion lens, and images were captured using a Leica DFC 300 FX camera. Photographs were taken and images were scanned and processed using Adobe Photoshop software.

Human cell culture The SGBS cell line is a human preadipocyte cell line with a high capacity for adipose differentiation (Wabitsch et al., 2001). Cells were seeded at 3  104 cells/cm2 in a preadipocyte medium (Advancell, Barcelona, Spain). Confluent cells were induced to differentiate to mature adipocytes in adipocyte differentiation medium (Advancell) containing 0.25 mM isobutylmethylxanthine, 1 mM dexametasone, 1 nM human insulin and a peroxisome proliferator-activated receptor-g (PPARg) agonist. To differentiate cells, two series of 3 days of differentiation induction with adipocyte differentiation medium were carried out. Then, 50% of the medium was replaced every 2 days for 10 days, with an adipocyte medium (Advancell). Adipocyte cells were collected and used for RNA extraction.

Statistical analysis Statistical analysis was performed using the SPSS/ PC + statistical package (v 15.0 for Windows; SPSS, Chicago, IL, USA). Data are expressed as mean value  standard deviation (SD). Statistical differences between samples were compared using the General Linear Model Repeated Measures Test. Statistical significance occurred if the computed two-tailed probability value (p) was < 0.05.

Results Resveratrol induces ROS accumulation in yeast cells Despite the role of resveratrol as a protecting agent against oxidative stress, some recent studies highlight that low doses of resveratrol may increase ROS levels. We evaluated ROS concentration in response to short-term exposure to resveratrol using a general and unspecific dye for ROS, dihydrorhodamine 1,2,3 (DHR). Resveratrol at low doses (5 mM) increased total ROS levels, as detected in wild-type cells by fluorescence microscopy and flow cytometry Yeast 2012; 29: 251–263. DOI: 10.1002/yea

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(Figure 1A, B). In contrast, high doses of resveratrol (50 mM) promoted a decrease in total ROS (Figure 1B), acting as an antioxidant agent. In addition, we used DHET to assess elevated levels of superoxide radical (O–2), although it is not totally specific for this ROS, obtaining similar results (data not shown). In a time course experiment, we observed a recovery to the initial ROS levels after 40 min of resveratrol exposure (Figure 1C). These results confirm that cells accumulate ROS in response to low doses of resveratrol in a timedependent manner, which could indicate an adaptation process to this new scenario.

yap1Δ cells are sensitive to resveratrol Usually, adaptation to new stimuli requires changes in transcriptional activity. For this reason, we investigated the role of different transcription factors after resveratrol exposure at physiological concentrations. We tested the growth of knockout strains for different transcription factors in rich medium (YPD plates) or rich medium plus resveratrol (resveratrol plates) (Figure 2A). Compared to the appropriate wild-type, none of the strains revealed greater sensitivity to resveratrol, except for the YAP1 knockout strain. Moreover, we checked the resveratrol effect on the skn7 and no effect was observed (data not shown). Therefore, we focused our interest on the transcription factor Yap1p. We confirmed the sensitivity of yap1Δ cells to resveratrol in minimalmedium plates (Figure 2B) as well as in liquid media (Figure 2C). Interestingly, the sensitivity of yap1Δ cells to resveratrol could be detected within the first 70 min after treatment (Figure 2C).

Yap1p deletion aggravates ROS accumulation in response to resveratrol Next, we further characterized the effects of resveratrol in a YAP1 knockout strain. As was expected, we observed a dramatic impact on ROS levels in the absence of the transcription factor Yap1p (Figure 3A–C for total ROS). In addition, yap1Δ cells presented elevated levels of superoxide radicals (data not shown). Moreover, exposure of exponentially growing yap1Δ cells to 5 mM resveratrol revealed a clear accumulation of ROS in a timedependent manner (Figure 3D), which is more prominent with long-term exposure. These results imply that low doses of resveratrol increase ROS Copyright © 2012 John Wiley & Sons, Ltd.

