Bcl-2 sustains hormetic response by inducing Nrf-2 nuclear translocation in L929 mouse fibroblasts

Free Radical Biology & Medicine 49 (2010) 1192–1204

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Free Radical Biology & Medicine j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f r e e r a d b i o m e d

Original Contribution

Bcl-2 sustains hormetic response by inducing Nrf-2 nuclear translocation in L929 mouse fibroblasts Armando Luna-López a,b, Francisco Triana-Martínez a,b, Norma E. López-Diazguerrero a, José L. Ventura-Gallegos c,d, María C. Gutiérrez-Ruiz a, Pablo Damián-Matsumura e, Alejandro Zentella c,d, Luis E. Gómez-Quiroz a, Mina Königsberg a,⁎ a

Departamento de Ciencias de la Salud, División de Ciencias Biológicas y de la Salud, Universidad Autónoma Metropolitana Iztapalapa, CP 09340 México, DF, México Posgraduate Program in Biología Experimental, Universidad Autónoma Metropolitana Iztapalapa, CP 09340 México, DF, México c Departamento Medicina Genómica y Toxicología Ambiental, IIB, UNAM, México, DF, México d Departamento Bioquímica, Instituto Nacional de Ciencias Médicas y Nutrición “Salvador Zubirán,” México, DF, México e Departamento de Biología de la Reproducción, División de Ciencias Biológicas y de la Salud, Universidad Autónoma Metropolitana Iztapalapa, CP 09340 México, DF, México b

a r t i c l e

i n f o

Article history: Received 16 March 2010 Revised 28 June 2010 Accepted 7 July 2010 Available online 14 July 2010 Keywords: Bcl-2 Hormesis GST γGCS Nrf-2 Oxidative stress Free radicals

a b s t r a c t Hormesis is the process whereby exposure to a low dose of a chemical agent induces an adaptive effect on the cell or organism. This response evokes the expression of cytoprotective and antioxidant proteins, allowing prooxidants to emerge as important hormetic agents. The antiapoptotic protein Bcl-2 is known to protect cells against death induced by oxidants; it has been suggested that Bcl-2 might also modulate steady-state reactive oxygen species levels. The aim of this work was to find out if Bcl-2 might play a role during the hormetic response and in Nrf-2 activation. We have established a model to study the oxidative conditioning hormesis response (OCH) by conditioning the cell line L929 with 50 μM H2O2 for 9 h. This condition did not induce oxidative damage nor oxidative imbalance, and OCH cells maintained a 70–80% survival rate after severe H2O2 treatment compared to nonconditioned cells. When cells were pretreated with the Bcl-2 inhibitor HA14-1 or were silenced with Bcl-2siRNA, both the hormetic effect and the Nrf-2 nuclear translocation previously observed were abrogated. Our results suggest a sequence of causal events related to increase in Bcl-2 expression, induction of Nrf-2 activation, and sustained expression of cytoprotective proteins such as GST and γGCS. © 2010 Elsevier Inc. All rights reserved.

During evolution, living organisms have always had to cope with adverse environmental conditions and, to survive, they have developed complex mechanisms to overcome them. For several years many biological subdisciplines have identified an evolutionarily preserved process in which a low dose of a stressful stimulus activates an adaptive response that enhances the resistance of the cell or organism to a severe level of stress. To unify the concepts and terminology for this kind of response in cells and organisms after the disruption in their homeostasis, the phenomenon has been called “hormesis” [1]. Hormesis can be defined as “a process in which exposure to a low dose of a chemical agent or environmental factor that is damaging at higher doses induces an adaptive beneficial effect on the cell or organism” [2–5]. The main hormetic agents identified so far are irradiation, heat, heavy metals, antibiotics, ethanol, prooxidants, exercise, and food restriction [5–7]. The hormetic response

Abbreviations: ARE, antioxidant response element; γGCS, γ-glutamylcysteine synthetase; GSH, reduced glutathione; GST, glutathione-sulfhydryl-transferase; Nrf-2, nuclear factor erythroid-derived 2-like 2; OCH, oxidative conditioning hormesis; ROS, reactive oxygen species. ⁎ Corresponding author. Fax: + 52 55 5804 4727. E-mail address: [email protected] (M. Königsberg). 0891-5849/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2010.07.004

involves the expression of genes that encode cytoprotective proteins such as chaperones, for example, heat-shock proteins; antioxidant enzymes; and growth factors [3,8,9]. Bcl-2 is the prototype of a family of proteins that regulate cell survival [10–12]. It was first discovered as an oncogene product that interferes with apoptosis in multiple cell contexts [13]. It is also known that Bcl-2 overexpression prevents oxidative stress damage and cell death [14,15], probably by increasing reduced glutathione (GSH)1 content [16], particularly at the mitochondrial level [17], as well as superoxide dismutase and proteasome activities [18]. Bcl-2 might also be implicated in regulating the cell cycle to allow damage repair [19,20]. Hence, all the previous survival properties attributed to Bcl-2 make it a suitable participant throughout the hormetic response against an oxidative insult. Modifications in redox state are known to modulate transcription factors [21,22]. A transcription factor that responds to this kind of variation in redox state is the nuclear factor erythroid-derived 2-like 2 (Nrf-2). Nrf-2 is a member of the Cap'n’ Collar family of bZIP proteins and a central regulator of antioxidant and phase II detoxifying enzymes through the activation of the antioxidantresponse element (ARE) [23–25]. Nrf-2 regulation occurs posttranslationally [25–29]; under basal conditions, it is constitutively

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produced and sequestered in the cytoplasm, where it is targeted for ubiquitin-mediated proteolysis [30–32]. Under oxidative stress, Nrf-2 is stabilized and translocated to the nucleus where it activates AREresponsive genes leading to cytoprotection. The persistent accumulation of Nrf-2 in the nucleus is harmful, and several studies have demonstrated that Nrf-2 is inactivated and released after a short time [33]. Hence, Nrf-2 might also play an important role during the oxidative hormetic response [34]. It has been suggested that Bcl-2 might act through a pro-oxidant mechanism, by raising or modifying steady-state reactive oxygen species (ROS) levels [12,20,35,36], and because Nrf-2 is a transcription factor that responds to changes in redox state, the hypothesis for this work was that Bcl-2 levels might augment the response to a low oxidative insult as part of the oxidative hormetic response and that this increase might be accomplished by enhancing Nrf-2 activation. L929 murine fibroblasts were used for this purpose, because previous work from one of the authors had shown this was a good model to study survival and oxidative responses [37]. Fifty micromolar H2O2 for 9 h was chosen as the most advantageous treatment for studying oxidative conditioning hormesis (OCH) and under these conditions cells were protected when they were reexposed to severe oxidative insults. This hormetic effect was abrogated when cells were pretreated with the Bcl-2 inhibitor ethyl-[2-amino-6-bromo-4-(1cyano-2-ethoxy-2-oxoethyl)]-4H-chromene-3-carboxylate (HA14-1) or when Bcl-2 was silenced using Bcl-2-siRNA. Nrf-2 nuclear translocation was observed at early times after OCH and then again at 8.5–9.0 h, correlating with Bcl-2 maximal overexpression; Nrf-2 nuclear second translocation and activation were abrogated when HA14-1 was used. Our data show that Bcl-2 contributes to the survival pathway during the hormetic response and that this participation correlates with redox modulation and Nrf-2 nuclear translocation.

Experimental procedures Chemicals All chemicals and reagents were of the highest analytical grade and most of them were purchased from Sigma (St. Louis, MO, USA). Reagents obtained from other suppliers are detailed in the text.

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GSH/GSSG determination by HPLC GSH and glutathione disulfide (GSSG) contents were determined in OCH and NO-H cells by HPLC as described by Fariss and Reed [38] with some modifications by Gómez-Quiroz and co-workers [39]. DNA extraction, enzymatic hydrolysis, and analysis of 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodGuo) by HPLC/electrochemical (EC) detection DNA was isolated using the chaotropic-NaI method [40], as modified by Matos and co-workers [41]. This technique avoids DNA parasite oxidation during the isolation process. DNA concentration was determined spectrophotometrically at 260 nm and its purity was assessed by ensuring that the A260/A280 ratio was N1.75. DNA was enzymatically digested with nuclease P1 and Escherichia coli acid phosphatase as described elsewhere [41]. One hundred micrograms of digested DNA was injected into an HPLC/EC system consisting of a Waters 600 C pump (Waters, Milford, MA, USA) connected to a Supelcosil LC-18 (Supelco, Bellefonte, PA, USA) reverse-phase column (250 × 4.6 mm i.d., particle size 5 μm). The isocratic eluent was 50 mM potassium phosphate buffer, pH 5.5, with 8% methanol at 1 ml/min flow rate. EC detection was performed by a INTRO detector (Antec Leyden, Leiden, The Netherlands) operated at 290 mV. Elution of unmodified nucleosides was simultaneously monitored by a 486 Waters UV spectrometer set at 254 nm. The molar ratio of 8-oxodGuo to desoxyguanosine (dGuo) in each DNA sample was determined based on EC detection at 290 mV for 8oxodGuo and absorbance at 254 nm for dGuo [41]. Bcl-2 expression during OCH To determine if Bcl-2 expression was induced during OCH, cells were treated for various periods of time with the indicated H2O2 concentrations. L929 cells were seeded at density of 5 × 103 cells per well on a 48well plate (Corning). After 24 h the cells were incubated with 50 μM H2O2 dissolved in the culture medium, for 0, 3, 6, 9, and 12 h at 37 °C and 5% CO2. Cells were also treated with various H2O2 concentrations (0, 50, 150, 100, and 200 μM) for 9 h and total protein and total RNA were isolated to determine Bcl-2 and Bax expression. Cellular functionality and viability were also assessed to verify cellular integrity. RNA isolation, reverse transcription, and PCR analysis

Cell culture Mouse L929 lung fibroblasts were cultured at 37 °C in an atmosphere of 95% air and 5% CO2 as described elsewhere [37].

Oxidative survival limit and OCH L929 cells were seeded at 1 × 105 density into 24-well plates (Corning, Acton, MA, USA). Cells were treated with 0.025, 0.05, 0.1, 0.2, 0.5, 1.0, 5.0, or 10.0 mM H2O2 for 24 h and cellular viability was assessed. The 0.05 and 0.2 mM H2O2 concentrations were further examined at shorter time intervals: 0, 3, 6, 9, and 12 h. The H2O2 concentration of 0.05 mM was chosen as an OCH inducer and it was compared in some experiments with 0.2 mM H2O2, a nonhormetic condition (NO-H).

Cellular viability After H2O2 treatment cell viability was quantified using the trypan blue assay, counting the living cells in an hemocytometer under a phase-contrast optical microscope, as described previously [20].