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production and, since the absence of YAP1 leads to greater resveratrol sensitivity, as cells are not able to respond efficiently to this oxidative injury, this suggests that YAP1 transcription factor activity is probably induced.

Resveratrol activates Yap1p transcriptional activity To assess whether Yap1p suffered a post-translational modification in response to resveratrol, we constructed a chromosomal tagged version of YAP1. Protein extracts from cells exposed to resveratrol showed a shift in Yap1p mobility compared to the vehicle (Figure 4A). Moreover, this change in the mobility of Yap1p was comparable to the shift observed when cells were exposed to H2O2 (Figure 4B, C). Finally, we checked whether the mobility changes were a consequence of phosphorylation, observing that Yap1p showed similar behaviour as in media containing H2O2, and these mobility changes disappeared when protein extracts were treated with alkaline phosphatase (Figure 4C). Moreover, in a time course where cells were treated with resveratrol or H2O2 and the extracts were treated (or not) with alkaline phosphatase, we observed a similar behaviour in both treatments, which indicates that Yap1 is phosphorylated after resveratrol treatment in a similar way to that with H2O2 (Figure 4D). In this respect, we checked the subcellular localization of Yap1, observing increased signal in the nucleus after resveratrol treatment, but it is less apparent when compared to H2O2-treated cells (Figure 4E). To ascertain whether Yap1 expression is induced after resveratrol treatment, we performed a time course to analyse the mRNA levels of Yap1 by RT–PCR, and no significant changes in the YAP1 mRNA levels were observed, which indicates that accumulation observed after resveratrol treatment is a consequence of protein regulation (data not shown). Therefore, these data indicate that Yap1 is regulated at protein level after resveratrol treatment by phosphorylation.

Resveratrol induces an antioxidant response In order to explore a possible change induced by resveratrol in Yap1p transcriptional regulatory activity, we next examined some Yap1p gene targets that respond to oxidative stress. We demonstrated that expression levels of TRX2, TRR1 and Yeast 2012; 29: 251–263. DOI: 10.1002/yea

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Figure 1. Exposure to resveratrol induces ROS accumulation in yeast cells. (A) Wild-type cells were grown in exponential phase in rich liquid medium and were exposed to 5 mM resveratrol or 300 mM H2O2 for 20 min. Whole cells were stained with DHR. (B) Wild-type cells were exposed for 20 min to DMSO (□), 5 ( ) or 50 mM (■) resveratrol, or 300 mM H2O2 ( ), and ROS were evaluated by cytometry, using DHR. (C) Time course of wild-type cells exposed to 0 (white bars), 5 (grey bars) or 50 mM (black bars) of resveratrol at the indicated times, and ROS were evaluated by cytometry. Data  SD from three independent experiments are shown (#p < 0.001, **p < 0.005, *p < 0.05)

Copyright © 2012 John Wiley & Sons, Ltd.

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Figure 2. yap1Δ cells are sensitive to resveratrol. (A) Resveratrol sensitivity tests with various yeast knockout strains. Dilutions of wild-type and knockout strains were inoculated for 3 days at 30  C on YPD plates without (control) and with 5 mM resveratrol; images are representative of three independent experiments. (B) Wild-type and yap1Δ cell growth were scored on minimal medium plates inoculated with different dilutions in the absence or presence of 5 mM resveratrol. (C) yap1Δ cells were grown in exponential phase on rich medium and subjected to 5 mM resveratrol for the times indicated and the optical density was measured at 660 nm. Control (open bars) and resveratrol (closed bars). Data are expressed as relative units  SD from three independent experiments

AHP1 were clearly induced upon resveratrol treatment in wild-type cells in the first minutes and tended to return to the initial expression level after a longer time (Figure 5A–C). In contrast, in the yap1-deleted strain, this effect was completely abolished (Figure 5A–C), even observing clear downregulation in the expression of the TRX2 and in the TRR1 genes but not in AHP1. Therefore, these results suggest that resveratrol promotes the Copyright © 2012 John Wiley & Sons, Ltd.

expression of typical oxidative stress-induced genes, such as TRR1, TRX2 and AHP1, in a Yap1p-dependent manner.

C-terminal region of Yap1p is responsible for its resveratrol sensitivity To discern the molecular basis for the sensitivity of Yap1 to the presence of resveratrol, we transformed Yeast 2012; 29: 251–263. DOI: 10.1002/yea

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Figure 3. ROS accumulation in response to resveratrol is increased in yap1Δ cells. (A) Wild-type and yap1Δ cells were grown in exponential phase in rich liquid medium and were exposed (or not) to 5 mM resveratrol for 20 min. Whole cells were stained with DHR. The images are the result of merging ROS staining and bright field; 300 cells were evaluated and the percentage of ROS-positive was indicated. (B) Wild-type and yap1Δ cells were exposed to 5 mM resveratrol for 20 min and ROS were evaluated by cytometry, using DHR. (C) Wild-type and yap1Δ cells were grown in exponential phase in minimal medium and were exposed to 5 mM resveratrol for 20 min, and ROS were evaluated by cytometry, using DHR. □, DMSO; ■, resveratrol; data  SD from three independent experiments are shown. (D) Time course of yap1Δ cells exposed to 5 mM resveratrol and cells were taken at indicated times and ROS were evaluated by cytometry. Data  SD from three independent experiments are shown Copyright © 2012 John Wiley & Sons, Ltd.

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Figure 4. Resveratrol induces Yap1p phosphorylation. (A) Yap1p is modified under resveratrol exposure. Cells were grown in a liquid medium, subjected for 20 min to 5 mM resveratrol and measured by Western blot. Hog1 was detected as a control of non-phosphorylated protein. (B) As in (A), cells were treated with resveratrol or 300 mM H2O2 and Yap1p mobility was assayed by Western blot. (C) As in (A), cells were treated with 5 mM resveratrol or 300 mM H2O2 and Yap1p mobility was assayed by Western blot, and protein extracts were treated with phosphatase alkaline. (D) As in (A), cells were treated with resveratrol or H2O2 for 15 or 30 min and Yap1p mobility was assayed by Western blot, and protein extracts were treated with phosphatase alkaline. (E) A yap1Δ strain was transformed with GFP–YAP1 plasmid. This strain was treated as in (A). Nucleocytoplasmic redistribution of Yap1p after 20 min of treatment (DMSO, resveratrol or H2O2) is shown

yap1Δ cells with plasmids containing different versions of the gene YAP1. As expected, yap1Δ cells transformed with the empty plasmid presented sensitivity to resveratrol, whereas these cells transformed with a full-length version of gene YAP1 were Copyright © 2012 John Wiley & Sons, Ltd.

able to completely recover growth (Figure 6A). Moreover, the same result was obtained by transforming the cells with a plasmid containing the gain-of-function allele YAP1–11 (a single mutation in gene YAP1 resulting in a C620F Yeast 2012; 29: 251–263. DOI: 10.1002/yea

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resveratrol treatment might prepare cells for exposure to harsh oxidants by inducing ROS to pre-activate the antioxidant defence mechanisms through Yap1. Wild-type cells were pretreated with resveratrol (20 min with 0, 5 or 50 mM resveratrol) and then H2O2 was added (Figure 6C). We observed that upon H2O2 exposure, cells treated previously with resveratrol accumulated less ROS than non-treated cells (Figure 6C). Finally, we checked by means of plate growth assays that wild-type cells pretreated with resveratrol can proliferate better in the presence of H2O2. In contrast, yap1Δ cells were not able to proliferate in the presence of H2O2 even when treated previously with resveratrol (Figure 6D).