Total RNA was extracted from H2O2-treated cells with 1 ml Trizol reagent (Invitrogen, Carlsbad, CA, USA) as previously described [42]. RNA integrity was confirmed by agarose gel electrophoresis. Total RNA from each sample was reverse-transcribed into cDNA, using a Superscript preamplification system (Invitrogen) according to the manufacturer's instructions. PCR was performed with the following primers: bcl-2 (amplification product, 250 bp), 5′-CGGCTTTGCAGAGATGTCCA-3′ (forward) and 5′-ATGCCGGTTCAGGTACTCAG-3′ (reverse); bax (amplification product, 230 bp), 5′-GAACAGATCATGAAGACAGG-3′ (forward) and 5′GCAAAGTAGAAGAGGGCAAC-3′ (reverse); cyclophilin (used as a housekeeping control; amplification product, 450 bp), 5′-CCCCAGCGTGTTCTTCGACAT-3′ (forward) and 5′-GCTGGTCTTGCCATTCCTGGA-3′ (reverse). The reaction was completed in 5 μl PCR buffer, 1.5 μl 50 mM MgCl2, 1 μl 10 mM dNTP master mix, 0.3 μl 20 mM cyclophilin primer, 1 μl 20 mM Bcl-2 or Bax primer, 1 U GoTaq polymerase (Promega, Madison, WI, USA), 5 μl retrotranscription product, and 34.6 μl ultrapure water for PCR. PCR amplifications were performed as follows: 94 °C for 5 min (hot start) and 35 cycles of denaturation at 94 °C for 30 s, annealing at 57 °C for 30 s, elongation at 72 °C for 30 s, with a final step at 72 °C for 3 min. The PCR products were electrophoresed in 1% agarose gels containing 0.05 mg/ml ethidium bromide. The mRNA expression was quantified using a Kodak IMAGEN GEL DOC, with Kodak 3.1 software,

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and was standardized to the cyclophilin housekeeping gene signal to correct any variability in gel loading. Data were expressed as arbitrary units.

fection Bcl-2 expression was verified by Western blot as previously described. Western blot analysis

Bcl-2 inhibition by HA14-1 L929 cells were seeded at density of 5 × 103 cells per well on a 48well plate (Corning). After 24 h, the cells were exposed to 50 μM H2O2 (OCH) or 200 μM H2O2 (NO-H) dissolved in the culture medium, for 9 h at 37 °C and 5% CO2. Subsequently, the cells were exposed again to the oxidative stressor (200, 300, or 400 μM H2O2) for another 3 h, and cellular viability was determined. The experimental design for this assay is represented in the inset on Fig. 2. To determine Bcl-2 participation, the Bcl-2 inhibitor HA14-1 (Calbiochem, La Jolla, CA, USA) [43] was used. HA14-1 (10 μM in 0.2% dimethyl sulfoxide) was added to the culture medium 4 h before H2O2 pretreatment. Bcl-2 silencing L929 were grown in a 35-mm cell culture dish to 30% confluence, and then the medium was changed to an antibiotic and serum-free medium and OPTI-MEM, 1 ml/dish (Invitrogen). After 24 h, the cells were transfected with 100 pmol/dish Bcl-2 siRNA (SC-29215) using the Lipofectamine RNAiMAX reagent (13778-075) according to the manufacturer's protocol (Invitrogen). Twenty-four hours after trans-

Treated cells were trypsinized and resuspended in lysis buffer (50 mM Tris–HCl, pH 8.0, 120 mM NaCl, 0.5% NP-40, 100 mM NaF, 0.2 mM NaVO3, 1 μg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin). Cell homogenates were incubated at 4 °C for 5– 10 min and centrifuged at 22,000 g, 4 °C, for 20 min. Protein concentration in the supernatant was determined using a commercial Bradford reagent (Bio-Rad, Hercules, CA, USA) [44]. Cell lysates were separated on 13% SDS–PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Invitrogen) and probed with anti-Bcl-2, anti-Bax, anti-GST, or anti-γGCS (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Membranes were washed three times with TBS–Tween and incubated with a horseradish peroxidase-conjugated anti-mouse IgG secondary antibody (Pierce, Rockford, IL, USA) for 1 h. After three consecutive washes, the blots were developed using a commercial chemiluminescence reagent (Supersignal; Pierce). Nrf-2 nuclear translocation Nrf-2 nuclear translocation was assessed by two methodologies, the first determining Nrf-2 subcellular localization after OCH, either in

Fig. 1. OCH establishment. (A) L929 oxidative survival limit after 24 h treatment with various H2O2 concentrations (0–10 mM). (B) Cell survival during a time course (0–12 h) of 50 (light gray bars) and 200 μM (dark gray bars) H2O2 treatment. (C) Cellular redox homeostasis calculated as GSH/GSSG ratio determined as described under Experimental procedures after 50 and 200 μM H2O2 treatment for 9 h. (D) DNA damage quantified as the molar ratio of 8-oxodGuo to dGuo. DNA was obtained from cells treated with 50 or 200 μM H2O2 for 9 h as described under Experimental procedures. Each point represents the mean ± SD of nine determinations performed in three independent experiments. Statistical significance with respect to control cells: *p b 0.05, **p b 0.001; ***p b 0.00001.