Resveratrol mimics ROS accumulation in human cells

Figure 5. Resveratrol induces a YAP1–dependent response. (A–C) Wild-type (■) and yap1Δ (□) cells were grown in exponential phase in a liquid YPD. Then the cells were subjected (or not) to 5 mM resveratrol at the times indicated and analysed by real-time PCR. Expression levels for TRR1, AHP1 and TRX2 were normalized using CDC28 as the reference gene. Data are expressed as mean  SD from three independent experiments

exchange in the Yap1 protein) which did not affect growth in the presence of resveratrol (Figure 6A). In contrast, a C-terminal truncated version of Yap1p did not restore the effect of yap1Δ cells in resveratrol-containing media (Figure 6A). Similar results were obtained with higher resveratrol concentrations (Figure 6B). These results point to the presence of the last 212 amino acids of Yap1p at the C-terminal region, which seems to be important under resveratrol exposition.

Resveratrol prepares cells for exposure to oxidants Until now, resveratrol has been described as an antioxidant agent. We ascertained whether Copyright © 2012 John Wiley & Sons, Ltd.

We studied whether this system was evolutionarily conserved. For this purpose, we aimed to analyse the effect in human adipocyte cells (SGBS cell line) as a putative target for resveratrol. Under fluorescence microscopy, resveratrol-treated cells showed a clear increase in total ROS accumulation in comparison with non-treated cells (Figure 7A). This result suggests that the oxidative effect of resveratrol observed in S. cerevisiae is evolutionarily conserved from yeast to human cells. To discern whether resveratrol induces such a transcriptional response in human adipocytes as that observed in yeast cells, we used quantitative RT–PCR to verify mRNA levels of some genes in cells exposed (or not) to low doses of resveratrol: cyclo-oxygenase-2 (PTGS2), which is induced by oxidative stress; Sirt1 deacetylase (SIRT1), which is activated by resveratrol; g-glutamylcysteine synthetase heavy chain (GCL), which is an AP-1 target (a Yap1p-related protein); and leptin (LEP), which is able to induce oxidative stress, were analysed (Figure 7B). As expected, mRNA levels of SIRT1 and PTGS2 were clearly upregulated under resveratrol treatment. Moreover, GCL was also induced in resveratrol-treated adipocytes. In contrast, LEP expression was downregulated in resveratrol-treated cells (Figure 7B). Taken together, these results suggest that in human cells the oxidative effect of resveratrol induces the transcription of stress response genes in a similar manner to that observed in yeast cells. Yeast 2012; 29: 251–263. DOI: 10.1002/yea

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Figure 6. Yap1p sensitivity to resveratrol is restricted to the C-terminal region of Yap1. (A) After 3 days at 30  C, yap1Δ growth was scored on minimal media plates inoculated with different dilutions in the absence or presence of 5 mM resveratrol. yap1Δ cells were transformed with the empty vector or with plasmids carrying different versions of gene YAP1 (YAP1, full-length gene YAP1; YAP1–Cter truncated, gene YAP1 lacking the C-terminal region encoding for the first 438 amino acids; YAP1–C620F, allele YAP1–11 carrying a single mutation in gene YAP1, resulting in a C620F exchange in the Yap1 protein). Wild-type transformed with the empty vector was shown as a control. (B) As in (A), strains were spread on plates containing 50 mM resveratrol. (C) Wild-type cells were grown in exponential phase in rich liquid medium and were exposed to 0 (■), 5 ( ) or 50 mM ( ) resveratrol for 20 min. Then these cells were treated with 300 mM H2O.2for 20 min. Whole cells were stained with DHR. Data  SD from three independent experiments are shown (**p < 0.005, *p < 0.05). (D) Wild-type and yap1Δ cells were grown in exponential phase in rich liquid medium and were exposed to 5 mM resveratrol for 20 min. Then the cells were inoculated into YPD plates with different dilutions in the absence or presence of H2O2 Copyright © 2012 John Wiley & Sons, Ltd.