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the nuclear or in the cytoplasmic fraction, by Western blot and the second by performing immunofluorescence experiments. Both methodologies are described below. Nrf-2 subcellular localization L929 cells (5 × 106) were treated with either 300 μM H2O2 (for 15, 30, 45, 60, 90, or 120 min) or 50 μM H2O2 (for 8.5, 10, 11, and 12 h). After treatment, nuclear and cytoplasmic proteins were obtained as described by Gómez-Quiróz et al. [39]. Cytoplasmic and nuclear proteins were independently separated on 13% SDS–PAGE and transferred to a PVDF membrane as described before. Membranes were blocked and incubated with a monoclonal anti-Nrf-2 antibody (Santa Cruz Biotechnology) for 2 h. The membranes were washed and incubated with a horseradish peroxidase-conjugated anti-mouse IgG secondary antibody (Pierce) Blots were developed using a commercial chemiluminescence reagent (Supersignal; Pierce). Equal loading was demonstrated by probing the same membranes with anti-actin antibody (donated by Dr. A. Hernández, CINVESTAV, IPN México) for cytoplasmic proteins and histone H1 antibody (NeoMarker, Fremont, CA, USA) for nuclear proteins. Immunofluorescence experiments Treated L929 cells were fixed and then incubated in blocking buffer. Cells were washed and incubated with the primary antibody anti-Nrf-2 (Santa Cruz Biotechnology), followed by another incubation with the secondary antibody (FITC–anti-rabbit, dilution 1:200) and with propidium iodide (10 ng/ml), to stain the DNA and mark the nucleus. Cells were mounted with fluorescence mounting medium (Dako Cytomation, Glostrup, Denmark). Images were obtained with a confocal microscope (LSM-META-Zeiss Axioplan).

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OH, USA) and [γ-32P]ATP (3000 Ci/mmol; MP Biomedical, Irving, CA, USA) and purified using Bio-Spin 30 chromatography columns (BioRad). The reaction mixture contained nuclear protein extract (20 μg) in 5 μl of incubation buffer (50 mM Tris–HCl, pH 7.5, 200 mM NaCl, 5 mM EDTA, 5 mM β-mercaptoethanol, 20% glycerol, 1 μg dI–dC) and 32 P-labeled probe. In competition experiments, 100-fold molar excess of unlabeled oligonucleotide was included in the reaction mixture 5 min before addition of the labeled probe. For the supershift assay, a 1:2 concentration of monoclonal anti-Nrf-2 antibody (Santa Cruz Biotechnology) was added to the reaction mixture for 30 min. The reactions were electrophoresed on 6% polyacrylamide native gels. The gels were exposed in a Storage Phosphor Screen (Amersham Bioscience, Arlington Heights, IL, USA) and after 24 h were analyzed in a variable-mode imager (Typhon 9400; Amersham Bioscience) using the software Image Quant TL (Amersham Bioscience). Measurement of ROS production Intracellular ROS production was measured using the fluorescent dichlorofluorescein (DCF) assay [46,47]. L929 cells (1× 106) were treated for OCH with 50 μM H2O2 for 8.5 and 10 h. After that time the cells were trypsinized and loaded with 4 μg/ml 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA; Molecular Probes, Eugene, OR, USA) for 15 min in the dark to allow the intracellular deesterification of H2DCFDA to H2DCF. The cells were centrifuged at 800 g for 3 min to eliminate the unincorporated H2DCFDA, and the subsequent oxidation of this substrate by the intracellular ROS into the fluorescent DCF was quantified with a flow cytometer (FACScan; Becton–Dickinson, San Jose, CA, USA), using an excitation wavelength of 480 nm, an emission wavelength of 520 nm, and CellQuest software for data analysis.

Electrophoretic mobility shift assay (EMSA) Data analysis Nuclear extracts were prepared with Igepal CA-630 according to Gómez-Quiroz et al. [39]. Protein concentration was determined in the supernatant using a commercial Bradford reagent (Bio-Rad) [44]. Nrf-2–DNA binding activity was assayed using the Nrf-2 consensus oligonucleotide: 5′-TTTTCTGCTGACTCAAGGTCCG-3´ [45] (Promega). The probe was labeled with T4 polynucleotide kinase (USB, Cleveland,

Data are reported as the means ± SD for at least three independent experiments performed in triplicate. The ANOVA followed by the Tukey variance analysis was used to compare data. A 0.05 level of probability was used as a minimum criterion of significance.

Fig. 2. Cell survival against severe oxidative treatment after OCH. Cell survival was determined after 3 h of severe oxidative treatment (200, 300, or 400 μM H2O2) after OCH or NO-H. The inset represents the experimental design used to induce OCH. OCH, oxidative conditioning hormesis (50 μM H2O2, 9 h); NO-H, nonhormetic condition (200 μM H2O2, 9 h); NT, nontreated. Each point represents the mean ± SD of nine determinations performed in three independent experiments. Statistical significance with respect to control cells: *p b 0.05, **p b 0.001, ***p b 0.00001. &Different with respect to control and control + H2O2 cells; #different from all others.

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Results Oxidative conditioning hormesis induction To establish the oxidative survival limit, L929 fibroblasts were subjected to increasing concentrations of H2O2 for 24 h. As shown in Fig. 1A, 200 μM was the highest concentration at which cell survival was permissible (15%), and at 50 μM 42% of the population survived (Fig. 1A). These two concentrations (50 and 200 μM) were chosen, and viability was further examined at shorter times (0–12 h), in which 100% of the L929 fibroblasts survived after 9 h 50 μM H2O2 treatment, and 48% of cells survived after the same exposure time to 200 μM H2O2 treatment (Fig. 1B). Fig. 1C shows that redox cellular homeostasis in L929 cells treated with 50 μM H2O2 was not different from that obtained for control untreated cells, in contrast to the 200 μM H2O2 treatment, which decreased GSH by 32% (p b 0.05). No DNA oxidative damage was found when cells were treated with the lower H2O2 concentration, whereas an increase of 65% of 8-oxodGuo adducts was detected for the 200 μM H2O2 treatment (p b 0.05; Fig. 1D). Because these results correlate with the survival rate shown in Fig. 1B, 50 μM H2O2 for 9 h was chosen as the most advantageous to study OCH, and it was compared in some experiments with a nonhormetic condition of 200 μM H2O2. To verify the hormetic physiological effect and actual cell protection, L929 cells exposed to either 50 (OCH) or 200 μM H2O2 (NO-H) were further subjected to a severe oxidative insult of 200, 300, or 400 μM H2O2 for another 3 h (experimental design in Fig. 2 inset). Fig. 2 shows the percentage of living cells after those treatments. OCH cells presented a statistically significant survival rate of 80% after 200 μM H2O2 treatment and 68% after 300 and 400 μM H2O2, in comparison with nonconditioned control cells (p b 0.001). Only a 10–15% survival rate was observed when the cells were treated with those same concentrations after the 200 μM NO-H conditioning (p b 0.00001).

Bcl-2 induction in response to OCH To determine if Bcl-2 expression increases during OCH as a survival response against low oxidative insults, cells were treated with 50 μM H2O2 for diverse periods of time and Bcl-2 expression was determined. The maximal expression was observed at 9 h (Fig. 3A), coinciding with the hormetic response. Fig. 3B shows that when cells were treated with higher H2O2 concentrations (100, 150, 200 μM) Bcl-2 expression was not induced, suggesting that the cells promote this protein expression only in response to mild oxidative changes. To determine if this response was part of the survival reaction, Bax expression was also determined under the same experimental conditions (50, 100, 150, 200 μM H2O2 for 9 h). Our results indicated that only the 200 μM H2O2 treatment (the highest concentration assessed) induced Bax mRNA expression in a statistically significant manner (Fig. 3B). To find out whether these mRNA increments were effectively translated into proteins, Western blots were performed with protein from cells treated with 50 or 200 μM H2O2 for 9 h. Fig. 3C shows that Bcl-2 increased after the 50 μM treatment but it remained unchanged after 200 μM treatment, and conversely, Bax did not change with the mild treatment, but was augmented after the severe treatment. These changes were statistically significant (p b 0.05) according to the densitometric analysis (Fig. 3C) and were Fig. 3. Bcl-2 induction during OCH. (A) Bcl-2 mRNA was assessed for the period of 0 to 12 h of OCH as described under Experimental procedures. (B) Bcl-2 mRNA (dark gray bars) and Bax mRNA (light gray bars) were determined after 9 h of 50, 100, 150, and 200 μM H2O2 treatment. (C) Representative blot of Bcl-2 and Bax after 9 h treatment. Densitometric analysis was normalized against the actin control. Bax is represented in the graph by the light bars and Bcl-2 by the dark bars. Each point represents the mean ± SD of three determinations performed in independent experiments. Statistical significance with respect to controls: *p b 0.05, **p b 0.001.

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used to obtain the Bcl-2/Bax index, which was 1.120 ± 0.038 for the 50 μM treatment (OCH) and 0.798 ± 0.056 for the 200 μM treatment. A Bcl-2/Bax index ≥ 1.0 might suggest cellular survival, whereas Bcl2/Bax ≤ 1.0 might imply cellular death. To validate the Bcl-2/Bax index, the living cells remaining after both treatments were scored, and as expected, there was not a significant difference between control and OCH cell survival, but only 45–50% of cells survived after 9 h 200 μM H2O2 treatment (data not shown).

Bcl-2 participates during OCH To address whether Bcl-2 indeed participated during OCH, two strategies were utilized. The first was silencing of Bcl-2 expression with siRNA, and the second was using the small organic compound HA14-1, which strongly inhibits Bcl-2 by preventing the interactions Bcl-2/Bax and Bcl-2/Bim [48]. The inset in Fig. 4 shows that when 100 and 150 pmol siRNA were used, almost all Bcl-2 expression was abolished. Therefore 100 pmol was used to silence Bcl-2 in the OCH experiment. According to the experimental design, L929 cells were pretreated for 4 h with the Bcl-2 inhibitor HA14-1 and then conditioned for 9 h with 50 μM H2O2 (OCH) and then subjected again to 300 μM H2O2 for another 3 h. In regard to the silenced cells, 24 h after silencing of Bcl-2, the cells were treated in exactly the same way as described before except for the HA14-1 pretreatment. The results presented in Fig. 4 reveal that the protective effect previously observed was abrogated when Bcl-2 was inhibited with HA14-1 (black bar, p b 0.0001) or when it was silenced (striped bar, p b 0.001), demonstrating the importance of Bcl-2 during the hormesis response. It is worth noticing that the pretreatment with HA14-1 decreased cell survival by 24% prior to any further treatment, confirming Bcl-2 participation in cellular homeostasis. Because the inhibition with HA14-1 exhibited the same behavior as the Bcl-2-siRNA in abrogating the Bcl-2 effect, further experiments were done only with HA14-1.