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Figure 7. Exposure to resveratrol induces ROS accumulation and AP-1 response in human cells. (A) Adipocytes were grown as described in Materials and methods and treated (or not) with 5 mM resveratrol for 4 h. The images shown are representative of three independent experiments. Total ROS were stained with DHR. (B) Human differentiated adipocytes were grown as in (A) and mRNA expression of indicated genes was evaluated by RT–PCR; □, DMSO; ■, resveratrol

Discussion Oxidative stress, a ROS-antioxidant imbalance, occurs when the net amount of ROS exceeds the antioxidant capacity, due to either an increase in ROS generation, a depletion of the antioxidant systems or both. When excess ROS cannot be inactivated by the cellular antioxidant systems, they can react with cellular macromolecules and enhance the processes of cellular damage (Roberts and Sindhu, 2009). It is generally thought that ageing is caused by the accumulation of macromolecular damage. Increased ROS or decreased antioxidant pools may negatively impact the vascular function, the ageing process, and lead to a decrease in lifespan (Douglas and Haddad, 2008). The exposure of cells to oxidative stress leads to a stress-activated response induced by Yap1p/ AP-1 (Delaunay et al., 2000). Yap1p activation is Copyright © 2012 John Wiley & Sons, Ltd.

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transient, so elucidating the biological roles of the transcription factor is complex. To improve knowledge of the mechanistic functioning of resveratrol, a poly-phenolic phytoalexin, we have characterized the eukaryotic responses to low doses and short exposures to this compound. However, the antioxidant protective role of high-dose resveratrol against stronger oxidant stresses is extensively addressed in other studies (Dani et al., 2008). Moreover, we have observed that resveratrol promoted ROS accumulation, which led to the induction of Yap1/AP-1 transcriptional activity. It is usually accepted that resveratrol promotes antioxidant effects in part by modulating several enzymes involved in oxidative stress response. However, some authors have demonstrated that low doses of resveratrol may induce an accumulation of O–2 (Ahmad et al., 2003). In this sense, our data with DHET are in agreement with this observation, although DHET is not totally specific for this ROS. Furthermore, other studies tend to show protective cell signalling associated with higher resveratrol concentrations (Baur et al., 2006). Thus, one may speculate that resveratrol-induced oxidative stress could promote a response that allows cells to adapt properly and to prevent damage in further situations of oxidative stress, acting as a hormetic agent. Hormesis is defined as the beneficial effects of low levels of stress, referring to an adaptive response of cells and organisms to a moderate stress to cope with more severe stresses. In fact, exposure to low levels of one type of hormetic agent can protect cells/organisms against more than one type of stress (Mattson, 2008). The cellular and molecular mechanisms of hormesis are being revealed and include the activation of growth factor signalling pathways, protein chaperones, cell survival genes and enzymes called sirtuins (Mattson, 2008). It has been extensively described that the depletion of a single transcription factor may induce sensitivity to different stresses, due to the lack of adaptation to the new condition by inducing gene transcription (Gasch and Werner-Washburne, 2002). We observed that only yap1Δ cells showed sensitivity to resveratrol, indicating that Yap1p presence is essential for viability in these conditions. Yap1p activity is regulated post-translationally and by its subcellular localization (Wood et al., 2004). Under an oxidative stress situation, Yap1p is modified, increasing its nuclear concentration and activating the expression of several genes. Yeast 2012; 29: 251–263. DOI: 10.1002/yea

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In resveratrol-treated cells, Yap1p is phosphorylated, which might regulate Yap1p transcription activity. In this line, our results confirm the possibility that Yap1 was also oxidized under resveratrol treatment. Thus, in further studies it would be interesting to analyse the role of the transducer enzyme Orp1 (Gpx3) and the helper protein Ybp1 in resveratrol-treated cells. Moreover, interaction of the C-terminal domain of Yap1p with the export receptor Crm1p could also regulate its activity. Indeed, Yap1p transcriptional activity in response to resveratrol is rapidly modified, as shown by the induction of the previously described, Yap1p-regulated genes TRX2, TRR1 and AHP1 (Morgan et al., 1997). However, this induction is modest when is compared with the induction observed with H2O2 (our data not shown). Consequently, in the yap1Δ strain, this effect is completely abolished or downregulated, which might at least partially explain its sensitivity to resveratrol. Taken together, these results provide evidence for the first time that ROS accumulated by resveratrol induce Yap1p transcriptional activity as the necessary element for proper adaptation to the new circumstances. Finally, we checked the relevance of this system in mammalian cells. Resveratrol again induced a clear ROS accumulation in cell cytoplasm. This may indicate an evolutionary conservative response helping the cell to fight against oxidative stress stimuli. Moreover, when we analysed a possible resveratrol-dependent transcriptional response, we observed similar results to those found in the yeast cells. PTGS2, an oxidative target gene (Kennedy et al., 2009), and SIRT1, an NAD-deacetylase directly activated by resveratrol (Yoshizaki et al., 2009), were significantly upregulated under resveratrol treatment. In contrast, LEP (an adipokine that controls food intake, which has also been described as a pro-oxidant gene; Konstantinidis et al., 2009), was downregulated. These changes may be interpreted as an attempt to avoid excess oxidative stress and are in line with a recent report in which leptin secretion was inhibited by resveratrol in isolated rat adipocytes (Szkudelska et al., 2009). Additionally, an AP-1 gene target, such as GCL (Morales et al., 1997), was upregulated, mimicking the response observed in Yap1p target genes in yeast. Taken together, the oxidative effect of resveratrol in human cells promotes a protective transcriptional response in a similar manner to that observed in yeast cells Copyright © 2012 John Wiley & Sons, Ltd.

and suggests that resveratrol might also act as a hormetic agent in human cells. What might the physiological impact of resveratrol on mammalian cells be? It might protect cells from hazardous oxidative stress, precisely by previously creating the corresponding intracellular environment. There are several limitations in this work. First, although it was not an objective of this work, the lack of identification of the specific species of ROS produced by exposure to low doses of resveratrol restricts the impact of the work. Therefore, future studies should focus on these specific species that are created by the presence of resveratrol. Second, although it was not a target of this study, to study the role of the cysteines located in the n-CRD region (C303, C310 and C315) at low doses of resveratrol. Third, the use of antioxidant compounds such as scavengers can help to understand the hormetic role of resveratrol, although their use in combination with resveratrol could be a far from easy interpretation. In summary, we suggest that under resveratrol exposure reactive oxygen species are increased, which in turn induces AP-1 activity in order to diminish ROS levels. In yap1Δ cells, exposure to resveratrol increases ROS, which, however, cannot be reduced via Yap1p transcriptional activity, which yields an accumulation leading to toxic levels and eventually producing growth delay. Thus, we propose that resveratrol, by inducing ROS and acting via AP-1, may prepare cells for further oxidant aggressions. Acknowledgements We thank F.Posas for his valuable advice, plasmids and strains. This study was supported by Instituto de Salud Carlos III (ISCIII) grants FIS08/1195, FIS10/00967, FIS11/00085 and CIBERDEM de Diabetes y Enfermedades Metabólicas Asociadas (CB07/08/0012). CIBERDEM is an initiative of the Instituto de Salud Carlos III. We acknowledge the invaluable technical assistance provided by Verònica Alba.

Supporting information on the internet The following supporting information may be found in the online version of this article: Table S1. Oligonucleotides used in this study Abbreviations DHET DHR

dihydroethidium dihydrorhodamine 123 Yeast 2012; 29: 251–263. DOI: 10.1002/yea

Yap1p activation by resveratrol

GCL H2O2 LEP NES PPAR PTGS ROS SIRT1 SD O2– YPD

g-glutamylcysteine synthetase heavy chain hydrogen peroxide leptin nuclear export signal gperoxisome proliferator-activated receptor-g cyclooxygenase-2 radical oxygen species Sirt1 deacetylase standard deviation superoxide yeast extract peptone dextrose medium.

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Yeast 2012; 29: 251–263. DOI: 10.1002/yea