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Bcl-2 induces Nrf-2 nuclear translocation as part of the hormetic response During hormesis, the adaptive response typically conditions the affected biological system to display a heightened resistance to a subsequent and more massive exposure by enhancing repair and defense systems. Nrf-2 is a transcription factor that responds to redox changes and induces the expression of antioxidant enzymes and phase II proteins. Therefore, Nrf-2 nuclear translocation was evaluated in the first 120 min of the H2O2 preconditioning, as a primary reply to the oxidative treatment (Fig. 5A), and then at longer times during OCH (7.5– 10 h), as a response associated with Bcl-2 increment (Fig. 5B). Fig. 5A shows that major Nrf-2 nuclear translocation is attained very early after the H2O2 preconditioning (between 15 and 30 min), and then it decreases, suggesting that Nrf-2 might migrate to the nucleus as part of a primary response to the change in steady-state ROS levels, but it abandons the nucleus or it is degraded after a short time. This result was confirmed by the immunofluorescence experiments in which Nrf-2 was found in the nucleus at 30 min (Fig. 6, row 2) and then outside the nucleus at 2 h (Fig. 6, row 3). To determine if Bcl-2 increase was able to activate Nrf-2 again, nuclear translocation was evaluated for a second time from 7.5 to 10 h of OCH. As illustrated in Fig. 5B, maximal Nrf-2 nuclear translocation was observed at 8.5 h, correlating very closely with the highest increase in Bcl-2 at 9 h. The immunofluorescence experiments also showed Nrf-2 presence in the nucleus at 8.5 h (Fig. 6, row 4), whereas it was outside again, at the cytoplasm, at 10 h (Fig. 6, row 5). To verify whether Nrf-2 nuclear translocation is associated with Bcl-2 increase, L929 cells were pretreated with Bcl-2 inhibitor before OCH. The results in Fig. 5C show that when the cells were pretreated with HA14-1, Nrf-2 nuclear translocation was abrogated. Plateletderived growth factor (PDGF) and H2O2 were used as positive controls for Nrf-2 nuclear translocation. Last, to confirm that when Nrf-2 was translocating into the nucleus, it was actually binding to its DNA consensus sequence, an EMSA was performed at intervals from 1 to 10 h. Fig. 7A shows that at 1 h Nrf-2 binds to the DNA, correlating with the time of the first

Fig. 4. Bcl-2 participation in cell survival during OCH response. Cell survival was determined after 3 h of severe oxidative treatment (300 μM H2O2) after OCH and compared to survival of cells that were pretreated with the Bcl-2 inhibitor HA14-1 4 h before OCH (black bars) or with Bcl-2-siRNA cells (striped bars). The lipo bar stands for the Lipofectamine transfection control. The inset is a representative Western blot performed to verify effective Bcl-2 silencing. Each point represents the mean ± SD of six determinations performed in three independent experiments. Statistical significance with respect to control cells: **p b 0.001, ***p b 0.0001. &Different with respect to control and OCH cells.

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Fig. 5. Bcl-2 correlates with Nrf-2 nuclear translocation. (A) Nrf-2 nuclear translocation was determined by quantifying nuclear and cytosolic Nrf-2 by Western blot as described under Experimental procedures at early times during OCH (0–120 min). (B) Nrf-2 nuclear translocation was determined as mentioned before at a period of time that correlates with Bcl-2 maximal expression (7.5–10 h) during OCH. (C) Nrf-2 nuclear translocation determined at 8.5 h during OCH. 0.3 mM H2O2 for 30 min and 30 ng/ml PDGF for 1 h were used as positive controls and 10 μM HA14-1 was used to inhibit Bcl-2. N, nuclear (black bars); C, cytosolic (white bars). The blots are representative of three independent experiments each. The densitometric analyses were normalized against histone H1 (nuclear) and actin (cytosol) controls.

translocation in response to the oxidative treatment, and it decreases with time. Nrf-2 binds to the DNA a second time at 8.5 h; this second translocation is the one that has been associated with Bcl-2 induction and was abrogated when HA14-1 was used. At 10 h no Nrf-2 DNA binding was observed. Fig. 7B shows the supershift, corroborating that Nrf-2 was indeed the protein assessed. The results presented here indicate that Nrf-2 is activated twice during the OCH response. The first time is almost certainly a consequence of the change in the redox state due to the H2O2 treatment, and the second is probably associated with Bcl-2 increment, as part of the survival response sustained by Bcl-2.

GST and γGCS increment is sustained at longer time courses during OCH, probably because of Bcl-2 participation The proteins GST and γGCS were evaluated as part of the antioxidant defense system activated by Nrf-2 through their AREs. Both enzymes increased significantly (p b 0.05) after 3 h preconditioning and continued their increase until 9 h (data not shown). GST and γGCS protein levels were determined at longer times after OCH (Fig. 8). GST sustained its elevated levels up to 15 h and increased to higher levels on reexposure to severe oxidative stress, whereas γGCS did not, perhaps because no GSH de novo synthesis was needed after that time.

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Fig. 6. Nrf-2 nuclear translocation by immunofluorescence. Representative images obtained as described under Experimental procedures with a confocal microscope (LSM-METAZeiss Axioplan 2) imaging at 30× original magnification are shown. Nrf-2 was dyed with FITC and propidium iodide was used to detect the nuclear area. L929 cells were treated with 50 μM H2O2 for 30 min (row 2), 2 h (row 3), 8.5 h (row 4), or 10 h (row 5). Cells in row 1 did not receive any treatment (NT).

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Fig. 7. Nrf-2 nuclear binding. Nrf-2 nuclear binding was determined by EMSA as described under Experimental procedures. (A) Nrf-2 translocation after the oxidative treatment at 1, 2, 4, 6, 8.5, and 10 h. HA14-1 was used only for the 8.5-h point, in column 8. The cold probe (CP) was performed 1 h after H2O2 treatment using 50% unlabeled probe and 50% labeled probe. (B) NT, nontreated. The cold probe (CP) was performed using 90% unlabeled probe and 10% labeled probe. In the last column a Nrf-2 supershift was performed. Representative gels from two independent experiments are shown.

When OCH cells were pretreated with HA14-1 no induction of GST or γGCS was observed (Figs. 8A and B), therefore suggesting that Bcl-2 is necessary for the expression of these enzymes. The increment found in GST and γGCS during OCH correlates with the contribution of GSH as part of the antioxidant response and suggests that the expression of these enzymes might be part of the hormesis response mediated by Nrf-2. Bcl-2 overexpression induces low ROS production and buffers oxidative stress To obtain more information on Bcl-2 participation in hormesis response and Nrf-2 activation, we determined ROS production by flow cytometry at 8.5 and 10 h after OCH. As shown in Fig. 8C, at 8.5 and 10 h, no statistically significant change was observed. Interestingly, when L929 cells were pretreated with the inhibitor HA14-1 before OCH, ROS levels increased, supporting the idea that Bcl-2 modulates ROS levels (black bar in Fig. 8C). Discussion The term hormesis has been recognized as accepted terminology to unify and describe the main mechanism that preconditioning and adaptive responses have in common: exposure to low levels of stress will activate existing cellular and molecular pathways that will enhance the ability of cells and organisms to withstand increased levels of stress [1–8]. Bcl-2 has been proposed here as a possible mediator or facilitator of hormesis because of its known cytoprotec-

tive and antioxidant functions as well as its antiapoptotic role [11,12,14–16,49]. Although Bcl-2 effects have been extensively studied, the exact mechanism by which they take place is still not well understood. These results suggest a sequence of causal events related to increase in Bcl-2 expression, induction of Nrf-2 activation, and sustained expression of cytoprotective proteins such as GST and γGCS. We have established a model to study OCH response by conditioning the cell line L929 for 9 h with 50 μM H2O2. These conditions did not induce either oxidative damage or oxidative imbalance, i.e., the GSH/GSSG ratio was sustained. Most notably, cells conditioned with 50 μM H2O2 for 9 h manifested a significant statistical survival rate of 70–80% after severe H2O2 treatment. The induction of enzymes devoted to reestablishing homeostasis is a central feature of the hormetic response. Hence, two important enzymes in the GSH antioxidant system were analyzed: γGCS, the key enzyme in GSH synthesis [50], and GST, which is crucial during detoxification and phase II response [51,52]. The genes encoding γGCS and GST are regulated by the ARE, which is a cis-acting regulatory element of genes encoding phase II detoxification enzymes and antioxidant proteins. It has been reported that Nrf-2 regulates a wide array of ARE-driven genes in various cell types [25–28], including γGCS and GST. Here we found that the increase in both enzymes (γGCS and GST) observed at 9 h was apparently enough to protect the cells against a major oxidative insult, as demonstrated by the survival rate observed in Fig. 2. Calabrese and others [2,6] have suggested that the overcompensation or stimulation during the hormetic response should be modest, with a maximum of 30 to 60%

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Fig. 8. Antioxidant response after OCH and oxidative severe treatment. (A) GST and (B) γGCS were determined by Western blot as described under Experimental procedures. Densitometric analyses were normalized against the actin control. Each point represents the mean ± SD of three determinations performed in independent experiments. Statistical significance with respect to control: *p b 0.05, **p b 0.001. (C) ROS production determined by flow cytometry using DCF as described under Experimental procedures at 8.5 and 10 h of OCH. H2O2 (0.3 mM for 30 min) and PDGF (30 ng/ml for 1 h) were used as positive controls and 10 μM HA14-1 was used to inhibit Bcl-2. Each point represents the mean ± SD of nine determinations performed in three independent experiments. Statistical significance with respect to control: *p b 0.05.

above the controls, because the goal of hormesis is to reestablish the homeostatic condition, in accordance with our data. During OCH, these enzymatic increases might function as a priming for a major oxidative insult. The Nrf-2–ARE pathway is important in the cellular antioxidant defense system [25–30,34]. Nrf-2 is a ubiquitous cytosolic protein that is continuously degraded during cellular homeostasis. However, in response to an increment in the oxidative state, Nrf-2 is released from its repressor and translocated into the nucleus. Nrf-2 is known to induce γGCS and GST expression [53]; here we found that the increase in these enzymes correlates with Nrf-2 activation, because Nrf-2 nuclear translocation was found at early times after OCH, coinciding with the rapid Nrf-2 activation as reported by Kaspar and co-workers [26]. Nrf-2 phosphorylation is a key event in the control of mediated antioxidant response, providing for both its activation and its eventual degradation [32,54].

The hypothesis that Bcl-2 might play a role during the OCH response was based in the original idea by Hockenbery et al. [14], who suggested that Bcl-2 increases cellular redox capacity and protects cells against death induced by oxidants. The previous hypothesis is supported by the findings of many investigators that demonstrated that Bcl-2 overexpression protects cells against oxidant-mediated damage [15–20,35,36], through elevation of antioxidant defense mechanisms, particularly the GSH system, as observed by our group and others [16,17,56]. It has been suggested that Bcl-2 induces mitochondrial ROS liberation [35,36], even though the actual mechanism is currently unknown. Therefore it was interesting to study the mechanistic interaction between Bcl-2 and Nrf-2 in support of a possible survival pathway. Interestingly, Bcl-2 induction to maintain cell survival was also found in other hormetic responses such as in pharmacological conditioning of bone marrow-derived rat mesenchymal stem cells

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treated with trimetazidine and then exposed to oxidative lethal conditions [57] as well as against apoptosis induced by H2O2 in pretreated PC12 cells [55]. Bcl-2 was also induced as part of the neuroprotective effect of cycloheximide [58]. To address whether Bcl2 increment also encompasses antioxidant properties related to the hormetic response in this model, cells that were known to present a hormetic behavior and survival after a major oxidative insult were pretreated with HA14-1 or silenced with Bcl-2 siRNA, and in both situations the protective effect was abrogated, supporting the idea of Bcl-2 participation. These experiments also support the fact that HA14-1 is an appropriate Bcl-2 inhibitor as previously described [45,59,60]. Here we found that Nrf-2 translocates into the nucleus at early times after OCH and then at 8.5 h. The first Nrf-2 activation at 1 h might be a response to the 50 μM H2O2 treatment and would not be related to further increases in Bcl-2. The Bcl-2 increment as a response to this conditioning might be worth exploring further. Interestingly, when Nrf-2 nuclear translocation was screened again from 7.5 to 10 h after the OCH, which was the range of time in which Bcl-2 maximal endogenous overexpression was obtained, the major Nrf-2 nuclear translocation was observed at 8.5 h, correlating very closely with highest Bcl-2 overexpression at 9 h. These results suggest that Bcl-2 increment could modify cellular redox state and induce antioxidant response throughout Nrf-2 activation. When cells were pretreated with HA14-1, this second Nrf-2 nuclear translocation was also abrogated. Despite the fact that our results do not reflect a significant change, neither in redox state (GSH/GSSG) nor in ROS levels (cytometry with H2DCF), Nrf-2 activation suggests a change in redox balance that was undetectable by the analytic determinations performed. In contrast to Nrf-2 activation through variations in redox state, Nrf-2 activation can be modulated by posttransductional modifications such as serine– threonine phosphorylation by several kinases such as phosphatidy-

linositol 3-kinase, protein kinase C, c-Jun N-terminal kinase, or the extracellular signal-regulated kinase. The phosphorylation by these enzymes apparently facilitates Nrf-2 dissociation from Keap-1 and Nrf-2 nuclear translocation and transcriptional activation [61,62]. Nevertheless, the ROS increment found when Bcl-2 was inhibited with HA14-1 (Fig. 8C) suggests an oxidative ongoing buffering effect dependent on Bcl-2. If Bcl-2 is able to sustain the hormesis response by activating Nrf-2, it must be reflected in the enzymatic content within the cell. Our results showed that mainly GST sustains its high levels up to 15 h after OCH. The increment was higher in those cells that were challenged with a major oxidative insult and was abrogated when HA14-1 was used, in support of the concept that Bcl-2 might help to sustain the hormetic response. Our data are in agreement with the fact that Bcl-2 expression induces protection against the depletion of cellular GSH [63,64]. In conclusion, low doses of ROS probably increase Bcl-2 expression as part of a survival response that modulates redox state, activating the Nrf-2–ARE pathway and thus contributing to sustaining the OCH response (Fig. 9). However, this might not be the only mechanism by which Bcl-2 physiological overexpression contributes to GSH increment and cell survival. Bcl-2 has been shown to directly interact with GSH via the BH3 groove, regulating the mitochondrial GSH pool [17]. Mitochondrial GSH has been demonstrated to be essential for cell functioning and survival [65]. Our data document a mechanistic relationship between Bcl-2 and Nrf-2 as part of the cellular survival response to a mild oxidative insult during OCH. This is important because Bcl-2 increment and Nrf-2 sustained activation have been related to immortalization and tumorigenesis [66,67]. Because numerous cancer chemotherapeutic agents generate ROS as a manifestation of their toxicity, it is essential to consider that cells might respond by increasing Bcl-2 and Nrf-2 survival pathways, contributing to either the adaptation/resistance process or the reactivation of oncogenic progression. This observation

Fig. 9. OCH response. Oxidative conditioning hormesis activates the Nrf-2 pathway to induce the antioxidant response and at the same time increases Bcl-2 levels, which contribute to sustaining cellular survival.

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is a valuable clinical tool for physicians, because an enhanced oxidative hormetic response involving Bcl-2 and Nrf-2 as a collateral side effect of these agents might play a role in the choice of these drugs. Our findings contribute to the understanding of the hormetic response mechanisms and concomitantly allow for new studies of the potential therapeutic uses of these survival proteins when applied to devastating entities associated with changes in cellular redox state as seen during ischemia–reperfusion, cancer, and aging.

Acknowledgments The authors thank Dr. Edith Cortés from UAMI for her help with the cytometry and Karina Chimal Ramírez, M. en C., from INR for her help with the confocal microscopy; M. in SC. J.C. Conde-Pérezprina for his help with the figures; Dr. A. Hernández from CINVESTAV for generously donating the actin antibody; Kipi Turok, Lic., for his help with the language; and Dr. Victor Fainstein from Methodist Hospital (Houston, TX, USA) for his editorial review. This work was supported by CONACyT Grants CB-2006-1-59659 and CB-2006-1-61544. A. Luna-López and F. Triana-Martínez are CONACyT scholarship holders.

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